CN113125419A - Donor reagent and application thereof - Google Patents
Donor reagent and application thereof Download PDFInfo
- Publication number
- CN113125419A CN113125419A CN201911420699.6A CN201911420699A CN113125419A CN 113125419 A CN113125419 A CN 113125419A CN 201911420699 A CN201911420699 A CN 201911420699A CN 113125419 A CN113125419 A CN 113125419A
- Authority
- CN
- China
- Prior art keywords
- donor
- reagent
- particles
- group
- agent
- 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.)
- Granted
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 113
- 239000002245 particle Substances 0.000 claims abstract description 149
- 238000009826 distribution Methods 0.000 claims abstract description 53
- 239000007853 buffer solution Substances 0.000 claims abstract description 22
- 235000000346 sugar Nutrition 0.000 claims abstract description 22
- 230000009870 specific binding Effects 0.000 claims abstract description 19
- 150000004676 glycans Chemical class 0.000 claims description 29
- 229920001282 polysaccharide Polymers 0.000 claims description 29
- 239000005017 polysaccharide Substances 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 25
- 238000001514 detection method Methods 0.000 claims description 24
- 125000000524 functional group Chemical group 0.000 claims description 20
- 239000003795 chemical substances by application Substances 0.000 claims description 17
- 108090001008 Avidin Proteins 0.000 claims description 16
- 108010090804 Streptavidin Proteins 0.000 claims description 14
- 239000011616 biotin Substances 0.000 claims description 12
- 229960002685 biotin Drugs 0.000 claims description 12
- 238000009739 binding Methods 0.000 claims description 11
- 230000027455 binding Effects 0.000 claims description 9
- 239000007791 liquid phase Substances 0.000 claims description 9
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 claims description 8
- 125000003172 aldehyde group Chemical group 0.000 claims description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
- 108010087904 neutravidin Proteins 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 239000003642 reactive oxygen metabolite Substances 0.000 claims description 5
- 108010058846 Ovalbumin Proteins 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 4
- 229940092253 ovalbumin Drugs 0.000 claims description 4
- 238000012123 point-of-care testing Methods 0.000 claims description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 3
- 108010017596 Vitellins Proteins 0.000 claims description 2
- 125000003368 amide group Chemical group 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 61
- 238000006243 chemical reaction Methods 0.000 description 29
- 239000004005 microsphere Substances 0.000 description 24
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 20
- 239000000523 sample Substances 0.000 description 20
- 239000007987 MES buffer Substances 0.000 description 18
- 239000000126 substance Substances 0.000 description 17
- 229920002307 Dextran Polymers 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 238000002835 absorbance Methods 0.000 description 14
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 13
- 102100036859 Troponin I, cardiac muscle Human genes 0.000 description 13
- 101710128251 Troponin I, cardiac muscle Proteins 0.000 description 13
- 238000002156 mixing Methods 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 239000000725 suspension Substances 0.000 description 12
- 238000003018 immunoassay Methods 0.000 description 11
- 235000020958 biotin Nutrition 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 239000002086 nanomaterial Substances 0.000 description 9
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 239000003550 marker Substances 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 7
- 150000001299 aldehydes Chemical class 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 239000000839 emulsion Substances 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000005281 excited state Effects 0.000 description 6
- 239000003504 photosensitizing agent Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 229920002527 Glycogen Polymers 0.000 description 4
- 229910020889 NaBH3 Inorganic materials 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229940096919 glycogen Drugs 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 229920002223 polystyrene Polymers 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 238000002038 chemiluminescence detection Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000891 luminescent agent Substances 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 208000010125 myocardial infarction Diseases 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- -1 vitelloavidin Proteins 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 description 2
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 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 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 229920002670 Fructan Polymers 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- WZELXJBMMZFDDU-UHFFFAOYSA-N Imidazol-2-one Chemical group O=C1N=CC=N1 WZELXJBMMZFDDU-UHFFFAOYSA-N 0.000 description 2
- 229920001202 Inulin Polymers 0.000 description 2
- 229920000057 Mannan Polymers 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 102000013394 Troponin I Human genes 0.000 description 2
- 108010065729 Troponin I Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 229960002086 dextran Drugs 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 230000005283 ground state Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 2
- 229940029339 inulin Drugs 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 230000003680 myocardial damage Effects 0.000 description 2
- 208000037891 myocardial injury Diseases 0.000 description 2
- 210000002381 plasma Anatomy 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 229940032147 starch Drugs 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 238000001132 ultrasonic dispersion Methods 0.000 description 2
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 206010008479 Chest Pain Diseases 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010008177 Fd immunoglobulins Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 102000015728 Mucins Human genes 0.000 description 1
- 108010063954 Mucins Proteins 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 101100537532 Rattus norvegicus Tnni3 gene Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical group C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- 239000001752 chlorophylls and chlorophyllins Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000023077 detection of light stimulus Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000000835 electrochemical detection Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- RBTKNAXYKSUFRK-UHFFFAOYSA-N heliogen blue Chemical compound [Cu].[N-]1C2=C(C=CC=C3)C3=C1N=C([N-]1)C3=CC=CC=C3C1=NC([N-]1)=C(C=CC=C3)C3=C1N=C([N-]1)C3=CC=CC=C3C1=N2 RBTKNAXYKSUFRK-UHFFFAOYSA-N 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000008235 industrial water Substances 0.000 description 1
- 229920000592 inorganic polymer Polymers 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000012048 reactive intermediate Substances 0.000 description 1
- 229930187593 rose bengal Natural products 0.000 description 1
- 229940081623 rose bengal Drugs 0.000 description 1
- STRXNPAVPKGJQR-UHFFFAOYSA-N rose bengal A Natural products O1C(=O)C(C(=CC=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 STRXNPAVPKGJQR-UHFFFAOYSA-N 0.000 description 1
- VDNLFJGJEQUWRB-UHFFFAOYSA-N rose bengal free acid Chemical compound OC(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C(O)=C(I)C=C21 VDNLFJGJEQUWRB-UHFFFAOYSA-N 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Images
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a donor reagent and application thereof. The donor reagent comprises a buffer solution and, suspended therein, donor particles comprising a support, the interior of which is filled with a sensitizer, the surface of which is directly or indirectly attached to one of the specific binding partner members, characterized in that the donor particles have a coefficient of variation of particle size distribution C.V value in the donor reagent of not less than 5% and a sugar content per gram mass of the donor particles of not more than 25 mg.
Description
Technical Field
The invention relates to the field of chemiluminescence detection, and in particular relates to a donor reagent and application thereof.
Background
In Vitro Diagnosis (IVD) technology generally refers to products and services that help to determine diseases or body functions by detecting samples of the body, including blood, body fluids, and tissues, to obtain relevant clinical diagnosis information outside the human body. The nano materials have unique size-dependent physical or chemical properties, the optical, magnetic, electric, thermal and biological properties of the nano materials can be adjusted by changing the size, shape, chemical composition, surface functional groups and the like of the nano materials in a nano scale, and particularly, the nano materials can provide a large amount of space for modifying different molecules on the surfaces of the nano materials due to the fact that the nano materials have the specific surface area far higher than that of macroscopic materials, so that the nano materials have important roles in the aspects of application of bioanalysis, biosensors and the like. The nano materials with different molecules modified on the surfaces can be used for selectively detecting small molecules, nucleic acids, proteins, microorganisms and the like. Obviously, the combined nano material and clinical diagnosis and analysis technology will push the clinical in vitro diagnosis subject to new development and growth point.
Immunoassays have evolved over half a century, with a number of detection categories appearing in succession. Heterogeneous immunoassays and homogeneous immunoassays can be classified according to whether or not a substance to be measured is to be separated from a reaction system in a measurement process. Heterogeneous immunoassay refers to the operation process of introducing a probe for labeling, wherein various related reagents are required to be separated after mixed reaction, and an object to be detected is separated from a reaction system and then detected, and is the mainstream method in the existing immunoassay. Such as enzyme-linked immunosorbent assay (ELISA method) and magnetic particle chemiluminescence method. Homogeneous immunoassay refers to direct measurement after mixing and reacting an analyte with a relevant reagent in a reaction system in the measurement process, and no redundant separation or cleaning step is needed. Up to now, various sensitive detection methods are applied to homogeneous immunoassays, such as chemiluminescence detection methods, electrochemical detection methods, and the like.
A Light-activated chemiluminescence Assay (LiCA) is a typical homogeneous immunoassay. The method is based on the reaction of an antigen coated on the surface of one nano microsphere and an antibody coated on the surface of the other nano microsphere in a liquid phase to draw the two nano microspheres closer, thereby forming a 'double-sphere' immune complex. That is, the donor particle and the acceptor particle together form a pair of "two-ball" systems by virtue of antigen-antibody binding. The double spheres are nano microspheres which supplement each other in a light-activated chemiluminescence system, interact with each other, are matched with each other and influence each other, and the two cannot be used. The two nano microspheres have good suspension characteristics in a liquid phase, and the liquid dynamic characteristics are completely met when the microspheres meet antigens or antibodies in the liquid phase. Under 680nm laser irradiation, the photosensitizer of the donor particle is responsible for exciting oxygen in the surrounding environment to singlet oxygen molecules. When singlet oxygen molecules diffuse to receptor particles in a 'double-sphere' system, a series of chemiluminescence reactions are generated with chemiluminescence agents in the receptor particles, so that light signals with emission wavelengths of 610nm to 620nm are generated, and the number of photons is converted into the concentration of target molecules through a photon counter and mathematical fitting, so that 'separation-free' homogeneous immunoassay is realized. When the sample to be detected does not contain target molecules, immune complexes cannot be formed between the two nano microspheres, and the distance between the two nano microspheres exceeds the propagation range of singlet oxygen within 200nm, so that the singlet oxygen is rapidly quenched in a liquid phase, and no high-level red light signal is generated during detection. The method has the characteristics of high speed, homogeneous phase (no flushing), high sensitivity and simple operation.
However, the "double-ball" in the prior art has the defects of poor homogeneity in a liquid phase, poor repeatability and unstable luminescence effect when used for immunoassay, and the like, and is difficult to realize high sensitivity, wide detection range and the like.
Disclosure of Invention
In view of the drawbacks of the prior art, one of the present invention provides a donor reagent comprising a buffer solution and donor particles suspended therein, the donor particles being capable of generating reactive oxygen species when excited in a liquid phase, wherein the donor particles have a variation coefficient of size distribution C.V value of not less than 5% in the donor reagent and a sugar content of not more than 25mg per gram mass of the donor particles.
In a specific embodiment, the donor particle comprises a carrier, the interior of which is filled with a sensitizer, and the surface of which is attached to one of the specific pair binding members.
In one embodiment, the donor particles have a coefficient of variation of particle size distribution C.V value in the donor reagent of no greater than 20%.
In one embodiment, the donor particles have a coefficient of variation of particle size distribution C.V value in the donor reagent of no more than 15%.
In one embodiment, the donor particles have a size distribution variation coefficient C.V value of not less than 8% in the donor reagent.
In a particular embodiment, the sugar content per gram mass of the donor particles is not higher than 15 mg.
In a particular embodiment, the sugar content per liter of volume of said buffer solution is not lower than 0.2g and not higher than 2 g.
In a particular embodiment, the sugar content per liter of volume of said buffer solution is not lower than 0.5g and not higher than 1.5 g.
In one embodiment, the sugar content is determined by the anthrone method.
In one embodiment, the surface of the support carries a bonding functionality for chemically bonding one of the members of the specific binding pair to the surface of the support.
In a specific embodiment, the bonding functional group is selected from at least one of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group, and a thiol group; preferably, the bonding functional group is selected from an aldehyde group and/or a carboxyl group.
In a specific embodiment, the donor particles exhibit a particle size distribution in the donor agent that exhibits polydispersity.
In one embodiment, at least two mean size distributions of donor particles are included in the donor agent.
In one embodiment, the specific binding partner is an avidin-biotin system, the avidin being selected from the group consisting of ovalbumin, vitelloavidin, streptavidin, neutravidin and an avidin-like molecule, preferably streptavidin.
In a specific embodiment, the avidin is selected from streptavidin.
The second aspect of the invention provides a chemiluminescent detection kit comprising a donor reagent according to any one of the first aspect of the invention.
In one embodiment, the kit comprises a plurality of reagent strips, each reagent strip is provided with a plurality of reagent holes for containing reagents, and one of the reagent holes is used for containing the donor reagent.
The third invention provides the use of the donor reagent of any one of the first inventions or the kit of any one of the second inventions in a chemiluminescence analyzer.
The fourth aspect of the present invention provides a use of the donor reagent of any one of the first aspects of the present invention or the kit of any one of the second aspects of the present invention in a POCT apparatus.
The donor reagent has the effect that donor particles in the reagent can generate singlet oxygen after being excited by external exciting light, and the singlet oxygen can transfer energy to the donor particles within 200nm of the acceptor particles to generate a chemiluminescent signal finally, so that the detection of an unknown substance is realized.
The term "active oxygen" as used herein refers to a general term for a substance which is composed of oxygen, contains oxygen, and is active in nature, and is mainly an excited oxygen molecule, including superoxide anion (O) which is an electron reduction product of oxygen2(-) and the two-electron reduction product hydrogen peroxide (H)2O2) The three-electron reduction product hydroxyl radical (. OH) and nitric oxide and singlet oxygen (1O)2) And the like.
In the present invention, the term "directly or indirectly linked" means that a specified substance is capable of chemically or physically bonding to another substance (direct linkage); or the specified substance is chemically or physically bonded to another substance (indirect linkage) through an intermediate substance (compound, polymer, polysaccharide).
The term "acceptor particle" as used herein refers to a particle containing a compound capable of reacting with reactive oxygen species to produce a detectable signal. The donor particles are induced by energy or an active compound to activate and release reactive oxygen species in a high energy state that are captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles.
In one embodiment, the acceptor particle comprises a luminescent agent and a carrier, wherein the luminescent agent is filled in the carrier and/or coated on the surface of the carrier. The "carrier" according to the present invention is selected from the group consisting of tapes, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which may be microspheres or microparticles known to those skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which may have any density, but preferably has a density close to that of water, preferably is capable of floating in water, and which are composed of a transparent, partially transparent or opaque material. The carrier may or may not have a charge, and when charged, is preferably a negative charge. The carrier may be a latex particle or other particle containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes.
In the present invention, the "luminescent agent", i.e. a compound called a label, may undergo a chemical reaction in order to cause luminescence, for example by being converted into another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly, or may return to the ground state itself by transferring excitation energy to an emission energy acceptor. In this process, the energy acceptor particle will be transitioned to an excited state to emit light.
The term "donor particles" as used herein refers to particles containing a sensitizer capable of generating a reactive intermediate, such as a reactive oxygen species, upon activation by energy or a reactive compound, to react with the acceptor particles. The donor particles may be light activated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In one embodiment, the donor particles are polymeric microspheres filled with a photosensitizer, which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. patent No. 5709994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or more hydrophilic and/or as a linker group to a member of a specific binding pair. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference.
In one embodiment, the surface of the support is coated with at least two successive polysaccharide layers, wherein the polysaccharide layers are spontaneously associated with the second polysaccharide layer.
In one embodiment, each of the successive polysaccharide layers is spontaneously associated with each of the preceding polysaccharide layers.
In one embodiment, the polysaccharide has pendant functional groups, the pendant functional groups in the continuous polysaccharide layer being oppositely charged from the pendant functional groups in the previous polysaccharide layer.
In one embodiment, the polysaccharide has pendant functional groups, and the continuous polysaccharide layer is covalently linked to the preceding polysaccharide layer by a reaction between the pendant functional groups and the pendant functional groups of the preceding polysaccharide layer.
In one embodiment, the pendant functional groups in two adjacent polysaccharide layers alternate between amine functional groups and amine reactive functional groups in the continuous polysaccharide layer. I.e., where the pendant functional group in one layer is an amine functional group, the pendant functional group in the other layer is an amine-reactive functional group.
In one embodiment, the amine-reactive functional group is an aldehyde group or a carboxyl group.
In one embodiment, the first polysaccharide layer (i.e., the innermost layer) is spontaneously associated with the support.
In one embodiment, the polysaccharide in the outermost polysaccharide layer of the coating has at least one pendant functional group.
In one embodiment, the pendant functional groups of the polysaccharide in the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl, and maleic groups; preferably selected from aldehyde groups and/or carboxyl groups.
In one embodiment, the pendant functional groups of the polysaccharide in the outermost polysaccharide layer of the coating are directly or indirectly bound by one of the members of the specific binding pair chemically bonded.
In one embodiment, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.
In one embodiment, the particle size of the carrier is selected from 100 to 400nm, preferably 150 to 350nm, more preferably 180 to 220 nm.
In a particular embodiment, the concentration of the donor particles in the donor agent is from 10 μ g/ml to 1mg/ml, preferably from 20 μ g/ml to 500 μ g/ml, more preferably from 50 μ g/ml to 200 μ g/ml.
In one embodiment, the donor reagent further comprises a buffer solution having a PH of 7.0 to 9.0, wherein the donor particles are suspended in the buffer solution.
In a particular embodiment, the buffer solution contains a polysaccharide selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from dextran, starch, glycogen and polyribose.
In a particular embodiment, the polysaccharide (e.g. dextran) has a molecular weight distribution Mw selected from 10000 to 1000000Da, preferably from 100000 to 800000Da, more preferably from 300000 to 700000 Da.
In a particular embodiment, the sugar content per liter of volume of said buffer solution is not lower than 0.2g and not higher than 2 g.
In a particular embodiment, the sugar content per liter of volume of said buffer solution is not lower than 0.5g and not higher than 1.5 g.
In the present invention, the "C.V value of the variation coefficient of particle size distribution" refers to the variation coefficient of the particle size in the gaussian distribution in the results of measurement by a nanometer particle analyzer. The coefficient of variation is calculated as: the coefficient of variation C.V value (standard deviation SD/Mean) x 100%. The Standard Deviation (SD), also known as the Standard Deviation, describes the mean of the distances of the data from the mean (mean Deviation), which is the square of the Deviation and the root of the mean, expressed as a. The standard deviation is the arithmetic square root of the variance. The standard deviation reflects the degree of dispersion of a data set, and the smaller the standard deviation, the less the values deviate from the mean, and vice versa. The standard deviation σ is a distance between an inflection point (0.607 times the peak height) on the normal distribution curve and a vertical line between the peak height and the time axis, that is, a distance between two inflection points on the normal distribution curve is half. The peak width at half height (Wh/2) is the width of the peak at half the peak height, Wh/2 ═ 2.355 σ. The tangent is drawn by the inflection points on both sides of the normal distribution curve, the intercept at the base line is called the peak width or base line width, and W is 4 sigma or 1.699 Wh/2.
The inventor has found through extensive research that the value of the variation coefficient C.V of the particle size distribution of the particles in the liquid phase influences the detection signal of the light-activated chemiluminescence. If a large batch of donor particles with qualified quality and stable performance is required to realize the commercial application of the light-activated chemiluminescent system in clinical immunodiagnosis, the C.V value of the donor particle size distribution in the donor reagent must be strictly controlled within a proper range. It should be noted that, unlike the particle size distribution of the conventional blank polystyrene microsphere (i.e. the carrier of the present invention, which is not filled with functional substances therein, nor is the surface modified with avidin molecules or polysaccharides), the C.V value of the present invention refers to the C.V value of the variation coefficient of the particle size distribution of the whole donor particles in the donor agent. Since the donor particle size distribution variation coefficient C.V value is continuously changed from the carrier in the preparation process of the donor particle, especially after the donor particle is coated with polysaccharide or avidin, the change of the particle size distribution variation coefficient C.V value of the nano-microsphere is more obvious and unstable, and the requirements of medical device product registration and clinical application reagents are difficult to meet. Therefore, through a great deal of experimental research, the applicant gradually searches and discovers a range and a method for strictly controlling the variation coefficient C.V value of the particle size distribution of the donor particles in the liquid phase, and finally realizes the commercial application of the light-activated chemiluminescence technology in clinical examination.
The term "test sample" as used herein refers to a mixture to be tested that contains or is suspected of containing a target molecule to be tested. The test sample that can be used in the present invention includes body fluids such as blood (which may be anticoagulated blood commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be tested can be diluted with a diluent as required before use. For example, to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before the on-line detection and then detected on the detection instrument.
The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected will be used for the detection of the target molecule. The target molecule to be detected may be a protein, a peptide, an antibody or a hapten which allows it to bind to an antibody. The target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected may be any other substance that can form a member of a specific binding pair. Other examples of typical target molecules to be detected include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteroazoids, nucleic acids, and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a receptor. Analytes also include cells, viruses, bacteria, and fungi.
The term "antibody" as used herein is used in the broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair member), and the like.
The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect.
The term "binding" as used herein refers to direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.
The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure. Under the technical idea disclosed by the invention, the detection method of the specific binding reaction comprises but is not limited to the following steps: double antibody sandwich, competition, neutralization competition, indirect or capture.
The term "specific binding pair member" as used herein refers to a pair of molecules that are capable of specifically binding to each other, e.g., enzyme-substrate, antigen-antibody, ligand-receptor. An example of a specific binding pair member is the biotin-streptavidin system, where "biotin" is widely present in animal and plant tissues and has two cyclic structures on the molecule, an imidazolone ring and a thiophene ring, respectively, where the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be conjugated to almost any biological macromolecule known, including proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by a protein cross-linking agent; the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin, preferably neutravidin and/or streptavidin. Avidin is a glycoprotein extracted from egg white, has a molecular weight of 60kD, and each molecule consists of 4 subunits, so that avidin can be closely combined with 4 biotin molecules and plays an important role in an immune mechanism. The avidin mainly comprises ovalbumin, streptavidin, yolk avidin, neutravidin and the like. Streptavidin is a protein secreted by streptomyces, and the "streptavidin" molecule consists of 4 identical peptide chains, each of which can bind a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby creating a "tentacle effect" that increases assay sensitivity. Any reagent used in the present invention, including antigens, antibodies, acceptor particles or donor particles, may be conjugated to any member of the biotin-streptavidin specific binding pair as desired.
In the light-activated chemiluminescent system, in addition to the donor reagent, other reagents may be included as necessary for the test object or test method, such as: receptor reagent, biotin-coated secondary antibody, diluent and the like. In the field of in vitro diagnostics, in particular in the field of immunoassays, the various manufacturers, in order to simplify the naming of the different components of the commercial kits, generally label or simply refer to the components of the different bottles of the kit as reagent 1 or R1, reagent 2 or R2, reagent 3 or R3, … …, and so on, which facilitates the identification, assembly and use by the client, also for technical secrecy purposes. Therefore, the kit products of different in vitro diagnostic manufacturers may contain reagent 1, reagent 2, reagent 3 and reagent … …, but the reagent components of different manufacturers are different.
The invention has the beneficial effects that:
the liquid homogeneous mode enables consistency among tests to be better, and products have better repeatability and consistency. And the method is free from washing, avoids introducing unnecessary interferents and other uncertain factors in the reaction, and ensures that the detection result is more stable. The unique chemiluminescence technology can be introduced into the field of rapid diagnosis, so that the overall detection performance is better.
Drawings
Fig. 1 shows the gaussian distribution of donor particles a prepared in example 1.
Fig. 2 shows the gaussian distribution of donor particles b prepared in example 2.
FIG. 3 shows the relationship between glucose concentration and corresponding absorbance.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not to be construed as limiting the invention in any way.
The reagents used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 preparation of Donor particles a and Donor reagent A containing such Donor particles a
1.1 preparation of the vector
a) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10min, N was introduced thereinto2 30min。
b) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step a), and continuously introducing N2 30min。
c) The reaction system is heated to 70 ℃ and reacted for 15 hours to obtain emulsion.
d) And cooling the emulsion after the reaction to room temperature, filtering the emulsion by using a proper filter cloth, centrifuging, settling and cleaning the obtained filtered emulsion by using deionized water for multiple times until the conductivity of the supernatant obtained after centrifugation is close to that of the deionized water, and diluting the supernatant by using water to obtain the final emulsion aldehyde group polystyrene microsphere liquid.
e) The size of the carrier was measured by a nanometer particle size analyzer to have a gaussian distribution, an average particle diameter of 201.3nm, and a coefficient of variation (C.V.) -8.0%.
1.2 sensitizer-filled Carrier
a) A25 ml round bottom flask was prepared, 0.11g of copper phthalocyanine and 10ml of N, N-dimethylformamide were added, magnetic stirring was carried out, and the temperature in the water bath was raised to 75 ℃ to obtain a photosensitizer solution.
b) A100 ml three-necked flask was prepared, 10ml of 95% ethanol, 10ml of water, and 10ml of the 10% concentration carrier prepared in step 1.1 were added thereto, magnetically stirred, and heated to 70 ℃ in a water bath.
c) Slowly dripping the photosensitizer solution obtained in the step a) into the three-neck flask obtained in the step b), stirring at 70 ℃ for reacting for 2 hours to fill the sensitizer into the aldehyde polystyrene microspheres, stopping stirring, and naturally cooling to obtain the filled emulsion.
d) The filled emulsion 30000g was centrifuged for 1 hour, after which the supernatant was discarded and resuspended in 50% ethanol. After repeating the centrifugal washing three times, the suspension was resuspended in 50mM CB buffer solution having a pH of 10 to give a final concentration of 20mg/ml aldehyde-based polystyrene microspheres filled with a sensitizer, to obtain a microsphere suspension.
1.3 preparation of Donor reagent A
a) Treating microsphere suspension: and (5) centrifuging the microsphere suspension prepared in the step (II) in a high-speed refrigerated centrifuge, removing the supernatant, adding MES buffer solution, performing ultrasound on an ultrasonic cell disruption instrument until particles are resuspended, and adding MES buffer solution to adjust the mass concentration of the microspheres to 100mg/ml to obtain the treated microsphere suspension.
b) Preparing an avidin solution: a certain amount of streptavidin was weighed and dissolved in MES buffer to 8 mg/ml.
c) Mixing: mixing the treated microsphere suspension, 8mg/ml avidin and MES buffer solution in a volume ratio of 2:5:1, and quickly mixing to obtain a reaction solution.
d) Reaction: preparing 25mg/ml NaBH by MES buffer solution3CN solution according to NaBH3Adding the CN solution and the reaction solution in a volume ratio of 1:25, and quickly and uniformly mixing. The reaction was rotated at 37 ℃ for 48 hours.
e) And (3) sealing: 75mg/ml glycine (Gly) solution and 25mg/ml NaBH solution are respectively prepared by MES buffer solution3CN solution according to glycine solution, NaBH3Adding the CN solution and the reaction solution in a volume ratio of 2:1:10 into the solution obtained in the step d), uniformly mixing, and carrying out a rotary reaction at 37 ℃ for 2 hours. Then, 200mg/ml BSA solution (prepared with MES buffer) was added at a volume ratio of 5:8, and the mixture was rapidly mixed and reacted at 37 ℃ for 16 hours with rotation.
f) Cleaning: adding MES buffer solution into the solution reacted in the step e), centrifuging by using a high-speed refrigerated centrifuge, discarding the supernatant, then adding fresh MES buffer solution again, performing ultrasonic suspension, centrifuging again, washing for 3 times, finally performing suspension by using a small amount of buffer solution, measuring the solid content, and adjusting the solid content to the concentration of 150 mu g/ml by using the buffer solution to obtain the donor reagent A containing the donor particles a, wherein the composition of the buffer solution is as follows: 0.1mol Tris-HCl, 0.3mol NaCl, 25mmol EDTA, 0.1% dextran, 0.01% gentamicin and 15ppm ProClin-300, pH 8.00.
g) The donor particles a in the donor reagent a were gaussian distributed by a nano-particle size analyzer, the average particle size was 227.7nm, and the coefficient of variation c.v. value was 6.5%, as shown in fig. 1.
EXAMPLE 2 preparation of Donor particles B and Donor reagent B containing such Donor particles B
The preparation of the carrier used in this example and the filling of the sensitizer are the same as in steps 1.1 and 1.2 of example 1.
2.1 coating the surface of the support with dextran
a) 50mg of the aminodextran solid was placed in a 20mL round-bottom flask, and 5mL of 50mM/pH 10 carbonate buffer was added and dissolved with stirring at 30 ℃ in the dark.
b) 100mg of donor particles a were added to the aminodextran solution and stirred for 2 hours to obtain a reaction solution.
c) After 10mg of sodium borohydride was dissolved in 0.5mL of 50mM/pH 10 carbonate buffer solution, the solution was added dropwise to the reaction solution, and the mixture was reacted overnight at 30 ℃ in the dark to obtain a mixture of the aminodextran-coated carrier.
d) 30000g of the mixture from step c) was centrifuged and the supernatant discarded, and then added to 50mM/pH 10 carbonate buffer for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted with 50mM/pH 10 carbonate buffer solution to a final concentration of 20mg/ml of the aminodextran-coated carrier, thereby obtaining an aminodextran carrier solution.
e) 100mg of aldehyde dextran solid was placed in a 20mL round-bottom flask, 5mL of 50mM/pH 10 carbonate buffer was added, and the mixture was dissolved with stirring at 30 ℃ in the dark.
f) The aminodextran carrier fluid was added to the aldehyde dextran solution and stirred for 2 hours.
g) After 15mg of sodium borohydride was dissolved in 0.5mL of 50mM/pH 10 carbonate buffer solution, the solution was added dropwise to the reaction solution, and the mixture was reacted overnight at 30 ℃ in the dark to obtain a mixed solution of the aldehyde dextran-coated carrier.
h) 30000g of the mixture from step g) was centrifuged and the supernatant discarded, and then added to 50mM/pH 10 carbonate buffer for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted with 50mM/pH 10 carbonate buffer solution to a final concentration of 20mg/ml of the aldehyde dextran-coated carrier, thereby obtaining a dextran-coated carrier solution.
i) The dextran-coated carrier had a gaussian distribution of size, as measured by a nano-particle sizer, with an average particle size of 235.6nm and a coefficient of variation (C.V.) -8.1%.
2.2 preparation of Donor reagent B
a) Treatment of dextran-coated carrier liquid: the dextran-coated carrier fluid obtained in step h) of step 2.1 of this example was centrifuged in a high-speed refrigerated centrifuge, the supernatant was discarded, MES buffer was added, sonication was performed on an ultrasonic cell disruptor until the microspheres were resuspended, and MES buffer was added to adjust the mass concentration of the carrier to 100 mg/ml.
b) Preparing an avidin solution: a certain amount of neutravidin was weighed and dissolved to 8mg/ml by adding MES buffer.
c) Mixing: mixing the treated microsphere suspension, 8mg/ml avidin and MES buffer solution in a volume ratio of 2:5:1, and quickly mixing to obtain a reaction solution.
d) Reaction: preparing 25mg/ml NaBH by MES buffer solution3CN solution according to NaBH3Adding the CN solution and the reaction solution in a volume ratio of 1:25, and quickly and uniformly mixing. The reaction was rotated at 37 ℃ for 48 hours.
e) And (3) sealing: 75mg/ml Gly solution and 25mg/ml NaBH were prepared by MES buffer distribution3CN solution, as Gly solution, NaBH3Adding the CN solution and the reaction solution in a volume ratio of 2:1:10 into the solution obtained in the step d), uniformly mixing, and carrying out a rotary reaction at 37 ℃ for 2 hours. Then, 200mg/ml BSA solution (prepared with MES buffer) was added at a volume ratio of 5:8, and the mixture was rapidly mixed and reacted at 37 ℃ for 16 hours with rotation.
f) Cleaning: adding MES buffer solution into the solution reacted in the step e), centrifuging by using a high-speed refrigerated centrifuge, discarding the supernatant, then adding fresh MES buffer solution again, performing ultrasonic suspension, centrifuging again, washing for 3 times, finally performing suspension by using a small amount of buffer solution, measuring the solid content, and adjusting the solid content to 150 mu g/ml by using the buffer solution to obtain donor reagent B containing donor particles B, wherein the composition of the buffer solution is as follows: 0.1mol Tris-HCl, 0.3mol NaCl, 25mmol EDTA, 0.1% dextran, 0.01% gentamicin and 15ppm ProClin-300, pH 8.00.
g) The donor particles B in the donor reagent B were gaussian distributed by a nano-particle sizer, the average particle size was 249.9nm, and the coefficient of variation C.V was 11.6%, as shown in fig. 2.
Example 3 determination of sugar content in Donor particles
3.1 determining the relationship between glucose concentration and corresponding absorbance value by the anthrone method
a) Preparing a glucose standard solution: a1 mg/mL glucose stock solution was prepared as a standard solution curve at 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.075mg/mL, 0.10mg/mL, 0.15mg/mL with purified water.
b) Preparing an anthrone solution: the solution is prepared into 2mg/mL by 80 percent sulfuric acid solution (the solution is stable within 24 hours at room temperature, and the solution is prepared as before use).
c) 0.1mL of the glucose standard solution at each concentration was added to each centrifuge tube, and 1mL of the anthrone test solution was added to each tube.
d) Incubating at 85 ℃ for 30 min; the supernatant was then centrifuged at 15000g for 40min and the clear liquid was aspirated from the bottom of the tube with a pipette tip to avoid aspiration of the supernatant.
e) After the aspirated clear liquid was returned to room temperature, the absorbance at 620nm (preferably, the measurement was performed within 2 hours) was measured and repeated twice, and the average value of the two repetitions was taken as the final absorbance, and the results are shown in Table 1.
f) Taking the concentration of the standard substance as an X value and the average absorbance as a Y value, and performing linear regression once to obtain a result shown in figure 3, so as to obtain the sugar concentration of the sample to be detected.
TABLE 1
| Serial number | Concentration mg/mL | Absorbance A | Absorbance B | Absorbance mean value |
| 1 | 0.15 | 0.415 | 0.411 | 0.4130 |
| 2 | 0.1 | 0.293 | 0.302 | 0.2975 |
| 3 | 0.075 | 0.214 | 0.227 | 0.2205 |
| 4 | 0.05 | 0.146 | 0.153 | 0.1495 |
| 5 | 0.025 | 0.101 | 0.098 | 0.0995 |
| 6 | 0 | 0.032 | 0.031 | 0.0315 |
3.2 determination of sugar content in Donor particles
a) And taking a donor reagent containing 1mg of donor particles, centrifuging for 40min at 20000g, pouring out supernatant, ultrasonically dispersing by using purified water, repeatedly centrifuging and dispersing for three times, and fixing the volume to 1mg/mL by using the purified water to prepare a sample to be detected.
b) Preparing an anthrone solution: the solution is prepared into 2mg/mL by 80 percent sulfuric acid solution (the solution is stable within 24 hours at room temperature, and the solution is prepared as before use).
c) 0.1mL of sample to be tested and 1mL of anthrone test solution are sequentially added into the centrifuge tube.
d) Incubating at 85 ℃ for 30 min; centrifuging at 15000g for 40min, and sucking clarified liquid from the bottom of the tube with pipette tip to avoid sucking suspended matter from the upper part.
e) After the aspirated clear liquid was returned to room temperature, the absorbance at 620nm (the measurement was preferably performed within 2 h) was measured, and the absorbance was repeated twice, and the average of the two repetitions was taken as the final absorbance, and then the sugar content in the donor particles was calculated according to the curve equation obtained in FIG. 3, and the results are shown in Table 2.
TABLE 2
| Serial number | Absorbance A | Absorbance B | Absorbance mean value | Sugar concentration mg/g |
| Donor particle a | 0.0572 | 0.0564 | 0.0568 | 11.5 |
| Donor particle b | 0.172 | 0.165 | 0.1685 | 53.6 |
Example 4
The donor particles and donor reagent preparation method described above in example 1 were used to prepare a donor reagent comprising a series of donor particles as follows:
donor reagent 1: the average particle size of the donor particles in the gaussian distribution curve is 226.5nm, and the value of the variation coefficient C.V of the particle size distribution is 3.8; nicomp distribution is unimodal.
Donor reagent 2: the average particle size of the donor particles in the Gaussian distribution curve is 225.3nm, and the value of the variation coefficient C.V of the particle size distribution is 4.6; nicomp distribution is unimodal.
Donor reagent 3: the average particle size of the donor particles in the Gaussian distribution curve is 225.2nm, and the value of the variation coefficient of the particle size distribution C.V is 5.0; nicomp distribution is unimodal.
Donor reagent 4: the average particle size of the donor particles in the Gaussian distribution curve is 226.7nm, and the value of the variation coefficient of the particle size distribution C.V is 8.1; nicomp distribution is unimodal.
Donor reagent 5: the average particle size of the donor particles in the Gaussian distribution curve is 227.8nm, and the value of the variation coefficient C.V of the particle size distribution is 15.6; nicomp distribution is unimodal.
Donor reagent 6: the average particle size of the donor particles in the Gaussian distribution curve is 225.9nm, and the value of the variation coefficient of the particle size distribution C.V is 26.1; nicomp distribution is unimodal.
Troponin I quantitative determination detection kits 1 to 6 were prepared using the above donor reagents 1 to 6.
The kit of this example was composed of a reagent 1(R1 ') comprising acceptor particles coated with a first anti-cTnI antibody, a reagent 2(R2 ') comprising a biotin-labeled second anti-cTnI antibody, and additionally comprised any one of the reagents (R3 ') of the donor reagents 1 to 6. Notably, R1' is an acceptor agent; r3' is a donor reagent.
The chemiluminescence detection process is completed on a full-automatic light-activated chemiluminescence analysis system (LiCA HT) developed by Boyang Biotechnology (Shanghai) Limited company and a detection result is output, and the specific experimental steps are as follows:
(1) adding 40 mu l of a sample to be detected containing a cTnI marker with a known concentration, 15 mu l R1 'and 15 mu l R2' into an 8X 12 96-well plate, and uniformly mixing;
(2) incubating at 37 ℃ for 8 min;
(3) add 160. mu.l of general solution (R3');
(4) incubating at 37 ℃ for 2 min;
(5) the sample was placed in a LiCA HT apparatus to excite the reading, and the specific detection results are shown in Table 3 below. The sensitivity and upper limit of detection of the kits 1 to 6 were then analyzed according to the detection results.
TABLE 3
As can be seen from Table 3, when the variation coefficient of the particle size distribution of the donor particles is greater than or equal to 5%, the method has both appropriate sensitivity and appropriate detection range, and the production cost of the reagent raw materials and the kit is relatively low, so that the requirement of clinical in vitro diagnosis application can be met.
Example 5
The donor particles and the preparation methods of the donor particles and the donor reagents in examples 1 and 2 above were used to prepare donor reagents comprising a series of donor particles, and the sugar content in the donor particles was measured using the anthrone method in example 3.
With reference to the method given in example 4 above, troponin I quantitative determination test kits A to E containing donor reagents A to E were prepared, and the chemiluminescence test procedure was carried out on a fully automatic light-activated chemiluminescence assay system (LiCA HT) developed by Boyang Biotechnology, Inc. and the test results were outputted, and the specific experimental procedures were as given in example 4 and are shown in Table 4.
TABLE 4
| Name of Donor reagent | Sugar content of the Donor particles mg/g | Light-activated chemiluminescent detection signal |
| Donor reagent A in example 1 | 11.5 | 1117775 |
| Donor reagent B in example 2 | 53.6 | 42500 |
| Donor reagent C | 23.8 | 1079654 |
| Donor reagent D | 45.3 | 32642 |
| Donor reagent E | 60.3 | 25056 |
As can be seen from Table 4, the detection of light-activated chemiluminescence was better when the sugar content of the donor particles in the donor reagent was less than 25 mg/g.
Example 6
This example used the homogeneous chemiluminescence POCT assay device disclosed in CN208568604U to detect CRP.
The kit prepared by the embodiment comprises a plurality of reagent cup strips, wherein the reagent cup strips are provided with a plurality of hole sites for containing reagents, the hole sites include but are not limited to a to-be-detected sample hole site, a first reagent hole site and a second reagent hole site, and the to-be-detected sample hole site is used for containing a to-be-detected sample containing target molecules to be detected. The first reagent aperture site is for holding a donor reagent comprising a donor particle capable of producing singlet oxygen in an excited state. The second reagent well site is for holding an acceptor reagent comprising an acceptor particle, the acceptor particle capable of reacting with singlet oxygen to produce a chemiluminescent signal, and the donor particle having a particle size greater than the acceptor particle. The hole site can be selected and the function is expanded according to the actual need.
The donor reagent contained in the first reagent well site of this example was donor reagent A prepared in example 1, in which the donor particles had a coefficient of variation of particle size distribution C.V value of 6.5% and a sugar content of 11.5 mg/g. 50 mu L of clinical samples (containing serum and whole blood) with different CRP concentrations are added into one hole site of the reagent cup strip, the mean value of parallel tubes of each hole is taken, 50 mu L of biotinylated anti-CRP antibody and 50 mu L of acceptor reagent containing coupled CRP acceptor particles are reacted for 7.5min at 37 ℃, 50 mu L of donor reagent A prepared in example 1 is further added, the reaction is carried out for 5min at 37 ℃, and the light excitation detection is carried out, wherein the experimental results are shown in Table 5. As can be seen in table 5, the correlation coefficient between serum and whole blood reached 0.9988. The experimental result shows that the donor particle greatly reduces the nonspecific adsorption in the sample, has very good correlation between the measurement results aiming at the serum and the whole blood, greatly enhances the adaptability of the donor reagent to the clinical sample, and can be directly used for the detection of the clinical whole blood sample.
TABLE 5
Example 7
In this example, 40 clinical samples including 13 negative samples (numbered 1 to 13) and 27 positive samples (numbered 14 to 40) were tested, and the cTnI quantitative determination test kit (light-activated chemiluminescence method) used included: reagent 1(R1) comprising acceptor particles coated with a first anti-cTnI monoclonal antibody, reagent 2(R2) comprising a biotin-labeled second anti-cTnI monoclonal antibody, and a photo-activated chemiluminescence analysis system-general liquid (R3) containing donor particles. Wherein, the concentration of the donor particles in the reagent R3 is 100 mug/ml, the variation coefficient of the particle size distribution of the donor particles in the reagent R3 is C.V percent, and the sugar content of the donor particles is 14.5 mg/g.
The detection process is completed on a full-automatic light-activated chemiluminescence analysis system (LiCA HT) developed by Boyang Biotechnology (Shanghai) Inc., and the detection result is output, and the specific detection steps are shown in example 4.
When the cTnI marker exists in a clinical sample, the cTnI marker is specifically combined with the receptor particles coated with the first anti-cTnI monoclonal antibody and a biotin-labeled second anti-cTnI monoclonal antibody at the same time, and a double-antibody sandwich complex is formed on the surface of the receptor particles; at this time, if a donor particle modified by streptavidin is added, biotin and streptavidin are combined to enable the two particles to approach each other, under the excitation of an excitation light source, the donor particle releases singlet oxygen, and chemiluminescence is generated after the donor particle touches an acceptor particle in a solution, so that a fluorophore on the same particle is further excited to generate a cascade amplification reaction to generate fluorescence. At this time, the more the content of the cTnI marker is present, the stronger the fluorescence intensity is, and the amount of the cTnI marker in the serum of the patient is quantitatively measured according to the intensity of the luminescence, and the specific detection result of the content of the cTnI marker is shown in table 6.
TABLE 6
The data were compared and the correlation between Abbott and Boyang measurements was 0.9973, with a slope of 1.0495. Sample No. 1-13 is a patient with normal physical examination, the distribution range is 1.77pg/ml to 25.3pg/ml, and the median value is 6.77 pg/ml; samples No. 14-40 identified patients with myocardial injury, with a distribution range of 30.94pg/ml to 29896.88pg/ml, with a median of 450.54 pg/ml.
The concentration of cardiac troponin I (cTnI) in serum or plasma of healthy people is low, 4-8 hours after chest pain of patients, a large amount of cTNI is released by necrotic myocardial cells to enter a blood circulation system, the peak value is reached within 12-48 hours, and the cTnI is still maintained at a high level after several days of severe myocardial infarction patients, so that the cardiac troponin I (cTnI) is an optimal marker for diagnosing myocardial injury and myocardial infarction. The data according to example 7 of the present invention show that the use of the donor reagent of the present invention in the preparation of a kit for use in a method for in vitro diagnosing whether a subject has myocardial damage is feasible, and that the quantitative results of the cTnI marker in the body fluid of a subject measured using the donor reagent of the present invention and the corresponding method can be used for diagnosing myocardial damage and myocardial infarction.
Claims (18)
1. A donor reagent comprising a buffer solution and, suspended therein, donor particles capable of generating reactive oxygen species upon excitation in a liquid phase, characterized in that the donor particles have a coefficient of variation of particle size distribution C.V value in the donor reagent of not less than 5% and a sugar content per gram mass of the donor particles of not more than 25 mg.
2. The donor reagent of claim 1, wherein the donor particle comprises a carrier, the interior of the carrier being filled with a sensitizer, and a surface of the carrier having attached thereto one of the specific binding partner members.
3. The donor agent according to claim 1 or 2, wherein the donor particles have a coefficient of variation C.V of the particle size distribution in the donor agent of not higher than 20%, preferably not lower than 15%.
4. The donor agent of any one of claims 1 to 3, wherein the donor particles have a variation coefficient of size distribution C.V value of not less than 8% in the donor agent.
5. The donor agent of any one of claims 1 to 4, wherein the sugar content per gram mass of the donor particles is not higher than 15 mg.
6. The donor agent according to any one of claims 1 to 5, wherein the sugar content per liter volume of the buffer solution is not lower than 0.2g and not higher than 2g, preferably not lower than 0.5g and not higher than 1.5 g.
7. The donor reagent according to any one of claims 1 to 6, wherein the sugar content is detected by an anthrone method.
8. The donor reagent of any one of claims 2 to 7, wherein the surface of the support carries a binding functionality for chemically binding one of the members of the specific binding pair to the surface of the support.
9. The donor reagent of claim 8, wherein the surface of the support is free of a coating polysaccharide and one of the specific binding pair members is directly chemically bonded to the bonding functionality on the surface of the support.
10. The donor reagent of claim 8 or 9, wherein the bonding functional group is selected from at least one of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group, and a thiol group; preferably, the bonding functional group is selected from an aldehyde group and/or a carboxyl group.
11. The donor agent of any one of claims 1 to 10, wherein the donor particles exhibit a polydispersity in their size distribution in the donor agent.
12. The donor agent of any one of claims 1 to 11, wherein the donor agent comprises at least two mean size distributions of donor particles.
13. The donor reagent of any one of claims 2 to 12, wherein the surface of the support is directly bound to one of the specific pair binding members.
14. The donor agent of any one of claims 2 to 13, wherein the specific binding partner is an avidin-biotin system, said avidin being selected from the group consisting of ovalbumin, vitellin, streptavidin, neutravidin and an avidin-like molecule, preferably streptavidin.
15. A chemiluminescent detection kit comprising the donor reagent of any one of claims 1 to 14.
16. The kit of claim 15, wherein the kit comprises a plurality of reagent strips, each reagent strip having a plurality of reagent wells for holding reagents, wherein one of the reagent wells is adapted to hold the donor reagent.
17. Use of the donor reagent of any one of claims 1 to 14 or the kit of claim 15 or 16 in the use of a chemiluminescent analyzer.
18. Use of the donor reagent of any one of claims 1 to 14 or the kit of claim 15 or 16 in a POCT instrument.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911420699.6A CN113125419B (en) | 2019-12-31 | 2019-12-31 | Donor reagent and application thereof |
| CN202310605655.0A CN116400074A (en) | 2019-12-31 | 2019-12-31 | CRP detection kit and application thereof |
| CN202310612239.3A CN116449001A (en) | 2019-12-31 | 2019-12-31 | cTnI detection kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911420699.6A CN113125419B (en) | 2019-12-31 | 2019-12-31 | Donor reagent and application thereof |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310605655.0A Division CN116400074A (en) | 2019-12-31 | 2019-12-31 | CRP detection kit and application thereof |
| CN202310612239.3A Division CN116449001A (en) | 2019-12-31 | 2019-12-31 | cTnI detection kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113125419A true CN113125419A (en) | 2021-07-16 |
| CN113125419B CN113125419B (en) | 2023-06-23 |
Family
ID=76769458
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310612239.3A Pending CN116449001A (en) | 2019-12-31 | 2019-12-31 | cTnI detection kit and application thereof |
| CN201911420699.6A Active CN113125419B (en) | 2019-12-31 | 2019-12-31 | Donor reagent and application thereof |
| CN202310605655.0A Pending CN116400074A (en) | 2019-12-31 | 2019-12-31 | CRP detection kit and application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310612239.3A Pending CN116449001A (en) | 2019-12-31 | 2019-12-31 | cTnI detection kit and application thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310605655.0A Pending CN116400074A (en) | 2019-12-31 | 2019-12-31 | CRP detection kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN116449001A (en) |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7179660B1 (en) * | 2000-03-06 | 2007-02-20 | Dade Behring Marburg Gmbh | Carriers coated with polysaccharides, their preparation and use |
| US20080044926A1 (en) * | 2004-12-24 | 2008-02-21 | Daiichi Pure Chemicals Co., | Reagent for Assaying Antigen and Method of Assaying Antigen |
| US20100267071A1 (en) * | 2009-02-27 | 2010-10-21 | Beckman Coulter, Inc. | Solution phase homogeneous assays |
| CN101981057A (en) * | 2008-03-31 | 2011-02-23 | 旭化成纤维株式会社 | Cellulose derivative fine particles, fluid dispersions of the same, solid dispersions thereof, and diagnostic drugs |
| CN102759631A (en) * | 2012-08-02 | 2012-10-31 | 南京诺尔曼生物技术有限公司 | Latex enhanced turbidimetric immunoassay kit of quantitatively detecting procalcitonin PCT |
| CN102892896A (en) * | 2010-05-14 | 2013-01-23 | 贝克曼考尔特公司 | Homogeneous chemiluminescence assay methods with increased sensitivity |
| CN105126714A (en) * | 2015-08-05 | 2015-12-09 | 上海交通大学 | Functional nano particle composite microsphere, preparation and applications thereof |
| CN105758835A (en) * | 2016-05-04 | 2016-07-13 | 成都爱兴生物科技有限公司 | Homogeneous phase immunoassay POCT detection technique and system using same |
| CN106268810A (en) * | 2016-07-29 | 2017-01-04 | 华南理工大学 | A kind of surface recombination modifies particle aggregate carrier material and preparation method and application |
| US20170305983A1 (en) * | 2015-02-10 | 2017-10-26 | Shenzhen New Industries Biomedical Engineering Co., Ltd | Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor |
| CN108445239A (en) * | 2018-02-11 | 2018-08-24 | 北京科美生物技术有限公司 | Detect the homogeneous immunological detection reagent suit and its preparation method and application of β human chorionic gonadotrophins |
| WO2019053328A1 (en) * | 2017-09-12 | 2019-03-21 | Helsingin Yliopisto | A method for assaying presence of an antibody in a sample and a kit therefor |
| CN109991405A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | A kind of immunity detection reagent and its application |
| CN110161250A (en) * | 2018-02-11 | 2019-08-23 | 博阳生物科技（上海）有限公司 | A kind of homogeneous human cardiac troponin I quick detection kit, system, detection method and application |
| US20190353664A1 (en) * | 2016-11-22 | 2019-11-21 | Beijing Chemclin Biotech Co., Ltd. | Method, system, reagent kit, and device for determining hd-hook-effect sample and immunoassay |
-
2019
- 2019-12-31 CN CN202310612239.3A patent/CN116449001A/en active Pending
- 2019-12-31 CN CN201911420699.6A patent/CN113125419B/en active Active
- 2019-12-31 CN CN202310605655.0A patent/CN116400074A/en active Pending
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7179660B1 (en) * | 2000-03-06 | 2007-02-20 | Dade Behring Marburg Gmbh | Carriers coated with polysaccharides, their preparation and use |
| US20080044926A1 (en) * | 2004-12-24 | 2008-02-21 | Daiichi Pure Chemicals Co., | Reagent for Assaying Antigen and Method of Assaying Antigen |
| CN101981057A (en) * | 2008-03-31 | 2011-02-23 | 旭化成纤维株式会社 | Cellulose derivative fine particles, fluid dispersions of the same, solid dispersions thereof, and diagnostic drugs |
| US20100267071A1 (en) * | 2009-02-27 | 2010-10-21 | Beckman Coulter, Inc. | Solution phase homogeneous assays |
| CN102892896A (en) * | 2010-05-14 | 2013-01-23 | 贝克曼考尔特公司 | Homogeneous chemiluminescence assay methods with increased sensitivity |
| US20130084652A1 (en) * | 2010-05-14 | 2013-04-04 | Beckman Coulter, Inc. | Homogeneous Chemiluminescence Assay Methods with Increased Sensitivity |
| CN102759631A (en) * | 2012-08-02 | 2012-10-31 | 南京诺尔曼生物技术有限公司 | Latex enhanced turbidimetric immunoassay kit of quantitatively detecting procalcitonin PCT |
| US20170305983A1 (en) * | 2015-02-10 | 2017-10-26 | Shenzhen New Industries Biomedical Engineering Co., Ltd | Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor |
| CN105126714A (en) * | 2015-08-05 | 2015-12-09 | 上海交通大学 | Functional nano particle composite microsphere, preparation and applications thereof |
| CN105758835A (en) * | 2016-05-04 | 2016-07-13 | 成都爱兴生物科技有限公司 | Homogeneous phase immunoassay POCT detection technique and system using same |
| CN106268810A (en) * | 2016-07-29 | 2017-01-04 | 华南理工大学 | A kind of surface recombination modifies particle aggregate carrier material and preparation method and application |
| US20190353664A1 (en) * | 2016-11-22 | 2019-11-21 | Beijing Chemclin Biotech Co., Ltd. | Method, system, reagent kit, and device for determining hd-hook-effect sample and immunoassay |
| WO2019053328A1 (en) * | 2017-09-12 | 2019-03-21 | Helsingin Yliopisto | A method for assaying presence of an antibody in a sample and a kit therefor |
| CN109991405A (en) * | 2017-12-29 | 2019-07-09 | 博阳生物科技（上海）有限公司 | A kind of immunity detection reagent and its application |
| CN108445239A (en) * | 2018-02-11 | 2018-08-24 | 北京科美生物技术有限公司 | Detect the homogeneous immunological detection reagent suit and its preparation method and application of β human chorionic gonadotrophins |
| CN110161250A (en) * | 2018-02-11 | 2019-08-23 | 博阳生物科技（上海）有限公司 | A kind of homogeneous human cardiac troponin I quick detection kit, system, detection method and application |
Non-Patent Citations (1)
| Title |
|---|
| 林冠峰;邹丽萍;刘天才;侯经远;任志奇;吴英松;: "人促黄体生成素光激化学发光免疫分析法的建立", 热带医学杂志 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113125419B (en) | 2023-06-23 |
| CN116400074A (en) | 2023-07-07 |
| CN116449001A (en) | 2023-07-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN116429757A (en) | Homogeneous chemiluminescence detection kit and application thereof | |
| CN110736739A (en) | homogeneous phase chemiluminescence detection kit and application thereof | |
| CN110736735A (en) | homogeneous phase chemiluminescence detection kit and application thereof | |
| CN116754757A (en) | Donor particle for homogeneous chemiluminescence analysis and application thereof | |
| CN113125419B (en) | Donor reagent and application thereof | |
| CN113125711B (en) | Acceptor reagent and application thereof | |
| CN113125709A (en) | Donor reagent and application thereof | |
| CN112240928B (en) | Homogeneous chemiluminescence analysis method and application thereof | |
| CN113125715A (en) | Human immunodeficiency virus antibody detection kit and application thereof | |
| CN113125703B (en) | Myoglobin homogeneous detection kit and application thereof | |
| CN113125705B (en) | Myoglobin homogeneous detection kit and application thereof | |
| CN112114131A (en) | Homogeneous phase chemiluminescence detection method and application thereof | |
| CN113125730B (en) | Homogeneous detection kit for interleukin 6 and application thereof | |
| CN113125721B (en) | Homogeneous detection kit for creatine kinase isozymes and application thereof | |
| CN112240930A (en) | Homogeneous phase chemiluminescence analysis method and application thereof | |
| CN112114130A (en) | Receptor reagent for homogeneous phase chemiluminescence detection and application thereof | |
| CN113125700B (en) | Homogeneous detection kit for creatine kinase isozymes and application thereof | |
| CN113125732B (en) | Homogeneous detection kit for interleukin 6 and application thereof | |
| CN112240936A (en) | Application of donor reagent in diagnosis of myocardial damage of main body | |
| CN113125701B (en) | Homogeneous chemiluminescence detection kit and application thereof | |
| CN113125417A (en) | Receptor reagent and application thereof | |
| CN113125702A (en) | Homogeneous phase chemiluminescence detection kit and application thereof | |
| CN113125698A (en) | Receptor reagent and application thereof | |
| CN112240937A (en) | Application of donor reagent in diagnosis of main body infected bacterial inflammatory disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 201210 third and fifth floors, building 1, No. 88, Cailun Road, pilot Free Trade Zone, Pudong New Area, Shanghai Applicant after: Kemei Boyang diagnostic technology (Shanghai) Co.,Ltd. Applicant after: Kemei Diagnostic Technology Co.,Ltd. Address before: 201210 third and fifth floors, building 1, No. 88, Cailun Road, pilot Free Trade Zone, Pudong New Area, Shanghai Applicant before: BEYOND DIAGNOSTICS (SHANGHAI) Co.,Ltd. Applicant before: Kemei Diagnostic Technology Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |