CA2050340A1 - Method and diagnostic test kit for detection of autoimmune antibody - Google Patents
Method and diagnostic test kit for detection of autoimmune antibodyInfo
- Publication number
- CA2050340A1 CA2050340A1 CA002050340A CA2050340A CA2050340A1 CA 2050340 A1 CA2050340 A1 CA 2050340A1 CA 002050340 A CA002050340 A CA 002050340A CA 2050340 A CA2050340 A CA 2050340A CA 2050340 A1 CA2050340 A1 CA 2050340A1
- Authority
- CA
- Canada
- Prior art keywords
- coating
- antigen
- rnp
- antibody
- dsdna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 72
- 238000001514 detection method Methods 0.000 title claims abstract description 32
- 230000001363 autoimmune Effects 0.000 title claims abstract description 15
- 238000002405 diagnostic procedure Methods 0.000 title claims description 13
- 239000000427 antigen Substances 0.000 claims abstract description 91
- 108091007433 antigens Proteins 0.000 claims abstract description 91
- 102000036639 antigens Human genes 0.000 claims abstract description 91
- 230000003100 immobilizing effect Effects 0.000 claims abstract 5
- 108020004414 DNA Proteins 0.000 claims description 70
- 238000000576 coating method Methods 0.000 claims description 63
- 239000011248 coating agent Substances 0.000 claims description 61
- 102000053602 DNA Human genes 0.000 claims description 50
- 108010077055 methylated bovine serum albumin Proteins 0.000 claims description 45
- 239000005018 casein Substances 0.000 claims description 31
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 31
- 235000021240 caseins Nutrition 0.000 claims description 31
- 239000007787 solid Substances 0.000 claims description 29
- 101710163270 Nuclease Proteins 0.000 claims description 25
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 230000027455 binding Effects 0.000 claims description 17
- 230000009871 nonspecific binding Effects 0.000 claims description 14
- 210000000987 immune system Anatomy 0.000 claims description 6
- 108010042407 Endonucleases Proteins 0.000 claims description 5
- 238000003149 assay kit Methods 0.000 claims description 3
- 230000002411 adverse Effects 0.000 claims description 2
- 230000003429 anti-cardiolipin effect Effects 0.000 claims description 2
- 102000004533 Endonucleases Human genes 0.000 claims 2
- 230000001464 adherent effect Effects 0.000 claims 1
- 230000000593 degrading effect Effects 0.000 claims 1
- 238000002965 ELISA Methods 0.000 abstract description 21
- 238000003018 immunoassay Methods 0.000 abstract description 4
- 238000013096 assay test Methods 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 152
- 239000000523 sample Substances 0.000 description 66
- 239000003085 diluting agent Substances 0.000 description 52
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 51
- 239000002953 phosphate buffered saline Substances 0.000 description 51
- 238000003556 assay Methods 0.000 description 50
- 210000002966 serum Anatomy 0.000 description 49
- 239000008186 active pharmaceutical agent Substances 0.000 description 32
- 230000000694 effects Effects 0.000 description 32
- 239000000758 substrate Substances 0.000 description 32
- 238000012360 testing method Methods 0.000 description 31
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 29
- 238000011534 incubation Methods 0.000 description 24
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 24
- 238000002835 absorbance Methods 0.000 description 23
- 239000000872 buffer Substances 0.000 description 23
- 239000003153 chemical reaction reagent Substances 0.000 description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 21
- 108020004682 Single-Stranded DNA Proteins 0.000 description 19
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 15
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 15
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 14
- 239000012895 dilution Substances 0.000 description 13
- 238000010790 dilution Methods 0.000 description 13
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 11
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 10
- 201000003066 Diffuse Scleroderma Diseases 0.000 description 10
- 201000009594 Systemic Scleroderma Diseases 0.000 description 10
- 206010042953 Systemic sclerosis Diseases 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 230000009260 cross reactivity Effects 0.000 description 10
- 230000002583 anti-histone Effects 0.000 description 9
- 239000012888 bovine serum Substances 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 9
- 239000012530 fluid Substances 0.000 description 8
- 150000002500 ions Chemical class 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 241000283707 Capra Species 0.000 description 7
- 210000001124 body fluid Anatomy 0.000 description 7
- 239000010839 body fluid Substances 0.000 description 7
- 230000029087 digestion Effects 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 7
- 239000003446 ligand Substances 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- 239000004033 plastic Substances 0.000 description 7
- 229920003023 plastic Polymers 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000012224 working solution Substances 0.000 description 7
- 208000023275 Autoimmune disease Diseases 0.000 description 6
- 208000003250 Mixed connective tissue disease Diseases 0.000 description 6
- 239000004793 Polystyrene Substances 0.000 description 6
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- 230000002860 competitive effect Effects 0.000 description 6
- 239000012470 diluted sample Substances 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000001046 green dye Substances 0.000 description 6
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 229920002223 polystyrene Polymers 0.000 description 6
- 229910000160 potassium phosphate Inorganic materials 0.000 description 6
- 235000011009 potassium phosphates Nutrition 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 239000002981 blocking agent Substances 0.000 description 5
- LEOJDCQCOZOLTQ-UHFFFAOYSA-N dibutylcarbamothioyl n,n-dibutylcarbamodithioate Chemical compound CCCCN(CCCC)C(=S)SC(=S)N(CCCC)CCCC LEOJDCQCOZOLTQ-UHFFFAOYSA-N 0.000 description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 229940124272 protein stabilizer Drugs 0.000 description 5
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 5
- 229940033663 thimerosal Drugs 0.000 description 5
- 210000001541 thymus gland Anatomy 0.000 description 5
- 238000009007 Diagnostic Kit Methods 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000000951 immunodiffusion Effects 0.000 description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
- 108010039627 Aprotinin Proteins 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 241000222712 Kinetoplastida Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102000019040 Nuclear Antigens Human genes 0.000 description 3
- 108010051791 Nuclear Antigens Proteins 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 208000021386 Sjogren Syndrome Diseases 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 230000003172 anti-dna Effects 0.000 description 3
- 229960004405 aprotinin Drugs 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 201000004997 drug-induced lupus erythematosus Diseases 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000012898 sample dilution Substances 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 description 3
- 235000011330 Armoracia rusticana Nutrition 0.000 description 2
- 240000003291 Armoracia rusticana Species 0.000 description 2
- 241000205762 Crithidia luciliae Species 0.000 description 2
- 108010033040 Histones Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 108010007568 Protamines Proteins 0.000 description 2
- 102000007327 Protamines Human genes 0.000 description 2
- 101150089644 Rnls gene Proteins 0.000 description 2
- 108091006629 SLC13A2 Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 150000001540 azides Chemical class 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229950008679 protamine sulfate Drugs 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000163 radioactive labelling Methods 0.000 description 2
- 238000003118 sandwich ELISA Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000182988 Assa Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 241000222716 Crithidia Species 0.000 description 1
- 102000012677 DET1 Human genes 0.000 description 1
- 101150113651 DET1 gene Proteins 0.000 description 1
- 101000968267 Drosophila melanogaster Protein dachsous Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 241000252067 Megalops atlanticus Species 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 101710154508 Purine nucleoside phosphorylase 1 Proteins 0.000 description 1
- 101710084347 Purine nucleoside phosphorylase DeoD-type 1 Proteins 0.000 description 1
- 108010076622 SS-A antigen Proteins 0.000 description 1
- 108010004487 SS-B antigen Proteins 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 241000473945 Theria <moth genus> Species 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 108091032917 Transfer-messenger RNA Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003476 anti-centromere Effects 0.000 description 1
- 230000003460 anti-nuclear Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000002820 assay format Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000009266 disease activity Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 235000020030 perry Nutrition 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 208000005987 polymyositis Diseases 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000002764 solid phase assay Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- SRVJKTDHMYAMHA-WUXMJOGZSA-N thioacetazone Chemical compound CC(=O)NC1=CC=C(\C=N\NC(N)=S)C=C1 SRVJKTDHMYAMHA-WUXMJOGZSA-N 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Rehabilitation Therapy (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method and apparatus optimized as an autoimmune diagnostic sandwich assay test kit, which is capable of immobilizing antigen onto precoated support wells for the purpose of detection of autoimmune antibodies specific to such compounds by either ELISA (Enzyme Linked Immunosorbent Assay) or FIA (Fluorescent Immunoassay) techniques.
Description
WO 90/10~29 2~ 3~ Pcr/~s90/o1029 ,,.. ~ , .
IRETHOD AND DIAGNOSTIC TEST Kl~ FOR~;.', ;~;
DET1::TION OF AUTOIMMWNE ANTIBODY
BACKGROUN~ OF TIIE iNVENTlON
The immune system is the body's defense mechanism against foreign substances and5 invading microorganisms. The underlying operating principle of the immune system is a self/nonself recognition pattern. If the invader organism is recognized as not being part of the "se1f", then a defensive immune response is mounted against it. In the case of autoimmune diseases (See Figure 7) the immune system fails to properly recognize "sel~" and mounts a defense immune response against its own norrnal body components. Figure 7 is 10 a list of autoimmune diseases and the antigens associated with them.
Antibodies generated by the immune system to diverse tissue and cellular components have been used to diagnose and monitor autoimmune disease activity. These antibodies include anti-dsDNA, anti-RNP (ribonucleoprotein3, anti-DNP (Deoxynucleoprotein), anti-Cardiolipin, anti-histones (symptomatic of drug induced lupus), anti-Sm (Smith), anti-ENA (extractable 15 nuclear antigen), which are often found in patients with Systemic Lupus Erythematosus (SLE), anti-RNP, and anti-ENA are also symptomatic of Mixed Connective Tissue Disease (MCTD).
Other antibodies, such as anti-RNA (ribonucleic acicl), anti-Scl-70 (Scleroderma-70) are often found in patients with Progressive Systemic Sclerosis (PSS); and anti-SS-A and B (Sjogren's Syndrome Antibody A and B) which are frequently found in patients with Sjogren's Syndrome.
~o In SLE (a type of autoimmune disease) one of the antibodies produced reacts with DNA that is found widely distributed in cell nuclei in a multitude of body tissues. Formation of antibodies to double-stranded or native deoxyribcnucleic acid tanti-dsDNA), is relatively specific to SLE. Although other disorders, such as Mixed Connective Tissue Disease (MCTD), Drug Induced Lupus (DIL)1 Rheumatoid Arthritis, Sclerodermal PSS, and Sjogren 2~ Syndrome, produce similar clinical manifestations as SLE high levels of anti-dsDNA are seldom associated with these disorders. Therefore, detecting anti-dsDNA is useful in specifically diagnosing SLE. Anti-dsDNA levels correlate well with the disease activlty of the patient; thus making It a good monitoring tool.
Also in SLE one of the antibodies produced reacts with Sm antigen that is tound widely 30 distributed in cell nuclei in a mul!itude of body tissues. Formation of antibodies to Sm antigen is r~latively specHiG to SLE. Detecting anti-Sm antibodies which are specific for SLE
is useful in diaynosing SLE. Anti~Sm antibodies are frequently accompanied by another antibo~y anti-RNP. Antibodies directed against the RNP antigen are found in the absence :
.. .. .... ...... . . . . . .
~ . ~,, .
wo 90~1022g ' i'` ! -' ~ PCI'/US90/01029 2~ 2- `
of anti-Sm antibodies in MCTD, which is an aoverlap syndrome~ that combines features of SLE, PSS, and Polymyositis. Therefore, detecting anti-Sm/RNP antibodies is useful in diagnosing two different autoimmune diseases, SLE and MCTD.
Many different techniques and different diagnostic kits have been developed in the search for a standardized, accurate, rapid, and stable method for the detection of anti-dsDNA, anti-Sm, and anti-Sm/RNP antibociies. Most of the methods have been somewhat successful, but due to unacceptably high levels of cross-reactivity (such as with single-stranded DNA [ssDNA]~, the slow run time of the assays, and the short shelf iife of the kit components, no method is fully adequate. i=xamples of the limitations of the prior methods follow.
A number of techniques have been developed to detect antibodies to dsDNA, including immunofluorescent assays (FIA), radioimrnunoassay (RiA), and en~yme-linked immunosorbent assays (ELISA).
Firstiy, the RIA technique has been developed in a variety of formats to measure levels of anti-dsDNA in sera. A RIA diagnostic test kit (using the Farr technique) has been developed 1~ for commercial sale to clinical laboratories. This test precipitates the bound labelled DNA
which is then retained ~or counting. This test is sensitive to high levels of anti-DNA but has Iess specificity in lower levels of anti-dsDNA activity, thus resulting in false negatives.
Unfortunately, this test has a run time of approximately two hours and fifteen minutes, and a limited stable shelf life. Furthermore, this test kit has the same draw-backs which are inherent in all RlAs; the expense associated with radioactive rnaterial, which also have a !imited sheif life and can be potentially dangerous.
Secondly, Crithidia luciliae immunofluorescencs tests have been developed as diagnostic test , kits ior the detection of anti-dsDNA. This test kit is based on a staining method associated with the kinetoplast of the protozoan. A positive reaction is demonstrated by detection of specific fluorescence in the kinetoplast of most cells. The kinetoplast, within the protozoan, contains many other components besides dsDNA which can cross react with anti-dsDNA or other antibodies, thus rendering false positive results. This test is extremely subjective, as it i9 based on an individual's abiiity to recognize a positive fluorescent pattern. Fur~hermore, this rnethod, which has a run time of approximately two hours, has a lack of sensitivity in low levels of concentration of anti-isDNA resulting in false negatives. The false negative and false positive results make this method more useful when used together with other tests in clinical practice.
~ .
.; .. , , . : i , . .. ... .. . . . ... . .. . .. ... . .
WO 90~102~9 ;~ 3~ PCI'/US90/010~9 ~:.
IRETHOD AND DIAGNOSTIC TEST Kl~ FOR~;.', ;~;
DET1::TION OF AUTOIMMWNE ANTIBODY
BACKGROUN~ OF TIIE iNVENTlON
The immune system is the body's defense mechanism against foreign substances and5 invading microorganisms. The underlying operating principle of the immune system is a self/nonself recognition pattern. If the invader organism is recognized as not being part of the "se1f", then a defensive immune response is mounted against it. In the case of autoimmune diseases (See Figure 7) the immune system fails to properly recognize "sel~" and mounts a defense immune response against its own norrnal body components. Figure 7 is 10 a list of autoimmune diseases and the antigens associated with them.
Antibodies generated by the immune system to diverse tissue and cellular components have been used to diagnose and monitor autoimmune disease activity. These antibodies include anti-dsDNA, anti-RNP (ribonucleoprotein3, anti-DNP (Deoxynucleoprotein), anti-Cardiolipin, anti-histones (symptomatic of drug induced lupus), anti-Sm (Smith), anti-ENA (extractable 15 nuclear antigen), which are often found in patients with Systemic Lupus Erythematosus (SLE), anti-RNP, and anti-ENA are also symptomatic of Mixed Connective Tissue Disease (MCTD).
Other antibodies, such as anti-RNA (ribonucleic acicl), anti-Scl-70 (Scleroderma-70) are often found in patients with Progressive Systemic Sclerosis (PSS); and anti-SS-A and B (Sjogren's Syndrome Antibody A and B) which are frequently found in patients with Sjogren's Syndrome.
~o In SLE (a type of autoimmune disease) one of the antibodies produced reacts with DNA that is found widely distributed in cell nuclei in a multitude of body tissues. Formation of antibodies to double-stranded or native deoxyribcnucleic acid tanti-dsDNA), is relatively specific to SLE. Although other disorders, such as Mixed Connective Tissue Disease (MCTD), Drug Induced Lupus (DIL)1 Rheumatoid Arthritis, Sclerodermal PSS, and Sjogren 2~ Syndrome, produce similar clinical manifestations as SLE high levels of anti-dsDNA are seldom associated with these disorders. Therefore, detecting anti-dsDNA is useful in specifically diagnosing SLE. Anti-dsDNA levels correlate well with the disease activlty of the patient; thus making It a good monitoring tool.
Also in SLE one of the antibodies produced reacts with Sm antigen that is tound widely 30 distributed in cell nuclei in a mul!itude of body tissues. Formation of antibodies to Sm antigen is r~latively specHiG to SLE. Detecting anti-Sm antibodies which are specific for SLE
is useful in diaynosing SLE. Anti~Sm antibodies are frequently accompanied by another antibo~y anti-RNP. Antibodies directed against the RNP antigen are found in the absence :
.. .. .... ...... . . . . . .
~ . ~,, .
wo 90~1022g ' i'` ! -' ~ PCI'/US90/01029 2~ 2- `
of anti-Sm antibodies in MCTD, which is an aoverlap syndrome~ that combines features of SLE, PSS, and Polymyositis. Therefore, detecting anti-Sm/RNP antibodies is useful in diagnosing two different autoimmune diseases, SLE and MCTD.
Many different techniques and different diagnostic kits have been developed in the search for a standardized, accurate, rapid, and stable method for the detection of anti-dsDNA, anti-Sm, and anti-Sm/RNP antibociies. Most of the methods have been somewhat successful, but due to unacceptably high levels of cross-reactivity (such as with single-stranded DNA [ssDNA]~, the slow run time of the assays, and the short shelf iife of the kit components, no method is fully adequate. i=xamples of the limitations of the prior methods follow.
A number of techniques have been developed to detect antibodies to dsDNA, including immunofluorescent assays (FIA), radioimrnunoassay (RiA), and en~yme-linked immunosorbent assays (ELISA).
Firstiy, the RIA technique has been developed in a variety of formats to measure levels of anti-dsDNA in sera. A RIA diagnostic test kit (using the Farr technique) has been developed 1~ for commercial sale to clinical laboratories. This test precipitates the bound labelled DNA
which is then retained ~or counting. This test is sensitive to high levels of anti-DNA but has Iess specificity in lower levels of anti-dsDNA activity, thus resulting in false negatives.
Unfortunately, this test has a run time of approximately two hours and fifteen minutes, and a limited stable shelf life. Furthermore, this test kit has the same draw-backs which are inherent in all RlAs; the expense associated with radioactive rnaterial, which also have a !imited sheif life and can be potentially dangerous.
Secondly, Crithidia luciliae immunofluorescencs tests have been developed as diagnostic test , kits ior the detection of anti-dsDNA. This test kit is based on a staining method associated with the kinetoplast of the protozoan. A positive reaction is demonstrated by detection of specific fluorescence in the kinetoplast of most cells. The kinetoplast, within the protozoan, contains many other components besides dsDNA which can cross react with anti-dsDNA or other antibodies, thus rendering false positive results. This test is extremely subjective, as it i9 based on an individual's abiiity to recognize a positive fluorescent pattern. Fur~hermore, this rnethod, which has a run time of approximately two hours, has a lack of sensitivity in low levels of concentration of anti-isDNA resulting in false negatives. The false negative and false positive results make this method more useful when used together with other tests in clinical practice.
~ .
.; .. , , . : i , . .. ... .. . . . ... . .. . .. ... . .
WO 90~102~9 ;~ 3~ PCI'/US90/010~9 ~:.
3 ~
Thirdly, the immunological community has developed diagnostic test kits for the determination of anti-dsDNA levels in the standard ELISA format. Although a variety of these kits have been ~eveloped, the same limitations of stability, cross reactivity with ssDNA, and length of run time plague each assay. Many of these kits claim a low level of cross r~activity with ssDNA which should render good sensitivity to low, medium, and high levels of anti-dsDNA; however, as evidenced by the kit instructions, there is a lack of sensitivity in the sera sample ~,vhich are borderline positives. As a consequence, these kits cannot be us~d to accurately monitor patients who are borderline positives. In fact, most kits require that low positives must be retested to assure confidence in the results, thereby increasing the cost to ~0 the patient. Development ol an assay which is sensitive to all levels of anti-dsGNA can only be achieved by reducing non-specific binding by sueh components as ssDNA.
A number of techniques including immunodiffusion or Ouchterlony analysis have been developed to detect antibodies to Sm antigen and Sm/RNP antigen often called ENA(Extractable nuclear antigens). Examples of the limitations of the prior methods follow.
Firstly, the immunodiffusion technique has a long incubation time, typically 24 hours or more.
This time span is of course inconvenient for both the patient and the lab personnel.
Secondly, the results gathered from Ouchterlony analysis are subjective. This results in different lab technlcians reporting varied results based on individual data interpretation.
The imrnunological community has developed diagnostic test kits for the determination of !0 anti-Sm antibody levels in the standard ELISA format. These ELlSAs have shown certain limitations in stability, cross reactivity, and length of run time.
it is therefore an objective of the prasent invention to provide compositions, methods, articles, and a diagnostic tast kit for thc selective adsorption, or affixing of dsDNA, Sm antigen, er Sm/RNP antigen, or any af the various antigens to the autoimmune antibodies previously listed, to a pre-coated plate for the effective detection of the specific autoimmune antibodies present in sera or plasma. It is a further objective of this invention to overcome the aforemantioned sensitivity, subjectivity, stability, and shelf life disadvantages inherent in many of the previously dascribed assays. It is, moreover, also an objective of this invention to provid~ in the ~orm of a kit a noval, readily utilizabie maans for quantitative and qualitative O detection of anti-dsDNA, anti-Sm or anti-Sm/RNP. This will provide a method and apparatus for the purpose of c!inical detection of anti-dsDNA, or tha anti-Sm antibody, or the anti-:
''''''' " ' ~', ;' , ,, ' ' WO 90/10229 ~ P(~/US90/01029 ;~a~3~ 4- ~
Srn/RNP antibody, or any of the listed autoimmune antibodies listed, or the detection of the specific antibody for any other purpose associated with human or animal medical testing.
It is believed that with little modification the present invention can be used to detect anti-histone, anti-RNA, anti-SS-A, anti-SS-B, anti-Scl-70, and anti-DNP.
BRIEF SUMNIA~Y OF THE INVENTION
The present invention includes novel compositions, novel methods and articles for the direct selective absorption, adsorption or attachment, by whatever mechanism, of the anti-dsDNA, or the anti-Sm or the anti-Sm/RNP antibody, or any of the listed autoimmune antibodies, from the body fluid sample to the dsDNA, the Sm-antigPn, or the Srn-RNP antigen, or possibly for ~10 any of the autoimrnune antigens coated onto any suitable solid support, such as test tubes, plates or wells (hereinafter referred to as wells or microwells~, for the purpose of quantitative and qualitative identification. The present invention utilizes immobilized native DNA, Sm, Sm-RNP, or possibly for any listed autoimmune antigen from whatever source, which has an affinity for attachment of anti-dsDNA, anti-Sm, or anti-Sm/RNP, or the other antibodies of the listed autoimmune antibQdies.
This invention utilizes a sandwich ELISA format which includes pre-coated wells made of any suitable material such as plastic, glass, etc., and the various reagents and antibodies necessary to run a highly sensitive, 45 minute assay for the presence of anti-dsDNA, or for the presence of anti-Sm, or for the presence of anti-Sm/RNP.
The various reagents and the method of coating these reagents to the rnicrowells have been employed advantageously in the practice of the present invention to formulate a test which has minimized or eliminated the problems associated with previous assays. The coating protocol has advantageously utilized methylated Bovine Serum Albumin (mBSA) for two main purposes; namely, the mBSA provides a positively charged surface which enhances the adherence of the antigen such as dsDNA, Sm, and Sm/RNP to the polystyrene wells, and second to eliminate the binding of anti-histone antibodies, which can create false positive results.
The use of mBSA as a coating prior to the application of DNA antigen for use in solid phase assays for anti-dsDNA was flrist explored by R. Rubin (J. Immunology 63, 359-366 l1983]).
In the practice of this invention, protamine sulfate as well as other functionally equivalent substltutes have been found to be capable of forming a positiveiy charged surface; however, WO 90/10229 ;~ 4C~ P(~/US90/0~iO29 ,". ,` ` ; ' '; , " ,' ';," :.
~5-due to the reactivity of these coatings with other components such as ssDNA and anti-histone antibodies, mBSA is preFerred. It was highly surprisin~ to find that a coating of mBSA on a solid support underlying the DNA antigen, or the Sm antigen, or the Sm-RNP antigen provides the pre-coated wells with a consistently high level of reproducibility even over a 5 period of one year.
Methylated Bovine Serum Albumin is utilized for the previously mentioned reasons, the second of which is directly related to the specificity and sensitivity of these varlous assays.
The lack of good correlation between ELlSAs, RlAs and Crithidia luciliae staining procedures and other tests in some cases appears to be due to binding by anti-histone antibodies which 10 gives the ELISA an elevated antibody reading. The mBSA pre-coating appears to have alleviated this area of non-specific binding.
A second factor which contributes to the lack of sensitivity and non-specHicity is the cross reactivity. This is particularly a problem when detecting antibodies to dsDNA, because of the cross reactivity hetween ssDNA and anti-dsDNA. To eliminate the potential binding of the ~15 anti-dsDNA antibodies to ssDNA the microwells which have been coated with dsDNA were treated with an endonuclease, S, nuclease, which is an enzyme specific for the breakdown degradation of ssDNA and has no affect on dsDNA. Although other anti-dsDNA tests utilize S, nuclease ffor example see A.G. Tzioufas, Clinical and Experimental Rheumatology 5, 247-253 [1987]) their sensitivity levels are still substantially lower than acceptable levels for ~2û patients with borderline positives. The use of S, nuclease in a digestion buffer with an acid pH, combined wi~h the use of a mBSA pre-eoating eliminates ssDNA activity and anti-histone activity providing a highly sensitive assay which renders few ~alse positives. The S, nuclease is of course only necessary in the detection of anti-dsDNA antibodies and not in anti-Sm or anti-Sm/RNP antibody detection.
The shelf life and the reproducibility of results from the ELISA is associated not only with the mBSA pre-coating, and the S, nuclease treatment (in the anti-dsDNA kit), but also with the next two steps of forming the pre-treated wells; and tha addition of a hydrolyzed casein blocker and the drying.
A casein-type blocker has been used in various ELISA techniques ~Robert F. Bogt; J.
Imrnunological Methods, 101, 43-50 ~1987l) to block non-specific hinding to ptastic through a protein-plastic interaction. It is an unexpected realization that a coating of hydrolyzed casein blocker maintained a consistent inhibition of non-specific binding over an extended , . . .
, . . .. , . ; . .. ... ~ .
WO 90/10229 `~ ; PCl'/US9~/0102~
3~) 6- ~
period of time, when the wells were stored at 4 degrees C in a sealed plastic bag.
Although the direct mechanisms by which the drying process and the blocker increases the stability and shelf life of the wells is not fully understood, it is obvious that the storage time is increased by these processes. Little to no ssDNA, or loss of Sm or Sm-RNP antigen activity is generated by this method during the storage condltions in a period of up to one year.
A variety of differing blocking agents could be utilized which are ~unctionally equivalent to or chemically relatsd to the casein blocking agent. For example, BSA and porcine thyroglobulin, dried milk, whole goat serum, etc., however the most preferable is the hydrolyzed casein 0 (commercially available from Sigma) due to its high level of inhibition of non-specific binding and its storage stability.
' This invention's use of mBSA, the S, nuclease treatment (in the anti-dsDNA klt), combined with the casein blocker and the drying process have unexpectedly resulted in novel assays whlch are characterlzed by a low level of cross-reactivity, a high level of Inhibition of non-specific bindlng, and a long shelf life.
The pre-coated wells are then used to detect the presence of anti-dsDNA antibody, anti-Sm antibody, or the anti-Sm/RNP antibody in the sample. The plasma or serum samples are prepared with a sarnple diluent and are then assayed for their components by an immunoassay technique, the ELISA and the fluorescent immunoassay (FIA) formats being 0 the preferred methods, though it is possible to perform a RIA or a luminescent assay with little modification.
:, The assays depicted in the following examples have an approximate run time of 45 minutes.
Th~ wells, when exposed to the samples, are provlded with approximately 15 minutes at ;~ room temperature to allow the blnding of the antlbody to the antigen to go to completion.
5 Then the labelled goat anti-human antibodies are exposed to the wells and a similar 15 minute incubation at room temperature is provided for. If the enzyme is utilized a substrate ` can ba added (although this is not necessary) and 10 minutes is allotted for the production of the color. If a fluorescent marker is used on the goat anti-human antibody then no substrate is needed, therefore the run time is shortened by 10 minutes reducing it to 35 minutc-.
WO 90/10~29 ~5~3~3 PCI'/US90/01029 .; ", . ,,, ,", ,. . I
Subsequent qualitative and quantitative detection of the antibody is relatively simple if the forrnat is either an ELISA or a FIA. Numerous enzyme-conjugated antibodies and fluorescent-labelled antibodies specific for any of the immuno~3lobulin classes can be utilized in this invention. The quantitation of the antibodies present is accomplished by the related instruments. The ELISA technique utilizes a spectrophotometer, and the FIA technique utilizes a microfluorometer. Use of the ELISA techniques were flrst described by Engvall and Perlman ([1971] Irnmunochemistry 8, 871-874 and [1972} J. Immunology 109, 129-135), and The Enzyme Linked Immunosorbent Assay (ELISA) by Voller, A., Bidwell, D.E., and Bartlett, A., (1979) Dynatech Laboratories, Inc., Alexandria, Virginia, both of which are, in their totality, ; 10 incorporated herein by re~erence.
BRIEF DESCRIPTION OF THE GF~APH
Figure 1 - This graph depicts a standardized curve based on arbitrary units/ml. This curve was generated by using the ELISA ~ormat of this invention. The Center for Disease Control (CDC) ANA Human Reference Serum #1 has an antigen binding capacity of .59 micro grams 1~ DNA bound per ml of serum. This is equivalent to 100 AU per ml.
Figure 2 - This graph depicts a standardized curve obtained ~rom the data in Example 3.
Figure 3 - This graph depicts a standardized curve based on arbitrary RE~DS~ units/ml. This curve was generated by using the ELISA format of this invention. The Center for Disease Control (CDC~ ANA Human Reference Lot #82-001 #5 Serum ffor Sm antigen) was run on an immunodiffusion assay with median titer of 1:64. This standard was serially diluted with normal sera and graphed. The 1:1 or neat dilution was assigned arbitrarily a value of 400 RE~DS units.
:
Figure 4 - This graph dspicts the range of negative and positive human sera control values expected for the anti-Sm tast kit.
:, 2~ Figure 5 - This graph depicts a standardked curve based on arbitrary RE~DS ED units/ml. This curve was generated by using the ELISA format of this invention. The Center for Disease Control (CDC;) ANA Human Reference Lot ~82-001 ~5 Serum (for Sm antigen) and the ANA
Human Referencs Lot #82-011 #4 Serurn ffor RNP antigen) was run on an immunodiffusion assay with median titer of 1:64. An equal volume of reference sera #4 and ~5 are combined to 1Orm the standard. This .standard was serially diluted with normal sera an~ graphed. The .
:
. ~ .
WO 90/10229 ' PCT/llS90/01029 ~ 8 -1:1 or neat dilution was assigned arbitrarily a value of 140C RE~DS units.
Figure 6 - This graph depicts the range of negative and positive hurnan sera control values expected for the anti-Sm/RNP test kit.
Figure 7 - This charl shows autoimmune disease and their associated antigens which could possii~ly be detected by the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions are supplied for the purpose of clarifying the invention and are not intended to limit the scope of the invention:
Methylated Bovine Serum Albumin Soiution: Unless otherwise specified, is intended to mean a solution of 1 milliliter of water or PBS with 20 micrograms of methylated bovine serum Albumin (mBSA) dissolved in it. A substitute for mBSA is protamine sulfate or any other chemical or chemical process capable of producing a slighily positively charged coating which is evenly distributed over the surface of the microtitre well. (It is believed that the anti~
histone test may not ~mploy methylated bovine serum coating on the microtiter plate.) ~5 PBS Solution: A .01 molar solution ot buffer containing 1.43 9 potassiurn phosphate, dibasic, " .~5 g potassium phosphate, monobasic, and ~.5 g sodium chloride in one liter of water. The ' pH is 7.3 +/- .1.
. , ~, dsDNA Solution: Purified dsDNA solution from cali~ thymus was utilized. Altcrnative sources ~î of DNA include, but are not limited to, native linear DNA from E. Coli, and circular DNA from ~0 plasmid, virus, crithidia, and synthetic polynucleotides (poly (dA.dT)).
Sm Solution: Puriiied Sm antigen solution from calf thymus was utilized. Alternative source can be employed.
Srn/RNP Svlution: Purified Srn/RNP antigen solution from calf thymus was utilized.
Alternativ~ source can be empioyed.
~,5 ~N~C~ tion: 15 rniliigrams of hydrolyzed casein blocker (Sigma), 2 mi glyercol, 10 grams sucro~e was dissolved in TEN buffer sufficient to brin9 the final volume , ': ' .,,.. ,.. , . : . . , . :. . .: .. ... ... .
WO 90/1022~ i a'l~'C~/US90/01029 ~ \ ~ 9 _ ' to 100 mls. The solution is adjusted to pH 7.3 +/- .1, (Sm and SmlRNP) Caseln Blocker Solution: 25 milligrams of hydrolyzed casein blocker (Sigma), 2 ml glyercol, 10 grams sucrose was dissolv~ed in TEN buffer sufficient to bring the final volume to 100 mls. The solution is adjusted to pH 7.3 +/- .1.
TEN Buffer: Is made by adding 6.1 g TRIS, .38 g EDTA, 8.B g NaCL, 3.8 mL of concentrated HCL to 900 rnL deionized water. Adjust pH to 7.3 and add deionized water sufficient to give iOOO milliliter total volume.
Anti-dsDNA Ant bodv: Circulating autoantibodies directed against dsDNA.
Anti-Smith Antibodv: Circulating autoantibodies directed against Sm antigen.
Io Anti-Sm/RNP Antibody: Circulating autoantibodies directed against Sm/RNP antigen.
Anti-histone Antibody: Circulating autoantibodies directed against histone antigen.
Anti-DNP Anhbodv: Circulating autoantibodies directed against DNP antigen.
~: Circulating autoantibodies directed against ENA antigen.
Anti-~NA: Circulatiny autoantibodies directed against RNA antigen.
Anti-Scl-70: Circulating autDantibodies directed against Sci-70 antigen.
Anti-SS-A: CirculaUng autoantibodies directed against SS-A antigen.
:`
: Circulating autoantibodies directed against SS-B antigen.
,~ S, Nuclease: An endonuclease (enzyme) which is capable of digestion of ssDNA antigen.
;, oub ~ Ar L~Q~LISA or F A: A solid support is coa~ed with material which detects and blnds the antibody of interest to the coated surface. To render a signal, a second conjugated aniibody with an affinity for tha previously bound antibody is exposed to the coated surface. This antibody binding to the original antibody makes the sandwich. If . .
WO 90tl0~29 ;~ 34~ ! PCI/U~90/01029 ,.. .. ~
-~iO- ~.
the sandwich assay is an ELISA then the second antibody is conjugated with an enzyme and substrate is used to produce a color. If the assay is an FIA then the second antibody is marked with a fluorescent tag ancl a substrate is unnecessary.
Buffer for S, Nuc~ea$e Digestion: 95 mls of acetate/acetic acid buffer, pH 4.6, is mixed with 5 mls glycerol, .29 ~ NaCL, .û29g ZnS04, for a final volume of 100 mls.
. ~
S, Nuclease i3uffer Solution: Buffer for S1 nuclease digestion plus 100 units (Sigma) S1 nuclease per ml of buffer. One unit of S, nuclease is defined as: Causing 1.0 microgram of ssDNA per minute to become perchloric acid solubl~ at pH 4.6 and 37 degrees C.
Serum: Is intended to mean the fluid component of any body fluid remaining after cells and coaguiable proteins such as fibrin which may be present in such body fluidic components have been removed by appropriate physical, chemical, or physicochemical means. Typically, this term refers to the residual watery fluid remaining after clotting of blood and removal of the clot, but in its broad sense is intended to include the fluidic component of cerebrospinal fluid, urine, interstitial fluid, cellular cytoplasm, and the like.
Sample Di!uent: A 1 liter solution has 100 mls of native bovine serum, 1.42 grams of potassium phosphate (dibasic), .26 9 of potassium phosphate (monobasic), 1 gram of ~I sodium azide, and 8.6 grams of sodium chloride dissolved in 900 mls of water. If an ELISA
is run, then 1 ml of stock green dye is added to the solution. If ths FIA format is used then the dye is unnecessary. The solution is then filtered through a .2 micron filter and stored at û 4 degrees C.
.., Diluted ldsDNA3 Sample Solution: 10 microliters of sera dissolved in 500 microliters of sample diluent (in dsDNA).
- Diluted (Sm and Sm~RNP) Sample Solution: 10 microliters of sera dissolved in 490 . microliters of sample diluent.
S ~Y93tLe~: A phosphate buffer, and protein stabilizer, plus .02% thimerosal adjusted to a pH of 7.5 (commercially available from Medix i3iotech Inc.) into which is added a protease inhibitor, aprotinin, (commercially available from Miles Pentex) at .01% of the volume ol the buffor.
, , .
.
, :
... .......... . . .. . . . . . . .. . .. . .... .. . . . .
WO 90/10229 ~ ' PCr/US90/nl029 ~11~ ` ~ ;;' (dsDNA) Workina ConJugated Antibody Solution: 1 volume of concentratecl conjugated antibodies/3000 volumes of conjugate ~iluent. The dilution is subject to change based on the concentration Isvel of the conjugated antibody (dsDNA).
(Sm and Srn/RNP) Workina_onjugated Antibody Solution: 1 volume of concentrated conjugated IgG antibodies/400G volumes of conjugate diluent, and 1 volume IgM to 1500 vol.
The dilution is subject to change based on the concentration level of the conjugated antibody.
Coniugated Antibodies: For an ~LISA, antibodies were chemically conjugated with horseradish peroxidase. For FIA, antibodies were chemically conjugated with Fluorescein Isothiocyanate.
, Imm nogLobu!in: Any member of the gammaglobulin fraction of serum possessing the ability to bind another agent.
j .~
Antiaen: Molecules ffrom whatever source, nature or man-made) which induce an immune - , - reaction when recognized by the host's immune system.
-- Antibodv: A class of serum proteins which specifically bind to an antigen which induced the formation of the antibody.
,' ~
Imrnuno~lobulin Classes: Antibodies separated by electrophorectic rnobility specifically, IgG
and IgM.
Substrat~ Solution: To quantitate the horseradish peroxidase, 100 microliters of buffered . (3,3',5,5'~ Tetramethylbenzidine/ hydrogen peroxide (commercially available from Kirkegaard Perry) was used.
Labelleci Antibodies: Any antibody substance which has been covalently or otherwise oornbined with a molecule or ion for the purpose of selectively identifying that group of ~, antibodles. Such adduct molecules or ions include enzymes, fluorescen~ substances, ,1 tadionuclides, and the like.
` ''15 Labelled Antigens: Any antigen substance which has been covalently or otherwise combined with a molecule or ion for the purpose of selectiveiy identifying ~hat group of antigens. Such adciuct molecules or ions include enzymes, fluorescent substances, radionucli~es, and the . .
~, , . . ., : . .: .
, . . . ' .; . . ~ . ~ . , . ~
WO 90~10229 ~ PC~`/IJS90/01029 ;` - 12-~ 2~5~33~LCl like.
: OpticaL De sity (OD~ Absorbance: A number which refers to the color absorbance of a sample. Optical density is related to the percent cf light transmitteci through the sample by the following formuia: OD = 2-log(percent transmittance).
Test Kit tor Anti-dsDNA
,t.
~; The preferred ernbodiment of the method and apparatus for the detection of anti-dsDNA in sera is a diagnostic test kit. The optimized kit contains:
.
5 vials (200 microliters) Assay Calibrators for anti-DNA containing these levels of anti-dsDNA
i ~ ~ activity: 120, 60, 30, 15, 7.5, 3.8 A.U./rnl (quantitative format only) ,, .
1 vial (30 ml) Sample Diluent - green solution: contains 0.1% sodium azide.
1 vial Human Negative Serum Control (about 3 A.U./ml).
, 1 vial High Human Serum Positive Controls - about 100 A.U./ml with antigen binding capaeity of 59 x 10-4 microgram of DNA.
1 vial Moderate Human Serum Positive Control - about 45 A.U./rnl.
12 coated 8-well Microwell Strips with frame holder.
., : 1 vial (15 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase conjugated anti-human IgG, and IgM.
~, , 1 bottle (8 mi) TMB Substrate Solution A - contains 3,3',5,5' Tetramethylbenzidine.
~ ~ .
1 bottle (8 rnl) TMB Substrate Solution B - contains hydrogen peroxide.
,, 0 1 botUe (12 ml) Stop Reagent: contains 2.5 N H2S04. (1.0 N HC1 can be used as a . substitute) 1 packet Phosphate Buffered Saline (PBS) - reconstitutes to 2 liters ot 0.01 M PBS, pH 7.4.
c, ~
~, ~ . . , : , , . . , , ,: ........................ .. . . .
:. ; , ,. , . . , , . , , .:
Wo 90/10229 2~5(~34~ Pcr/us9o/olo2~
r' ~ -13-Plate Template This kit for the measurement of anti-dsDNA in serum samples has been designed for use in a clinical laboratory. To determine the AU/ml of the anti-dsDNA present in the sample the kit includes calibrators and controls for generating a standard curve. The selection of the ievels of anti-dsDNA activity in these controls and c:alibrators can be varied without affecting perFormance of the assay.
Optimization of the process has yielded kits with pre-coated test wells and reagents, which produce low levels of variation between assays and within assays. Furthermore, these reagents and test wells are stable for extended periods of time. This kit has been optimized . 10 to have a 45 minute run time at room temperatur0, which is significantly shorter than other test kits, presently rnarketed for anti-dsDNA detection.
What ~ollows is a description of a preferred embodiment of the pre-coated wells, and the : method of coating the wells of the present invention, along with the preferred msthod for preparation of and ulilization of the various elements of an anti-dsDNA diagnostic test kit.
Step 1 - Affixation of the Coatina on the Microwells: Methylated Bovine Serum Albumin (mBSA) is dissolved in distilled water or PBS at a ratio of ~0 ug/ml. An aliquot of 100 micrograms of this prepared solution is placed in each microwell, to produce a surface which is slightly positiveiy charged. The mBSA servesi two important functions; one is to provide a stable evenly coated rnicrowell surface, and second is to inhibit non-specific binding such as anti-histone activity. Ten mls of mBSA solution will coat 96 microtiter wells, such as a Dynatech immulon 2, Dynatech Immulon 4, or Nunc Maxisorp. The coated wells are incubated overnight at 4 degrees C.
~, Unbound mBSA solution is shaken from the w911s, and they are rinsed with PBS solution, pH
7.3, and drained thoroughly.
'. .
Next, the ligand or antigen is pre,i~ared and exposed to the receiving surfaces of the microwalls. Purified dsDNA (calf thymus) is dissolved into .01 I\A PBS, pH 7.4 in a ratio of 5 ,~ micrograms/ml (weight per volume). Th~ buffered ligand is then dispensed into each rnicrowell, 100 microliters of buffered ligand per well. The bindin9 is enhanced by an ~` incubation period ot 1B-24 hours at 4 degrees C. The excess solution is shaken from the ~,. .
.,;j;. .
WO 90/10229 ~ ~ ` PCl'tUS90/01029 rr~ ~
3~
wells.
The next coating is applied to eliminate the cross reactivity with ssDNA. A buffer is prepared for S1 nuclease digestion. To prepare the buifer .03M acetic acid was titrated together with .03M sodium acetate in a ratio of 1.l part acetic acid to 1 part sodiurn acetate to a final pH
of 4.6. 95 mls of the prepared acetate/acetic acid buff~r is mixed with 5 mls of glycerol, .29 g NaC1, .029 g ZnS04, for a final volurne of 100 rnls. Added to this buffer was 100 units of S, nuclease (Sigma) per ml of digestion buffer solution to form a S, nuclease buffered solution which is dispensed in 100 microliter increments into each individual receiving well.
Thus, 10 units of S, nuclease is contacted with each individual well. This solution is of suHicient concentration to digest the unwanted ssDNA in the previously coated ligand material aiter a two hour incubation period at 37 degrees C. S, nuclease eliminates ssDNA which is a source of false positives in many assays. A~ter the incubation period the wells are invertecl to remove excess solution, then the wells are thoroughly rin~ed twice with P8S solution. The wells are again inverted on a paper towel and allowed to drain.
The next coating step is contacting casein blocker (pH of 7.3) with the microwells in aliquots of 200 microliters. This step decreases the non-specific binding that can occur due to protein-plaslic interaction. The hydrolyzed casein used in the blocker solution can be commercially obtained from Sigma. The casein biocker solution is prepared by mixing 2 rnl glycerol, 10 ~ sucrose, and 15 mg of hydrolyzed casein, and adding sufficient TEN buffer to ~0 make 100 ml of solution. The wells containing casein blocker solution are again incubated at 4 degrees C overnight, after which time the wells are inverted and allowed to drain for 15 minutes. Then the wells are uprighted and allowed to dry at room temperature for at least 24 hours.
:
This compietes ~he process of coating the wells and each diagnostic kit is then supplied with -5 96 coated wells. The shelf life of coated microwells when stored at 4 degrees C in a seaied plastic bag is up to one year.
St~p 2 - Adherin~ AnU-dsDNA to the Prepared Wells From Step 1: Tha sample diluent is supplied in the kit as a 30 ml green solution. To prepare a 1000 ml solution of Sample Dilusnt, 100 milliliters of nativ~ bovine serum, 1.42 9 of Potassium Phosphate (dibasic), .26 1 9 of Potassium Phosphate (rnonobasic), 1 gram of sodium azid0, and 8.6 9 of sodium -~' chloride, and 1 ml of stock green dye are dissolved in deionized water sufficient to make 1000 milliliters of solution. This solution is then filtered through a .2 micron filter.
.7 .
; "
WIO 90/10229 2~ ) PCr/US90/01029 5 ~
The Sample Diluent acts as a blocking agent similar to the casein blocker which was previously coated on the wells. The principle blocking component in the Sample Diluent is the native bovine serum which acts to inhibit the binding of any BSA-reactive antibodies to the mBSA coating on the surtace of the well.
Prior to contacting the body fluid with the prepared l~late, the serum is diluted by adding aliquots of sera to the sample diluent in a 1:50 ratio (volume of serum:volume of sample diluent), although this exact dilution is not critical and depends upon the nature of the body fluid and the assay techniqu0s employed. To form the dihlted sample solution the body fluid is aliquoted in 10 microliter proportions into 500 microliters of Sample Diluent. In an individual well, 100 microliters of the diluted sample is dispensed, and the affixation of the anti-dsDNA is enhanced by 15 minutes incubation at room temperature.
Following the affixation of the anti-dsDNA to the coated wells, the wells are thoroughly washed four times with PBS to remove the free unbound antibodies that are present in the sample, which if allowed to remain, would elevate the backgrourld absorbance.
Slep 3 - Assay for the Anti-dsDNA Affixed to the Wells: Standard enzyme-linkëd immunoassay techniques, previously described, are used for this assay, although any suitable means of detection such as radioactive labeling, fluorescence, or the like can be ernployed.
For the examples described hereinafter, anti-human IgG and anti-human IgM induced in goats were used to ascertain whether anti-dsDNA IgG and igM antibodies were present. Please ?O note that other species of animal can be used to produce anti-human antibodies. These antisera were linked to horseradish peroxidase, an enzyme which yieids a colored product whenever one of its substrates is present together with hydrogen peroxide. The substrate should be chosen to be consistent with the enzyme conjugated to the antibody. For the examples ciescribed hereinafter, th~ substrate was (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
The kit contains one 15 rnl vial of conjugated antibody solution with anti hurnan I~M and IgG
antibody conjugat~d to horseradish peroxidase. To prepare a working conjugated antibody solution, a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4 (commercially availabls frorn Medix) was mixed with aprotinin, a protease inhibitor 0 (commercially available from Miles Pentex) at a .01% ratio of inhibitor to voiume of buffer.
;, This diluent enhances the stability of the conjugated antibody. The solution is mixed at a , .
.. ~ . - . , ~. . . , ................. , , - , ......... . .
" ', ~ . ! . , ' . ', ,: ', ' .. ~ . . . .......................... ' ' ;, : ! . . ~ ' . ' ' ' ' ~ .
WO ~0/10229 ~ ~; Pcr/VS~)/01029 2~ 33~
ratio of 1/3000; one part of concentrated conjugated IgM and IgG antibodies is aliquoted into 3000 parts conjugate diluent. The ratio of concentrated conjugated antibody to conjugate diluent is subject to wide latitudes of dilutions based on the manufacturer's concentration of conjugated antibody used in the assay.
Next 100 microliters of the enzyme conjugated goat anti-hurnan antibody working solution, (prepared as described) is added to each microtiter well. Binding of these antibodies to the anti-dsDNA is permitted for at least 15 minutes at room temperature, then the rnicrotiter wells are emptied of their contents, washed four times with PBS, and allowed to draln before the next step.
.
The presence of a label and antibody, as previously described, is determined by incubating the wells with a solution of buffered (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
- This solution is supplied in the kit in two 8 ml via!s; one contains (3,3',5,5') Tetramethylbenzidine; the olher vial contains hydrogen peroxide. The separate vials are necessary due to the interaction between the two solutions. The two solutions are mixed in -i~ a one to one ratio just before use, and 100 rnicroliters of the mixed solution is dispensed into each microwell. The reaction is permitted to continue for 10 minutes at room temperature, or until sufficient color appears to be read on the spectrophotometric device used. The reaction is subsequently stopped through the addition of an equal volume of 2.5 normal sulfuric acid, and the intensity of color (the optical density, "OD", or absorbance) is read by a spectrophotometric device such as a Dynatech MR600 or ~he like.
As with any enzyme-linked immune assay, the resultant color of the reaction product is ,; proportional to the number of conjugated antibodies which have bound to the anti-dsDNA.
For most cases, the number of bound conjugated antibodies is linearly related to the number of anti-dsDNA antibodies. Hence, as the amount of anti~dsDNA bound to the wells increas0s, ~5 so does the optical density, or absorbance of the enzyme reaction.
Test K~t ~or Anti~Sm or ~nU-Srt~ NP
., This prefetred embodiment of the method and apparatus for the detection of anti Sm anUbodies in a sera is a diagnostic test kit. Similarly ~he preferred embodiment of the method `', and apparatus for the detection of anti-Sm/RNP antibociies in sera is a diagnostic test kit, with ~0 the following components (qualitative format).
~' .', .
,: . ... .. :, . . . . . . .. . .
WO 90/102~9 ~ P~/US~0/01029 1 vial (40 ml) Sample Diluent - green solution- contains 0.1% sodium azide.
1 vial Human Negative Serum Control (about 100 microliters).
1 vial High Human Serum Positive Controls (about 100 microliters).
12 coated 8-well Microwell Strips with frame holer.
. . .
1 vial (12 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase conjugated anti-human IgG, and IgM.
1 bottle (8 ml) TMB Substrate Solution A - contains 3,3',5,5' Tetramethylbenzidine.
1 bottle ~8 ml) TMB Substrate Solution B - contains hydro~en peroxide.
1 bottle (12 ml) Stop Reagent: contains 2.5 N H2S04. (1.0 N HC1 can be used as a substitute) (not supplied).
1 packet Phosphate Buffered Saline (PBS) - reconstitutes to 2 liters of O.O i M PBS, pH 7.4.
Plate Temp!ate This kit for the measurement of anti-Sm antibodies in serum samples has been eiesigned for use in a clinical laboratory. To determine the RE~DS units/ml of the anti-Sm antibodies or the anti-Sm/RNP antib~dies present in the sample kit can include calibrators and controls for generating a standarci curve. The selection of the levels of anti-Sm or anti-Sm/RNP antibody activity in these controls and calibrators can be varied without affecting performance of the assay.
What foilows is a description of a preferred embodiment of the pre-coatecl wells, and the 2Q method of coating the wells of llhe present invention, along with the preferred method for preparation of and utilization of the various elements of the anti Sm or the anti-Sm/RNP
antibody diagno~tic test kit. The differerlce bet~veen the anti Sm and the anti Sm/RNP kits ~' is only the concentration of antigen coated to the microwells.
Step 1:~ion s;)f the oating,on~Q~y~: Mathylated Bovine Serum Albumin .
,.
,, ... , .. ~ . ,. .. , .. . - . , WO 90/10229 ';; PC.`T/I)S91)/01029 ~i~33~
(mBSA) is dissolved in distilled water or PBS at a ratio of 20 ug/ml. An aliquot of 100 microliters of this prepared solution is placed in each rnicrowell, to produce a surface which is slightly positively charged. The mBSA serves two important functions; one is to provide a stable evenly coateci microwell surface, and second is to inhibit non-specific binding such 5 as anti-histone activity. Ten mls of mBS~ solution will coat 96 microtiter wells, such as a Dynatech Imrnulon 2, Dynatech Immulon 4, or Nunc Maxisorp. The coateci wells areincubated overnight at 4 degrees C.
Unbound mBSA solution is shaken from the wells and drained thoroughly.
Next, the ligand or antigen is prepared and exposed to the receiving surfaces of the microwells. Purified Sm antigen or the purified Sm/RNP antigen (calf thymus) is dissolved into .01M PBS, pH 7.4 in a ratio of 5 units/ml, and for Sm/PNP 1 unit/ml. The buffered ligand is then dispensed into each microwell, 100 microliters of buffered ligand per well. The binding is enhanced by an incubation period of 18-24 hours at 4 degrees C.
';
The next coating step is contacting casein blocker (pH of 7.3) with the microwells in aliquots of 200 microliters. This step decreases the non-specific binding that can occur due to protein-plastic interaction. The hydrolyzed casein used in the blocker solution can be commercially obtained from Sigma. The casein blocker solution is prepared by mixing 2 ml glycerol, 10 g sucrose, and 25 mg of hydrolyzed casein, and adding sufficient TEN buffer to make 100 ml of solution. The wells containing casein blocker solution are again incubated -~!0 at 4 degrees C overnight, after which time the wells are inverted and allowed to drain for 15 minutes. Then the wells are uprighted and allowed to dry at room temperature for at least 24 hours.
This compietes the process of coating the wells and each diagnostic kit is then supplied with 96 coated wells. The shelf life of coated mierowells when stored at 4 degrees C in a sealed ;5 plastic bag is up to one year.
Step 2: Adhering Anti-Sm or Anti-SrnLRNP antibodies to the Prepared Wells From Step 1:
The sample diluent is supplied in the kit as a 40 ml green solution. To prepare a 1000 ml `1 solution of Sample Diluent, 100 milliliters of native bovine serum, 1.42 g of Potassium Phosphate (dibasic), .26 g ot Potassiurn Phosphate (monobasic), 1 gram of sodium azide, 0 and 8.6 9 of sodium chloride, and 1 ml of stock green dye are dissolved in deionized water sufficient to make 1 OOû milliliters of solution. This solution is then filtered through a .2 micron . ~
. ~,, .. ,,, , . ~. . . ..
,. . ~ . .: . . , ~ ..
. . ~ - . , : :, -'~ ' ' .' " ~ ' ' ':' . ' WO 90/10229 ;~ 3D~ PCr/U~90/0102~
~ ` .. .. .. .
fllter.
The Sample Diluent acts as a blocking agent similar to the casein blocker which was previously coated on the wells. The principle biocking component in the Sample Diluent is the native bovine serum which acts to inhibit the bincling of any BSA-reactive antibodies to the mBSA coating on the surface of the well.
Prior to contacting the body fiuid with the prspared ~late, lhe serum is diluted by adding aliquots of sera to the sarnple diluent in a 1:50 ratio (volume of serum:volume of sample diluent), although this exact dilution is not critical and depends upon the nature of the body fluid and the assay techniques employed. To ~orrn the diluted sample solution the body fluid is aliquoted in 10 microliler proportions into 490 microliters of Sample Diluent. In an individual well, 100 microliters of the diluted sample is dispensed, and the affixation of the anti-Sm or anti-Sm/RNP antibody is enhanced by 15 rninutes incubation at room temperature.
Following the affixation of the anti-Sm or anti-Sm/RNP antibody to the coated wells, the wells j 15 are thoroughly washed four times with PBS to remove the free unbound antibodies that are present in the sample, which if ailowed to remain, would elevate the background absorbance.
.
Step 3: Assax for the Anti-Sm or Anti-SmlRNP antibod~/ Affixed to the Wells: Standard enzyme-linked immunoassay techniques, previously described, are used for this assay, although any suitable means of detsction such as radioactive labeling, fluorescence, or the like can be employed. For the examples describecl hereinafter, anti-human IgG and anti-h~man IgM induced in goats were used to ascertain whether anti-Sm IgG and IgM antibodies or anti-Sm/RNP IgG and IgM antibodies were present. Please note that other species of animal can be used to produce anti-human antibodies. These antisera were linked to horseradish peroxidase, an en~yme which yields a colored product whenever one of its substrates is present together with hydrogen peroxide. Tho substrate should be chosen to be consistent with the enzyme conJugated to the antibody. For the examples described hereinafter, the substrate was (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
The kit contains one 12 ml vial of conjugated antibody solution with anti-human IgM and IgG
antibody conjugated to horseradish peroxidase. To prepare a working conjugat d antibody solution, a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4 (commercially available from Medix~ was mixed wilh aprotinin, a protease inhibitor ' ., ,~
,. , . .: : .. . . . ..
WO ~0/102~g PCI'/US~0/~)1029 (commercially available from Miles Psntex) at a .01% ratio of inhibitor to volume of buffer.
This diluent enhances the stability of the conjugated antibody. The solution is mixed at a ratio of 1/~000 for IgG and 1/1500 for IgM; one part of concentrated conjugated IgM and IgG
antibodies is aliquoted into 4000 and 1500 parts conjugate diluent, respectively. The ratio of concentrated conjugated antibody to conjugate diluent is subject to wide latitudes of dilutions based on the manufacturer's concentration of conjugated antibody used in the assay.
. Next 100 microliters of the enzyme conjugated goat anti-human antibody working solution, (prepared as described) is added to each microtiter well. 8inding of these antibodies to the anti-Sm or anti-Sm/RNP antibody is permitted for at least 15 minutes at room temperature, then the microtiter wells are emptied of their contents, washed four times with PBS, and allowed to clrain before the next step.
The presence of a label and antibody, as previously described, is determined by incubating the wells with a solution of buffered (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
This solution is supplied in the kit in two 8 ml ~ials; one contains (3,3',5,5')Tetramethylbenzidine; the other vial contains hydrogen peroxide. The separate vials are necessary due to the interaction between the two solutions. The two solutions are mixed in a one to one ratio just before use, and 100 microliters of the mix~d solution is dispensed into each microwell. The reaction is permitted to continue for 10 minutes at room temperature, or until sufficient color appears to be read on the spectrophotometric device used. The reaction is subsequentty stopped through the addition of an equal volume oi 2.5 normal sulfuric acid, and the intensity of color (the optical density, ~OD", or absorbance) is read by a spectrophotometric device such as a Dynatech MR600 or the like.
As with any enzyme-linked immune assay, the resultant color of the reaction product is proportional to the number of conjugated antibodies which have bound to the antibody. For 2~ most cases, the number of bound conjugated antibodies is linearly related to the number of bound antibodies. Hence, as the amount of anti-Sm or anti-Sm/RNP antibody bound to the wells Increases, so does the optlcal density, or absorbance of the enzynne reactlon.
:.
., .~ , . ~ . . . . .
,,~, , :
.
Wo 90/10229 ~3~ rcr/us90/0l029 E~MPLE 1 Protocol for Pr~Co~tin~ Microwells with Purlfled dsDNA
Polystyrene wells were coated with (native) double stranded deoxyribonucleic acid (dsDNA) by the following procedure:
1. Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was dissolved at a ratio of 20 micrograms/ml in distilled water or PBS.
2. 100 microliters of the mBSA solution was placed in each microwell1 and incubated overnight at 4 degrees C.
.
10 3. The excess coating solution was shaken from the plate after incubation. The plate was rinsed once with a solution of .01 M PBS, pH 7.3 to remove unbound mBSA. Thewells were inverted to drain thoroughly.
Thirdly, the immunological community has developed diagnostic test kits for the determination of anti-dsDNA levels in the standard ELISA format. Although a variety of these kits have been ~eveloped, the same limitations of stability, cross reactivity with ssDNA, and length of run time plague each assay. Many of these kits claim a low level of cross r~activity with ssDNA which should render good sensitivity to low, medium, and high levels of anti-dsDNA; however, as evidenced by the kit instructions, there is a lack of sensitivity in the sera sample ~,vhich are borderline positives. As a consequence, these kits cannot be us~d to accurately monitor patients who are borderline positives. In fact, most kits require that low positives must be retested to assure confidence in the results, thereby increasing the cost to ~0 the patient. Development ol an assay which is sensitive to all levels of anti-dsGNA can only be achieved by reducing non-specific binding by sueh components as ssDNA.
A number of techniques including immunodiffusion or Ouchterlony analysis have been developed to detect antibodies to Sm antigen and Sm/RNP antigen often called ENA(Extractable nuclear antigens). Examples of the limitations of the prior methods follow.
Firstly, the immunodiffusion technique has a long incubation time, typically 24 hours or more.
This time span is of course inconvenient for both the patient and the lab personnel.
Secondly, the results gathered from Ouchterlony analysis are subjective. This results in different lab technlcians reporting varied results based on individual data interpretation.
The imrnunological community has developed diagnostic test kits for the determination of !0 anti-Sm antibody levels in the standard ELISA format. These ELlSAs have shown certain limitations in stability, cross reactivity, and length of run time.
it is therefore an objective of the prasent invention to provide compositions, methods, articles, and a diagnostic tast kit for thc selective adsorption, or affixing of dsDNA, Sm antigen, er Sm/RNP antigen, or any af the various antigens to the autoimmune antibodies previously listed, to a pre-coated plate for the effective detection of the specific autoimmune antibodies present in sera or plasma. It is a further objective of this invention to overcome the aforemantioned sensitivity, subjectivity, stability, and shelf life disadvantages inherent in many of the previously dascribed assays. It is, moreover, also an objective of this invention to provid~ in the ~orm of a kit a noval, readily utilizabie maans for quantitative and qualitative O detection of anti-dsDNA, anti-Sm or anti-Sm/RNP. This will provide a method and apparatus for the purpose of c!inical detection of anti-dsDNA, or tha anti-Sm antibody, or the anti-:
''''''' " ' ~', ;' , ,, ' ' WO 90/10229 ~ P(~/US90/01029 ;~a~3~ 4- ~
Srn/RNP antibody, or any of the listed autoimmune antibodies listed, or the detection of the specific antibody for any other purpose associated with human or animal medical testing.
It is believed that with little modification the present invention can be used to detect anti-histone, anti-RNA, anti-SS-A, anti-SS-B, anti-Scl-70, and anti-DNP.
BRIEF SUMNIA~Y OF THE INVENTION
The present invention includes novel compositions, novel methods and articles for the direct selective absorption, adsorption or attachment, by whatever mechanism, of the anti-dsDNA, or the anti-Sm or the anti-Sm/RNP antibody, or any of the listed autoimmune antibodies, from the body fluid sample to the dsDNA, the Sm-antigPn, or the Srn-RNP antigen, or possibly for ~10 any of the autoimrnune antigens coated onto any suitable solid support, such as test tubes, plates or wells (hereinafter referred to as wells or microwells~, for the purpose of quantitative and qualitative identification. The present invention utilizes immobilized native DNA, Sm, Sm-RNP, or possibly for any listed autoimmune antigen from whatever source, which has an affinity for attachment of anti-dsDNA, anti-Sm, or anti-Sm/RNP, or the other antibodies of the listed autoimmune antibQdies.
This invention utilizes a sandwich ELISA format which includes pre-coated wells made of any suitable material such as plastic, glass, etc., and the various reagents and antibodies necessary to run a highly sensitive, 45 minute assay for the presence of anti-dsDNA, or for the presence of anti-Sm, or for the presence of anti-Sm/RNP.
The various reagents and the method of coating these reagents to the rnicrowells have been employed advantageously in the practice of the present invention to formulate a test which has minimized or eliminated the problems associated with previous assays. The coating protocol has advantageously utilized methylated Bovine Serum Albumin (mBSA) for two main purposes; namely, the mBSA provides a positively charged surface which enhances the adherence of the antigen such as dsDNA, Sm, and Sm/RNP to the polystyrene wells, and second to eliminate the binding of anti-histone antibodies, which can create false positive results.
The use of mBSA as a coating prior to the application of DNA antigen for use in solid phase assays for anti-dsDNA was flrist explored by R. Rubin (J. Immunology 63, 359-366 l1983]).
In the practice of this invention, protamine sulfate as well as other functionally equivalent substltutes have been found to be capable of forming a positiveiy charged surface; however, WO 90/10229 ;~ 4C~ P(~/US90/0~iO29 ,". ,` ` ; ' '; , " ,' ';," :.
~5-due to the reactivity of these coatings with other components such as ssDNA and anti-histone antibodies, mBSA is preFerred. It was highly surprisin~ to find that a coating of mBSA on a solid support underlying the DNA antigen, or the Sm antigen, or the Sm-RNP antigen provides the pre-coated wells with a consistently high level of reproducibility even over a 5 period of one year.
Methylated Bovine Serum Albumin is utilized for the previously mentioned reasons, the second of which is directly related to the specificity and sensitivity of these varlous assays.
The lack of good correlation between ELlSAs, RlAs and Crithidia luciliae staining procedures and other tests in some cases appears to be due to binding by anti-histone antibodies which 10 gives the ELISA an elevated antibody reading. The mBSA pre-coating appears to have alleviated this area of non-specific binding.
A second factor which contributes to the lack of sensitivity and non-specHicity is the cross reactivity. This is particularly a problem when detecting antibodies to dsDNA, because of the cross reactivity hetween ssDNA and anti-dsDNA. To eliminate the potential binding of the ~15 anti-dsDNA antibodies to ssDNA the microwells which have been coated with dsDNA were treated with an endonuclease, S, nuclease, which is an enzyme specific for the breakdown degradation of ssDNA and has no affect on dsDNA. Although other anti-dsDNA tests utilize S, nuclease ffor example see A.G. Tzioufas, Clinical and Experimental Rheumatology 5, 247-253 [1987]) their sensitivity levels are still substantially lower than acceptable levels for ~2û patients with borderline positives. The use of S, nuclease in a digestion buffer with an acid pH, combined wi~h the use of a mBSA pre-eoating eliminates ssDNA activity and anti-histone activity providing a highly sensitive assay which renders few ~alse positives. The S, nuclease is of course only necessary in the detection of anti-dsDNA antibodies and not in anti-Sm or anti-Sm/RNP antibody detection.
The shelf life and the reproducibility of results from the ELISA is associated not only with the mBSA pre-coating, and the S, nuclease treatment (in the anti-dsDNA kit), but also with the next two steps of forming the pre-treated wells; and tha addition of a hydrolyzed casein blocker and the drying.
A casein-type blocker has been used in various ELISA techniques ~Robert F. Bogt; J.
Imrnunological Methods, 101, 43-50 ~1987l) to block non-specific hinding to ptastic through a protein-plastic interaction. It is an unexpected realization that a coating of hydrolyzed casein blocker maintained a consistent inhibition of non-specific binding over an extended , . . .
, . . .. , . ; . .. ... ~ .
WO 90/10229 `~ ; PCl'/US9~/0102~
3~) 6- ~
period of time, when the wells were stored at 4 degrees C in a sealed plastic bag.
Although the direct mechanisms by which the drying process and the blocker increases the stability and shelf life of the wells is not fully understood, it is obvious that the storage time is increased by these processes. Little to no ssDNA, or loss of Sm or Sm-RNP antigen activity is generated by this method during the storage condltions in a period of up to one year.
A variety of differing blocking agents could be utilized which are ~unctionally equivalent to or chemically relatsd to the casein blocking agent. For example, BSA and porcine thyroglobulin, dried milk, whole goat serum, etc., however the most preferable is the hydrolyzed casein 0 (commercially available from Sigma) due to its high level of inhibition of non-specific binding and its storage stability.
' This invention's use of mBSA, the S, nuclease treatment (in the anti-dsDNA klt), combined with the casein blocker and the drying process have unexpectedly resulted in novel assays whlch are characterlzed by a low level of cross-reactivity, a high level of Inhibition of non-specific bindlng, and a long shelf life.
The pre-coated wells are then used to detect the presence of anti-dsDNA antibody, anti-Sm antibody, or the anti-Sm/RNP antibody in the sample. The plasma or serum samples are prepared with a sarnple diluent and are then assayed for their components by an immunoassay technique, the ELISA and the fluorescent immunoassay (FIA) formats being 0 the preferred methods, though it is possible to perform a RIA or a luminescent assay with little modification.
:, The assays depicted in the following examples have an approximate run time of 45 minutes.
Th~ wells, when exposed to the samples, are provlded with approximately 15 minutes at ;~ room temperature to allow the blnding of the antlbody to the antigen to go to completion.
5 Then the labelled goat anti-human antibodies are exposed to the wells and a similar 15 minute incubation at room temperature is provided for. If the enzyme is utilized a substrate ` can ba added (although this is not necessary) and 10 minutes is allotted for the production of the color. If a fluorescent marker is used on the goat anti-human antibody then no substrate is needed, therefore the run time is shortened by 10 minutes reducing it to 35 minutc-.
WO 90/10~29 ~5~3~3 PCI'/US90/01029 .; ", . ,,, ,", ,. . I
Subsequent qualitative and quantitative detection of the antibody is relatively simple if the forrnat is either an ELISA or a FIA. Numerous enzyme-conjugated antibodies and fluorescent-labelled antibodies specific for any of the immuno~3lobulin classes can be utilized in this invention. The quantitation of the antibodies present is accomplished by the related instruments. The ELISA technique utilizes a spectrophotometer, and the FIA technique utilizes a microfluorometer. Use of the ELISA techniques were flrst described by Engvall and Perlman ([1971] Irnmunochemistry 8, 871-874 and [1972} J. Immunology 109, 129-135), and The Enzyme Linked Immunosorbent Assay (ELISA) by Voller, A., Bidwell, D.E., and Bartlett, A., (1979) Dynatech Laboratories, Inc., Alexandria, Virginia, both of which are, in their totality, ; 10 incorporated herein by re~erence.
BRIEF DESCRIPTION OF THE GF~APH
Figure 1 - This graph depicts a standardized curve based on arbitrary units/ml. This curve was generated by using the ELISA ~ormat of this invention. The Center for Disease Control (CDC) ANA Human Reference Serum #1 has an antigen binding capacity of .59 micro grams 1~ DNA bound per ml of serum. This is equivalent to 100 AU per ml.
Figure 2 - This graph depicts a standardized curve obtained ~rom the data in Example 3.
Figure 3 - This graph depicts a standardized curve based on arbitrary RE~DS~ units/ml. This curve was generated by using the ELISA format of this invention. The Center for Disease Control (CDC~ ANA Human Reference Lot #82-001 #5 Serum ffor Sm antigen) was run on an immunodiffusion assay with median titer of 1:64. This standard was serially diluted with normal sera and graphed. The 1:1 or neat dilution was assigned arbitrarily a value of 400 RE~DS units.
:
Figure 4 - This graph dspicts the range of negative and positive human sera control values expected for the anti-Sm tast kit.
:, 2~ Figure 5 - This graph depicts a standardked curve based on arbitrary RE~DS ED units/ml. This curve was generated by using the ELISA format of this invention. The Center for Disease Control (CDC;) ANA Human Reference Lot ~82-001 ~5 Serum (for Sm antigen) and the ANA
Human Referencs Lot #82-011 #4 Serurn ffor RNP antigen) was run on an immunodiffusion assay with median titer of 1:64. An equal volume of reference sera #4 and ~5 are combined to 1Orm the standard. This .standard was serially diluted with normal sera an~ graphed. The .
:
. ~ .
WO 90/10229 ' PCT/llS90/01029 ~ 8 -1:1 or neat dilution was assigned arbitrarily a value of 140C RE~DS units.
Figure 6 - This graph depicts the range of negative and positive hurnan sera control values expected for the anti-Sm/RNP test kit.
Figure 7 - This charl shows autoimmune disease and their associated antigens which could possii~ly be detected by the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions are supplied for the purpose of clarifying the invention and are not intended to limit the scope of the invention:
Methylated Bovine Serum Albumin Soiution: Unless otherwise specified, is intended to mean a solution of 1 milliliter of water or PBS with 20 micrograms of methylated bovine serum Albumin (mBSA) dissolved in it. A substitute for mBSA is protamine sulfate or any other chemical or chemical process capable of producing a slighily positively charged coating which is evenly distributed over the surface of the microtitre well. (It is believed that the anti~
histone test may not ~mploy methylated bovine serum coating on the microtiter plate.) ~5 PBS Solution: A .01 molar solution ot buffer containing 1.43 9 potassiurn phosphate, dibasic, " .~5 g potassium phosphate, monobasic, and ~.5 g sodium chloride in one liter of water. The ' pH is 7.3 +/- .1.
. , ~, dsDNA Solution: Purified dsDNA solution from cali~ thymus was utilized. Altcrnative sources ~î of DNA include, but are not limited to, native linear DNA from E. Coli, and circular DNA from ~0 plasmid, virus, crithidia, and synthetic polynucleotides (poly (dA.dT)).
Sm Solution: Puriiied Sm antigen solution from calf thymus was utilized. Alternative source can be employed.
Srn/RNP Svlution: Purified Srn/RNP antigen solution from calf thymus was utilized.
Alternativ~ source can be empioyed.
~,5 ~N~C~ tion: 15 rniliigrams of hydrolyzed casein blocker (Sigma), 2 mi glyercol, 10 grams sucro~e was dissolved in TEN buffer sufficient to brin9 the final volume , ': ' .,,.. ,.. , . : . . , . :. . .: .. ... ... .
WO 90/1022~ i a'l~'C~/US90/01029 ~ \ ~ 9 _ ' to 100 mls. The solution is adjusted to pH 7.3 +/- .1, (Sm and SmlRNP) Caseln Blocker Solution: 25 milligrams of hydrolyzed casein blocker (Sigma), 2 ml glyercol, 10 grams sucrose was dissolv~ed in TEN buffer sufficient to bring the final volume to 100 mls. The solution is adjusted to pH 7.3 +/- .1.
TEN Buffer: Is made by adding 6.1 g TRIS, .38 g EDTA, 8.B g NaCL, 3.8 mL of concentrated HCL to 900 rnL deionized water. Adjust pH to 7.3 and add deionized water sufficient to give iOOO milliliter total volume.
Anti-dsDNA Ant bodv: Circulating autoantibodies directed against dsDNA.
Anti-Smith Antibodv: Circulating autoantibodies directed against Sm antigen.
Io Anti-Sm/RNP Antibody: Circulating autoantibodies directed against Sm/RNP antigen.
Anti-histone Antibody: Circulating autoantibodies directed against histone antigen.
Anti-DNP Anhbodv: Circulating autoantibodies directed against DNP antigen.
~: Circulating autoantibodies directed against ENA antigen.
Anti-~NA: Circulatiny autoantibodies directed against RNA antigen.
Anti-Scl-70: Circulating autDantibodies directed against Sci-70 antigen.
Anti-SS-A: CirculaUng autoantibodies directed against SS-A antigen.
:`
: Circulating autoantibodies directed against SS-B antigen.
,~ S, Nuclease: An endonuclease (enzyme) which is capable of digestion of ssDNA antigen.
;, oub ~ Ar L~Q~LISA or F A: A solid support is coa~ed with material which detects and blnds the antibody of interest to the coated surface. To render a signal, a second conjugated aniibody with an affinity for tha previously bound antibody is exposed to the coated surface. This antibody binding to the original antibody makes the sandwich. If . .
WO 90tl0~29 ;~ 34~ ! PCI/U~90/01029 ,.. .. ~
-~iO- ~.
the sandwich assay is an ELISA then the second antibody is conjugated with an enzyme and substrate is used to produce a color. If the assay is an FIA then the second antibody is marked with a fluorescent tag ancl a substrate is unnecessary.
Buffer for S, Nuc~ea$e Digestion: 95 mls of acetate/acetic acid buffer, pH 4.6, is mixed with 5 mls glycerol, .29 ~ NaCL, .û29g ZnS04, for a final volume of 100 mls.
. ~
S, Nuclease i3uffer Solution: Buffer for S1 nuclease digestion plus 100 units (Sigma) S1 nuclease per ml of buffer. One unit of S, nuclease is defined as: Causing 1.0 microgram of ssDNA per minute to become perchloric acid solubl~ at pH 4.6 and 37 degrees C.
Serum: Is intended to mean the fluid component of any body fluid remaining after cells and coaguiable proteins such as fibrin which may be present in such body fluidic components have been removed by appropriate physical, chemical, or physicochemical means. Typically, this term refers to the residual watery fluid remaining after clotting of blood and removal of the clot, but in its broad sense is intended to include the fluidic component of cerebrospinal fluid, urine, interstitial fluid, cellular cytoplasm, and the like.
Sample Di!uent: A 1 liter solution has 100 mls of native bovine serum, 1.42 grams of potassium phosphate (dibasic), .26 9 of potassium phosphate (monobasic), 1 gram of ~I sodium azide, and 8.6 grams of sodium chloride dissolved in 900 mls of water. If an ELISA
is run, then 1 ml of stock green dye is added to the solution. If ths FIA format is used then the dye is unnecessary. The solution is then filtered through a .2 micron filter and stored at û 4 degrees C.
.., Diluted ldsDNA3 Sample Solution: 10 microliters of sera dissolved in 500 microliters of sample diluent (in dsDNA).
- Diluted (Sm and Sm~RNP) Sample Solution: 10 microliters of sera dissolved in 490 . microliters of sample diluent.
S ~Y93tLe~: A phosphate buffer, and protein stabilizer, plus .02% thimerosal adjusted to a pH of 7.5 (commercially available from Medix i3iotech Inc.) into which is added a protease inhibitor, aprotinin, (commercially available from Miles Pentex) at .01% of the volume ol the buffor.
, , .
.
, :
... .......... . . .. . . . . . . .. . .. . .... .. . . . .
WO 90/10229 ~ ' PCr/US90/nl029 ~11~ ` ~ ;;' (dsDNA) Workina ConJugated Antibody Solution: 1 volume of concentratecl conjugated antibodies/3000 volumes of conjugate ~iluent. The dilution is subject to change based on the concentration Isvel of the conjugated antibody (dsDNA).
(Sm and Srn/RNP) Workina_onjugated Antibody Solution: 1 volume of concentrated conjugated IgG antibodies/400G volumes of conjugate diluent, and 1 volume IgM to 1500 vol.
The dilution is subject to change based on the concentration level of the conjugated antibody.
Coniugated Antibodies: For an ~LISA, antibodies were chemically conjugated with horseradish peroxidase. For FIA, antibodies were chemically conjugated with Fluorescein Isothiocyanate.
, Imm nogLobu!in: Any member of the gammaglobulin fraction of serum possessing the ability to bind another agent.
j .~
Antiaen: Molecules ffrom whatever source, nature or man-made) which induce an immune - , - reaction when recognized by the host's immune system.
-- Antibodv: A class of serum proteins which specifically bind to an antigen which induced the formation of the antibody.
,' ~
Imrnuno~lobulin Classes: Antibodies separated by electrophorectic rnobility specifically, IgG
and IgM.
Substrat~ Solution: To quantitate the horseradish peroxidase, 100 microliters of buffered . (3,3',5,5'~ Tetramethylbenzidine/ hydrogen peroxide (commercially available from Kirkegaard Perry) was used.
Labelleci Antibodies: Any antibody substance which has been covalently or otherwise oornbined with a molecule or ion for the purpose of selectively identifying that group of ~, antibodles. Such adduct molecules or ions include enzymes, fluorescen~ substances, ,1 tadionuclides, and the like.
` ''15 Labelled Antigens: Any antigen substance which has been covalently or otherwise combined with a molecule or ion for the purpose of selectiveiy identifying ~hat group of antigens. Such adciuct molecules or ions include enzymes, fluorescent substances, radionucli~es, and the . .
~, , . . ., : . .: .
, . . . ' .; . . ~ . ~ . , . ~
WO 90~10229 ~ PC~`/IJS90/01029 ;` - 12-~ 2~5~33~LCl like.
: OpticaL De sity (OD~ Absorbance: A number which refers to the color absorbance of a sample. Optical density is related to the percent cf light transmitteci through the sample by the following formuia: OD = 2-log(percent transmittance).
Test Kit tor Anti-dsDNA
,t.
~; The preferred ernbodiment of the method and apparatus for the detection of anti-dsDNA in sera is a diagnostic test kit. The optimized kit contains:
.
5 vials (200 microliters) Assay Calibrators for anti-DNA containing these levels of anti-dsDNA
i ~ ~ activity: 120, 60, 30, 15, 7.5, 3.8 A.U./rnl (quantitative format only) ,, .
1 vial (30 ml) Sample Diluent - green solution: contains 0.1% sodium azide.
1 vial Human Negative Serum Control (about 3 A.U./ml).
, 1 vial High Human Serum Positive Controls - about 100 A.U./ml with antigen binding capaeity of 59 x 10-4 microgram of DNA.
1 vial Moderate Human Serum Positive Control - about 45 A.U./rnl.
12 coated 8-well Microwell Strips with frame holder.
., : 1 vial (15 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase conjugated anti-human IgG, and IgM.
~, , 1 bottle (8 mi) TMB Substrate Solution A - contains 3,3',5,5' Tetramethylbenzidine.
~ ~ .
1 bottle (8 rnl) TMB Substrate Solution B - contains hydrogen peroxide.
,, 0 1 botUe (12 ml) Stop Reagent: contains 2.5 N H2S04. (1.0 N HC1 can be used as a . substitute) 1 packet Phosphate Buffered Saline (PBS) - reconstitutes to 2 liters ot 0.01 M PBS, pH 7.4.
c, ~
~, ~ . . , : , , . . , , ,: ........................ .. . . .
:. ; , ,. , . . , , . , , .:
Wo 90/10229 2~5(~34~ Pcr/us9o/olo2~
r' ~ -13-Plate Template This kit for the measurement of anti-dsDNA in serum samples has been designed for use in a clinical laboratory. To determine the AU/ml of the anti-dsDNA present in the sample the kit includes calibrators and controls for generating a standard curve. The selection of the ievels of anti-dsDNA activity in these controls and c:alibrators can be varied without affecting perFormance of the assay.
Optimization of the process has yielded kits with pre-coated test wells and reagents, which produce low levels of variation between assays and within assays. Furthermore, these reagents and test wells are stable for extended periods of time. This kit has been optimized . 10 to have a 45 minute run time at room temperatur0, which is significantly shorter than other test kits, presently rnarketed for anti-dsDNA detection.
What ~ollows is a description of a preferred embodiment of the pre-coated wells, and the : method of coating the wells of the present invention, along with the preferred msthod for preparation of and ulilization of the various elements of an anti-dsDNA diagnostic test kit.
Step 1 - Affixation of the Coatina on the Microwells: Methylated Bovine Serum Albumin (mBSA) is dissolved in distilled water or PBS at a ratio of ~0 ug/ml. An aliquot of 100 micrograms of this prepared solution is placed in each microwell, to produce a surface which is slightly positiveiy charged. The mBSA servesi two important functions; one is to provide a stable evenly coated rnicrowell surface, and second is to inhibit non-specific binding such as anti-histone activity. Ten mls of mBSA solution will coat 96 microtiter wells, such as a Dynatech immulon 2, Dynatech Immulon 4, or Nunc Maxisorp. The coated wells are incubated overnight at 4 degrees C.
~, Unbound mBSA solution is shaken from the w911s, and they are rinsed with PBS solution, pH
7.3, and drained thoroughly.
'. .
Next, the ligand or antigen is pre,i~ared and exposed to the receiving surfaces of the microwalls. Purified dsDNA (calf thymus) is dissolved into .01 I\A PBS, pH 7.4 in a ratio of 5 ,~ micrograms/ml (weight per volume). Th~ buffered ligand is then dispensed into each rnicrowell, 100 microliters of buffered ligand per well. The bindin9 is enhanced by an ~` incubation period ot 1B-24 hours at 4 degrees C. The excess solution is shaken from the ~,. .
.,;j;. .
WO 90/10229 ~ ~ ` PCl'tUS90/01029 rr~ ~
3~
wells.
The next coating is applied to eliminate the cross reactivity with ssDNA. A buffer is prepared for S1 nuclease digestion. To prepare the buifer .03M acetic acid was titrated together with .03M sodium acetate in a ratio of 1.l part acetic acid to 1 part sodiurn acetate to a final pH
of 4.6. 95 mls of the prepared acetate/acetic acid buff~r is mixed with 5 mls of glycerol, .29 g NaC1, .029 g ZnS04, for a final volurne of 100 rnls. Added to this buffer was 100 units of S, nuclease (Sigma) per ml of digestion buffer solution to form a S, nuclease buffered solution which is dispensed in 100 microliter increments into each individual receiving well.
Thus, 10 units of S, nuclease is contacted with each individual well. This solution is of suHicient concentration to digest the unwanted ssDNA in the previously coated ligand material aiter a two hour incubation period at 37 degrees C. S, nuclease eliminates ssDNA which is a source of false positives in many assays. A~ter the incubation period the wells are invertecl to remove excess solution, then the wells are thoroughly rin~ed twice with P8S solution. The wells are again inverted on a paper towel and allowed to drain.
The next coating step is contacting casein blocker (pH of 7.3) with the microwells in aliquots of 200 microliters. This step decreases the non-specific binding that can occur due to protein-plaslic interaction. The hydrolyzed casein used in the blocker solution can be commercially obtained from Sigma. The casein biocker solution is prepared by mixing 2 rnl glycerol, 10 ~ sucrose, and 15 mg of hydrolyzed casein, and adding sufficient TEN buffer to ~0 make 100 ml of solution. The wells containing casein blocker solution are again incubated at 4 degrees C overnight, after which time the wells are inverted and allowed to drain for 15 minutes. Then the wells are uprighted and allowed to dry at room temperature for at least 24 hours.
:
This compietes ~he process of coating the wells and each diagnostic kit is then supplied with -5 96 coated wells. The shelf life of coated microwells when stored at 4 degrees C in a seaied plastic bag is up to one year.
St~p 2 - Adherin~ AnU-dsDNA to the Prepared Wells From Step 1: Tha sample diluent is supplied in the kit as a 30 ml green solution. To prepare a 1000 ml solution of Sample Dilusnt, 100 milliliters of nativ~ bovine serum, 1.42 9 of Potassium Phosphate (dibasic), .26 1 9 of Potassium Phosphate (rnonobasic), 1 gram of sodium azid0, and 8.6 9 of sodium -~' chloride, and 1 ml of stock green dye are dissolved in deionized water sufficient to make 1000 milliliters of solution. This solution is then filtered through a .2 micron filter.
.7 .
; "
WIO 90/10229 2~ ) PCr/US90/01029 5 ~
The Sample Diluent acts as a blocking agent similar to the casein blocker which was previously coated on the wells. The principle blocking component in the Sample Diluent is the native bovine serum which acts to inhibit the binding of any BSA-reactive antibodies to the mBSA coating on the surtace of the well.
Prior to contacting the body fluid with the prepared l~late, the serum is diluted by adding aliquots of sera to the sample diluent in a 1:50 ratio (volume of serum:volume of sample diluent), although this exact dilution is not critical and depends upon the nature of the body fluid and the assay techniqu0s employed. To form the dihlted sample solution the body fluid is aliquoted in 10 microliter proportions into 500 microliters of Sample Diluent. In an individual well, 100 microliters of the diluted sample is dispensed, and the affixation of the anti-dsDNA is enhanced by 15 minutes incubation at room temperature.
Following the affixation of the anti-dsDNA to the coated wells, the wells are thoroughly washed four times with PBS to remove the free unbound antibodies that are present in the sample, which if allowed to remain, would elevate the backgrourld absorbance.
Slep 3 - Assay for the Anti-dsDNA Affixed to the Wells: Standard enzyme-linkëd immunoassay techniques, previously described, are used for this assay, although any suitable means of detection such as radioactive labeling, fluorescence, or the like can be ernployed.
For the examples described hereinafter, anti-human IgG and anti-human IgM induced in goats were used to ascertain whether anti-dsDNA IgG and igM antibodies were present. Please ?O note that other species of animal can be used to produce anti-human antibodies. These antisera were linked to horseradish peroxidase, an enzyme which yieids a colored product whenever one of its substrates is present together with hydrogen peroxide. The substrate should be chosen to be consistent with the enzyme conjugated to the antibody. For the examples ciescribed hereinafter, th~ substrate was (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
The kit contains one 15 rnl vial of conjugated antibody solution with anti hurnan I~M and IgG
antibody conjugat~d to horseradish peroxidase. To prepare a working conjugated antibody solution, a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4 (commercially availabls frorn Medix) was mixed with aprotinin, a protease inhibitor 0 (commercially available from Miles Pentex) at a .01% ratio of inhibitor to voiume of buffer.
;, This diluent enhances the stability of the conjugated antibody. The solution is mixed at a , .
.. ~ . - . , ~. . . , ................. , , - , ......... . .
" ', ~ . ! . , ' . ', ,: ', ' .. ~ . . . .......................... ' ' ;, : ! . . ~ ' . ' ' ' ' ~ .
WO ~0/10229 ~ ~; Pcr/VS~)/01029 2~ 33~
ratio of 1/3000; one part of concentrated conjugated IgM and IgG antibodies is aliquoted into 3000 parts conjugate diluent. The ratio of concentrated conjugated antibody to conjugate diluent is subject to wide latitudes of dilutions based on the manufacturer's concentration of conjugated antibody used in the assay.
Next 100 microliters of the enzyme conjugated goat anti-hurnan antibody working solution, (prepared as described) is added to each microtiter well. Binding of these antibodies to the anti-dsDNA is permitted for at least 15 minutes at room temperature, then the rnicrotiter wells are emptied of their contents, washed four times with PBS, and allowed to draln before the next step.
.
The presence of a label and antibody, as previously described, is determined by incubating the wells with a solution of buffered (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
- This solution is supplied in the kit in two 8 ml via!s; one contains (3,3',5,5') Tetramethylbenzidine; the olher vial contains hydrogen peroxide. The separate vials are necessary due to the interaction between the two solutions. The two solutions are mixed in -i~ a one to one ratio just before use, and 100 rnicroliters of the mixed solution is dispensed into each microwell. The reaction is permitted to continue for 10 minutes at room temperature, or until sufficient color appears to be read on the spectrophotometric device used. The reaction is subsequently stopped through the addition of an equal volume of 2.5 normal sulfuric acid, and the intensity of color (the optical density, "OD", or absorbance) is read by a spectrophotometric device such as a Dynatech MR600 or ~he like.
As with any enzyme-linked immune assay, the resultant color of the reaction product is ,; proportional to the number of conjugated antibodies which have bound to the anti-dsDNA.
For most cases, the number of bound conjugated antibodies is linearly related to the number of anti-dsDNA antibodies. Hence, as the amount of anti~dsDNA bound to the wells increas0s, ~5 so does the optical density, or absorbance of the enzyme reaction.
Test K~t ~or Anti~Sm or ~nU-Srt~ NP
., This prefetred embodiment of the method and apparatus for the detection of anti Sm anUbodies in a sera is a diagnostic test kit. Similarly ~he preferred embodiment of the method `', and apparatus for the detection of anti-Sm/RNP antibociies in sera is a diagnostic test kit, with ~0 the following components (qualitative format).
~' .', .
,: . ... .. :, . . . . . . .. . .
WO 90/102~9 ~ P~/US~0/01029 1 vial (40 ml) Sample Diluent - green solution- contains 0.1% sodium azide.
1 vial Human Negative Serum Control (about 100 microliters).
1 vial High Human Serum Positive Controls (about 100 microliters).
12 coated 8-well Microwell Strips with frame holer.
. . .
1 vial (12 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase conjugated anti-human IgG, and IgM.
1 bottle (8 ml) TMB Substrate Solution A - contains 3,3',5,5' Tetramethylbenzidine.
1 bottle ~8 ml) TMB Substrate Solution B - contains hydro~en peroxide.
1 bottle (12 ml) Stop Reagent: contains 2.5 N H2S04. (1.0 N HC1 can be used as a substitute) (not supplied).
1 packet Phosphate Buffered Saline (PBS) - reconstitutes to 2 liters of O.O i M PBS, pH 7.4.
Plate Temp!ate This kit for the measurement of anti-Sm antibodies in serum samples has been eiesigned for use in a clinical laboratory. To determine the RE~DS units/ml of the anti-Sm antibodies or the anti-Sm/RNP antib~dies present in the sample kit can include calibrators and controls for generating a standarci curve. The selection of the levels of anti-Sm or anti-Sm/RNP antibody activity in these controls and calibrators can be varied without affecting performance of the assay.
What foilows is a description of a preferred embodiment of the pre-coatecl wells, and the 2Q method of coating the wells of llhe present invention, along with the preferred method for preparation of and utilization of the various elements of the anti Sm or the anti-Sm/RNP
antibody diagno~tic test kit. The differerlce bet~veen the anti Sm and the anti Sm/RNP kits ~' is only the concentration of antigen coated to the microwells.
Step 1:~ion s;)f the oating,on~Q~y~: Mathylated Bovine Serum Albumin .
,.
,, ... , .. ~ . ,. .. , .. . - . , WO 90/10229 ';; PC.`T/I)S91)/01029 ~i~33~
(mBSA) is dissolved in distilled water or PBS at a ratio of 20 ug/ml. An aliquot of 100 microliters of this prepared solution is placed in each rnicrowell, to produce a surface which is slightly positively charged. The mBSA serves two important functions; one is to provide a stable evenly coateci microwell surface, and second is to inhibit non-specific binding such 5 as anti-histone activity. Ten mls of mBS~ solution will coat 96 microtiter wells, such as a Dynatech Imrnulon 2, Dynatech Immulon 4, or Nunc Maxisorp. The coateci wells areincubated overnight at 4 degrees C.
Unbound mBSA solution is shaken from the wells and drained thoroughly.
Next, the ligand or antigen is prepared and exposed to the receiving surfaces of the microwells. Purified Sm antigen or the purified Sm/RNP antigen (calf thymus) is dissolved into .01M PBS, pH 7.4 in a ratio of 5 units/ml, and for Sm/PNP 1 unit/ml. The buffered ligand is then dispensed into each microwell, 100 microliters of buffered ligand per well. The binding is enhanced by an incubation period of 18-24 hours at 4 degrees C.
';
The next coating step is contacting casein blocker (pH of 7.3) with the microwells in aliquots of 200 microliters. This step decreases the non-specific binding that can occur due to protein-plastic interaction. The hydrolyzed casein used in the blocker solution can be commercially obtained from Sigma. The casein blocker solution is prepared by mixing 2 ml glycerol, 10 g sucrose, and 25 mg of hydrolyzed casein, and adding sufficient TEN buffer to make 100 ml of solution. The wells containing casein blocker solution are again incubated -~!0 at 4 degrees C overnight, after which time the wells are inverted and allowed to drain for 15 minutes. Then the wells are uprighted and allowed to dry at room temperature for at least 24 hours.
This compietes the process of coating the wells and each diagnostic kit is then supplied with 96 coated wells. The shelf life of coated mierowells when stored at 4 degrees C in a sealed ;5 plastic bag is up to one year.
Step 2: Adhering Anti-Sm or Anti-SrnLRNP antibodies to the Prepared Wells From Step 1:
The sample diluent is supplied in the kit as a 40 ml green solution. To prepare a 1000 ml `1 solution of Sample Diluent, 100 milliliters of native bovine serum, 1.42 g of Potassium Phosphate (dibasic), .26 g ot Potassiurn Phosphate (monobasic), 1 gram of sodium azide, 0 and 8.6 9 of sodium chloride, and 1 ml of stock green dye are dissolved in deionized water sufficient to make 1 OOû milliliters of solution. This solution is then filtered through a .2 micron . ~
. ~,, .. ,,, , . ~. . . ..
,. . ~ . .: . . , ~ ..
. . ~ - . , : :, -'~ ' ' .' " ~ ' ' ':' . ' WO 90/10229 ;~ 3D~ PCr/U~90/0102~
~ ` .. .. .. .
fllter.
The Sample Diluent acts as a blocking agent similar to the casein blocker which was previously coated on the wells. The principle biocking component in the Sample Diluent is the native bovine serum which acts to inhibit the bincling of any BSA-reactive antibodies to the mBSA coating on the surface of the well.
Prior to contacting the body fiuid with the prspared ~late, lhe serum is diluted by adding aliquots of sera to the sarnple diluent in a 1:50 ratio (volume of serum:volume of sample diluent), although this exact dilution is not critical and depends upon the nature of the body fluid and the assay techniques employed. To ~orrn the diluted sample solution the body fluid is aliquoted in 10 microliler proportions into 490 microliters of Sample Diluent. In an individual well, 100 microliters of the diluted sample is dispensed, and the affixation of the anti-Sm or anti-Sm/RNP antibody is enhanced by 15 rninutes incubation at room temperature.
Following the affixation of the anti-Sm or anti-Sm/RNP antibody to the coated wells, the wells j 15 are thoroughly washed four times with PBS to remove the free unbound antibodies that are present in the sample, which if ailowed to remain, would elevate the background absorbance.
.
Step 3: Assax for the Anti-Sm or Anti-SmlRNP antibod~/ Affixed to the Wells: Standard enzyme-linked immunoassay techniques, previously described, are used for this assay, although any suitable means of detsction such as radioactive labeling, fluorescence, or the like can be employed. For the examples describecl hereinafter, anti-human IgG and anti-h~man IgM induced in goats were used to ascertain whether anti-Sm IgG and IgM antibodies or anti-Sm/RNP IgG and IgM antibodies were present. Please note that other species of animal can be used to produce anti-human antibodies. These antisera were linked to horseradish peroxidase, an en~yme which yields a colored product whenever one of its substrates is present together with hydrogen peroxide. Tho substrate should be chosen to be consistent with the enzyme conJugated to the antibody. For the examples described hereinafter, the substrate was (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
The kit contains one 12 ml vial of conjugated antibody solution with anti-human IgM and IgG
antibody conjugated to horseradish peroxidase. To prepare a working conjugat d antibody solution, a phosphate buffer with protein stabilizer and .02% thimerosal solution at pH 7.4 (commercially available from Medix~ was mixed wilh aprotinin, a protease inhibitor ' ., ,~
,. , . .: : .. . . . ..
WO ~0/102~g PCI'/US~0/~)1029 (commercially available from Miles Psntex) at a .01% ratio of inhibitor to volume of buffer.
This diluent enhances the stability of the conjugated antibody. The solution is mixed at a ratio of 1/~000 for IgG and 1/1500 for IgM; one part of concentrated conjugated IgM and IgG
antibodies is aliquoted into 4000 and 1500 parts conjugate diluent, respectively. The ratio of concentrated conjugated antibody to conjugate diluent is subject to wide latitudes of dilutions based on the manufacturer's concentration of conjugated antibody used in the assay.
. Next 100 microliters of the enzyme conjugated goat anti-human antibody working solution, (prepared as described) is added to each microtiter well. 8inding of these antibodies to the anti-Sm or anti-Sm/RNP antibody is permitted for at least 15 minutes at room temperature, then the microtiter wells are emptied of their contents, washed four times with PBS, and allowed to clrain before the next step.
The presence of a label and antibody, as previously described, is determined by incubating the wells with a solution of buffered (3,3',5,5') Tetramethylbenzidine and hydrogen peroxide.
This solution is supplied in the kit in two 8 ml ~ials; one contains (3,3',5,5')Tetramethylbenzidine; the other vial contains hydrogen peroxide. The separate vials are necessary due to the interaction between the two solutions. The two solutions are mixed in a one to one ratio just before use, and 100 microliters of the mix~d solution is dispensed into each microwell. The reaction is permitted to continue for 10 minutes at room temperature, or until sufficient color appears to be read on the spectrophotometric device used. The reaction is subsequentty stopped through the addition of an equal volume oi 2.5 normal sulfuric acid, and the intensity of color (the optical density, ~OD", or absorbance) is read by a spectrophotometric device such as a Dynatech MR600 or the like.
As with any enzyme-linked immune assay, the resultant color of the reaction product is proportional to the number of conjugated antibodies which have bound to the antibody. For 2~ most cases, the number of bound conjugated antibodies is linearly related to the number of bound antibodies. Hence, as the amount of anti-Sm or anti-Sm/RNP antibody bound to the wells Increases, so does the optlcal density, or absorbance of the enzynne reactlon.
:.
., .~ , . ~ . . . . .
,,~, , :
.
Wo 90/10229 ~3~ rcr/us90/0l029 E~MPLE 1 Protocol for Pr~Co~tin~ Microwells with Purlfled dsDNA
Polystyrene wells were coated with (native) double stranded deoxyribonucleic acid (dsDNA) by the following procedure:
1. Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was dissolved at a ratio of 20 micrograms/ml in distilled water or PBS.
2. 100 microliters of the mBSA solution was placed in each microwell1 and incubated overnight at 4 degrees C.
.
10 3. The excess coating solution was shaken from the plate after incubation. The plate was rinsed once with a solution of .01 M PBS, pH 7.3 to remove unbound mBSA. Thewells were inverted to drain thoroughly.
4. 5 ug/ml of purified dsDNA was diluted in PBS, .01M, pH 7.3, and 100 microliters of the diluted dsDNA solution was dispensed into each microwell and allowed to -16 incu~bate for 18-24 hours at 4 degrees C.
5. ~ A~er ineubation, the excess dsDNA solution was shaken from the wells and 100 microliters of S-1 nuclease in digestion buffer was added to each well.
. .
. .
6. To form S-1 nuclease buffer solution, .03M sodium acetate and .û3M acetic acid in a 1.1 to 1 ratio at pH 4.6 was pr~pared. Then 95 mls of this buffer was mixed with 5 mls ~ ~ of ~Iycerol, .29 9 NaC1 and .029 9 ZnS04 for a flnal volume of 100 rnls at pH 4.6.
S-1 nuclease (Sigma) was dissolved into this solution at a concentration of 100 units/ml buffer. (One unit of S-1 nuclease is defined as causing 1.0 microgram of ssDNA per minute to beeome perchloric acid soluble at pH 4.6 and 37 degrees C.) 7. 100 microliters of S 1 nuclease buffer solution was contacted with each well for two : 35 hours at 37 degrees C. ~ter incubation, the wells were emptied, rinsed twic~ with 1~ PBS, and draineci thoroughly.
.
S-1 nuclease (Sigma) was dissolved into this solution at a concentration of 100 units/ml buffer. (One unit of S-1 nuclease is defined as causing 1.0 microgram of ssDNA per minute to beeome perchloric acid soluble at pH 4.6 and 37 degrees C.) 7. 100 microliters of S 1 nuclease buffer solution was contacted with each well for two : 35 hours at 37 degrees C. ~ter incubation, the wells were emptied, rinsed twic~ with 1~ PBS, and draineci thoroughly.
.
8. ~ Casein blocker solution (pH 7.3) was dispensed in 200 microliter increments into each ,, .
~.:
W~ 90/10~2~ PC~/US90/01029 2~ 33~o ` f~
- 22 ~
well. The casein bloek~r solution consists of 2 ml glycerol, 10 g sucrose, 15 mgcasein and sufficient TEN buffer to bring the volume to -00 ml.
~.:
W~ 90/10~2~ PC~/US90/01029 2~ 33~o ` f~
- 22 ~
well. The casein bloek~r solution consists of 2 ml glycerol, 10 g sucrose, 15 mgcasein and sufficient TEN buffer to bring the volume to -00 ml.
9. The wells coated with the blocker solution were incubated overnight at 4 degrees C.
After incubation, the solution was shaken from the wells and the wells were inverted for 15 minutes. Then the weils were uprighteci and allowed to dry 24 hours at room temperature.
;
The coated dsDNA wells were then used to determine the presence of anti-dsDNA in serum samples obtained from individuals with:
1. no apparent pathology (normal);
2. Systemic Lupus Er~nhematosus.
These sera were drawn frorn patients with known pathological status. Described hereinafter, is a method of performing an anti-dsDNA assay, using the dsDNA coated wells in a sandwich ELISA format.
1. Standards, Sera from patients with no apparent pathology and patients with SLE, a i~ high positive human serum control (100 AU/ml), a moderate positive human serum control (45 AU/ml), and a negative hurnan serum control ~3 AU/ml) were diluted with Sample Diluent in a 1:50 ratio; 1 part serum to 50 parts Sample Diluent. The Sample Diluent was a solution consisting of 100 mls of native bovine serum in a phosphate buffered saline consisting of 1.42 9 of potassium phosphate (dibask~,), .26 g potassium phosphate (monobasic), 8.6 9 sodium chloride, .1% sedium azide, 1 ml stock greendye, and distilled water added to bring the volume to 1000 mls. The Sample Diluent was then filtered through a .2 micron filter.
2. 100 microliters of the Sample Diluent solutions were placed in the designated wells at room temp~rature for 15 minutes to allow completion of the antigen-antibody binding process. Following incubation, the wells were emptied and washed four times ` with PBS solution.
3. The welis were expo~ed to a working conjugateci antibody solution eonsistint~ of 1 part per volume horseradish peroxidase conjugated IgG and IgM speoific antibodies, and , . . . f . ~ ............ .. . . . . .
.~ . . : ~ , . . , . ..... . . , . , :, .. ; . . .
. . ' . ` !
~, . ~' ' ' . . ' . . I '~
WO 90/10~29 PCI/lJS90/01029 - ~3 -3000 part per volurne conjugate diluent (Medix) consisting of phosphate buffer, protein stabilizer, and .02% thimerosai, to which was added .01% by volume a protease inhibitor (commercially available from Miles Pentex).
4. 100 microliter of the working conjugated antibody solution was placed in eachmicrotiter well and allowed to incubate at room temperature for 15 minutes. After incubation, the wells were rinsed four times with PBS solution to remove unboundconjugated antibodies.
5. Each well was assayed for horseradish peroxidase activity by mixing equal volumes (3,3',5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and ~10 dispensing 100 microliters of this substrate solution into each well. The presence o~
the anti-dsDNA was detected by a blue color appearing after the 10 minute room temperature incubation. 100 microliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color. The yellow color was quantitated at 450 nm using a Dynatech MR600 plate reading spectrophotometer.
Deteotion of anti-dsDNA (IgG & IgM) Source Mean O.D.
; CDC Ref S~d. 1.0~
anti DNA pos 1.13 pos SLE #1 .54 pos SLE #2 1.75 ` pos anti nucleolar .10 normal serum .09 .
1 . :
~, .
WO9(1/lOZ~ 2~- PCr/US90/0102 Adsorp~ion ot anti-dsDNA f:rom Serum by Uncoated Polystyrene Wells To demonstrate the beneflcial effect of the coating tr0atment, polystyrene wells were left 5 totally uncoated but exposed to the sera and the standards as described in the previous example. The wells were then exposed to horseradish peroxide conjugated IgG and IgM
antibodies, and assayed lor peroxidase activity. As expected, c?nly slight selectivity was exhibited by untreated wells and high amounts of non-specific binding led to very high background absorbance.
G.D. Coated O.D.
Source AU/ML?~as in txample 1 Uncoated Wells Calibrator 120.0 1.38 1.28 2 60.0 1.05 1.08 ,15 3 30.0 .55 .77 - 4 15.0 .37 .57 7.5 .29 .67 - 6 3.8 .22 .50 Normal Serum 1 8.0 .28 .84 Normal Serum 2 9.0 .29 .97 Normal Serum 3 3.0 .19 .65 I ligh Positive SLE
112.0 1.34 .54 . 2 50.0 .89 .51 3 120.0 1.96 1.67 Low Positive SLE
, ~
~ 1 13.0 .35 1.76 *AU/ml vaiues calculated In previous experiment.
~ .
:, :
. I
,,~
~ ~ . : . ' ' ' ' wa:) 90/I~û229 27~ 3~ ; P~/U~90/û1029 r .
. . .
- 2i~ -EXAiV'PLE 3 A Diagnostie Test Kit Assa,y Procer''ure The kit contains pre-coatf?d microwells and, 1 vial (30 ml) of Sample Diluent (green solution containing 0.1% sodium az3de);
5 6 vials (200 ul each) of Assay Standards for anti-dsDNA, containing these levels of anti-dsi~NA activity 120, 60, 30, 15, 7.5, 3.8 AU/ml (anti-dsDNA activity 100 AU/ml of this assay calibrator has an antigen binding capacity of 59 x 10-4 ug of DNA), 1 vial of high positive hurnan serum control (about 100 AU/rnl), 1 vial of moderate positive human serum control (about 25 AU/ml), 1 vial of negativZs human serum eontrol (about 3 AU/ml). Also included in the diagnostic kit was a vial (15 ml) of conjugated antibody working solution Z~iontaining horseraciish peroxidase conjugated antihuman IgG, and IgM
1 bottle (8 ml) of TMB Substrate; solution A containing 3,3',5,5', tetramethylbenzidine, 1 bottle (8 ml) of TiYiB Substrate; Solution B containinZ~, hydrogen peroxide. When rnixed ~15 with equal parts Solution A will form a substrate capable of generating a colored product.
i 1 bottle (12 ml) of Stop Reagent containing 2.5 N H2S04 (1 N HCL can be substituted), 1 packet of Phosphate Buffere,d Saline (PBS) which reconstitutes to 2 liters of 0.01 M PBS, pH 7.3, which is utilized as a wash solution Plate Template.
., .
~0 The various reagents of th~ kit were utili~ed to perform the assay. The directions for the , methoci of assaying for anti~dsDNA were included in the kit and these directions were followed precisely during this experiment.
, .
The piate templates were labelled for sample placement in the microwells. A 1:50 dilution of :
, .
:.::
2~3~ P~/US90/0102~
the standards, controls and patient sarnples was prepared in Sample Diluent (green solution).
After incubation, the solution was shaken from the wells and the wells were inverted for 15 minutes. Then the weils were uprighteci and allowed to dry 24 hours at room temperature.
;
The coated dsDNA wells were then used to determine the presence of anti-dsDNA in serum samples obtained from individuals with:
1. no apparent pathology (normal);
2. Systemic Lupus Er~nhematosus.
These sera were drawn frorn patients with known pathological status. Described hereinafter, is a method of performing an anti-dsDNA assay, using the dsDNA coated wells in a sandwich ELISA format.
1. Standards, Sera from patients with no apparent pathology and patients with SLE, a i~ high positive human serum control (100 AU/ml), a moderate positive human serum control (45 AU/ml), and a negative hurnan serum control ~3 AU/ml) were diluted with Sample Diluent in a 1:50 ratio; 1 part serum to 50 parts Sample Diluent. The Sample Diluent was a solution consisting of 100 mls of native bovine serum in a phosphate buffered saline consisting of 1.42 9 of potassium phosphate (dibask~,), .26 g potassium phosphate (monobasic), 8.6 9 sodium chloride, .1% sedium azide, 1 ml stock greendye, and distilled water added to bring the volume to 1000 mls. The Sample Diluent was then filtered through a .2 micron filter.
2. 100 microliters of the Sample Diluent solutions were placed in the designated wells at room temp~rature for 15 minutes to allow completion of the antigen-antibody binding process. Following incubation, the wells were emptied and washed four times ` with PBS solution.
3. The welis were expo~ed to a working conjugateci antibody solution eonsistint~ of 1 part per volume horseradish peroxidase conjugated IgG and IgM speoific antibodies, and , . . . f . ~ ............ .. . . . . .
.~ . . : ~ , . . , . ..... . . , . , :, .. ; . . .
. . ' . ` !
~, . ~' ' ' . . ' . . I '~
WO 90/10~29 PCI/lJS90/01029 - ~3 -3000 part per volurne conjugate diluent (Medix) consisting of phosphate buffer, protein stabilizer, and .02% thimerosai, to which was added .01% by volume a protease inhibitor (commercially available from Miles Pentex).
4. 100 microliter of the working conjugated antibody solution was placed in eachmicrotiter well and allowed to incubate at room temperature for 15 minutes. After incubation, the wells were rinsed four times with PBS solution to remove unboundconjugated antibodies.
5. Each well was assayed for horseradish peroxidase activity by mixing equal volumes (3,3',5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and ~10 dispensing 100 microliters of this substrate solution into each well. The presence o~
the anti-dsDNA was detected by a blue color appearing after the 10 minute room temperature incubation. 100 microliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color. The yellow color was quantitated at 450 nm using a Dynatech MR600 plate reading spectrophotometer.
Deteotion of anti-dsDNA (IgG & IgM) Source Mean O.D.
; CDC Ref S~d. 1.0~
anti DNA pos 1.13 pos SLE #1 .54 pos SLE #2 1.75 ` pos anti nucleolar .10 normal serum .09 .
1 . :
~, .
WO9(1/lOZ~ 2~- PCr/US90/0102 Adsorp~ion ot anti-dsDNA f:rom Serum by Uncoated Polystyrene Wells To demonstrate the beneflcial effect of the coating tr0atment, polystyrene wells were left 5 totally uncoated but exposed to the sera and the standards as described in the previous example. The wells were then exposed to horseradish peroxide conjugated IgG and IgM
antibodies, and assayed lor peroxidase activity. As expected, c?nly slight selectivity was exhibited by untreated wells and high amounts of non-specific binding led to very high background absorbance.
G.D. Coated O.D.
Source AU/ML?~as in txample 1 Uncoated Wells Calibrator 120.0 1.38 1.28 2 60.0 1.05 1.08 ,15 3 30.0 .55 .77 - 4 15.0 .37 .57 7.5 .29 .67 - 6 3.8 .22 .50 Normal Serum 1 8.0 .28 .84 Normal Serum 2 9.0 .29 .97 Normal Serum 3 3.0 .19 .65 I ligh Positive SLE
112.0 1.34 .54 . 2 50.0 .89 .51 3 120.0 1.96 1.67 Low Positive SLE
, ~
~ 1 13.0 .35 1.76 *AU/ml vaiues calculated In previous experiment.
~ .
:, :
. I
,,~
~ ~ . : . ' ' ' ' wa:) 90/I~û229 27~ 3~ ; P~/U~90/û1029 r .
. . .
- 2i~ -EXAiV'PLE 3 A Diagnostie Test Kit Assa,y Procer''ure The kit contains pre-coatf?d microwells and, 1 vial (30 ml) of Sample Diluent (green solution containing 0.1% sodium az3de);
5 6 vials (200 ul each) of Assay Standards for anti-dsDNA, containing these levels of anti-dsi~NA activity 120, 60, 30, 15, 7.5, 3.8 AU/ml (anti-dsDNA activity 100 AU/ml of this assay calibrator has an antigen binding capacity of 59 x 10-4 ug of DNA), 1 vial of high positive hurnan serum control (about 100 AU/rnl), 1 vial of moderate positive human serum control (about 25 AU/ml), 1 vial of negativZs human serum eontrol (about 3 AU/ml). Also included in the diagnostic kit was a vial (15 ml) of conjugated antibody working solution Z~iontaining horseraciish peroxidase conjugated antihuman IgG, and IgM
1 bottle (8 ml) of TMB Substrate; solution A containing 3,3',5,5', tetramethylbenzidine, 1 bottle (8 ml) of TiYiB Substrate; Solution B containinZ~, hydrogen peroxide. When rnixed ~15 with equal parts Solution A will form a substrate capable of generating a colored product.
i 1 bottle (12 ml) of Stop Reagent containing 2.5 N H2S04 (1 N HCL can be substituted), 1 packet of Phosphate Buffere,d Saline (PBS) which reconstitutes to 2 liters of 0.01 M PBS, pH 7.3, which is utilized as a wash solution Plate Template.
., .
~0 The various reagents of th~ kit were utili~ed to perform the assay. The directions for the , methoci of assaying for anti~dsDNA were included in the kit and these directions were followed precisely during this experiment.
, .
The piate templates were labelled for sample placement in the microwells. A 1:50 dilution of :
, .
:.::
2~3~ P~/US90/0102~
the standards, controls and patient sarnples was prepared in Sample Diluent (green solution).
10 ul of sample was added to 500 ul sample diluent in a one volurne to 50 volume sample dilution. 1:50 dilutions of the assay standards and controls were diluted in the same manner.
100 ul of each diluted sample, control or standard was added to the appropriate mierowell(s).
5 The wells were allowed to incubate ior 15 minutes at room temp~rature. To perform the rinse step the contents of PBS packet was added to 2 lilters of reagent grade water, and the solution was mixed well until all the crystals were dissolved. Then th~ PBS buffer was used - ~ to wash the wells four times. The microwells were inverted between each wash to empty the fluid, after which the wells were drained and blotted on absorbent paper to remove residual ~10 wash fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
100 ul of working conjugated antibody (red solution) was added to each well, incubated for 15 minutes at room terrlperature, and then the wells were again washed ~our times with PBS
colution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TMB Substrate Solution A and TMB Substrate Solution B were combined to form the color generating substrate. The kit instructed that if properly combined this substrate solution would be colorless and it was colorless. Next a 100 ul of the colorless working substrate solution v~as added to each well and the wells were incubated for ten rninutes at room temperature.
~0 Next 100 ul stop reagent was added to each well to end the enzyme reaction, and the O.D.
of each well was read at 450 nm against a water blank. A standard curve graph was - generated by p!otting AU/ml of standards against O.D. of standards, and sample results were obtained from this standard curve.
Source O.D. AU/ml ;25 Calibrator 1 .2 3.8 Calibrator2 .3 7.5 Calibrator 3 .4 15.0 Calibrator 4 .57 30 0 Calibrator 5 .88 60.0 Calibrator 6 1.24 120.0 Refer to Graph 2 'i ' ~', WO 90/10229 2~ 34~ r~us90/0l029 t: ~
Controls O. D. AU~
high positive 1.11 104.0 moderate positive .55 24.0 negative .14 2.0 O.D. AUlm~
normal human sera #1 .14 2.0 norrnal human sera #2 .04 1.0 normal human sera #3 .18 3.0 SLE positive #1 .53 23.0 SLE positive #2 1.36 130.0 SLE positive #3 1.48 14Z.0 ~ ' , ':
: . .
' : ' ' :. .
.~
', . ...
., :
i ~, .
.' .~:
WO 90/10229 ;~Cl S ~34~ ' PCT/US90/01029 - 2E~ -Accelerated Stabliity Study DNA coated microwells were treated with S,-nuclease using the optimized protocol, then duplicate microwells were stored at 37 degrees C and 30 degrees C, and each set of wells ~` 5 was tested at various time intervals using anti-dsDNA and anti-ssDNA monoclonal antibodies.
The results of this experiment are shown in the follovving table:
' ~ Real Time Equivalent Time In Days~ In Mont,,hs**, % ssDN,A Activity O 0.0 3-4%
1 0 1 1 .6 3-~%
' ~ 2 3.0 ~-3%
7.5 3-4%
~, 7 1 0.5 4-5%
, g 13.5 3-4%
13 19.5 3-4%
', ~incubated at 37 degreés C
~; : **if incubated at 4 degrees C
,,1 ,'~ The dsDNA activity in these microwells remained unchanged during the accelerated stability study. The results obtained with the microwells stored at 30 degrees C were similar to those ,70 presented above and confirmed that the stability of the dsDNA coated on the microwells ~1 using this protocol was equivalent to more than one year without the generation of significant amounts of ~sDNA.
, ~!
!
,~
,, .
.~ ' .
,~
WO 90/10229 Z~ 5~ ¢3 PCr/US90/01029 Pro~ocol for Pr~Coatin~ Microwell~
with Purified Smith Antigen Polystyrene wells were coated with Sm antigen by the following procedure:
1. Methylated Bovine Serum Albumin (Sigma) (hereinafter ciesignated as mBSA) was dissolved at a ratio of 2û micrograms/ml in distilled water or PBS.
2. 100 microliters of the mBSA solution was placed in each microwell, and incubated overnight at 4 degrees C.
3. The excess coating solution was shaken from the plate after incubation. The wells were inverted to drain thoroughly.
;
4. 5 units/ml of purified Sm antigen vvas diluted in PBS, .OlM, pH 7.3, and 100microliters of the diluted Sm antigen solution was dispensecl into each microwell and allowed to incubate for 18-24 hows at ~ degrees C.
5. Casein blocker soluUon (pH 7.3) was dispensed in 200 microliter increments into each s well. The casein blocker solution consists of 2 ml glycerol, 10 g sucrose, 25 mg casein and sufficient TEN buffer to bring the volume to 100 ml.
:
6. Tha walls coated with the blocker solution wcre incubated overnight at 4 degrees C.
After incubation, the solution was haken from the wells and the wells were inverted for 15 minutes. Then the wells were uprighted and allowed to dry 24 hours at room :i~o ternpera~ure.
Tha coated Sm antigen wells ware then used to determin~ th~ presence of anti-Srn antibody in seven CDC reference sera for anti-nuclear antibodies. Thle r~sults show that only anti-Sm antibodies rsact in the assay.
( Describsd hereinafter, is a method of performing an anti-Sm antibody assay, using the Sm ~5 antigen coated walls in a s~ndwich ELISA format.
`, .
1. Standards, seven rieferance sera samples, a negative human serum control .', :
., : : . ... . . . . .. . . . ~ :.. . .
WO 90/102~ . PCr/US90/01029 RE~DS/ml, a high positive human s~rurn control (RE~DS/ml), a moderate positive human serum control (RE~nS/ml), and a low positive hurnan serum control (RE~DS/ml) were ciiluted with Sample Diluent in a 1:50 ratio; 1 part serum to 50 parts Sample Diluent. The Sample Diluent was a solution consisting of 100 mls of native bovine serum in a phosphate buffered saline consisting of 1.42 g of potassium phosphate (dibasic), .26 g potassium phosphate (monobasic), 8.6 9 sodium chloride, .1% ~odium azide, 1 ml stock green dye, anai distilled water added to bring the volume to 1000 mls. The Sample Diluent was then fiHered through a .2 rnicron filter.
2. 100 microliters of lhe Sample Diluent solutions were placed in the designated wells ~10 at room temperature for 15 minutes to allow completion of the antigen-antibody binding process. Following incubation, ths wells were emptied and washed fourtimes with PB5 soiution.
3. The wells were exposed to a working conjugated antibody colution consisting of 1 part per volume horseradish peroxidase conjugated Igt; and IgM specific antibodies, and 4000 and 1500 part per volume conjugate diluent, resp0ctively, ~Medix) consisting of phosphate buffer, protein stabili~er, and .02% thimerosal, to which was added .01%
by volume a protease inhibitor ~commercially available from Miles Pentex).
4. 100 microliter of the working conjugated antibody solution wac placed in each~, microtiter well and allowed to incubate at room temperature for 15 minutes. After ~o incubation, the wells were rinsed four times with PBS solution to remove unbound ~i conjugated antibodi~s.
.
5. i_ach w~ll was assayed for horseradish peroxidase activity by mixing equal volumes (3,3',5,5') Tetramethylbenzidine (K~rkegaard Perry) and hydrogen peroxide solution and . disp0nsing 100 rnicroliters of this substrate solution into each well. The presence of ~5 the anti-Sm antibodies was detected by a blue color appearing after the ~iO rninute - ~- room temperature incubation. i 00 microliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color. The yellow color was quantitated at 450 nm - using a Dynatech MF16û0 plate r0ading spectrophotom~ter.
Resuits are calcuiated by the following procedure:
~0 1. Calculatc tha mean absorbance values for the duplicates of the controls. The duplicate absorbance values of the positive control should be within 10% of ~he mean .
WO 90/1û229 ~ .PCr/VS90/01~2 absorbance value.
~.
2. The mean absorbance reading of the reagent blank should be less than 0.05.
Readings greater than 0.05 may indicate possible contaminated of the Substrate Solution.
3. Calculate the cut-off absorbance values for the assay, and write them in the appropriato blanks on the results graph.
.
a. The mean absorbance (O.D.) of negative control x 3.2 = negative cut-off absorbance, which is equal to 20 RE~DS units of anti-Sm activity. Sarnples with O.D. values below the cut-off have less than 20 RE~DS units of anti-Sm ~10 activity, and should be scored as ~negative~. Samples with O.D~ values equal to or higher than the cut-off have greater than 20 R~DS units anti-Sm activity, and should be scored as ~positive~. Samples with O.D. values close to the cut-off may warrant further testing (see Figure 4~.
The results were calcuiated by determining a cut-off O.D. The cut-off O.D. was determined ~1~ by running 200 normal sera samples and taking the mean of the O.D.s plus three standard d~viations. This cut-off was equal to 3.2 x the O.D. of the standardized negative control.
~i , `1 .~
:~' .
, '~'', ' :',. : ' . "
~ .
PC~I'/IJS90/01029 f~
- 3~ -Detection of antl-~m antibo~i~s (IgG & ~gM) SampLe O.D. St~tus normal cut-off 0.1 12 Iow positive serum 0.21 positive medium positive serum 0.40 positive high positive serum 0.89 positive anti Sm 1.22 positive anti-~NP 0.112 negative anti-SS-A 0.02 negalivs . anti-SS-B 0.04 negative anti-Sel-70 0.06 negative anti-nucleolus 0.04 negative .
:, :
-, .~
;: , . . . . . .. . . . .
. ,, ! . . . .
WO 90/102~9 . ` Pl-r/US~)0/0102~
~q33~
~ D~agnostic Tes~ Kit Assaly Procedure The kit contains pre-coated microwells (qualitative format) and, 1 vial (40 ml) of Sample Diluent (green solution containing 0.1% sodium azide);
1 vial ~100 ul) of high positive human serum control (about 20 28 RE~DS/ml), 1 vial (100 ul) of calibrator (optional), .~
1 vial ~100 ul) of negative human serum control (about ~.2 RE~DS/ml). The negative cut-off value is 20 RE~DS units. To calculate O.D.s into RE~DS units, the negative value 6.2 is divided by the mean value O.D. it produces; the resuHant number is multiplied by the O.D.s of the samples to give RE~DS units.
1 vial ~12 mi) Conjugated Antibody Working Solution - containing horseradish peroxidase eonjugated anti-human I~G and igM, 1 bottle (8 ml) of TMB Substrate, solution A containing 3,3i,5,5', tetramethylbenzidine, 1 bottle (8 ml) of TMB Substrate; Solution B containing hydrogen peroxide. When rnixed with equal parts Solution A will form a substrate capable of generating a colored product.
.
1 bottle ~12 ml) of Stop Reagent containing 2.5 N H2SO4 (I N HCL can be substituted) (not - necessarily supplied in kit), 1 packet of Phosphate Buffered Saline (PBS) which reconstitutes to 2 liters of 0.01 M PBS, pH 7.3, which is utilized as a wash solution Plate Template.
The various reagents of the kit were utilized to perforrn the assay. The direetions for the ~, method of assaying for anti-Sm antibodies were included in the kit and these directions were ~: followed precisely during this expçriment.
"
~ .
.
.'`
Wo 90/10229 ~ i3~ Pcr/usgo/01029 ~",-, - 3~- .
The plate templates were labelled for sample placement in the microwells. A 1:50 dilution of the controls and pa~ient samples was prepared in Sample Diluent (green solution) (no calibrators were used in this example). 10 ul of sarnpl~ was added to 490 ul sample diluent in a one volume to 50 volume sample dilution.
100 ul of each diluted sample and control was added lo lhe apprnpriate microwell(s). The wells were allowed to incubate for 15 minutes at room temperature. To perform the rinse step the contents of PBS packet was added to 2 liters of reagent grade water, and the solution was nnixed well until all the crystals were dissolved. Then th0 PBS buffer was used to wash the wells four times. The microwells were inverted between each wash to empty the fluid, after which the wells were drained and blotted on absorbent paper to remove residual wash fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
- 100 ul of working conjugated antibody (red solution) was added to each well, incubated for 15 minutes at room temperature, and then the wells were again washed four times with PBS
solution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TMB Substrate Solution A and TMB Substrate Solution B were combined to ~orm the coJor generating substrate. The kit instructed that if properly eombined this substrate solution would be colorless and it was colorless. Next a 100 ul of the colorless working substrate solution was added to each well and the wells were incubated for ten minutes at room temperature.
Next 100 ul stop reagent was added to each well to end the en~yme reaction, and the O.D.
of each well was read at 450 nm against a water blank. The final resulting data can be read as O.D.s. To determine if the sample is positive for anti-Sm antibodies, the sample O.D. is eompared to the cut-off C).D. value calculated for the assay (see Figure 4). For semi-~5 quantitative results the O.D. value can be converted to R~DSunits by rnultiplying the sample O.D. by the quantity (6.2 RE~DS units/negative control O.D.). 6.2 FlE~DS units is the anti-Sm activity of the negative c:ontrol, which was standardized against the CDC referenee serurn.
The cut-off Is 20 RE~DS units.
.~
.
- , ;
~ , ~ , . . .
WO 90/~()229 ~ PCI/U~;~0/011J2~
(, 'I
SLE ~era Sa~ Q~ RE~DSUnits 0.16 16.5 2 0.10 10.3 3 0.07 7.2 4 0.09 9.3 0.12 12.4 6 0.10 10.3 7 0.12 12.4 8 0.08 8.
10 ~ 009 93 0.12 12.4 11 0.19 19.6 12 0.18 18.5 13 0.27 27.8*
5 14 0.35 36.0*
0.29 ~9.9*
16 1.17 121.0 17 0.15 15.4 18 0.35 36.0*
2019 0.13 13.4 :: :
*positive result ~, : :
;
?
~~ :
:, ~
.~ l WO 90/10229 ~ PCr/US90/01029 ~ ,. . .
~' .,', , ,.. ,, ~
EXAI\APLE 7 Adsorptlon ot anti Sm antibodi~s From Serl~m by ~he Test Kit Invention and a Competitive Kit To demonstrate the sensitivity of the anti-Sm test kit samples from SLE patients were run in a competitive kit and were run in the test kit in accordancs with the present invention under the protocol demonslrated in Example 6, with the ~xception that only the negative serum was used to produce a cut-off level. The test was run as ia quaiitative test showing only negative or positive results. 19%-40% of SLE patients are expected to have anti-Sm antibodies present. With all the included samples (the data is only representative of a portion of the 'O data), approximately 28% of the sera samples were positive according to the present invention and only 21% were positiv0 according to the competitive kit. Note the present invention detects both IgG and IgM and the competitive kit only detects IgG.
The resulting O.D.s were obtained. The cut-off value for the present invention was 0.33 O.D.
The cut-off value of the competitive kit was the given by the kit instructions.
;
SLE Sera O.D. From O.D. From Samples Invention Status Competitive Kit Status 0.59 positive .94 positive 2 0.15 negative .05 negative 3 0.36 positive .12 negative 4 0.19 negative .08 negative 0.48 positive .61 positive 6 0.29 negative .13 negative 7 0.24 negativa .07 negative 8 0.21 negative .08 negative 9 0.21 negative .22 negative 0.19 negative .OB negative 11 0.25 negative . 1 0 negative 0.19 negative .13 n~gative 13 0.28 negative .14 negative ~;3 14 0.29 negative .16 negative V.08 negative .05 negative 18 0.86 positive 1.10 positive . ~ .. ~.. . . ,- ... - - , . - . .; . .
: , . : . . .
. - . ,: ~,: :. . . . . .
Wo 90/10229 ~ Pcr/vsso/olo29 ' :, ' ! ' .
XANiPLE 8 Accelerated Stability Study Sm antigen coated microwells were treated with the optimized protocol, then duplicate rnicrowells were stored at 37 clegrees C and 4 degre~es C, and each set of wells was tested on days 1, 4, 6, 8, and 12, using a high positive anti-Sm sera, a moderate positive anti-Sm sera, and a negative anti-Sm sera. The results of this experiment are shown in the following . table:
'.' .
O.D.s O.D.s O.D.s O.D.s O.D.s Dav 1 Day 4 Da.~ Day ~ Day 12 .~
;~0 High anti-Sm activity 4 1.12 1.14 1.12 1.23 1.00 37C (high) 1.15 1.05 1.08 1.09 1.12 -~, Moderate~ anti-Sm - l, activity `?~ 4 0.92 0.93 0.78 0.93 0.95 37~ 0.94 0.92 0.86 0.90 0.80 - Low anti-Sm activity 4 (negative) 0.07 0.07 0.05 0.06 3.05 , 37 0.06 0.09 0.0~ O.o~ 0.05 0 *(1:2 dilution of the hi~h anti-Sm sera) ~j The Sm antigen activity in these rnicrowells remained unchanged during tha accelerated ~', stability study. The results obtained with tha microwells stored at 37 degrees C wera similar l to those presented abov~ and confirmed that the stability of the Sm anti~en coated on the `~ microwells using this protocol was equivalent to more than one year shelf life.
.
.
`' ~
, WO 90/10229 , PClr/US~0/01029 2t~ i34L0 - 3B -Protocol for Pr~Coating Microw~lls with Purified Sm/RNP ;intlgen Polystyrene wells were coated with Sm/RNP antigen by the following procedure:
5 1. Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was dissolvad at a ratio of 20 micro~rams/ml in ciistilled water or PBS.
2. 100 microliters of th~ mBSA solution was placed in each microwell, and incubated - overnight at 4 degrees C.
3. The excess coating solution was shaken from the plate after incubation. The wells .0 were inverted to drain thoroughly.
: ~`
. 1 unit/ml of purified Sm/RNP antigen was diluted in PBS., .01M, pH 7.3, and 100 rnicroliters of the diluted Sm/RNP antigen solution was dispensed into each microwell and allowed to incubate for 18-24 hours at 4 degrees C.
. :
5. Casein blocker solution (pH 7.3) was ciispensed in 200 microliter increments into each . 5 well. The casein blocker solution consists of 2 ml glycerol, 10 g sucrose, ~5 mg .~ casein and sufficient TEN buffer to bring the volume to 100 ml.
6. The wells coatecl with the blocker solution were incubated overn,.ght at 4 degrees C.
After incubation, the solution was shaken from the wells and the wells were inverted for 15 minutes. Then the wells were uprighted and allowed to dry 24 hours at room o temperature.
Th~ coated Sm/RNP antigen wells were than used to determine the presence of anti-Sm/RNP antibody in serum samples obtained from individuals with Progressive SystemicSclerosis (PSS).
i~j Described hereinafter, is a rnethod of performing an anti-Sm/RNP antibody assay, using the ;:5 Sm/RNP antigen co~teci w~lls in a sandwlch ELiSA format.
,' :
WO 9~/1022~ PCrtUS90/01029 2~ i34~) , 1. Sera from patients with PSS, a high positive human serum control (RE~DS/ml), and a negative human serum control ~RE~DS/ml) were diluted with Sample Diluent in a ~:50 ratio; 1 part serum to 50 parts Sample Diluent. The Sample Diluent was a solution consisting of 100 mls of native bovine serum in a phosphate buffered saline consisting of 1.42 g of potassium phosphate (dibasic), .26 g potassium phosphate(monobasic), 8.6 9 sodium chloride, .1% sodium azide, 1 ml stock green dye, and distilled water added to bring the volume to 1000 mls. The Sample Diluent was then filtered through a .2 rnicron filter.
2. 100 microliters of the Sample Diluent solutions were placed in the designated wells at room temperature for 15 minutes to allow completion of the antigen-antibody binding process. Following incubation, the wells were emptied and washed four times with PBS solution.
3. The wells were exposed to a working conjugated antibody solution consisting of 1 part per volume horseradish peroxidase conjugated IgG and IgM specific antibodies, and 4000 and 1500 part per volume conjugate diluent, respectively, (Medix) consisting of phosphate buffer, protein stabilizer, and .02% thimerosal, to which was added .01%
by volume a protease inhibitor (commercially available from Miles Pentex).
, - 4. 100 microliter of the working conjugated antibody solution was placed in each ;~ microtiter weli and allowed to incubate at room temperature for 15 minutes. After ~o incubation, the wells were rinsed four times with PBS solution to remove unbound conjugated antibodies.
5. Each well was assayed for horseradish peroxidasa activity by mixing equal volumes (3,3',5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and dispensing 100 mlcroliters of this substrate solution into each well. Th0 presence of 2S the anti-Sm/RNP antibodies was detected by a blue color appearing after the 10 minute room temperature incubation. 100 rnicroliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color. The yellow color was quantitated at 45û nm using a Dynatech MR600 plate reading spectrophotometer.
R~sults 1. Calculate the mean absorbance values for the duplicates of the controls. The :i ., ~,: ; , , , , :
' ' ~ ;,:' ' , :.
WO !~0/10229 PCI'/lJS90/~1029 - 40 ~
duplicate absorbance values of the positive control should be within 10% of the mean absorbance value.
2. The rnean absorbance reading of the reagent blank should be less than 0.05.
Readings greater than 0.05 may indicate possible contamination of the substrate solution.
3. Calculate the cut-off absorbance values for the assay:
a. The mean absorbance (O.D.) of negative control x 2.8 = negative cut-off absorbance, which is equal to 17.5 RE~DS units of anti-SM/RNP activity.
Samples with O.D. values below the cut-off have less than 17.5 RE~DS units ~0 of anti-SM/RNP activity, and should be scored as "negative". Sarnples with O.D. values equal to or higher than the cut-off have greater than 17.5 RE~DS
units anti~SM/RNP activity, and should be scored as ~positive~. Samples with O.D. values very close to the cut-off may warrant further testing (see Figure 6).
':
The results were calculated by determining a cut-off O.D. The cut-off O.D. was determined by running 200 normal sera samples and taking the mean of the O.D.s plus three standard deviations. This cut-off was equal to 2.8 x the O.D. of the standardized negative control. The cut-off for the flrst 40 samples was 0.11 negative O.D. x 2.8 = 0.31 O.D. The cut-off for the second 40 samples was 0.09 x 2.8 = 0.25 O.D. See Figure 6.
:~, :. ~
,'~.
:, . .
:i ~' ' :
-~, : ,.: , : , , " " . . .
W090/10~9 Z05i~34~ Pcr/1590/~1029 Detection of anti-Sm/RNP antibodies (IgG, ~ IgM~
(PSS! SeraSample ~ Status 0. 14 negative 2 0.16 negative . 5 3 0.17 negative 4 0.17 negative 0.09 negative 6 0.10 negative 7 0.15 negative ~0 8 0.11 negative 9 0.19 negative 0.22 negative 11 0. 1 7 negative ` 12 0.19 negative .5 13 0.18 negative 14 0.13 negative 0.17 negative 16 0.11 negative , 17 0.15 negative ilo ~ 1B 0.13 negative -' 19 0.15 negative 0.15 negative 21 0.22 negative Z2 0.18 negative 23 0.15 negative i 24 0.15 negative 0.60 positive 26 0.1 1 negative , ~7 0.16 negative D 28 0.17 negative ', 29 0.13 negative 0.16 negative 31 0.18 negative 32 0.13 negative , . .
, ~
. .
, ., WO 90/10229 ` i `; P(:~US90/01029 3~ - 4~
~PSS) Sera Sample O.D. Status 33 0.?0 negative 34 0.24 negative 0.24 negative 36 0. 11 negative 37 0.19 negative 38 0.28 negative 39 0.12 negative 0.10 negative 41 0.18 negative 42 0.19 negative 43 0.1~ negative ~4 0.16 negative 0.08 negative J 5 46 0.18 negative 47 0. 11 neyative 48 0.38 positive 49 0.09 negative 0.25 positive ~0 51 0.23 negative 52 0.19 negative 53 0.21 negative 54 0.~7 positive 0.17 negative ` '5 56 0.15 negative - 57 0.21 negative : 58 1.23 positive sg 0.16 negative - 60 0.33 positive - '~ 61 0.12 neyative 62 0.11 negative 63 0.24 negative .~ 64 0.11 negative 0.09 negative ~'5 66 0.22 negative :~l 67 0.18 negativa , .
: 'j ', ., . ,.. : : . : . ,- :, .. .. I , . , . , . - . ..
;, .~ , ;' , ' , ' ' ,~ ! ' ' WO 9~/10229 ~ 3'L~ P~l/U590/ûl029 ~ :.
(PSS! Sera Sample O.D. Sta~
68 0.47 positive 69 0.09 negative 70 0.14 negative 71 0.18 negative 72 0.13 negative 73 0.20 negative 74 0.10 negative 0.17 negative 76 0.22 negative 77 0 07 negative 78 0.10 negative 79 o. ~ 7 negative 0.15 negative '.,, ~
` s , ~.
~, ~` ' .
~;~
i "~' "','` .
;~
, : ' , ., " ~ " ~ , ", ,, ,~"~ , ," ,,, ,~ " " ~ "" ,~,. . .
, : ,. . . , j :, .. . . :
, . . ., . , . ~ . . . ~ .. ,;- ~, . . . .. . .
Wo ~0~10229 . . Pc~r/us~o/0~02'~
~ 44 - t A Diagnostic Test Kit Assay Procedure (Oualitative Format) The kit contains pre-coated microwells and, 1 vial (40 ml) of Sample Diluent (green solution containing 0.1% sodium azide);
1 vial (100 ul) of high positive human serum control (about 100 RE~DS/rnl), 1 vial (100 ul) of calibrator (optional), , 1 vial (100 ul) of negative human serunn control (about 6.3 RE~DS/ml). The negative cut-off value is 17.5 RE~DS units. To calculate O.D.s into RE~DS units, the negative value 6.3 - ~ 10 is divided by the negative O.D. rnean value. The resultant number is muttiplied by the O.D.s of the samples to give RE~DS units.
1 vial (12 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase conjugated anti-human IgG and IgM, ~:;
1 bo~iile (8 ml3 of TMB Substrate; solution A comaining 3,3',5,5', tetramethylbenzidine, i15 1 bome (8 ml) of TMB Substrate; Solution B containing hydrogen peroxide. When mixed .; ~ with equal parls Solution A will form a substrate capable of generating a colored product.
1 bottle (12 ml) of Stop Reagent containing 2.5 N H2S04 (1 N HCL can be substituted), ~' 1 packet of Phosphate Buffered Saline (P8S) which reconstilutes to 2 liters of 0.01 M PBS, pH 7.3, which is utilized as a wash solution ; 20 Plate Template.
The various reagents of the kit werc utilized to perform the assay. The directions for the method of assaying for anti-Sm/RNP anlibodies were included in the kit and these directions - ' were followed preoisely during this experiment.
, .
. ,;
.
: ~ ~ ' ' .. :
',, ' : ~ ' . , ' ' ' '.
.
, . , , , . , , , ~ . .
wO go/]0229 Z~15~;~41~ . PCr/US~0/01029 ~, . ~ . , ,.., , i The plate templates were labelled for sample placement in the microwells. A 1:50 dilution of the controls and SLE patient samples was preparecl in Sample Diluent (green solu~ion). 10 ul of sample was added to 500 ul sample diluent in a one volume to 50 volume sample dilution.
100 ul of each diluted sample and controls was addled to the appropriate microwell(s). The wells were allowed to incubate for 15 minutes at room temperature. To perform the rinse step the contents of PBS packet was added to 2 liters ol reagent grade water, and the solution was mixed well until all the crystals were dissolved. Then the PBS buffer was used to wash the wells four times. The microwells were inverted between each wash to empty the fluid, after which the wells were drained and blotted on absorbent paper to remove residual wash fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
.
100 ul of working conjugated antibody (red solution) was added to each well, incubated for - 15 rninutes at room temperature, and then the wells wer~ again washed four times with PBS
solution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TM8 Substrate Solution A and TMB Substrate Solution B were combined to form the color generating substrate. The kit instructed that H properly combined this substrate solution would be colorless and it was colorless. Next a 1ûO ul of the colorless working substrate solution was added to each well and the wells were incubated for ten rninutes at room tsmperature.
. ~
Next 100 ul stop reagent was added to each well to end the enzyme reac~ion, and the O.D.
of each well was read at 450 nm against a water blank. For semi-quantitative results the conversion factor to convert O.D. of the sampla to RE~DS units was the quantity (6.3/mean - O.D. of negative control). 6.3 RE~DSunits is the anti-Sm/RNP activity of the negative control ~5 which was standardi~ed against the CDC reference serum. In this example the conversior factor was 6.3/.0~75 = 72.
:, : Controls RE~DS~m , cùt-off 17.5 negative 6.3 ., '.
~ ~.
... .
.. ,, ~ ,... . ` . .. .
WO !)0/10229 ' .~ ` .,PC~/US90/()1029 _LE Patient Sera O D. RE~DS Units - 1 0.24 17.3 2 0.18 12.6 3 0.19 13.7 0.11 7.9 0.66 48.2*
6 0.15 11.2 7 0.16 11.5 8 0.84 60.51t 9 . 0.01 0.36 0.44 32.0*
11 0.31 22.3*
12 0.53 38.2*
13 1.03 74.2*
14 0.31 22.3*
1.49 107.0*
: ~6 0.52 37.1*
17 1.42 102.0*
, 18 0.53 38. *
~; 20 *positive results . ~
-: :
' ., ,:
~ ~ , . I ;
'-:~
; '., : ,~ .
~ :!
, ., . .
.~
''~
`~ _ ~;.' ;'~ ' '-.'~ ' ' . ", '": .. ' ' I ' . . . , . ' . , .' :, ' "' ', , . , , ', . . . . ...
WO 90/10229 ~,3~ p~lUS90/01029 Adsorption ot anU-Sm/RNP ~ntibodies From Serum To demonstrate the sensitivity of this assay for anti-SM and anti-RNP, the test kit was used , in accordance with the protocol shown in Example 10. Nine CDC reference for nuclear antigen were tested. All were negative except for the anti-Sm and anti-RNP sera which were positive.
Sa,mple O.D.......... Stat,us normal cut-off 0.12 anti-Snn 0.98 positive 10 anti-RNP 0.56 positive anti-SS-A 0.03 negative anti-SS-B 0.06 negative ', anti-Scl-70 0.08 negative anti-nucleolus 0.06 negative .15 anti-centromere 0.06 negative - ` anti-dsDNA monoclonal 0.01 negative anti-ssDNA monoclonal 0.01 negative ~ .
~ ~ .
' 1 s '' .
:i ~, 1 ~,'................... .
WO ~0/10229 r ~ PCr/US~0/01029 3~
Accelerated Stabllity Study Sm/RNP antigen coated microwells were treated with the optimized protocol, then duplicate microwells were stored at 37 degrees C and 4 degrees C, and each set of wells was tested at on day 1, 4, 6, 8, and 12, using a high anti-Sm/RNP ssra sample, a moderate anti-Sm/RNP sera sample, and a negative anti-Sm/RlYP sera sample. The results of thisexperiment are shown in the following table:
O.D.s O.D.s O.D.s O.D.s O.D.s : Day 1 5~ Day 6 Day 8 Davl2 ES 22 4 1.39 1.26 1.35 1.42 1.33 ; ~high) 37 1.36 1.20 1.34 1.40 1.26 ES 22 (1:2) 4 1.09 1.08 1.13 1.17 1.00 (moderate) 37 1.08 1.01 1.07 1.16 1.06 ,~ I
negative 4 0.10 0.10 0.07 0.07 0.09 (low) 37 0.09 0.08 0.08 0.08 0.08 . . .
The significant Sm/P~NP antigen activity in these microwells remained unchanged during the accelerated stability study. The Sm/RNP antigen coated on the microwells using this protocol was equivalent to more than one year shelf life.
:, The foregoing examples serve to illustrate the efficiency and utility of the methylated BSA to 20 provide a coating which Inhibits non-specific binding and provides a coating capable of affixing antigen evenly over the solid support. Without being bound to the specific quantities given in the definitional section, it is possibl0 to utilize a wide latitude of concentrations of mBSA or other similar, functionally equivalent substitutes which have the capability to evenly attach antigen to the support solid by providin~ a charged surface or by any other like `25 mechanism to affix the and to continue to inhibit non-specific binding.
I Likewise, the blocker utilized in the preferred embodiment of this invention to stabiliz0 the 2~ shelf life and elirninate non-specific binding cannot be limited to the cornpounds or the ;
~' :
:
WO 90/10229 i . . .PCI/US90/01029 5~3L~ " ,", j ingredients or the concentrations thereof listed in the definitional section. A variety of functional equivalent blocking agents are known to those skilled in the art. A partial listing of some materials which could be utilized to perform a similar function is found in ~Robert F.
Bogt; J. Immunological Methods, 101, 43-50 [1987]) and is hereby incorporated herein by re~erence. A third element in the anti-dsDNA test kit which plays a function in elirninating non-specific binding is the S, nuclease usad to degrade the ssDNA and thus to avoid any cross reactivity between the anti-dsDNA and the undesired ssDNA. The described rnethod of limiting cross reactivity is not iinnited to the definition given but the method can be performed with varying concentrations of S, nuclease or other similar, endonuclease enzymes, or similar 0 functional equivalents which are capable of eliminating problems of cross reactivity without adversely affecting the antigen present in the invention.
The treatment of the solid support which can be any of a variety of formats, i.e. test tubes, plates, wells, etc., made of various suitable materials, i.e. glass, plastics, etc. with the aforementioned technology affords many important and useful approaches to the detection of the specific antibodies. The detection of antibodies need not be limited to the conjugation of enzymes. Addition of fluorescent chemicals such as fluorescence or the like to the antibody will impart fluorescence to the assay if the antibody is present. Similarly, conjugation of the antibody with a radionuclide will impart radioactivity to the assay if the antibodies are present- in the assay. Many other methods of detection of antibodies aiso 0 exist, and these methods will yield positive resul.s provided that the antibody exists in the assayed sera and is affixed according to the methods descrlbed herein.
The test kit and the underlying coating and detection methods herebefore descrlbed are not intended to be limited by the assay format described or by the volumes or the concentrations or specific ingredients given for the various reagents, controls, and calibrators. It should be understood that sirnilar chemicai equivaients or other functional equivalents of the components found in the coatings, or in any of the various reagents, controis, and calibrators can be utilized within the scope of this invention.
It is contemplated that the inventivo concepts herein described may have difFering embodiments and it is intended that the appended claims be eonstrued to include all such 3 alternative embodiments of the invention except insofar as they are limited by the prior art~
'' , ~ .
. , ~ .. , . . , . , ~ .. . . .:: ,. . .
100 ul of each diluted sample, control or standard was added to the appropriate mierowell(s).
5 The wells were allowed to incubate ior 15 minutes at room temp~rature. To perform the rinse step the contents of PBS packet was added to 2 lilters of reagent grade water, and the solution was mixed well until all the crystals were dissolved. Then th~ PBS buffer was used - ~ to wash the wells four times. The microwells were inverted between each wash to empty the fluid, after which the wells were drained and blotted on absorbent paper to remove residual ~10 wash fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
100 ul of working conjugated antibody (red solution) was added to each well, incubated for 15 minutes at room terrlperature, and then the wells were again washed ~our times with PBS
colution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TMB Substrate Solution A and TMB Substrate Solution B were combined to form the color generating substrate. The kit instructed that if properly combined this substrate solution would be colorless and it was colorless. Next a 100 ul of the colorless working substrate solution v~as added to each well and the wells were incubated for ten rninutes at room temperature.
~0 Next 100 ul stop reagent was added to each well to end the enzyme reaction, and the O.D.
of each well was read at 450 nm against a water blank. A standard curve graph was - generated by p!otting AU/ml of standards against O.D. of standards, and sample results were obtained from this standard curve.
Source O.D. AU/ml ;25 Calibrator 1 .2 3.8 Calibrator2 .3 7.5 Calibrator 3 .4 15.0 Calibrator 4 .57 30 0 Calibrator 5 .88 60.0 Calibrator 6 1.24 120.0 Refer to Graph 2 'i ' ~', WO 90/10229 2~ 34~ r~us90/0l029 t: ~
Controls O. D. AU~
high positive 1.11 104.0 moderate positive .55 24.0 negative .14 2.0 O.D. AUlm~
normal human sera #1 .14 2.0 norrnal human sera #2 .04 1.0 normal human sera #3 .18 3.0 SLE positive #1 .53 23.0 SLE positive #2 1.36 130.0 SLE positive #3 1.48 14Z.0 ~ ' , ':
: . .
' : ' ' :. .
.~
', . ...
., :
i ~, .
.' .~:
WO 90/10229 ;~Cl S ~34~ ' PCT/US90/01029 - 2E~ -Accelerated Stabliity Study DNA coated microwells were treated with S,-nuclease using the optimized protocol, then duplicate microwells were stored at 37 degrees C and 30 degrees C, and each set of wells ~` 5 was tested at various time intervals using anti-dsDNA and anti-ssDNA monoclonal antibodies.
The results of this experiment are shown in the follovving table:
' ~ Real Time Equivalent Time In Days~ In Mont,,hs**, % ssDN,A Activity O 0.0 3-4%
1 0 1 1 .6 3-~%
' ~ 2 3.0 ~-3%
7.5 3-4%
~, 7 1 0.5 4-5%
, g 13.5 3-4%
13 19.5 3-4%
', ~incubated at 37 degreés C
~; : **if incubated at 4 degrees C
,,1 ,'~ The dsDNA activity in these microwells remained unchanged during the accelerated stability study. The results obtained with the microwells stored at 30 degrees C were similar to those ,70 presented above and confirmed that the stability of the dsDNA coated on the microwells ~1 using this protocol was equivalent to more than one year without the generation of significant amounts of ~sDNA.
, ~!
!
,~
,, .
.~ ' .
,~
WO 90/10229 Z~ 5~ ¢3 PCr/US90/01029 Pro~ocol for Pr~Coatin~ Microwell~
with Purified Smith Antigen Polystyrene wells were coated with Sm antigen by the following procedure:
1. Methylated Bovine Serum Albumin (Sigma) (hereinafter ciesignated as mBSA) was dissolved at a ratio of 2û micrograms/ml in distilled water or PBS.
2. 100 microliters of the mBSA solution was placed in each microwell, and incubated overnight at 4 degrees C.
3. The excess coating solution was shaken from the plate after incubation. The wells were inverted to drain thoroughly.
;
4. 5 units/ml of purified Sm antigen vvas diluted in PBS, .OlM, pH 7.3, and 100microliters of the diluted Sm antigen solution was dispensecl into each microwell and allowed to incubate for 18-24 hows at ~ degrees C.
5. Casein blocker soluUon (pH 7.3) was dispensed in 200 microliter increments into each s well. The casein blocker solution consists of 2 ml glycerol, 10 g sucrose, 25 mg casein and sufficient TEN buffer to bring the volume to 100 ml.
:
6. Tha walls coated with the blocker solution wcre incubated overnight at 4 degrees C.
After incubation, the solution was haken from the wells and the wells were inverted for 15 minutes. Then the wells were uprighted and allowed to dry 24 hours at room :i~o ternpera~ure.
Tha coated Sm antigen wells ware then used to determin~ th~ presence of anti-Srn antibody in seven CDC reference sera for anti-nuclear antibodies. Thle r~sults show that only anti-Sm antibodies rsact in the assay.
( Describsd hereinafter, is a method of performing an anti-Sm antibody assay, using the Sm ~5 antigen coated walls in a s~ndwich ELISA format.
`, .
1. Standards, seven rieferance sera samples, a negative human serum control .', :
., : : . ... . . . . .. . . . ~ :.. . .
WO 90/102~ . PCr/US90/01029 RE~DS/ml, a high positive human s~rurn control (RE~DS/ml), a moderate positive human serum control (RE~nS/ml), and a low positive hurnan serum control (RE~DS/ml) were ciiluted with Sample Diluent in a 1:50 ratio; 1 part serum to 50 parts Sample Diluent. The Sample Diluent was a solution consisting of 100 mls of native bovine serum in a phosphate buffered saline consisting of 1.42 g of potassium phosphate (dibasic), .26 g potassium phosphate (monobasic), 8.6 9 sodium chloride, .1% ~odium azide, 1 ml stock green dye, anai distilled water added to bring the volume to 1000 mls. The Sample Diluent was then fiHered through a .2 rnicron filter.
2. 100 microliters of lhe Sample Diluent solutions were placed in the designated wells ~10 at room temperature for 15 minutes to allow completion of the antigen-antibody binding process. Following incubation, ths wells were emptied and washed fourtimes with PB5 soiution.
3. The wells were exposed to a working conjugated antibody colution consisting of 1 part per volume horseradish peroxidase conjugated Igt; and IgM specific antibodies, and 4000 and 1500 part per volume conjugate diluent, resp0ctively, ~Medix) consisting of phosphate buffer, protein stabili~er, and .02% thimerosal, to which was added .01%
by volume a protease inhibitor ~commercially available from Miles Pentex).
4. 100 microliter of the working conjugated antibody solution wac placed in each~, microtiter well and allowed to incubate at room temperature for 15 minutes. After ~o incubation, the wells were rinsed four times with PBS solution to remove unbound ~i conjugated antibodi~s.
.
5. i_ach w~ll was assayed for horseradish peroxidase activity by mixing equal volumes (3,3',5,5') Tetramethylbenzidine (K~rkegaard Perry) and hydrogen peroxide solution and . disp0nsing 100 rnicroliters of this substrate solution into each well. The presence of ~5 the anti-Sm antibodies was detected by a blue color appearing after the ~iO rninute - ~- room temperature incubation. i 00 microliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color. The yellow color was quantitated at 450 nm - using a Dynatech MF16û0 plate r0ading spectrophotom~ter.
Resuits are calcuiated by the following procedure:
~0 1. Calculatc tha mean absorbance values for the duplicates of the controls. The duplicate absorbance values of the positive control should be within 10% of ~he mean .
WO 90/1û229 ~ .PCr/VS90/01~2 absorbance value.
~.
2. The mean absorbance reading of the reagent blank should be less than 0.05.
Readings greater than 0.05 may indicate possible contaminated of the Substrate Solution.
3. Calculate the cut-off absorbance values for the assay, and write them in the appropriato blanks on the results graph.
.
a. The mean absorbance (O.D.) of negative control x 3.2 = negative cut-off absorbance, which is equal to 20 RE~DS units of anti-Sm activity. Sarnples with O.D. values below the cut-off have less than 20 RE~DS units of anti-Sm ~10 activity, and should be scored as ~negative~. Samples with O.D~ values equal to or higher than the cut-off have greater than 20 R~DS units anti-Sm activity, and should be scored as ~positive~. Samples with O.D. values close to the cut-off may warrant further testing (see Figure 4~.
The results were calcuiated by determining a cut-off O.D. The cut-off O.D. was determined ~1~ by running 200 normal sera samples and taking the mean of the O.D.s plus three standard d~viations. This cut-off was equal to 3.2 x the O.D. of the standardized negative control.
~i , `1 .~
:~' .
, '~'', ' :',. : ' . "
~ .
PC~I'/IJS90/01029 f~
- 3~ -Detection of antl-~m antibo~i~s (IgG & ~gM) SampLe O.D. St~tus normal cut-off 0.1 12 Iow positive serum 0.21 positive medium positive serum 0.40 positive high positive serum 0.89 positive anti Sm 1.22 positive anti-~NP 0.112 negative anti-SS-A 0.02 negalivs . anti-SS-B 0.04 negative anti-Sel-70 0.06 negative anti-nucleolus 0.04 negative .
:, :
-, .~
;: , . . . . . .. . . . .
. ,, ! . . . .
WO 90/102~9 . ` Pl-r/US~)0/0102~
~q33~
~ D~agnostic Tes~ Kit Assaly Procedure The kit contains pre-coated microwells (qualitative format) and, 1 vial (40 ml) of Sample Diluent (green solution containing 0.1% sodium azide);
1 vial ~100 ul) of high positive human serum control (about 20 28 RE~DS/ml), 1 vial (100 ul) of calibrator (optional), .~
1 vial ~100 ul) of negative human serum control (about ~.2 RE~DS/ml). The negative cut-off value is 20 RE~DS units. To calculate O.D.s into RE~DS units, the negative value 6.2 is divided by the mean value O.D. it produces; the resuHant number is multiplied by the O.D.s of the samples to give RE~DS units.
1 vial ~12 mi) Conjugated Antibody Working Solution - containing horseradish peroxidase eonjugated anti-human I~G and igM, 1 bottle (8 ml) of TMB Substrate, solution A containing 3,3i,5,5', tetramethylbenzidine, 1 bottle (8 ml) of TMB Substrate; Solution B containing hydrogen peroxide. When rnixed with equal parts Solution A will form a substrate capable of generating a colored product.
.
1 bottle ~12 ml) of Stop Reagent containing 2.5 N H2SO4 (I N HCL can be substituted) (not - necessarily supplied in kit), 1 packet of Phosphate Buffered Saline (PBS) which reconstitutes to 2 liters of 0.01 M PBS, pH 7.3, which is utilized as a wash solution Plate Template.
The various reagents of the kit were utilized to perforrn the assay. The direetions for the ~, method of assaying for anti-Sm antibodies were included in the kit and these directions were ~: followed precisely during this expçriment.
"
~ .
.
.'`
Wo 90/10229 ~ i3~ Pcr/usgo/01029 ~",-, - 3~- .
The plate templates were labelled for sample placement in the microwells. A 1:50 dilution of the controls and pa~ient samples was prepared in Sample Diluent (green solution) (no calibrators were used in this example). 10 ul of sarnpl~ was added to 490 ul sample diluent in a one volume to 50 volume sample dilution.
100 ul of each diluted sample and control was added lo lhe apprnpriate microwell(s). The wells were allowed to incubate for 15 minutes at room temperature. To perform the rinse step the contents of PBS packet was added to 2 liters of reagent grade water, and the solution was nnixed well until all the crystals were dissolved. Then th0 PBS buffer was used to wash the wells four times. The microwells were inverted between each wash to empty the fluid, after which the wells were drained and blotted on absorbent paper to remove residual wash fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
- 100 ul of working conjugated antibody (red solution) was added to each well, incubated for 15 minutes at room temperature, and then the wells were again washed four times with PBS
solution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TMB Substrate Solution A and TMB Substrate Solution B were combined to ~orm the coJor generating substrate. The kit instructed that if properly eombined this substrate solution would be colorless and it was colorless. Next a 100 ul of the colorless working substrate solution was added to each well and the wells were incubated for ten minutes at room temperature.
Next 100 ul stop reagent was added to each well to end the en~yme reaction, and the O.D.
of each well was read at 450 nm against a water blank. The final resulting data can be read as O.D.s. To determine if the sample is positive for anti-Sm antibodies, the sample O.D. is eompared to the cut-off C).D. value calculated for the assay (see Figure 4). For semi-~5 quantitative results the O.D. value can be converted to R~DSunits by rnultiplying the sample O.D. by the quantity (6.2 RE~DS units/negative control O.D.). 6.2 FlE~DS units is the anti-Sm activity of the negative c:ontrol, which was standardized against the CDC referenee serurn.
The cut-off Is 20 RE~DS units.
.~
.
- , ;
~ , ~ , . . .
WO 90/~()229 ~ PCI/U~;~0/011J2~
(, 'I
SLE ~era Sa~ Q~ RE~DSUnits 0.16 16.5 2 0.10 10.3 3 0.07 7.2 4 0.09 9.3 0.12 12.4 6 0.10 10.3 7 0.12 12.4 8 0.08 8.
10 ~ 009 93 0.12 12.4 11 0.19 19.6 12 0.18 18.5 13 0.27 27.8*
5 14 0.35 36.0*
0.29 ~9.9*
16 1.17 121.0 17 0.15 15.4 18 0.35 36.0*
2019 0.13 13.4 :: :
*positive result ~, : :
;
?
~~ :
:, ~
.~ l WO 90/10229 ~ PCr/US90/01029 ~ ,. . .
~' .,', , ,.. ,, ~
EXAI\APLE 7 Adsorptlon ot anti Sm antibodi~s From Serl~m by ~he Test Kit Invention and a Competitive Kit To demonstrate the sensitivity of the anti-Sm test kit samples from SLE patients were run in a competitive kit and were run in the test kit in accordancs with the present invention under the protocol demonslrated in Example 6, with the ~xception that only the negative serum was used to produce a cut-off level. The test was run as ia quaiitative test showing only negative or positive results. 19%-40% of SLE patients are expected to have anti-Sm antibodies present. With all the included samples (the data is only representative of a portion of the 'O data), approximately 28% of the sera samples were positive according to the present invention and only 21% were positiv0 according to the competitive kit. Note the present invention detects both IgG and IgM and the competitive kit only detects IgG.
The resulting O.D.s were obtained. The cut-off value for the present invention was 0.33 O.D.
The cut-off value of the competitive kit was the given by the kit instructions.
;
SLE Sera O.D. From O.D. From Samples Invention Status Competitive Kit Status 0.59 positive .94 positive 2 0.15 negative .05 negative 3 0.36 positive .12 negative 4 0.19 negative .08 negative 0.48 positive .61 positive 6 0.29 negative .13 negative 7 0.24 negativa .07 negative 8 0.21 negative .08 negative 9 0.21 negative .22 negative 0.19 negative .OB negative 11 0.25 negative . 1 0 negative 0.19 negative .13 n~gative 13 0.28 negative .14 negative ~;3 14 0.29 negative .16 negative V.08 negative .05 negative 18 0.86 positive 1.10 positive . ~ .. ~.. . . ,- ... - - , . - . .; . .
: , . : . . .
. - . ,: ~,: :. . . . . .
Wo 90/10229 ~ Pcr/vsso/olo29 ' :, ' ! ' .
XANiPLE 8 Accelerated Stability Study Sm antigen coated microwells were treated with the optimized protocol, then duplicate rnicrowells were stored at 37 clegrees C and 4 degre~es C, and each set of wells was tested on days 1, 4, 6, 8, and 12, using a high positive anti-Sm sera, a moderate positive anti-Sm sera, and a negative anti-Sm sera. The results of this experiment are shown in the following . table:
'.' .
O.D.s O.D.s O.D.s O.D.s O.D.s Dav 1 Day 4 Da.~ Day ~ Day 12 .~
;~0 High anti-Sm activity 4 1.12 1.14 1.12 1.23 1.00 37C (high) 1.15 1.05 1.08 1.09 1.12 -~, Moderate~ anti-Sm - l, activity `?~ 4 0.92 0.93 0.78 0.93 0.95 37~ 0.94 0.92 0.86 0.90 0.80 - Low anti-Sm activity 4 (negative) 0.07 0.07 0.05 0.06 3.05 , 37 0.06 0.09 0.0~ O.o~ 0.05 0 *(1:2 dilution of the hi~h anti-Sm sera) ~j The Sm antigen activity in these rnicrowells remained unchanged during tha accelerated ~', stability study. The results obtained with tha microwells stored at 37 degrees C wera similar l to those presented abov~ and confirmed that the stability of the Sm anti~en coated on the `~ microwells using this protocol was equivalent to more than one year shelf life.
.
.
`' ~
, WO 90/10229 , PClr/US~0/01029 2t~ i34L0 - 3B -Protocol for Pr~Coating Microw~lls with Purified Sm/RNP ;intlgen Polystyrene wells were coated with Sm/RNP antigen by the following procedure:
5 1. Methylated Bovine Serum Albumin (Sigma) (hereinafter designated as mBSA) was dissolvad at a ratio of 20 micro~rams/ml in ciistilled water or PBS.
2. 100 microliters of th~ mBSA solution was placed in each microwell, and incubated - overnight at 4 degrees C.
3. The excess coating solution was shaken from the plate after incubation. The wells .0 were inverted to drain thoroughly.
: ~`
. 1 unit/ml of purified Sm/RNP antigen was diluted in PBS., .01M, pH 7.3, and 100 rnicroliters of the diluted Sm/RNP antigen solution was dispensed into each microwell and allowed to incubate for 18-24 hours at 4 degrees C.
. :
5. Casein blocker solution (pH 7.3) was ciispensed in 200 microliter increments into each . 5 well. The casein blocker solution consists of 2 ml glycerol, 10 g sucrose, ~5 mg .~ casein and sufficient TEN buffer to bring the volume to 100 ml.
6. The wells coatecl with the blocker solution were incubated overn,.ght at 4 degrees C.
After incubation, the solution was shaken from the wells and the wells were inverted for 15 minutes. Then the wells were uprighted and allowed to dry 24 hours at room o temperature.
Th~ coated Sm/RNP antigen wells were than used to determine the presence of anti-Sm/RNP antibody in serum samples obtained from individuals with Progressive SystemicSclerosis (PSS).
i~j Described hereinafter, is a rnethod of performing an anti-Sm/RNP antibody assay, using the ;:5 Sm/RNP antigen co~teci w~lls in a sandwlch ELiSA format.
,' :
WO 9~/1022~ PCrtUS90/01029 2~ i34~) , 1. Sera from patients with PSS, a high positive human serum control (RE~DS/ml), and a negative human serum control ~RE~DS/ml) were diluted with Sample Diluent in a ~:50 ratio; 1 part serum to 50 parts Sample Diluent. The Sample Diluent was a solution consisting of 100 mls of native bovine serum in a phosphate buffered saline consisting of 1.42 g of potassium phosphate (dibasic), .26 g potassium phosphate(monobasic), 8.6 9 sodium chloride, .1% sodium azide, 1 ml stock green dye, and distilled water added to bring the volume to 1000 mls. The Sample Diluent was then filtered through a .2 rnicron filter.
2. 100 microliters of the Sample Diluent solutions were placed in the designated wells at room temperature for 15 minutes to allow completion of the antigen-antibody binding process. Following incubation, the wells were emptied and washed four times with PBS solution.
3. The wells were exposed to a working conjugated antibody solution consisting of 1 part per volume horseradish peroxidase conjugated IgG and IgM specific antibodies, and 4000 and 1500 part per volume conjugate diluent, respectively, (Medix) consisting of phosphate buffer, protein stabilizer, and .02% thimerosal, to which was added .01%
by volume a protease inhibitor (commercially available from Miles Pentex).
, - 4. 100 microliter of the working conjugated antibody solution was placed in each ;~ microtiter weli and allowed to incubate at room temperature for 15 minutes. After ~o incubation, the wells were rinsed four times with PBS solution to remove unbound conjugated antibodies.
5. Each well was assayed for horseradish peroxidasa activity by mixing equal volumes (3,3',5,5') Tetramethylbenzidine (Kirkegaard Perry) and hydrogen peroxide solution and dispensing 100 mlcroliters of this substrate solution into each well. Th0 presence of 2S the anti-Sm/RNP antibodies was detected by a blue color appearing after the 10 minute room temperature incubation. 100 rnicroliters per well of 2.5 N sulfuric acid terminated the reaction producing a yellow color. The yellow color was quantitated at 45û nm using a Dynatech MR600 plate reading spectrophotometer.
R~sults 1. Calculate the mean absorbance values for the duplicates of the controls. The :i ., ~,: ; , , , , :
' ' ~ ;,:' ' , :.
WO !~0/10229 PCI'/lJS90/~1029 - 40 ~
duplicate absorbance values of the positive control should be within 10% of the mean absorbance value.
2. The rnean absorbance reading of the reagent blank should be less than 0.05.
Readings greater than 0.05 may indicate possible contamination of the substrate solution.
3. Calculate the cut-off absorbance values for the assay:
a. The mean absorbance (O.D.) of negative control x 2.8 = negative cut-off absorbance, which is equal to 17.5 RE~DS units of anti-SM/RNP activity.
Samples with O.D. values below the cut-off have less than 17.5 RE~DS units ~0 of anti-SM/RNP activity, and should be scored as "negative". Sarnples with O.D. values equal to or higher than the cut-off have greater than 17.5 RE~DS
units anti~SM/RNP activity, and should be scored as ~positive~. Samples with O.D. values very close to the cut-off may warrant further testing (see Figure 6).
':
The results were calculated by determining a cut-off O.D. The cut-off O.D. was determined by running 200 normal sera samples and taking the mean of the O.D.s plus three standard deviations. This cut-off was equal to 2.8 x the O.D. of the standardized negative control. The cut-off for the flrst 40 samples was 0.11 negative O.D. x 2.8 = 0.31 O.D. The cut-off for the second 40 samples was 0.09 x 2.8 = 0.25 O.D. See Figure 6.
:~, :. ~
,'~.
:, . .
:i ~' ' :
-~, : ,.: , : , , " " . . .
W090/10~9 Z05i~34~ Pcr/1590/~1029 Detection of anti-Sm/RNP antibodies (IgG, ~ IgM~
(PSS! SeraSample ~ Status 0. 14 negative 2 0.16 negative . 5 3 0.17 negative 4 0.17 negative 0.09 negative 6 0.10 negative 7 0.15 negative ~0 8 0.11 negative 9 0.19 negative 0.22 negative 11 0. 1 7 negative ` 12 0.19 negative .5 13 0.18 negative 14 0.13 negative 0.17 negative 16 0.11 negative , 17 0.15 negative ilo ~ 1B 0.13 negative -' 19 0.15 negative 0.15 negative 21 0.22 negative Z2 0.18 negative 23 0.15 negative i 24 0.15 negative 0.60 positive 26 0.1 1 negative , ~7 0.16 negative D 28 0.17 negative ', 29 0.13 negative 0.16 negative 31 0.18 negative 32 0.13 negative , . .
, ~
. .
, ., WO 90/10229 ` i `; P(:~US90/01029 3~ - 4~
~PSS) Sera Sample O.D. Status 33 0.?0 negative 34 0.24 negative 0.24 negative 36 0. 11 negative 37 0.19 negative 38 0.28 negative 39 0.12 negative 0.10 negative 41 0.18 negative 42 0.19 negative 43 0.1~ negative ~4 0.16 negative 0.08 negative J 5 46 0.18 negative 47 0. 11 neyative 48 0.38 positive 49 0.09 negative 0.25 positive ~0 51 0.23 negative 52 0.19 negative 53 0.21 negative 54 0.~7 positive 0.17 negative ` '5 56 0.15 negative - 57 0.21 negative : 58 1.23 positive sg 0.16 negative - 60 0.33 positive - '~ 61 0.12 neyative 62 0.11 negative 63 0.24 negative .~ 64 0.11 negative 0.09 negative ~'5 66 0.22 negative :~l 67 0.18 negativa , .
: 'j ', ., . ,.. : : . : . ,- :, .. .. I , . , . , . - . ..
;, .~ , ;' , ' , ' ' ,~ ! ' ' WO 9~/10229 ~ 3'L~ P~l/U590/ûl029 ~ :.
(PSS! Sera Sample O.D. Sta~
68 0.47 positive 69 0.09 negative 70 0.14 negative 71 0.18 negative 72 0.13 negative 73 0.20 negative 74 0.10 negative 0.17 negative 76 0.22 negative 77 0 07 negative 78 0.10 negative 79 o. ~ 7 negative 0.15 negative '.,, ~
` s , ~.
~, ~` ' .
~;~
i "~' "','` .
;~
, : ' , ., " ~ " ~ , ", ,, ,~"~ , ," ,,, ,~ " " ~ "" ,~,. . .
, : ,. . . , j :, .. . . :
, . . ., . , . ~ . . . ~ .. ,;- ~, . . . .. . .
Wo ~0~10229 . . Pc~r/us~o/0~02'~
~ 44 - t A Diagnostic Test Kit Assay Procedure (Oualitative Format) The kit contains pre-coated microwells and, 1 vial (40 ml) of Sample Diluent (green solution containing 0.1% sodium azide);
1 vial (100 ul) of high positive human serum control (about 100 RE~DS/rnl), 1 vial (100 ul) of calibrator (optional), , 1 vial (100 ul) of negative human serunn control (about 6.3 RE~DS/ml). The negative cut-off value is 17.5 RE~DS units. To calculate O.D.s into RE~DS units, the negative value 6.3 - ~ 10 is divided by the negative O.D. rnean value. The resultant number is muttiplied by the O.D.s of the samples to give RE~DS units.
1 vial (12 ml) Conjugated Antibody Working Solution - containing horseradish peroxidase conjugated anti-human IgG and IgM, ~:;
1 bo~iile (8 ml3 of TMB Substrate; solution A comaining 3,3',5,5', tetramethylbenzidine, i15 1 bome (8 ml) of TMB Substrate; Solution B containing hydrogen peroxide. When mixed .; ~ with equal parls Solution A will form a substrate capable of generating a colored product.
1 bottle (12 ml) of Stop Reagent containing 2.5 N H2S04 (1 N HCL can be substituted), ~' 1 packet of Phosphate Buffered Saline (P8S) which reconstilutes to 2 liters of 0.01 M PBS, pH 7.3, which is utilized as a wash solution ; 20 Plate Template.
The various reagents of the kit werc utilized to perform the assay. The directions for the method of assaying for anti-Sm/RNP anlibodies were included in the kit and these directions - ' were followed preoisely during this experiment.
, .
. ,;
.
: ~ ~ ' ' .. :
',, ' : ~ ' . , ' ' ' '.
.
, . , , , . , , , ~ . .
wO go/]0229 Z~15~;~41~ . PCr/US~0/01029 ~, . ~ . , ,.., , i The plate templates were labelled for sample placement in the microwells. A 1:50 dilution of the controls and SLE patient samples was preparecl in Sample Diluent (green solu~ion). 10 ul of sample was added to 500 ul sample diluent in a one volume to 50 volume sample dilution.
100 ul of each diluted sample and controls was addled to the appropriate microwell(s). The wells were allowed to incubate for 15 minutes at room temperature. To perform the rinse step the contents of PBS packet was added to 2 liters ol reagent grade water, and the solution was mixed well until all the crystals were dissolved. Then the PBS buffer was used to wash the wells four times. The microwells were inverted between each wash to empty the fluid, after which the wells were drained and blotted on absorbent paper to remove residual wash fluid. Per the kit instructions, the wells were not allowed to dry-out between washes.
.
100 ul of working conjugated antibody (red solution) was added to each well, incubated for - 15 rninutes at room temperature, and then the wells wer~ again washed four times with PBS
solution.
The working substrate solution was prepared just before use, according to the kit instructions.
Equal volumes of TM8 Substrate Solution A and TMB Substrate Solution B were combined to form the color generating substrate. The kit instructed that H properly combined this substrate solution would be colorless and it was colorless. Next a 1ûO ul of the colorless working substrate solution was added to each well and the wells were incubated for ten rninutes at room tsmperature.
. ~
Next 100 ul stop reagent was added to each well to end the enzyme reac~ion, and the O.D.
of each well was read at 450 nm against a water blank. For semi-quantitative results the conversion factor to convert O.D. of the sampla to RE~DS units was the quantity (6.3/mean - O.D. of negative control). 6.3 RE~DSunits is the anti-Sm/RNP activity of the negative control ~5 which was standardi~ed against the CDC reference serum. In this example the conversior factor was 6.3/.0~75 = 72.
:, : Controls RE~DS~m , cùt-off 17.5 negative 6.3 ., '.
~ ~.
... .
.. ,, ~ ,... . ` . .. .
WO !)0/10229 ' .~ ` .,PC~/US90/()1029 _LE Patient Sera O D. RE~DS Units - 1 0.24 17.3 2 0.18 12.6 3 0.19 13.7 0.11 7.9 0.66 48.2*
6 0.15 11.2 7 0.16 11.5 8 0.84 60.51t 9 . 0.01 0.36 0.44 32.0*
11 0.31 22.3*
12 0.53 38.2*
13 1.03 74.2*
14 0.31 22.3*
1.49 107.0*
: ~6 0.52 37.1*
17 1.42 102.0*
, 18 0.53 38. *
~; 20 *positive results . ~
-: :
' ., ,:
~ ~ , . I ;
'-:~
; '., : ,~ .
~ :!
, ., . .
.~
''~
`~ _ ~;.' ;'~ ' '-.'~ ' ' . ", '": .. ' ' I ' . . . , . ' . , .' :, ' "' ', , . , , ', . . . . ...
WO 90/10229 ~,3~ p~lUS90/01029 Adsorption ot anU-Sm/RNP ~ntibodies From Serum To demonstrate the sensitivity of this assay for anti-SM and anti-RNP, the test kit was used , in accordance with the protocol shown in Example 10. Nine CDC reference for nuclear antigen were tested. All were negative except for the anti-Sm and anti-RNP sera which were positive.
Sa,mple O.D.......... Stat,us normal cut-off 0.12 anti-Snn 0.98 positive 10 anti-RNP 0.56 positive anti-SS-A 0.03 negative anti-SS-B 0.06 negative ', anti-Scl-70 0.08 negative anti-nucleolus 0.06 negative .15 anti-centromere 0.06 negative - ` anti-dsDNA monoclonal 0.01 negative anti-ssDNA monoclonal 0.01 negative ~ .
~ ~ .
' 1 s '' .
:i ~, 1 ~,'................... .
WO ~0/10229 r ~ PCr/US~0/01029 3~
Accelerated Stabllity Study Sm/RNP antigen coated microwells were treated with the optimized protocol, then duplicate microwells were stored at 37 degrees C and 4 degrees C, and each set of wells was tested at on day 1, 4, 6, 8, and 12, using a high anti-Sm/RNP ssra sample, a moderate anti-Sm/RNP sera sample, and a negative anti-Sm/RlYP sera sample. The results of thisexperiment are shown in the following table:
O.D.s O.D.s O.D.s O.D.s O.D.s : Day 1 5~ Day 6 Day 8 Davl2 ES 22 4 1.39 1.26 1.35 1.42 1.33 ; ~high) 37 1.36 1.20 1.34 1.40 1.26 ES 22 (1:2) 4 1.09 1.08 1.13 1.17 1.00 (moderate) 37 1.08 1.01 1.07 1.16 1.06 ,~ I
negative 4 0.10 0.10 0.07 0.07 0.09 (low) 37 0.09 0.08 0.08 0.08 0.08 . . .
The significant Sm/P~NP antigen activity in these microwells remained unchanged during the accelerated stability study. The Sm/RNP antigen coated on the microwells using this protocol was equivalent to more than one year shelf life.
:, The foregoing examples serve to illustrate the efficiency and utility of the methylated BSA to 20 provide a coating which Inhibits non-specific binding and provides a coating capable of affixing antigen evenly over the solid support. Without being bound to the specific quantities given in the definitional section, it is possibl0 to utilize a wide latitude of concentrations of mBSA or other similar, functionally equivalent substitutes which have the capability to evenly attach antigen to the support solid by providin~ a charged surface or by any other like `25 mechanism to affix the and to continue to inhibit non-specific binding.
I Likewise, the blocker utilized in the preferred embodiment of this invention to stabiliz0 the 2~ shelf life and elirninate non-specific binding cannot be limited to the cornpounds or the ;
~' :
:
WO 90/10229 i . . .PCI/US90/01029 5~3L~ " ,", j ingredients or the concentrations thereof listed in the definitional section. A variety of functional equivalent blocking agents are known to those skilled in the art. A partial listing of some materials which could be utilized to perform a similar function is found in ~Robert F.
Bogt; J. Immunological Methods, 101, 43-50 [1987]) and is hereby incorporated herein by re~erence. A third element in the anti-dsDNA test kit which plays a function in elirninating non-specific binding is the S, nuclease usad to degrade the ssDNA and thus to avoid any cross reactivity between the anti-dsDNA and the undesired ssDNA. The described rnethod of limiting cross reactivity is not iinnited to the definition given but the method can be performed with varying concentrations of S, nuclease or other similar, endonuclease enzymes, or similar 0 functional equivalents which are capable of eliminating problems of cross reactivity without adversely affecting the antigen present in the invention.
The treatment of the solid support which can be any of a variety of formats, i.e. test tubes, plates, wells, etc., made of various suitable materials, i.e. glass, plastics, etc. with the aforementioned technology affords many important and useful approaches to the detection of the specific antibodies. The detection of antibodies need not be limited to the conjugation of enzymes. Addition of fluorescent chemicals such as fluorescence or the like to the antibody will impart fluorescence to the assay if the antibody is present. Similarly, conjugation of the antibody with a radionuclide will impart radioactivity to the assay if the antibodies are present- in the assay. Many other methods of detection of antibodies aiso 0 exist, and these methods will yield positive resul.s provided that the antibody exists in the assayed sera and is affixed according to the methods descrlbed herein.
The test kit and the underlying coating and detection methods herebefore descrlbed are not intended to be limited by the assay format described or by the volumes or the concentrations or specific ingredients given for the various reagents, controls, and calibrators. It should be understood that sirnilar chemicai equivaients or other functional equivalents of the components found in the coatings, or in any of the various reagents, controis, and calibrators can be utilized within the scope of this invention.
It is contemplated that the inventivo concepts herein described may have difFering embodiments and it is intended that the appended claims be eonstrued to include all such 3 alternative embodiments of the invention except insofar as they are limited by the prior art~
'' , ~ .
. , ~ .. , . . , . , ~ .. . . .:: ,. . .
Claims (15)
1. A method for the detection of anti-double strand DNA in sera or plasma comprising the steps of:
providing a solid support capable of receiving a coating thereon, coating said solid support with methylated Bovine serum albumin, attaching native DNA to said coated support which native DNA has an affinity for the attachment thereto of specific anti-dsDNA, contacting said coated support having native dsDNA attached thereto with sera or plasma to be tested for the presence therein of anti-dsDNA and subsequently detecting the attachment of anti-dsDNA to the said coating on said support after contacting said support with sera or plasma to be tested.
providing a solid support capable of receiving a coating thereon, coating said solid support with methylated Bovine serum albumin, attaching native DNA to said coated support which native DNA has an affinity for the attachment thereto of specific anti-dsDNA, contacting said coated support having native dsDNA attached thereto with sera or plasma to be tested for the presence therein of anti-dsDNA and subsequently detecting the attachment of anti-dsDNA to the said coating on said support after contacting said support with sera or plasma to be tested.
2. The method of Claim 1 wherein said coated support containing native DNA or antigen is contacted with an endonuclease prior to being contacted with sera or plasma.
3. The method of Claim 2 wherein said endonuclease is S, nuclease.
4. The method of Claim 3 wherein said coated support after being contacted with S, nuclease is then contacted with a hydrolyzed casein.
5. A diagnostic test kit comprising a coated solid support capable of selectively attaching and immobilizing anti-dsDNA present in sera or plasma for subsequent detection containing:
solid support means, coating means for forming a coating on said solid support means, said coating means including methylated Bovine serum albumin having adherent thereon a DNA antigen specific for binding an anti-dsDNA, said coating means in addition being treated after dsDNA attachment with an enzyme capable of degrading any ssDNA
attached to said coated support without affecting the attached dsDNA, and indication means for providing an indication of the presence of attached anti-dsDNA on said coating.
solid support means, coating means for forming a coating on said solid support means, said coating means including methylated Bovine serum albumin having adherent thereon a DNA antigen specific for binding an anti-dsDNA, said coating means in addition being treated after dsDNA attachment with an enzyme capable of degrading any ssDNA
attached to said coated support without affecting the attached dsDNA, and indication means for providing an indication of the presence of attached anti-dsDNA on said coating.
6. A method for the detection of anti double strand DNA, or anti-Sm antibodies or anti-Sm/RNP antibodies in sera or plasma comprising the steps of:
providing a solid support capable of receiving a coating thereon, coating said solid support with methylated bovine serum albumin, attaching native DNA antigen or anti-Sm antibodies, or anti-Sm/RNP antibodies to said coated support which native DNA antigen or Sm antigen or Sm/RNP antigen has an affinity for the attachment thereto of specific anti-dsDNA antibodies, or anti-Sm antibodies, or anti-Sm/RNP antibodies contacting said coated support having native DNA or anti-Sm antibodies or anti-Sm/RNP antibodies attached thereto with sera or plasma to be tested for the presence therein of anti-dsDNA and subsequently detecting the attachment of anti-dsDNA to the said coating on said support after contacting said support with sera or plasma to be tested.
providing a solid support capable of receiving a coating thereon, coating said solid support with methylated bovine serum albumin, attaching native DNA antigen or anti-Sm antibodies, or anti-Sm/RNP antibodies to said coated support which native DNA antigen or Sm antigen or Sm/RNP antigen has an affinity for the attachment thereto of specific anti-dsDNA antibodies, or anti-Sm antibodies, or anti-Sm/RNP antibodies contacting said coated support having native DNA or anti-Sm antibodies or anti-Sm/RNP antibodies attached thereto with sera or plasma to be tested for the presence therein of anti-dsDNA and subsequently detecting the attachment of anti-dsDNA to the said coating on said support after contacting said support with sera or plasma to be tested.
7. A diagnostic test kit comprising a coated solid support capable of selectively attaching and immobilizing a specific autoimmune antibody present in sera or plasma for subsequent detection containing:
solid support means, coating means for forming a coating on said solid support means, said coating means including methylated Bovine serum albumin having attached thereon an autoimmune antigen specific for binding an autoimmune antibody, said coating means in addition being treated after autoimmune antibody attachment with a casein blocker capable of reducing non-specific binding to said solid support without adversely affecting the attached autoimmune antigen, and indication means for providing an indication of the presence of attached autoimmune antibody on said coating.
solid support means, coating means for forming a coating on said solid support means, said coating means including methylated Bovine serum albumin having attached thereon an autoimmune antigen specific for binding an autoimmune antibody, said coating means in addition being treated after autoimmune antibody attachment with a casein blocker capable of reducing non-specific binding to said solid support without adversely affecting the attached autoimmune antigen, and indication means for providing an indication of the presence of attached autoimmune antibody on said coating.
8. A method for the detection of anti-Sm/RNP antibodies in sera or plasma comprising the steps of:
- providing a solid support capable of receiving a coating thereon, - coating said solid support with methylated Bovine serum albumin, - attached Sm/RNP antigen to said coated support with Sm/RNP antigen has anaffinity for the attachment thereto of specific anti-Sm/RNP antibody, - contacting said coated support having Sm/RNP antigen attached thereto with sera or plasma to be tested for the presence therein of anti-Sm/RNP antibody and subsequently detecting the attachment of anti-Sm/RNP antibody to said coating on said support.
- providing a solid support capable of receiving a coating thereon, - coating said solid support with methylated Bovine serum albumin, - attached Sm/RNP antigen to said coated support with Sm/RNP antigen has anaffinity for the attachment thereto of specific anti-Sm/RNP antibody, - contacting said coated support having Sm/RNP antigen attached thereto with sera or plasma to be tested for the presence therein of anti-Sm/RNP antibody and subsequently detecting the attachment of anti-Sm/RNP antibody to said coating on said support.
9. The method of Claim 8 wherein said coated support is contacted with a hydrolyzed casein.
10. A diagnostic assay kit comprising a coated solid support capable of selectively attaching and immobilizing IgG and IgM anti-Sm/RNP present in sera or plasma for subsequent detection containing:
solid support means, coating means for forming a coating on said solid support means, said coating means including methylated bovine serum albumin having attached thereon an Sm/RNP antigen specific for binding an anti-Sm/RNP antibody, said coating means further being treated after Sm/RNP attachment with rinsing procedure capable of removing any unattached Sm/RNP antigen without affecting the attached Sm/RNP
antigen and indication means for providing an indication of the presence of attached anti-Sm/RNP antigen said coating.
solid support means, coating means for forming a coating on said solid support means, said coating means including methylated bovine serum albumin having attached thereon an Sm/RNP antigen specific for binding an anti-Sm/RNP antibody, said coating means further being treated after Sm/RNP attachment with rinsing procedure capable of removing any unattached Sm/RNP antigen without affecting the attached Sm/RNP
antigen and indication means for providing an indication of the presence of attached anti-Sm/RNP antigen said coating.
11. A method for the detection of anti-Sm antibodies in sera or plasma comprising the steps of:
- providing a solid support capable of receiving a coating thereon, - coating said solid support with methylated Bovine serum albumin, - attaching Sm antigen to said coated support which Sm antigen has an affinity for the attachment thereto of specific anti-Sm antibody, - contacting said coated support having Sm antigen attached thereto with sera or plasma to be tested for the presence therein of anti-Sm antibody and subsequently detecting the attachment of anti-Sm antibody to the said coating onsaid support.
- providing a solid support capable of receiving a coating thereon, - coating said solid support with methylated Bovine serum albumin, - attaching Sm antigen to said coated support which Sm antigen has an affinity for the attachment thereto of specific anti-Sm antibody, - contacting said coated support having Sm antigen attached thereto with sera or plasma to be tested for the presence therein of anti-Sm antibody and subsequently detecting the attachment of anti-Sm antibody to the said coating onsaid support.
12. The method of Claim 11 wherein said coated support is further coated with a hydrolyzed casein.
13. A diagnostic assay kit comprising a coated solid support capable of selectively attaching and immobilizing IgG and IgM anti-Sm present in sera or plasma for subsequent detection containing:
solid support means, coating means for forming a coating on said solid support means, said coating means including methylated Bovine serum albumin having attached thereon an Sm antigen specific for binding an anti-Sm antibody, said coating means further to being treated after Sm attachment with rinsing procedure capable of removing anyunattached Sm antigen without affecting the attached Sm antigen, and indication means for providing an indication of the presence of attached anti-Sm antigen on said coating.
solid support means, coating means for forming a coating on said solid support means, said coating means including methylated Bovine serum albumin having attached thereon an Sm antigen specific for binding an anti-Sm antibody, said coating means further to being treated after Sm attachment with rinsing procedure capable of removing anyunattached Sm antigen without affecting the attached Sm antigen, and indication means for providing an indication of the presence of attached anti-Sm antigen on said coating.
14. A method for the detection of an antibody generated by the human immune system in sera or plasma comprising the steps of:
- providing a solid support capable of receiving a coating thereon, - coating said solid support with methylated Bovine serum albumin, - attaching an antigen specific for a said antibody to said coated support which antigen has an affinity for the attachment thereto of specific human antibody, - contacting said coated support having a said antigen attached thereto with sera or plasma to be tested for the presence therein of an antibody specific for a said antigen and subsequently detecting the attachment of a said antibody to said coating on the support.
- providing a solid support capable of receiving a coating thereon, - coating said solid support with methylated Bovine serum albumin, - attaching an antigen specific for a said antibody to said coated support which antigen has an affinity for the attachment thereto of specific human antibody, - contacting said coated support having a said antigen attached thereto with sera or plasma to be tested for the presence therein of an antibody specific for a said antigen and subsequently detecting the attachment of a said antibody to said coating on the support.
15. A method according to Claim 14 wherein the antibody for whose presence is tested is a member selected from the group consisting of anti-dsDNA, anti-RNP, anti-DNP, anti-cardiolipin, anti-hesbone, anti-Sm, anti-ENA, anti-RNA, anti-Scl-70 and anti-SS-A and B.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US31556789A | 1989-02-27 | 1989-02-27 | |
| US315,567 | 1989-02-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2050340A1 true CA2050340A1 (en) | 1990-08-28 |
Family
ID=23225031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002050340A Abandoned CA2050340A1 (en) | 1989-02-27 | 1990-02-26 | Method and diagnostic test kit for detection of autoimmune antibody |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0460102A4 (en) |
| AU (1) | AU5186890A (en) |
| CA (1) | CA2050340A1 (en) |
| WO (1) | WO1990010229A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0568554T3 (en) * | 1991-01-22 | 1995-12-04 | Akzo Nobel Nv | Method for Detecting Anti-RNA Antibodies |
| US6030773A (en) * | 1992-07-08 | 2000-02-29 | Agnello; Vincent | Chemiluminescent assay for DSDNA antibodies |
| US5681700A (en) * | 1994-05-25 | 1997-10-28 | Oklahoma Medical Research Foundation | Assay for pathogenicity of anti-DNA antibodies |
| US6280944B1 (en) | 1994-05-25 | 2001-08-28 | Oklahoma Medical Research Foundation | Assay for pathogenicity of anti-DNA antibodies |
| CA2339030A1 (en) * | 2000-03-02 | 2001-09-02 | Ortho-Clinical Diagnostics, Inc. | Analytical elements |
| CN103575907B (en) * | 2013-10-30 | 2015-12-02 | 浙江理工大学 | The detection method of egg protein in a kind of ancient times jointing compound Wen Tianxiang |
| EP3712618B1 (en) * | 2019-03-18 | 2023-10-04 | Euroimmun Medizinische Labordiagnostika AG | Method for detecting a binding of antibodies of a patient sample to double-stranded dna using crithidia luciliae cells and fluorescence microscopy |
| CN113917136A (en) * | 2021-10-18 | 2022-01-11 | 北京和杰创新生物医学科技有限公司 | Detection method of anti-double-stranded deoxyribonucleic acid antibody immunoglobulin G |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4564597A (en) * | 1981-03-18 | 1986-01-14 | Research Corporation | Anti-Sm hybridoma |
| US4784942A (en) * | 1984-11-05 | 1988-11-15 | The Board Of Regents For The University Of Oklahoma | Monoclonal antibodies against autoimmune RNA proteins |
| US4738932A (en) * | 1985-12-03 | 1988-04-19 | Advanced Polymer Systems, Inc. | Reaginic test for syphilis |
| EP0460097A4 (en) * | 1989-02-27 | 1992-03-11 | Biostar Med Prod | Method and diagnostic test kit for detection of anti-cardiolipin |
-
1990
- 1990-02-26 CA CA002050340A patent/CA2050340A1/en not_active Abandoned
- 1990-02-26 EP EP19900904577 patent/EP0460102A4/en not_active Withdrawn
- 1990-02-26 WO PCT/US1990/001029 patent/WO1990010229A1/en not_active Application Discontinuation
- 1990-02-26 AU AU51868/90A patent/AU5186890A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0460102A4 (en) | 1992-01-15 |
| EP0460102A1 (en) | 1991-12-11 |
| AU5186890A (en) | 1990-09-26 |
| WO1990010229A1 (en) | 1990-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4469787A (en) | Immunoassay involving soluble complex of second antibody and labeled binding protein | |
| US4757024A (en) | Immune complex detection method and article using immunologically non-specific immunoglobulins | |
| EP0162914B1 (en) | Novel immunoassays and kits for use therein | |
| US4444879A (en) | Immunoassay with article having support film and immunological counterpart of analyte | |
| US4578361A (en) | Creatinine antibody | |
| US20020106708A1 (en) | Assays reagents and kits for detecting or determining the concentration of analytes | |
| JPS59208463A (en) | Solid-phase immunoassay method containing luminescent mark | |
| WO1985000663A1 (en) | Immunometric assay using polyclonal and monoclonal antibodies and a kit for use therein | |
| US7256008B2 (en) | Determination of concentration of FK778 by competitive immunoassay | |
| JP4197393B2 (en) | Test method for IgA nephropathy | |
| CA2050340A1 (en) | Method and diagnostic test kit for detection of autoimmune antibody | |
| JPH03229153A (en) | Detection of existence of specific anti- body or antigen useful for diagnosis of rheumatism and test kit used therefor | |
| US4612281A (en) | Immunoassay for detecting immunoglobulins and test kit | |
| JPWO1999050663A6 (en) | Testing methods for IgA nephropathy | |
| US4753893A (en) | Method and article for detection of immune complexes | |
| US5183735A (en) | Method and diagnostic test kit for detection of anti-dsDNA antibodies | |
| WO1990010227A1 (en) | Method and diagnostic test kit for detection of anti-cardiolipin | |
| US5210020A (en) | Immunoassay utilizing alginate to enhance signal to noise ratio | |
| JP2001511820A (en) | Cyclosporin derivatives and uses thereof | |
| US4713350A (en) | Hydrophilic assay reagent containing one member of specific binding pair | |
| US4732848A (en) | Process for the determination of an immunologically-bindable substance involving a Fab fragment | |
| US5277589A (en) | Process for the determination of antibodies | |
| EP0362284A1 (en) | Multiple antigen immunoassay | |
| CN115197294A (en) | Polypeptide, polypeptide composition, kit and related application | |
| CA1340439C (en) | Agent, for immunochemical assays, containing amine oxides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |