GB1589107A - Immunologic compositions - Google Patents
Immunologic compositions Download PDFInfo
- Publication number
- GB1589107A GB1589107A GB23243/78A GB2324378A GB1589107A GB 1589107 A GB1589107 A GB 1589107A GB 23243/78 A GB23243/78 A GB 23243/78A GB 2324378 A GB2324378 A GB 2324378A GB 1589107 A GB1589107 A GB 1589107A
- Authority
- GB
- United Kingdom
- Prior art keywords
- erythrocytes
- composition
- antigen
- bifunctional molecule
- glutaraldehyde
- 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.)
- Expired
Links
- 239000000203 mixture Substances 0.000 title claims description 91
- 230000001900 immune effect Effects 0.000 title claims description 18
- 210000003743 erythrocyte Anatomy 0.000 claims description 95
- 239000000427 antigen Substances 0.000 claims description 53
- 102000036639 antigens Human genes 0.000 claims description 52
- 108091007433 antigens Proteins 0.000 claims description 52
- 238000000034 method Methods 0.000 claims description 46
- AIJULSRZWUXGPQ-UHFFFAOYSA-N Methylglyoxal Chemical compound CC(=O)C=O AIJULSRZWUXGPQ-UHFFFAOYSA-N 0.000 claims description 42
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 210000004027 cell Anatomy 0.000 claims description 34
- 230000001588 bifunctional effect Effects 0.000 claims description 31
- 230000035931 haemagglutination Effects 0.000 claims description 31
- 239000000725 suspension Substances 0.000 claims description 23
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 21
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000012360 testing method Methods 0.000 claims description 20
- FRTGEIHSCHXMTI-UHFFFAOYSA-N dimethyl octanediimidate Chemical compound COC(=N)CCCCCCC(=N)OC FRTGEIHSCHXMTI-UHFFFAOYSA-N 0.000 claims description 19
- 210000002966 serum Anatomy 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 17
- 229960004407 chorionic gonadotrophin Drugs 0.000 claims description 16
- 230000007935 neutral effect Effects 0.000 claims description 16
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 239000005057 Hexamethylene diisocyanate Substances 0.000 claims description 14
- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 claims description 14
- 210000004369 blood Anatomy 0.000 claims description 13
- 239000008280 blood Substances 0.000 claims description 13
- 238000005119 centrifugation Methods 0.000 claims description 13
- PCSMJKASWLYICJ-UHFFFAOYSA-N Succinic aldehyde Chemical compound O=CCCC=O PCSMJKASWLYICJ-UHFFFAOYSA-N 0.000 claims description 12
- 230000006641 stabilisation Effects 0.000 claims description 11
- DVKJHBMWWAPEIU-UHFFFAOYSA-N toluene 2,4-diisocyanate Chemical compound CC1=CC=C(N=C=O)C=C1N=C=O DVKJHBMWWAPEIU-UHFFFAOYSA-N 0.000 claims description 11
- 102000003886 Glycoproteins Human genes 0.000 claims description 10
- 108090000288 Glycoproteins Proteins 0.000 claims description 10
- 239000008188 pellet Substances 0.000 claims description 10
- 210000002700 urine Anatomy 0.000 claims description 10
- 241001465754 Metazoa Species 0.000 claims description 9
- 239000008366 buffered solution Substances 0.000 claims description 9
- 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 claims description 9
- 229940015043 glyoxal Drugs 0.000 claims description 9
- 239000002953 phosphate buffered saline Substances 0.000 claims description 9
- 239000003146 anticoagulant agent Substances 0.000 claims description 8
- 229940127219 anticoagulant drug Drugs 0.000 claims description 8
- 239000007975 buffered saline Substances 0.000 claims description 8
- 150000002463 imidates Chemical class 0.000 claims description 8
- 229920001184 polypeptide Polymers 0.000 claims description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000013019 agitation Methods 0.000 claims description 7
- 230000000295 complement effect Effects 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 229960004509 serum gonadotrophin Drugs 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 241000271566 Aves Species 0.000 claims description 6
- 108010073521 Luteinizing Hormone Proteins 0.000 claims description 6
- 102000009151 Luteinizing Hormone Human genes 0.000 claims description 6
- 241001494479 Pecora Species 0.000 claims description 6
- 230000004520 agglutination Effects 0.000 claims description 6
- 229940040129 luteinizing hormone Drugs 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 claims description 5
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 claims description 5
- 102000003864 Human Follicle Stimulating Hormone Human genes 0.000 claims description 5
- 108010082302 Human Follicle Stimulating Hormone Proteins 0.000 claims description 5
- 206010070834 Sensitisation Diseases 0.000 claims description 5
- 102000011923 Thyrotropin Human genes 0.000 claims description 5
- 108010061174 Thyrotropin Proteins 0.000 claims description 5
- 239000000910 agglutinin Substances 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 230000035945 sensitivity Effects 0.000 claims description 5
- 230000008313 sensitization Effects 0.000 claims description 5
- 238000011105 stabilization Methods 0.000 claims description 5
- CBUWTGCATVNMJE-UHFFFAOYSA-N 6,6-dimethylheptanal Chemical compound CC(C)(C)CCCCC=O CBUWTGCATVNMJE-UHFFFAOYSA-N 0.000 claims description 4
- 241000270322 Lepidosauria Species 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 claims description 4
- 229940090343 human menopausal gonadotrophin Drugs 0.000 claims description 4
- FPGCYQVKNKEGRQ-UHFFFAOYSA-N methyl 6,17,18-trimethoxy-1,3,11,12,14,15,16,17,18,19,20,21-dodecahydroyohimban-19-carboxylate Chemical compound COC1=CC=C2C(CCN3CC4CC(C(C(C4CC33)C(=O)OC)OC)OC)=C3NC2=C1 FPGCYQVKNKEGRQ-UHFFFAOYSA-N 0.000 claims description 4
- 241000283707 Capra Species 0.000 claims description 3
- 229920002307 Dextran Polymers 0.000 claims description 3
- 108010010803 Gelatin Proteins 0.000 claims description 3
- 230000009260 cross reactivity Effects 0.000 claims description 3
- 229920000159 gelatin Polymers 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
- 229940088597 hormone Drugs 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 3
- VMPITZXILSNTON-UHFFFAOYSA-N o-anisidine Chemical compound COC1=CC=CC=C1N VMPITZXILSNTON-UHFFFAOYSA-N 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 2
- 102000004506 Blood Proteins Human genes 0.000 claims description 2
- 108010017384 Blood Proteins Proteins 0.000 claims description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 150000001299 aldehydes Chemical class 0.000 claims description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 241000283073 Equus caballus Species 0.000 claims 1
- NCKXRWJWVLCUEI-UHFFFAOYSA-N oxaldehyde;pentanedial Chemical compound O=CC=O.O=CCCCC=O NCKXRWJWVLCUEI-UHFFFAOYSA-N 0.000 claims 1
- 229920006316 polyvinylpyrrolidine Polymers 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 12
- 239000000872 buffer Substances 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 239000007822 coupling agent Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 229920001296 polysiloxane Polymers 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 2
- 241000283086 Equidae Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 125000005442 diisocyanate group Chemical group 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 2
- 238000009597 pregnancy test Methods 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 239000005297 pyrex Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- VWVRASTUFJRTHW-UHFFFAOYSA-N 2-[3-(azetidin-3-yloxy)-4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]pyrazol-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound O=C(CN1C=C(C(OC2CNC2)=N1)C1=CN=C(NC2CC3=C(C2)C=CC=C3)N=C1)N1CCC2=C(C1)N=NN2 VWVRASTUFJRTHW-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000588832 Bordetella pertussis Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 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
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 240000005265 Lupinus mutabilis Species 0.000 description 1
- 235000008755 Lupinus mutabilis Nutrition 0.000 description 1
- 108010057021 Menotropins Proteins 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 235000019095 Sechium edule Nutrition 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 238000005882 aldol condensation reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical class C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- RBHJBMIOOPYDBQ-UHFFFAOYSA-N carbon dioxide;propan-2-one Chemical compound O=C=O.CC(C)=O RBHJBMIOOPYDBQ-UHFFFAOYSA-N 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 238000009535 clinical urine test Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- SPJDSYVGUJARDI-UHFFFAOYSA-N diethyl propanediimidate;hydrochloride Chemical compound Cl.CCOC(=N)CC(=N)OCC SPJDSYVGUJARDI-UHFFFAOYSA-N 0.000 description 1
- KIQKWYUGPPFMBV-UHFFFAOYSA-N diisocyanatomethane Chemical compound O=C=NCN=C=O KIQKWYUGPPFMBV-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 150000002009 diols Chemical class 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000005534 hematocrit Methods 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 210000000548 hind-foot Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000001592 luteinising effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 230000016087 ovulation Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229940066827 pertussis vaccine Drugs 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000004053 quinones Chemical class 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- ZNCPFRVNHGOPAG-UHFFFAOYSA-L sodium oxalate Chemical compound [Na+].[Na+].[O-]C(=O)C([O-])=O ZNCPFRVNHGOPAG-UHFFFAOYSA-L 0.000 description 1
- 229940039790 sodium oxalate Drugs 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- DJMIIQXPYUKIMF-UHFFFAOYSA-N trihydroxy(octadecyl)silane Chemical compound CCCCCCCCCCCCCCCCCC[Si](O)(O)O DJMIIQXPYUKIMF-UHFFFAOYSA-N 0.000 description 1
- 150000003672 ureas Chemical class 0.000 description 1
Classifications
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- 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
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
- G01N33/555—Red blood cell
- G01N33/556—Fixed or stabilised red blood cell
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Mycology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
PATENT SPECIFICATION ( 11) 1589 107
j N ( 21) Application No 23243/78 ( 22) Filed 26 May 1978 0 ( 31) Convention Application No 806 563 ( 19)(, _ ( 32} Filed 14 June 1977 in C ( 33) United States of America (US) ( 44) Complete Specification published 7 May 1981 ( 51) INT CL 3 GON 33/50 ( 52) Index at acceptance G 1 B BR ( 72) Inventors MICHAEL ALLEN HIRSCH, DOUGLAS STALKER IRVINE and JOHN KRUPEY ( 54) IMMUNOLOGIC COMPOSITIONS ( 71) We, AMERICAN HOME PRODUCTS CORPORATION, a corporation organised and existing under the laws of the State of Delaware, United States of America, of 685, Third Avenue, New York, New York 10017, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly 5
described in and by the following statement:-
This invention relates to immunologic compositions.
The development of reliable immunologic compositions useful as reagents in immunoassays to determine the presence or absence of antigens or antibodies in a test fluid has and continues to be a sought after goal One approach which has received 10 a great deal of attention has been the development of reagents for haemagglutination and haemagglutination inhibition systems These systems employ red blood cells also known as erythrocytes which, for use, are joined with antigenic or antibody substances so as to provide an indicator system which can be used to detect the antibody or the antigenic substance itself 15 An early development in haemagglutination inhibition or passive haemagglutination tests was described by Wide and Gemzell, Acta Endocr, 35 261 ( 1960).
They established that the red blood cells (Rbc) coated by or attached to the antigen must be stabilised to be useful in haemagglutination inhibition tests, otherwise the red blood cells would need to be fresh daily While Wide et at stabilised the erthro 20 cytes before attachment of the antigen, others have stabilised following attachment of the antigen.
Stabilisation of Rbc was originally described by Boyden using formaldehyde, J.
Exp Med, 93 107 ( 1951) and the concept was extended by Wide, Acta Endocr, supp.
70, 41 ( 1962), when following pretreatment of formaldehyde treated Rbc with tannic 25 acid, it was shown that h CG could be adsorbed onto the cell surface of the Rbc.
Ling, Brit J Haemat, 7 229 ( 1961), also showed that pyruvic aldehyde could beneficially replace formaldehyde and that pretreatment with tannic acid was not necessary for antigen attachment However, for stabilisation, Ling decreed that 48 hours at + 40 C was necessary But, the coated erythrocytes are unstable and are also 30 time consuming to prepare.
One approach to improving stability and also of obtaining erythrocytes with higher haemagglutination titres was disclosed in U S Patents 3,714,345; 3, 715,427 and 3,925,541 There the erythrocytes were treated sequentially with pyruvic aldehyde and then formaldehyde for periods in excess of 12 hours for each treatment prior 35 to coating the thus stabilised erythrocytes with an antigen or antibody The double aldehyde treated erythrocytes were in some cases further subjected to a lengthy freezethaw cycle at very low temperatures.
Another approach to obtain antigen sensitised erythrocytes was through the use of bivalent reagents as coupling agents between antigens and erythrocytes, e g bis 40 diazotized benzidines illustrated in U S Patent 3,236,732; various diols and quinones described in U S Patent 3,322,634; toluene-2,4-diisocyantes described in Immunochemistry, 1, 43 ( 1964) Since even the coupling coated erythrocytes are still readily susceptible to decomposition, formaldehyde treatment was usually employed prior to coating with antigen or simultaneously as described in U S Patent 3,987, 159 where 45 the coupling agent employed was glutaraldehyde In still a different approach des-:
cribed in U S Patent 3,991,175, glutaraldehyde was also utilized as a coupling agent but gelatin was used to stabilize the composition.
The various compositions and methods already noted and' numerous others suffer individually from various disadvantages such as instability, lack of reproducibility, false positive results, low haemagglutination titres, cost or time of preparation, and others.
It has now been found that the compositions and methods of this invention, 5 demonstrated in part by use in haemagglutination tests, provide a superior grade of stabilised, sensitized cells The composition of this invention have higher haemagglutination titres, are stable for long periods of time, and give reproducible patterns and results Another advantage of the herein disclosed invention is the stability of results obtained For example, in utilizing a commercially available reagent the 10 haemagglutination patterns change with time Thus, in a pregnancy test, a negative result, i e complete haemagglutination within 2 hours, will change over 24 hours to show a quasi-inhibition pattern; results recorded from 2-24 hours at room temperature will therefore indicate a significant number of false positive results However, utilizing the composition and methods of this invention, results are obtained within 15 2 hours of testing and will not change for about 11 days thereafter or more Other advantages will become apparent as the description of the invention unfolds.
Accordingly the present invention provides an immunologic composition comprising pyruvic aldehyde or dimethylsuberimidate stabilised erythrocytes sensitised with a polypeptide or glycoprotein antigen, said stabilised erythrocytes being coupled 20 to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, a bifunctional imido ester, (e g diethyl malonimidate dihydrochloride or dimethyl suberimidate), cyanuric chloride, tetrazotized o-anisidine, and a water soluble carbodiimide (e g 1ethyl-3-( 3-dimethylaminopropyl)carbodiimide) with the proviso that when the 25 erythrocytes are dimethylsuberimidate stabilised the bifunctional molecule is other than dimethyl suberimidate and that when the erythrocytes are pyruvic aldehyde stabilised the bifunctional molecule is selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4 diisocyanate and a bifunctional imido ester.
Preferably the erythrocytes are pyruvic aldehyde stabilised and the polypeptide 30 or glycoprotein antigen is chorionic gonadotrophin, pregnant mare's serum gonadotrophin, carcino embryonic antigen, luteinising hormone, follicle stimulating hormone, human menopausal gonadotrophin or thyroid stimulating hormone Thus in a preferred aspect the pesent invention provides an immunologic composition comprising pyruvic aldehyde stabilised erythrocytes sensitized with a polypeptide or glycoprotein 35 antigen selected from chorionic gonadotrophin, pregnant mare's serum gonadotrophin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotropin, and thyroid stimulating hormone, said stabilised erythrocytes being coupled to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal succinaldehyde, hexamethylene diisocyanate, toluene-2,4 40 diisocyanate and bifunctional imido esters including diethylmalonimidate hydrochloride and dimethyl suberimidate It is preferred to use as the bifunctional molecule for coupling glutaraldehyde, hexamethylene diisocyanate or dimethyl suberimidate.
When the erythrocytes are dimethyl suberimidate stabilised the coupling agent or bifunctional molecule can be glutaraldehyde, glyoxal, succinaldehyde, hexa methy 45 lene diisocyanate, toluene 2,4-diisocyanate, a bifunctional imido ester other than dimethyl suberimidate, cyanuric chloride, tetrazotised o-anisidine or a water soluble carbodiiminide such as 1-ethyl-3-( 3-dimethylaminopropyl)carbodiimide.
In a further aspect the invention provides a method for preparing immunologic so 50 compositions of animal erythrocytes sensitized with antigens useful in passive haemag 50 glutination tests which comprises:
a) collecting animal blood directly into an isotonic sterile anticoagulant so that a final ratio of blood to anticoagulant is from 0 8:1 to 1 2:1 of the resulting mixture wherein the erythrocyte concentration packed cell volume of said blood is from about 15 % to about 25 %; 55 b) separating the erythrocytes from said mixture by centrifugation; c) washing said erythrocytes with normal saline and then suspending said erythrocytes in normal saline at a concentration of about 10 to 30 %; d) mixing the suspension of erythrocytes with a mixture of pyruvic aldehyde, neutral buffered solution and normal saline to obtain stabilization mixture; 60 e) incubating the stabilization mixture of step (d) at 30 to 55 C for one to five hours, and collecting the stabilised erythrocytes by centrifugation followed by washing with normal saline; f) suspending the stabilised erythrocytes in a neutral buffered solution of antigen; 65 1,589,107 g) forming a sensitization mixture by thorough agitation of the suspension of stabilised erythrocytesantigen with a solution of a bifunctional molecule in saline, the bifunctional molecule being selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate and a bifunctional imido ester; h) collecting the sensitised erythrocytes by centrifugation and subsequently 5 washing them with neutral buffered saline; i) suspending the sensitized erythrocytes in a neutral buffered saline containing normal animal serum wherein the complement of said serum has previously been fixed, said erythrocytes being mammalian, avian or reptile erythrocytes.
The above mentioned method may be employed to prepare the immunologic 10 compositions of the invention in which, for example, the antigen is a polypeptide or glycoprotein antigen selected from chorionic gonadotrophin, pregnant mare's serum ganadotrophin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotrophin and thyroid stimulating hormone and the bifunctional molecule is, for example, glutaraldehyde, glycoxal, succinaldehyde, hexa 15 methylene diisocyanate, toluene 2,4-diisocyanate, diethyl malonimidate dihydrochloride or dimethyl suberimidate Immunologic compositions containing dimethyl suberimidate stabilised erythrocytes may be prepared in an analogous manner.
A preferred manner of carrying out the method of the invention in which a composition of stabilised red blood cells (erythrocytes) sensitized with a polypeptide 20 or glycoprotein such as, human chorionic gonadotrophin (h CG) is prepared as one component in a two component reagent system is described below Stabilisation of the erythrocytes may be first accomplished by taking freshly drawn whole blood, generally from sheep, although turkeys, goats or horses can be used, and immediately mixing it with an isotonic sterile anticoagulant, such as Alsever's solution, so that a 25 final ratio of blood to anticoagulant is from 0 8:1 to 1 2:1, preferably 1:1, of the resulting mixture The erythrocytes are then separated by centrifugation and washed with physiological saline Pyruvic aldehyde reagent is prepared in physiological saline.
A neutral buffered solution, preferably phosphate buffer, and a suspension of washed erythrocytes in saline are added to the pyruvic aldehyde reagent The suspension is 30 then incubated at 30 to 55 C for one to five hours, preferably at 37 C for three hours, with occasional agitation Subsequently the stabilised red blood cells are thoroughly washed with saline The pyruvic aldehyde stabilised cells (PAC) may then be stored at 4 C as a 10 % suspension in saline containing 0 1 % sodium azide as a preservative, for at least a year, if desired To sensitize the stabilised cells with 35 h CG, the pyruvic aldehyde treated cells are washed with dilute saline and then suspended in a neutral buffered solution, preferably phosphate buffer, containing h CG.
To this suspension is added glutaraldehyde in normal saline The mixture is then incubated, for example, at room temperature for about 2 hours under constant agitation and the cells are removed by centrifugation and washed not less than 3 times with 40 neutral buffered saline, preferably phosphate buffered saline Optionally, the sensitized cells are suspended in a neutral buffered saline, preferably phosphate buffered saline, containing as little as 0 2 % normal rabbit serum in which complement had previously been fixed by heating at 56 C for about 30 minutes and from which nonspecific agglutinins, interfering proteins, had previously been removed by serial 45 adsorption with an equal volume of pyruvic aldehyde stabilised cells Alternatively, normal serum from goats, horses or sheep may be used Sensitized cells may be stored at 4 C Again alternatively, in place of normal rabbit serum, one could use any properly treated stabilisation agent including properly adsorbed, complement fixed so 50 serum proteins Illustrative of such stabilisation agents are gelatin, dextrans, poly 50 vinyl pyrrolidone, albumins and water soluble carboxymethyl celluloses.
The h CG used for censitization of the stabilised cells, illustrated above may be of various grades of purity For example, material which assays, biologically, at approximately 2,500 I U /mg may be employed Alternatively, more purified h CG preparations, for example, a preparation assaying at approximately 10,000 I U /mg 55 or 14,000-16,000 I U /mg may be utilized in the sensitization of the aforementioned stabilised red blood cells Again the beta sub-unit of h CG may also be utilized as the antigen on the red blood cell It is generally preferable to use the more purified h CG in the stabilised, sensitized erythrocyte compositions of this invention.
While the invention has been illustrated primarily using mammalian erythrocytes, 60 such as those from sheep, these stabilised, sensitized erythrocytes may also be prepared from avian or reptilian red blood cells, which being nucleated settle more rapidly than mammalian erythrocytes In this way the results from haemagglutination tests may be obtained within 30 minutes or sooner The speed of the haemagglutination reaction is proportional to the buoyant density of the nucleated cell; thus as the 65 1,589,107 buoyant density increases, less time is required to obtain results Furthermore, whereas the invention was illustrated using Alsever's solution as the isotonic sterile anticoagulant, the freshly drawn whole blood may be optionally mixed with heparin sodium EDTA, or sodium oxalate.
The described stabilised, sensitized erythrocyte compositions together with their 5 antiserum compositions are most useful in haemagglutination and haemagglutination inhibition (passive haemagglutination) methods for determining the presence or absence of a particular antigen or antibody in the test fluid being examined, usually a body fluid such as urine or serum Thus determining the presence or absence of a pregnant condition in a female animal can be based on a haemagglutination method for deter 10 mining the presence of chorionic gonadotrophin which comprises mixing the erythrocyte composition of this invention with a chorionic gonadotrophin antiserum and with the urine of test subject and, following a suitable incubation period, visually observing the results, whereby if said gonadotrophin is not present agglutination of the erythrocytes occurs upon standing whereas if said gonadotrophin is present, no such S 15 agglutination occurs A method for determining the presence of luteinizing hormone in a woman and thereby the day of ovulation may also be done utilizing the compositions of this invention wherein a urine sample is concentrated by ultrafiltration and luteinizing hormone detected by immunological means, the antigen being human chorionic gonadotrophin, as described in U S Specification No 4,123,510 Similiarly, 20 pregnancy in a mare may be determined utilizing as the antigen pregnant mare's serum gonadotrophin and a composition utilizing carcino embryonic antigen would be useful in a blood test for cancer Other uses for the composition of this invention are described therein.
One use of the composition of the invention comprises a method for determining 25 the presence or absence of human chorionic gonadotrophin in a human urine sample which comprises mixing the composition of the invention in which the antigen is human chorionic gonadotrophin and the erthyrocytes are mammalian, avian or reptile pyruvic aldehyde stabilised erthyrocytes with a highly purified h CG antiserum and with ultrafiltration concentrated human urine whereby if h CG is not present ag 30 glutination of the erythrocytes occurs upon standing whereas if h CG is present no such agglutination occurs.
As illustrated below utilizing as the antigen, h CG, it is hypothesized that the superior properties of the compositions of this invention are due to the coupling of the stabilised erythrocytes to the h CG by means of a chemical reaction between the 35 amino functions of the erythrocytes and the h CG and the difunctional glutaraldehyde.
The suggested course of reaction of glutaraldehyde is as follows:
(a) Aldol condensations:
CHO I OHC-CH 2-CH 2-CH 2-CHO OCH-CH 2-CH 2-CH 2-CH = C-CH 2-CH 2-CHO CHO CHO F I OHC-CH 2-CH 2-C-CH-CH = C-CH 2-C = CH-CH 2-CH 2-CH 2-CHO ,.
X C 4 'ACH CHO i H 2 c It CH 2 C CH 2 C H CH CH 2 c 2 (+ other polymeric compounds) 1,589,107 1,589,107 5 (b) Cross-linking reactions:
/red cell N CHO CH CH-CH-CH 2 C=CH-CH 2\ Red Cell N Hz+ h CG -NH 2 + X (PAC') h CG -NH CHO CHO CH-CH-CH -CH-OH h OCG-NH NH red cell The reaction with hexamethylene diisocyanate is thought to proceed in the following fashion:
Red Cell-NH, + O C = N-(CH 2) 6-N C O + NH-h CG O O II II Red Cell-NH-C-NH-(CH 2) 6-NH-C-NH-NH-h CG urea derivative In order to conduct the passive haemagglutination tests contemplated by this invention it is necessary to prepare an antiserum composition to be used in conjunction with the antigen sensitized erythocytes previously described Depending on the test h CG which assays biologically as low as 2500 I U /mg may be employed Alternatively, more purified h CG preparations that assay at approximately 10, 000 I U /mg, 10 15,000 I U /mg or higher may be employed It is preferred to utilize highly purified h CG and most preferred to use the p-sub-unit of h CG in preparing the antiserum of this invention to obtain greater specificity An example of highly purified p-h CG and a method of producing same is reported in our copending U K application 23299/78 (Serial No 1,589,108) (corresponding to U S specification 4,123, 343) 15
The antiserum compositions are generally prepared by immunizing a host animal with the desired antigen thereby producing antibodies to that antigen which may be obtained in the serum separated from the host animal A difficulty in the past has been that the h CG antigen has had other antigen impurities present, thus undesirable antibodies were also produced A haemagglutination method utilizing such an anti 20 serum composition would thus possess undesirable cross reactivity.
The antisera composition of this invention is diluted either in microtitre slides or in a tube system titration to determine the maximum dilution of antisera which can still agglutinate the red blood cell The dilution is then adjusted when utilized in a method for determining pregnancy so that the anti-h CG serum has a sensitivity 25 to h CG of about 100-150 m I U /test The acceptable antisera will also have a cross reactivity of less than 25 % particularly against other glycoprotein hormone antigens Thus an antisera sensitive to 100 m I U /test of h CG would at the same time have a sensitivity to 400 m I U /test or more of h LH or h MG This highly specific antiserum composition obtained using highly purified h CG as the antigen, 30 will detect early pregnancy via the presence of small amounts of h CG and also minimises the possibility of obtaining false positives in the passive haemagglutination inhibiton test A method and apparatus for use of the herein described reagents is described in U S Patent Specification No 4,123,509, U K Patent Applications Nos.
52640/76 (Serial No 1,537,538), 52639/76 (Serial No 1,537,537) and in U S 35 Patent No 4,033,723.
While the compositions of this invention may be utilized in aqueous compositions, it is preferred to lyophilize these compositions for use in the particular haemagglutinati 3 N method under consideration The compositions may be lyophilized into two separate pellets by methods known to those skilled in the art such as that described in U S Patent 3,862,302 That disclosure, however, does not teach how to 5 make lyophilized compositions containing in either the antiserum or the stabilised erythrocytes enough buffer for complexing the interfering calcium ions present in urine Rather it involves the use of a third lyophilized pellet containing the necessary buffers or as in a commercially available pregnancy test a separate buffer composition which is reconstituted at the time of its use 10 The compositions of this invention may also be Iyophilized into a single layered cake, as described in U S Patent 3,269,905 Also while a suitable chelating agent may be included within the lyophilized compositions of this invention to remove quantities of calcium present in a urine test sample, the compositions of this invention without added chelating agent may be utilized with urine and/or ultraconcentrated urine which 15 has been passed through a filter in which is placed a styrene divinyl benzene copolymer containing imide acetate functional groups (CHELEX 100 or DOWEX CHELATING RESIN A-1; "Dowex" is a Registered Trade Mark).
The physical characteristics of the vial in which the immunologic reagents e g.
the Rlbc preparation described in this invention together with a suitable antiserum and 20 appropriate buffers which are to be used in passive haemagglutination tests, must be properly defined Pyrex culture tubes ("Pyrex" is a Registered Trade Mark) have been subjected to various physical pretreatments prior to utilization of the immunologic reagents, described herein in passive haemagglutination tests We have found that round bottomned siliconized glass tubes having an inner diameter of 12-16 mm are 25 preferable in order to obtain reproducible flocculation times and reproducible agglutination patterns.
The invention is further illustrated in the following examples:
EXAMPLE 1.
Whole sheep blood was collected directly into Alserver's solution so that the 30 final ratio of sheep blood to Alserver's solution was 1: 1 (For the purpose of this invention it has been established that the Rbc concentration or packed cell volume (pcv) of such a mixture should be in the range of 15-25 % (i e a hematocrit of 30-50 %) and preferably, 15 to 25 % or more preferably 22 % The cells from 100 ml of such a mixture were collected by centrifugation and washed three times 35 with normal, saline solution, i e 0 9 % (w/v) sodium chloride in distilled water The preferred volume of saline per wash is 320 ml but may be in the range of 100-500 ml.
After washing, the cells are suspended in normal saline solution at a concentration of % A mixture of pyruvic aldehyde ( 64 ml of 25 % aqueous solution) and normal saline ( 120 ml) was adjusted to p H 7 0 using a 10 % w/v aqueous sodium carbonate 40 solution To this mixture of pyruvic aidehyde and saline was added the abovementioned cell suspension ( 100 ml at 20 %), followed by phosphate buffer ( 28 ml; 0.15 M, p H 8 0) This stabilization mixture was then incubated at 370 C for 3 hours, the mixture being shaken vigorously once every 30 minutes.
The cells were then collected by centrifugation and washed 4 times with normal 45 saline solution utilizing preferably 320 ml of saline per wash The stabilised cells may then be stored as a suspension, preferably 10 %, in normal saline containing, for example, 0 1 % sodium azide as a preservative This is stable for at least a year at + 4 CC.
EXAMPLE 2 50
Stabilized cells as described in Example 1 ( 10 ml of about a 10 % suspension) were collected by centrifugation and washed 3 times with normal saline solution, preferably 40 ml for each wash Alternatively, one may of course use unstored cells directly from Example 1 These washed cells were then suspended in a solution of h CG ( 100 lg in 100 gl of 0 5 M phosphate buffer) contained in 0 15 M phosphate 55 buffer ( 8 65 ml, p H 7 4) To this suspension was added a glutaraldehyde reagent ( 1.75 ml), prepared by diluting a 25 % aqueous solution of glutaraldehyde ( 1 ml) in normal saline ( 9 ml).
The sensitization mixture was thoroughly agitated and then gently mixed for 2 hours at room temperature The cells were then collected by centrifugation and 60 washed, preferably 4-5 times, with 0 15 M phosphate buffered saline (p H 7 4).
The sensitized cells are then suspended in 0 15 M phosphate buffered saline ( 39 ml, p H 7 4) containing 0 2 % of normal rabbit serum in which complement had 1,589 107 previously been fixed by incubating at 56 WC for 30 minutes and from which nonspecific agglutinins had been adsorbed by triple treatment with stabilised cells (Example 1) The removal of non-specific agglutinins is illustrated in Example 3.
EXAMPLE 3.
Prior to use for the removal of non-specific agglutinins from normal rabbit serum, 5 stabilised cells as prepared in Example 1, were washed with normal saline ( 5 volumes saline: 1 volume of 10 % cell suspension) and resuspended in saline solution at 10 %.
Equal volumes of rabbit serum and a 10 % suspension of stabilised red blood cells were then incubated at room temperature for 30 minutes The cells were collected by centrifugation, discarded and the process repeated 2 more times, using collected 10 cells as opposed to a 10 % suspension.
Alternatively, the last adsorption may be allowed to proceed at + 4 CC for 18 hours Under the conditions described in this example, the normal rabbit serum has been diluted 1: 1 with saline It should be understood, however, that for this adsorption it is possible to utilize collected cells as opposed to 10 % suspension which 15 would not result in a dilution of the normal rabbit serum.
EXAMPLE 4.
Utilizing the methods of Example 2, but with pregnant mare's serum gonadotrophin as the antigen, a stable sensitized composition was also prepared and satisfactorily evaluated 20 EXAMPLE 5.
Lyophilized cakes were prepared as follows A suspension of the stabilised, sensitized red blood cells (S-'PAGC) was washed about 3 times with a 0 15 M phosphate buffer solution adjusted to a p H of 7 4 The washed 5-PAGC were then resuspended as a 0 415 % v/v suspension in lyophilization buffer, LB, (LB used 25 was 10 g sucrose, 10 ml of 1 % merthiolate, 10 ml of NRS triply adsorbed, and q s.
to 1 litre with 0 15 M phosphate buffered saline containing about 0 2 % EDTA to a p H of 7 0) 300 AI of the cell suspension were then pipetted into different siliconized vials already in a test tube rack, and the rack containing the vials was immersed in an acetone-dry ice bath at about -700 C and frozen Dilutions of antisera and NRS, 30 (which had been pretitrated and adjusted to give a predetermined sensitivity) were made with LB such that the proper concentration of antisera in NRS is contained in a 200 AI aliquot, was then pipetted onto the frozen cell layers in the bath.
The frozen reagents may then be stored at about -100 C until lyophilization or lyophilized immediately Lyophilization of these reagents was then done in a freeze 35 drier for at least 18 hours at about 75-200 microns Hg, while gradually reaching ambient temperatures The vacuum was not permitted to be less than 75 microns to avoid spontaneous haemagglutination The lyophilized reagents when stored at 40 C are stable for at least 9 months.
EXAMPLE 6 40
Lyophilized pellets were prepared as follows:
A suspension of S-PAGC was washed as in Example 5 and then resuspended as a 2 5 % suspension in LB concentrated by a factor of 5 in all its components Thislatter suspension was constantly agitated while a proportioning pump repeatedly delivered about 50 AI of the S-PAGC suspension into a liquid N 2 bath at 196 CC 45 and the pellets were harvested in a petri dish.
The antisera pellets were prepared similarly but may optionally have incorporated within the LB 2 5 % of polyvinyl pyrrolidone or dextran The pellets may be stored as the cakes are stored in Example 4 or transferred immediately to the freeze drier Pellets have been dried satisfactorily at about 50-200 microns Hg for about 50 18 hours, with the temperature finally reaching room tem Derature or in some cases 350 C The pellets were stored in a dessicator at room temperature until packing into siliconized vials These pellets have been found to maintain their sensitivity and physical characteristics in long term stability studies at both 30 WC and 4 CC.
EXAMPLE 7 55
Pretreatment of tubes with silicone is most beneficial as exemplified in Example 8 below' The method used for pretreatment with silicone is described under e), whilst other pretreatments, used for comparative purposes, are described in a) to d).
a) Untreated tubes used directly from the manufacturer.
b) Pyrolyzed tubes prepared by heating at 560 VC (not more than 5 minutes) 60 1,589,107 8 1,589,107 8 c) BSA coated tubes i) washed in 1 % (w/v) bovine serum albumin with agitation, 20 sec.
ii) rinse in running distilled water, 3 times.
iii) dry overnight at 60 C.
d) BSA-coated acid washed tubes i) chromic-sulfuric acid wash, overnight soak.
ii) rinse in running tap water, 3 times.
iii) soak in 95 % ethanol one hour, 2 changes.
iv) rinse in running tap water, 2 times.
v) rinse in running distilled water, 3 times.
vi) dry overnight at 100 C.
vii) wash in 1 % BSA with agitation, 20 sec.
viii) rinse in running distilled water, 3 times.
ix) dry overnight at 60 C.
e) Siliconized tubes i) wash in 1 % (v/v) SILICLAD (Clay Adams), a silane in aqueous alcohol solution, with agitation, 20 sec.
ii) rinse in running distilled water, 6 times.
iii) dry overnight at 100 C.
EXAMPLE 8.
The following results were observed with the pretreated tubes of Example 7.
i) Untreated, pyrolized, BSA-treated and acid washed BSA-treated vials lead to collapsed cell matts, or matts of rough appearance, when a haemagglutination pattern should result using the reagents of this invention.
ii) With the vials pretreated as in (i) above, the transition from haemagglutination to inhibition of haemagglutination is irregular.
iii) Vials pretreated with silicone demonstrate smooth haemagglutination patterns.
iv) In vials pretreated with silicone, the transition from haemagglutination to inhibition of haemagglutination, in the presence of increasing amount of antigen, is regular and the degree to which haemagglutination is inhibited is proportional to the increasing amount of antigen present.
A method for preparing an antiserum composition is illustrated in the following Example 9.
EXAMPLE 9.
Antigen f-h CG subunit Initial Challenge Solution (ICS) Tubercle bacillus p-h CG saline complete Freund's adjuvant Subsequent Challenge Solution (SCS) f-h CG saline complete Freund's adjuvant 5.0 mg mg 2 ml 2 ml 1.0 mg ml ml Booster Solution (BS) p-h CG saline fg 1 ml Each of the ICS and SCS are homogenized well until the suspensions are thick and creamy.
Immunization Protocol Day 1 Three month old virgin female New Zealand white rabbits are injected with 2 0 ml of "ICS" at 30-50 intradermal sites Five hundred zl of crude Bordetella pertussis vaccine is injected at a separate intradermal site.
Day 14 Each rabbit is bled from the marginal ear vein.
Day 15 Each rabbit is injected with a total of 1 0 ml "SCS" in the hind foot pads.
Days 22, 29, 36, 43 Each rabbit is injected with a total of 1 0 ml "SCS" in multiple intradermal sites.
1,589,107 Day 49 Each rabbit is injected with 1 0 ml "BS" intravenously in the marginal ear vein.
Day 56 Each rabbit is bled from the marginal ear vein.
Thereafter, one ml of "BS" is injected travenously every 6 weeks and the rabbits bled 5-7 days after each booster The total time necessary to produce an antiserum 5 using this combined multiple intradermal site-foot pad method of immunization will vary and is considered complete when the antibody titre which is constantly being monitored reaches a plateau.
The antiserum is separated from the rabbit blood and complement may be fixed and the suspension adsorbed as described with the S-PAGC composition in Example 10 2 and Example 3, a single adsorption usually being sufficient Undiluted antisera or diluted antisera may be stored preferably at -100 C in small aliquots.
Claims (1)
- WHAT WE CLAIM IS: -1 An immunologic composition comprising pyruvic aldehyde or dimethylsuberimidate stabilised erythrocytes sensitized with a polypeptide or glycoprotein antigen, 15 said stabilized erythrocytes being coupled to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, a bifunctional imido ester, cyanuric chloride, tetrazotized oanisidine, and a water soluble carbodiimide, with the proviso that when the erythrocytes are dimethylsuberimidate stabilised the bifunctional molecule is other than 20 dimethyl suberimidate and that when the erythrocytes are pyruvic aldehyde stabilised the bifunctional molecule is selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate and a bifunctional imido ester.2 A composition as claimed in Claim 1 wherein the polypeptide or glycoprotein antigen is chorionic gonadotrophin, pregnant mare's serum gonadotrophin, carcino em 25 bryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotrophin or thyroid stimulating hormone and the bifunctional molecule is selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, diethyl malonimidate dihydrochloride, dimethyl suberimidate, cyanuric chloride, tetrazotized o-anisidine, and 1-ethyl-3-( 3dimethylaminopropyl)car 30 boxiimide.3 A composition as claimed in Claim 2 wherein the erythrocytes are pyruvic aldehyde stabilised.4 A composition as claimed in Claim 3 wherein the bifunctional molecule is selected from glutaraldehyde, glycoxal, succinaldehyde, hexamethylene diisocyanate, 35 toluene 2,4-diisocyanate and dimethylsuberimidate.A composition as claimed in Claim 3 or 4 wherein the erythrocytes are mammalian, avian or reptile erythrocytes.6 A composition as claimed in any one of Claims 3 to 5 wherein the antigen is chorionic gonadotrophin 40 7 A composition as claimed in Claim 6 wherein the antigen is human chorionic gonadotrophin.8 A composition as claimed in any one of Claims 3 to 7 wherein the bifunctional molecule is glutaraldehyde.9 A composition as claimed in any one of Claims 3 to 7 wherein the bifunctional 45 molecule is hexamethylene diisocyanate.A composition as claimed in any one of Claims 3 to 7 wherein the bifunctional molecule is dimethyl suberimidate.11 A composition as claimed in Claim 3 in which the antigen is pregnant mare's serum gonadotrophin and the bifunctional molecule is glutaraldehyde 50 12 A composition as claimed in any one of Claims 3 to 11 in which the composition is lyophilized.13 A composition as claimed in any one of Claims 3 to 12 wherein the antigen sensitized erythrocytes have been treated with phosphate buffered saline containing a serum protein in which complement had previously been fixed and from which non 55 specific agglutinins had previously been removed by serial adsorption with pyruvic aldehyde stabilized erythrocytes.14 A composition as claimed in any one of Claims 3 to 11 wherein the antigen sensitized erythrocytes have been treated with phosphate buffered saline containing a stabilisation agent selected from gelatin, dextrans, polyvinylpyrrolidine, and water 60 soluble carboxymethyl celluloses which had previously been treated by adsorption with pyruvic aldehyde stabilized erythrocytes.A composition as claimed in Claim 2 wherein the erythrocytes are dimethyl suberimidate stabilized.1,589,107 Q 16 A method for preparing immunologic compositions of animal erythrocytes sensitized with antigens useful in passive haemagglutination tests which comprises:a) collecting animal blood directly into an isotonic sterile anticoagulant so that a final ratio of blood to anticoagulant is from 0 8:1 to 1 2:1 of the resulting mixture wherein the erythrocyte concentration packed cell volume of said blood is from about 5 % to about 25 %; b) separating the erythrocytes from said mixture by centrifugation; c) washing said erythrocytes with normal saline and then suspending said erythrocytes in normal saline at a concentration of about 10 to 30 %; d) mixing the suspension of erythrocytes with a mixture of pyruvic aldehyde, 10 neutral buffered solution and normal saline to obtain stabilization mixture; e) incubating the stabilization mixture of step (d) at 30 to 55 C for one to five hours, and collecting the stabilized erythrocytes by centrifugation followed by washing with normal saline; f) suspending the stabilized erythrocytes in a neutral buffered solution of S 15 antigen; g) forming a sensitization mixture by thorough agitation of the suspension of stabilized erythrocytes-antigen with a solution of a bifunctional molecule in saline, the bifunctional molecule being selected from glutaraldehyde glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate and a bifunctional imido ester; 20 h) collecting the sensitized erythrocytes by centrifugation and subsequently washing them with neutral buffered saline; i) suspending the sensitized erythrocytes in a neutral buffered saline containing normal animal serum wherein the complement of said serum has previously been fixed, said erythrocytes being mammalian, avian or reptile erythrocytes 25 17 A method as claimed in Claim 16 for preparing a composition as claimed in Claim 3 wherein the antigen is a polypeptide or glycoprotein antigen selected from chorionic gonadotrophin, pregnant mare's serum gonadotrophin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotrophin and thyroid stimulating hormone, and the bifunctional molecule is glutar 30 aldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2, 4-diisocyanate, diethyl malonimidate dihydrochloride or dimethyl suberimidate.18 A method as claimed in Claim 17 wherein the neutral buffered solution in step (d) is phosphate buffer having a p H of about 8 0.19 A method as claimed in Claim 17 or 18 wherein the neutral buffered saline 35 in step (i) is phosphate buffered saline having a p H of about 724.A method as claimed in Claim 17 wherein in step (a) the isotonic sterile anticoagulant is Alserver's solution, the final ratio of blood to Alserver's solution is about 1:1 and the erythrocyte concentration cell volume is from about 15 % to about 25 % 40 21 A method as claimed in Claim 20 wherein in step (c) the concentration of the erythrocytes in normal saline is about 20 %.22 A method as claimed in Claim 21 wherein in step (d) the erythrocytes are mixed with a buffered mixture of pyruvic aldehyde and normal saline, buffered to about p H 8 0 45 23 A method as claimed in Claim 22 wherein in step (e) the incubation is effected at about 37 C for about 3 hours.24 A method as claimed in Claim 23 wherein in step (f) the neutral buffered solution is a phosphate buffered solution.25 A method as claimed in Claim 24 wherein after agitating the suspension so 50 of stabilized erythrocytes antigen with the solution of bifunctional molecule in step (g), the sensitization mixture is mixed for about a further 2 hours at room temperature before collecting the sensitized erythrocytes.26 A method as claimed in Claim 25 wherein in step (i) the neutral buffered saline is phosphate buffered saline 55 27 A method as claimed in Claim 26 wherein the antigen is human chorionic gonadotrophin.28 A method as claimed in Claim 26 or 27 wherein the bifunctional molecule is glutaraldehyde, hexamethylene diisocyanate or dimethyl suberimidate.29 A method as claimed in any one of Claims 17 to 28 wherein the erythrocytes 60 are rabbit, sheep, goat, horse or turkey erythrocytes.A method as claimed in any one of Claims 17 to 29 wherein the composition is lyophilized.31 A method for determining the presence or absence of human chorionic gonadotrophin in a human urine sample which comprises mixing the composition 65 1,589,107 of Claim 3 in which the antigen is human chorionic gonadotrophin and the erythrocytes are mammalian, avian or reptilian erythrocytes, with a highly purified h CG antiserum and with ultrafiltration concentrated human urine whereby if h CG is not present agglutination of the erythrocytes occurs upon standing whereas if h CG is present no such agglutination occurs 5 32 A method as claimed in Claim 31 wherein the antiserum is a /-h CG specific antiserum.33 A method as claimed in Claim 31 or 32 wherein the antiserum is adjusted to have a sensitivity to h CG of 100-150 m I U per test and a cross reactivity to other glycoprotein hormone antigens of less than 25 percent 10 34 A method as claimed in any one of Claims 31 to 34 wherein the mixing is conducted within a siliconized, round button, glass vial having an inner diameter of about 12-16 millimeters.A method as claimed in any one of Claims 31 to 34 wherein the composition is and the antiserum are each in the form of lyophilized pellets 15 36 A method as claimed in any one of Claims 31 to 34 wherein the composition and the antiserum are in the form of a single lyophilized stratified cake.37 An immunologic composition whenever prepared by the method claimed in any one of Claims 16 to 25, 29 and 30.38 An immunologic composition whenever prepared by the method claimed in 20 any one of Claims 26 to 28.39 An immunologic composition substantially as hereinbefore described with reference to Example 2 or 4.A method of preparing an immunologic composition substantially as hereinbefore described with reference to Example 2 or 4 25 K J S BROWN, Chartered Patent Agent.Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa, 1981.Published by the Patent Office, 25 Southampton Buildings, London, WC 2 A l AY, from which copies may be obtained.1,589,107
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US80656377A | 1977-06-14 | 1977-06-14 |
Publications (1)
Publication Number | Publication Date |
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GB1589107A true GB1589107A (en) | 1981-05-07 |
Family
ID=25194314
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB23243/78A Expired GB1589107A (en) | 1977-06-14 | 1978-05-26 | Immunologic compositions |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0000102B1 (en) |
JP (1) | JPS548719A (en) |
CA (1) | CA1108048A (en) |
DE (1) | DE2861050D1 (en) |
GB (1) | GB1589107A (en) |
IT (1) | IT1097807B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2138132A (en) * | 1983-04-12 | 1984-10-17 | Robin Royston Amos Coombs | Storage-stable antibody-linked erythrocytes |
US4543339A (en) * | 1982-03-16 | 1985-09-24 | Oneill Christopher | Early pregnancy detection by detecting enhanced blood platelet activation |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5665044A (en) * | 1979-10-31 | 1981-06-02 | Dainippon Ink & Chem Inc | Carbon fiber-reinforced resin composition |
JPS56135549A (en) * | 1980-03-26 | 1981-10-23 | Dainippon Ink & Chem Inc | Polyarylene sulfide resin composition |
EP0039195B1 (en) * | 1980-04-28 | 1986-06-18 | Montefiore Hospital and Medical Center | Antibody detection process |
EP0134868A1 (en) * | 1983-08-26 | 1985-03-27 | Anda Biologicals | Unicellular organisms activated by glutaraldehyde as solid carriers for sensitizing agents and uses of sensitized unicellular organisms |
DE3428984A1 (en) * | 1984-08-07 | 1986-02-20 | Bayer Ag, 5090 Leverkusen | METHOD FOR THE PRODUCTION OF HIGH MOLECULAR POLYARYL SULFIDES, BRANCHED IF NEEDED |
CA1239346A (en) * | 1985-06-04 | 1988-07-19 | Gursaran P. Talwar | Birth control vaccine |
US5494800A (en) * | 1987-01-29 | 1996-02-27 | Cytosignet, Inc. | Analyte detection in particulate-containing samples |
US4900685A (en) * | 1987-01-29 | 1990-02-13 | Cytosignet, Inc. | Analyte detection in particulate-containing samples |
JPH01107154A (en) * | 1987-10-20 | 1989-04-25 | Seitetsu Kagaku Co Ltd | Processed red blood-corpuscle and its manufacturing method |
US4910294A (en) * | 1988-06-20 | 1990-03-20 | Idemitsu Petrochemical Company Limited | Two-stage process for production of polyarylene sulfides with lithium compound |
US11722295B2 (en) | 2020-04-30 | 2023-08-08 | Musarubra Us Llc | Methods, apparatus, and articles of manufacture to securely audit communications |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3639558A (en) * | 1968-02-19 | 1972-02-01 | Louis Csizmas | Immunological reagent particles having proteinaceous materials covalently bonded thereto |
US3991174A (en) * | 1970-07-20 | 1976-11-09 | Rafa Laboratories Ltd. | Method of determining concentration of luteinizing hormone in body fluid |
FR2187909A1 (en) * | 1972-06-08 | 1974-01-18 | Merieux Inst | Stable antigens - by freeze-drying antigen bonded to red blood cells |
US3914400A (en) * | 1973-05-21 | 1975-10-21 | Us Health | Stable antigen-erythrocytes for measuring antibodies against toxoplasma organism |
-
1978
- 1978-05-26 GB GB23243/78A patent/GB1589107A/en not_active Expired
- 1978-06-02 CA CA304,707A patent/CA1108048A/en not_active Expired
- 1978-06-12 EP EP78300034A patent/EP0000102B1/en not_active Expired
- 1978-06-12 DE DE7878300034T patent/DE2861050D1/en not_active Expired
- 1978-06-14 JP JP7269678A patent/JPS548719A/en active Pending
- 1978-06-14 IT IT24572/78A patent/IT1097807B/en active
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4543339A (en) * | 1982-03-16 | 1985-09-24 | Oneill Christopher | Early pregnancy detection by detecting enhanced blood platelet activation |
GB2138132A (en) * | 1983-04-12 | 1984-10-17 | Robin Royston Amos Coombs | Storage-stable antibody-linked erythrocytes |
Also Published As
Publication number | Publication date |
---|---|
DE2861050D1 (en) | 1981-11-26 |
EP0000102B1 (en) | 1981-09-09 |
JPS548719A (en) | 1979-01-23 |
IT7824572A0 (en) | 1978-06-14 |
CA1108048A (en) | 1981-09-01 |
EP0000102A1 (en) | 1978-12-20 |
IT1097807B (en) | 1985-08-31 |
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PS | Patent sealed [section 19, patents act 1949] | ||
PCNP | Patent ceased through non-payment of renewal fee |