CN111856041A - 一种pct/il-6荧光定量快速检测试纸条及其制备方法和应用 - Google Patents
一种pct/il-6荧光定量快速检测试纸条及其制备方法和应用 Download PDFInfo
- Publication number
- CN111856041A CN111856041A CN202010688922.1A CN202010688922A CN111856041A CN 111856041 A CN111856041 A CN 111856041A CN 202010688922 A CN202010688922 A CN 202010688922A CN 111856041 A CN111856041 A CN 111856041A
- Authority
- CN
- China
- Prior art keywords
- pct
- pad
- detection
- test strip
- fluorescent
- 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.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 112
- 108090001005 Interleukin-6 Proteins 0.000 title claims abstract description 83
- 238000012360 testing method Methods 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000012528 membrane Substances 0.000 claims abstract description 53
- 239000004005 microsphere Substances 0.000 claims abstract description 42
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 30
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 30
- 210000004369 blood Anatomy 0.000 claims abstract description 21
- 239000008280 blood Substances 0.000 claims abstract description 21
- 238000003908 quality control method Methods 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000001914 filtration Methods 0.000 claims abstract description 16
- 238000010521 absorption reaction Methods 0.000 claims abstract description 13
- 241000283707 Capra Species 0.000 claims abstract description 7
- 102000004889 Interleukin-6 Human genes 0.000 claims description 79
- 239000011248 coating agent Substances 0.000 claims description 25
- 238000000576 coating method Methods 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 19
- 239000000427 antigen Substances 0.000 claims description 17
- 102000036639 antigens Human genes 0.000 claims description 17
- 108091007433 antigens Proteins 0.000 claims description 17
- 239000003085 diluting agent Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 12
- 238000006748 scratching Methods 0.000 claims description 12
- 230000002393 scratching effect Effects 0.000 claims description 12
- 238000007789 sealing Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 9
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 9
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 9
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 9
- 239000002299 complementary DNA Substances 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 8
- 108010010803 Gelatin Proteins 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000008273 gelatin Substances 0.000 claims description 6
- 229920000159 gelatin Polymers 0.000 claims description 6
- 235000019322 gelatine Nutrition 0.000 claims description 6
- 235000011852 gelatine desserts Nutrition 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 206010061218 Inflammation Diseases 0.000 claims description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 5
- 230000004054 inflammatory process Effects 0.000 claims description 5
- 238000002372 labelling Methods 0.000 claims description 5
- 239000003550 marker Substances 0.000 claims description 5
- 210000002381 plasma Anatomy 0.000 claims description 5
- 241000251468 Actinopterygii Species 0.000 claims description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 4
- 239000005018 casein Substances 0.000 claims description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 4
- 235000021240 caseins Nutrition 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims description 4
- 239000003365 glass fiber Substances 0.000 claims description 4
- 238000002615 hemofiltration Methods 0.000 claims description 4
- 230000003053 immunization Effects 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 claims description 4
- 238000002823 phage display Methods 0.000 claims description 4
- 235000018102 proteins Nutrition 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 239000013598 vector Substances 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000010931 gold Substances 0.000 claims description 3
- 229910052737 gold Inorganic materials 0.000 claims description 3
- 238000007857 nested PCR Methods 0.000 claims description 3
- 210000005259 peripheral blood Anatomy 0.000 claims description 3
- 239000011886 peripheral blood Substances 0.000 claims description 3
- 241001416177 Vicugna pacos Species 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 235000019688 fish Nutrition 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 238000003757 reverse transcription PCR Methods 0.000 claims description 2
- 239000012086 standard solution Substances 0.000 claims description 2
- 230000002463 transducing effect Effects 0.000 claims description 2
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 claims 9
- 239000013504 Triton X-100 Substances 0.000 claims 2
- 229920004890 Triton X-100 Polymers 0.000 claims 2
- 239000007987 MES buffer Substances 0.000 claims 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 abstract description 12
- 238000011282 treatment Methods 0.000 abstract description 11
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 25
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 230000000903 blocking effect Effects 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 241000282836 Camelus dromedarius Species 0.000 description 7
- 206010040047 Sepsis Diseases 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 239000004033 plastic Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000027455 binding Effects 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 5
- 208000022362 bacterial infectious disease Diseases 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 208000035143 Bacterial infection Diseases 0.000 description 4
- 102000011632 Caseins Human genes 0.000 description 4
- 108010076119 Caseins Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000004393 prognosis Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 206010051379 Systemic Inflammatory Response Syndrome Diseases 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000003317 immunochromatography Methods 0.000 description 3
- 230000028709 inflammatory response Effects 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 102000055006 Calcitonin Human genes 0.000 description 2
- 108060001064 Calcitonin Proteins 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 2
- 238000003287 bathing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 2
- 229960004015 calcitonin Drugs 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000004796 pathophysiological change Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- 206010048998 Acute phase reaction Diseases 0.000 description 1
- 241000282832 Camelidae Species 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010048233 Procalcitonin Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 206010040049 Sepsis neonatal Diseases 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 229920002359 Tetronic® Polymers 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 201000005008 bacterial sepsis Diseases 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 230000017306 interleukin-6 production Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008718 systemic inflammatory response Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 238000001132 ultrasonic dispersion Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000009385 viral infection Effects 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
- C07K16/248—IL-6
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
-
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
- G01N2333/5412—IL-6
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/585—Calcitonins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Endocrinology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供一种PCT/IL‑6荧光定量快速检测试纸条及其制备方法和应用,所述检测试纸条由背衬卡以及粘贴在背衬卡上的滤血膜样品垫、荧光微球结合垫、硝酸纤维素膜检测垫和吸水垫组成,所述硝酸纤维素膜检测垫上沿长度方向依次平行间隔设置有第一检测线、第二检测线和质控线,所述第一检测线和第二检测线上分别包被有IL‑6特异性纳米抗体和PCT特异性纳米抗体,质控线上包被有羊抗兔IgG。该试纸条可同时检测IL‑6、PCT两个标靶,有效避免单一指标对感染类别判断的误差,且具有检测范围宽、灵敏度高、准确度高、检测快速简便等特点,能及时帮助临床医师尽快确定患者的治疗方案,进而提高患者治疗成功率,具有重要的临床应用价值。
Description
技术领域
本发明属于生物医学和生物检测技术领域,具体涉及一种PCT/IL-6荧光定量快速检测试纸条及其制备方法和应用。
背景技术
感染性疾病是急诊科常见的疾病之一,由感染引起的SIRS是脓毒症最根本的病理生理学改变。脓毒症早期的病理生理改变是功能性的、可逆的。因此,如何运用更有效的手段对感染性疾病患者进行早期诊断、鉴别诊断、预后评估和治疗监测从而提高治疗效果、降低疾病死亡率是目前急诊医师共同关注的重点。
降钙素原(procalcitonin,简写为PCT)是血清降钙素(CT)的前肽物质,PCT在炎症刺激特别是细菌感染或脓毒血症状态下,机体各个组织、多种细胞类型均可产生PCT并释放进入血液循环系统。PCT在感染开始后的最初3小时即可测得,6-12小时后可达到最高峰值,与传统的生物标志物相比,PCT的半衰期接近24小时,且几乎不受肾功能状态、激素治疗的影响。独特的生物学特点,使PCT对机体感染的反映具有快速准确的特征。研究证明,在诊断感染性疾病时,PCT的敏感度和特异度均优于其他标志物。动态监测PCT水平有助于疾病早期诊断、指导抗生素治疗、评估病情进展程度、判断预后结果,其临床意义已经得到了广泛的认可。临床资料显示,PCT浓度大于0.1ng/ml时说明存在临床相关的细菌感染,需要采用抗生素进行治疗;当PCT浓度大于0.5ng/ml时,要考虑患者可能发展成重症败血症或败血症性休克的危险。PCT反映了全身炎症反应的活跃程度。而影响PCT水平的因素包括被感染器官的大小和类型、细菌的种类、炎症的程度和免疫反应的状况。检测病例中PCT浓度的变化,有利于判断机体细菌感染情况,评判机体健康状况,对临床和基础性研究均具有重要意义。
白介素6(IL-6)作为白细胞介素的一种细胞因子,主要由巨噬细胞、T细胞、B细胞等多种细胞产生,具有调节免疫应答、急性期反应及造血功能,并在机体的抗感染免疫反应中起重要作用。IL-6及其受体与炎症性疾病的有关,它是多功能炎性细胞因子,在炎症反应中起重要作用。IL-6水平上升可作为炎症反应的早期指标,并可用于评估系统性炎症反应综合征的严重程度(SIRS)。当炎症反应发生后,IL-6血清浓度的升高早于其他生物标志物,浓度升高后诱导PCT和CRP的产生。患者血清中IL-6的浓度水平在感染早期快速升高,2小时内达到峰值,相比PCT和CRP更为灵敏,在感染早期更有利临床预测,动态观察IL-6水平有助于了解疾病进展并确认疗效。一项内毒素刺激的人体试验显示,IL-6的血清浓度水平增高早于PCT和CRP,证明IL-6作为生物标志物可能具有早期诊断的价值。尽管IL-6半衰期只有1小时,但是能够在感染早期快速升高实现早期预测,同时其升高水平与疾病严重程度、死亡率相关,可作为有效的预后指标。在重症医学/急诊、早发型新生儿脓毒症、创伤危险分层/预后、术后风险等领域的应用,尤为突出。同时,IL-6也是评估脓毒症患者预后的优秀标志物。
值得注意的是,IL-6并不是感染的特异性指标,细菌感染、病毒感染、自身免疫、外伤等均会引起IL-6升高。在实际操作中,即使是在血标本采集后,内毒素和一些细胞因子可能诱导IL-6产生,标本最好采集在无内毒素的试管内,迅速分离血清、冷藏。IL-6仅是一个生物学指标,临床上只能帮助确诊,并不能作为排除脓毒症或高风险患者的诊断标准,所有的临床诊断及治疗策略的制订仍都必须结合患者的临床症状和其他检查结果进行综合判断。在脓毒症的全病程管理中,IL-6和PCT联合检测可以取长补短,提高诊断的准确率。PCT主要用于细菌感染性疾病的辅助诊断,IL-6主要用于监测机体的免疫状态、炎症反应等。联合检测IL-6和PCT可以避免单一指标对感染类别判断的误差,提高感染的早期诊断,及时帮助临床确定患者的治疗方案,进而提高患者治疗成功率,具有重要临床价值。但现有技术中炎症感染及脓毒症检测仅针对单一的炎症指标进行检测,另外IL-6检测试纸条较少,且测定IL-6/PCT的方法主要有免疫比浊法、荧光免疫层析法、胶体金法等。其中胶体金法具有操作简单快速的优点,但灵敏度低且定量不准确;免疫比浊法较为敏感、准确,可应用于全自动生化仪,但需要仪器且耗时较长,适合处理大量样本,无法满足快速检测的目的。
目前的免疫层析技术在包被及标记抗体的时候主要采用常规的小鼠单克隆抗体,少量使用其他种属的单克隆抗体,此类单克隆抗体的分子量约150KD,骆驼抗体天然缺失轻链,只含有重链,因此又称重链抗体(heavychainantibodies,HCAbs)。骆驼抗体中还缺少CH1。克隆重链抗体的可变区得到只由一个重链可变区组成的单域抗体,称为VHH抗体(variabledomainofheavychainofheavy-chainantibody,VHH)。VHH晶体直径2.5nm,长4nm,因此又称为纳米抗体(nanobody,Nb),纳米抗体体积很小,相对于常规抗体的6个互补决定区(complementaritydeterminingregion,CDR),Nb仅有的3个CDR就具备了特异的抗原结合能力和高亲合力,具有完整的抗原结合片段,如图1所示。与现有单克隆抗体相比,纳米抗体主要有如下优点:1)分子量小,仅有普通抗体的十分之一,可以自由进入到普通抗体所不能到达的区域,如实体瘤内部及穿越血脑屏障,另外小体积使得纳米抗体可以进入抗原的三维折叠构象内部,去特异性识别普通抗体不能识别的位点;2)高稳定性,能耐高温且具有高度的构像稳定性,在37℃孵育1周后纳米抗体仍具有80%的结合活性,它具有可逆的去折叠能力,将其长期存放在37℃的环境后仍能重新获得抗原结合能力,这一优点使得纳米抗体易于储存、运输,而不像普通抗体那样必须始终冷冻保存,另外在蛋白酶和极度pH值环境中纳米抗体仍然具有高度稳定性,可以开发口服类药物;3)对人的免疫原性弱,易于完全人源化,不引起病人体内排异反应,符合抗体药物的发展方向。纳米抗体的上述诸多优点使得其明显优于传统抗体,基于骆驼重链抗体的VHH单域抗体的特殊结构,兼具了传统抗体与小分子药物的优势,目前,纳米抗体已经发展成为有广泛生物应用价值的通用分子,其应用范围涉及到基础研究、新药开发、疾病的诊断和治疗等多个领域。
发明内容
针对现有技术存在的上述不足,本发明要解决的技术问题是:现有IL-6/PCT的测定方法存在灵敏度低、定量不准确,需要仪器且耗时较长,不适合处理大量样本,无法满足快速检测的问题。
为了解决上述技术问题,本发明采用如下技术方案:一种PCT/IL-6荧光定量快速检测试纸条,所述检测试纸条由背衬卡以及粘贴在背衬卡上的滤血膜样品垫、荧光微球结合垫、硝酸纤维素膜检测垫和吸水垫组成,所述背衬卡两端分别设有滤血膜样品垫和吸水垫;在背衬卡中部设有硝酸纤维素膜检测垫,在硝酸纤维素膜检测垫与滤血膜样品垫之间设有荧光微球结合垫;所述荧光微球结合垫一端与滤血膜样品垫互相叠加,另一端与硝酸纤维素膜检测垫互相叠加;所述荧光微球结合垫上包括检测微球和质控微球,检测微球为表面包被有PCT标记纳米抗体和IL-6标记纳米抗体的荧光微球,质控微球为表面包被有兔抗标记蛋白的荧光微球;所述硝酸纤维素膜检测垫上沿长度方向依次平行间隔设置有第一检测线、第二检测线和质控线,所述第一检测线和第二检测线上分别包被有IL-6特异性纳米抗体和PCT特异性纳米抗体,质控线上包被有羊抗兔IgG。
进一步,所述IL-6或PCT特异性纳米抗体的制备方法包括如下步骤:
1)分别以IL-6抗原或PCT抗原免疫骆驼科动物,取免疫后骆驼科动物的外周血,提取外周血淋巴细胞RNA,然后将所述外周血淋巴细胞RNA反转录为cDNA,运用逆转录PCR扩增所述cDNA;
2)以步骤1)得到的cDNA为模板,采用巢式PCR扩增VHH基因片段;
3)将步骤2)得到的VHH基因片段连接表达载体,得到IL-6或PCT特异性抗体的重组表达载体,再将所述重组表达载体转导至宿主菌,获得表达IL-6或PCT特异性抗体的重组工程菌,然后诱导所述重组工程菌表达重组蛋白,对所述重组蛋白进行分离纯化处理,即得到所述IL-6或PCT特异性纳米抗体。
进一步,所述骆驼科动物为羊驼,所述表达载体为噬菌体展示载体pHEN1,所述宿主菌为大肠杆菌。
本发明的目的还在于提供一种炎症检测剂盒,包括上述PCT/IL-6荧光定量快速检测试纸条。
本发明的目的还在于提供一种PCT/IL-6荧光定量快速检测试纸条的制备方法,包括以下步骤:
S1:制备IL-6特异性纳米抗体和PCT特异性纳米抗体;
S2:用包被稀释液将IL-6特异性纳米抗体、PCT特异性纳米抗体和羊抗兔IgG稀释成1mg/mL的包被液;然后分别于贴在背衬卡的硝酸纤维素膜上划膜间隔包被,分别得到第一检测线、第二检测线和质控线,划膜参数为1μL/cm,划膜结束后,置于37~40℃条件下干燥30~60min;
S3:将封闭液倒入玻璃加样槽中,将步骤S2烘干的包被板的硝酸纤维素膜的顶端贴上吸水垫,吸水垫与硝酸纤维素膜交叠1mm,随后将未贴吸水垫的一端放入盛装封闭液的加样槽中,20min后取出37℃烘干24h;
S4:取50μL荧光微球(固含量为1%),加入430μLMES缓冲液(50mM,pH6.0),再加入20μLEDC(10mg/mL),室温反应10min,16000g离心弃上清,加入500μLPBS(10mM,pH7.4)超声分散荧光微球离心沉淀,IL-6标记纳米抗体、PCT标记纳米抗体及兔抗标记蛋白各200μg,室温反应2h,16000g离心弃上清,加入标记稀释液500μL超声分散荧光微球离心沉淀,最后用喷金仪喷标记垫,标记垫为玻纤材料,37℃烘干24h,即得到荧光微球结合垫;
S5:将步骤S4得到的荧光微球结合垫贴于包被板上,荧光微球结合垫与硝酸纤维素膜交叠1mm,最后在带有背衬卡的包被板上贴上滤血膜样品垫,滤血膜样品垫与荧光微球结合垫交叠1mm,即得到所述检测试纸条。
作为优选的,所述包被稀释液为MOPSO、海藻糖和硫酸铵的混合溶液,所述包被液缓冲液的pH为7.0。
作为优选的,所述封闭液由MOPSO、海藻糖、硫酸铵、酪蛋白、鱼明胶、Tween20、TritonX-100和PVP40K组成,所述封闭液的pH为7.0。
作为优选的,所述标记稀释液的配方为:10mM/L的MOPSO、10mM/L的海藻糖、10mM/L的硫酸铵、0.2%(m/V)的酪蛋白、0.2%(m/V)的鱼明胶、0.2%(V/V)的Tween20、0.2%(V/V)的TritonX-100和0.2%(m/V)的PVP40K,所述标记稀释液的pH为7.0。
本发明的目的还在于提供一种PCT/IL-6荧光定量快速检测的方法,取待测样本加入检测缓冲液,混合10秒钟后,加到所述检测试纸条的样本垫上,将检测试纸条插入荧光分析仪的检测孔,放置15分钟,以检测线和质控线的荧光强度比值为纵坐标,PCT和IL-6标准溶液浓度为横坐标计算样品中PCT和IL-6的浓度值。
作为优选的,所述待测样本为全血、血清或血浆。
相比现有技术,本发明具有如下有益效果:
1、一个样本可同时检测IL-6、PCT两个标靶,PCT联合IL-6检测可以避免单一指标对感染类别判断的误差,及时帮助临床医师尽快确定患者的治疗方案,进而提高患者治疗成功率,具有重要的临床应用价值。且IL-6、PCT的炎症联合诊断更及时、更准确。
2、本发明可解决常规抗体无法识别的隐蔽表位或小表位、常规抗体和抗原结合后因为空间位阻遮蔽表位导致遮蔽表位结合抗体无法识别抗原的问题;同时,对包被后的NC膜进行封闭以提高检测的灵敏度。
3、本发明采用纳米抗体进行标记和包被标记,抗体包被后进行封闭,灵敏度更高。
附图说明
图1为常规抗体和纳米抗体的分子差异(A常规抗体;B骆驼血液中的重链抗体;C纳米抗体)。
图2为本发明检测试剂条的结构示意图。
图3为本发明检测试剂卡的结构示意图。
图4为本发明实施例所制备的检测试剂条与Cobas法检测IL-6的结果相关性比较。
图5为本发明实施例所制备的检测试剂条与Cobas法检测PCT的结果相关性比较。
具体实施方式
下面结合具体实施例对本发明作进一步详细说明。本实施案例在以本发明技术为前提下进行实施,现给出详细的实施方式和具体的操作过程来说明本发明具有创造性,但本发明的保护范围不限于以下的实施例。
实施例1IL-6/PCT联合检测荧光免疫层析定量检测试剂条
如图2所示,检测试纸条由背衬卡5以及粘贴在背衬卡5上的滤血膜样品垫1、荧光微球结合垫2、硝酸纤维素膜检测垫3和吸水垫4组成,所述背衬卡5两端分别设有滤血膜样品垫1和吸水垫4;在背衬卡5中部设有硝酸纤维素膜检测垫3,在硝酸纤维素膜检测垫3与滤血膜样品垫1之间设有荧光微球结合垫2;所述荧光微球结合垫2一端与滤血膜样品垫1互相叠加,另一端与硝酸纤维素膜检测垫3互相叠加;所述硝酸纤维素膜检测垫3上沿长度方向依次平行间隔设置有第一检测线、第二检测线和质控线,所述第一检测线和第二检测线上分别包被有IL-6特异性纳米抗体和PCT特异性纳米抗体,质控线上包被有羊抗兔IgG。
实施例2荧光定量检测IL-6/PCT的试剂条的制备方法
具体包括以下步骤:
1、纳米抗体的制备
(1)免疫骆驼
挑选健康强壮、精神状态良好的骆驼进行免疫实验,以IL-6及PCT为免疫抗原,每次在骆驼颈部淋巴结附近分左右两侧注射,每侧分2点注射,每次的免疫剂量为100μg。免疫后观察半小时确认骆驼状态良好,无不适症状。每1周免疫一次,免疫周期为28天。
(2)提取外周淋巴细胞
从免疫后的骆驼颈部静脉采集外周血20-30mL,在15mL的离心管中先加入3mL细胞分离液,然后缓慢加入3mL血液。室温400rpm离心30分钟,观察离心管中血液分离情况,用移液器小心吸取出中间环状乳白色淋巴细胞至含无菌RNAase-freePBS的离心管中,充分混合后,室温400rpm离心20分钟。去除上清液,重复洗涤两次即得所得淋巴细胞,使用RNAase-freePBS悬浮收集的淋巴细胞得到107/mL溶解液,-80度保存。
(3)提取总RNA,合成cDNA,PCR扩增VHH序列
RNA提取实验步骤如下:
取冻存的外周淋巴细胞溶液,加入700μLRLT试剂,充分混匀;
加入等体积70%酒精,移液器混匀后转移至吸附柱内,9000rpm离心15s,弃上清;
加入700μlBufferRW1于吸附柱内,9000rpm离心15s,弃上清;
加入500μlBufferRPE于吸附柱内,9000rpm离心15s,弃上清;
加入500μlBufferRPE于吸附柱内,9000rpm离心15s,弃上清;
将吸附柱转移至新的收集管,向吸附柱内加入30μlRNAase-free双蒸水,9000rpm离心1min,洗脱收集RNA;
cDNA合成步骤如下:
步骤1取无酶PCR反应管,配制以下反应体系:
试剂组分 | 体积(μl) |
TotalRNA | 10 |
OligodTPrimer | 1 |
RNase-Freewater | 3 |
混匀后,65℃水浴5min,冰浴5min;
步骤2向上述体系内依次加入下列组分至终体积为20μl。
试剂组分 | 体积(μl) |
RNase抑制剂 | 1 |
M-MLV5×ReactionBuffer | 3 |
dNTPs | 1 |
M-MLV逆转录酶 | 1 |
放置PCR仪内,37℃静置1h,然后70℃加热15min,所合成的cDNA放置于-20℃保存。
PCR扩增VHH序列:
以cDNA为模板,采用巢式PCR扩增VHH基因。使用外侧引物CALL001和CALL002进行第一轮PCR反应,获得约700bp左右片段。PCR反应条件为94℃、30s;56℃、30s;72℃、45s,以上过程32个循环;72℃延伸10min;PCR产物经过凝胶电泳进行回收并测定浓度。然后以700bp片段为模板,采用内侧引物VHH-Forward和VHH-Reverse进行第二轮PCR扩增,反应条件为94℃、40s;68℃、45s,32个循环;72℃、延伸10min。将第二轮PCR获得的400bpVHH片段利用胶回收试剂盒对其进行分离回收,保存于-20℃备用。
(4)构建纳米抗体库
用SfiⅠ/NotⅠ对回收得到的VHH基因和pHEN1噬菌体载体进行酶切,并将酶切产物通过T4DNA连接酶进行连接,构建重组载体,通过DNA回收试剂盒纯化连接产物。
挑取TG1大肠杆菌单菌落至500mL体积LB液体培养基中,于37℃,220rpm培养至OD600为0.8左右,于4℃,1200g转速离心10min,弃上清收集菌体;用预冷的100mL灭菌超纯水重悬菌体,于4℃,1000g离心10min,弃上清;用预冷的含10%甘油的50mL灭菌超纯水重悬菌体,于4℃,800g离心10min,弃上清,回收菌体;重复上一步;用含10%甘油的500μL灭菌超纯水重悬菌体,并分装成80μL每支,于-80℃超低温冰箱保存。
将酶连后的产物加入到TG1大肠杆菌感受态细胞中,于冰上静置30min;将混合物转移至电击杯中,电压设置2.45kV进行电击;电击后立即加入900μLLB培养基,轻柔重悬并收集菌液,37℃、100rpm培养1h。培养后的菌液浓缩后,取10μL梯度稀释,涂布在LB-A的平板上,于37℃培养箱倒置培养8h,对单菌落进行计数,计算得出噬菌体展示文库的库容。将剩余培养物全部涂布到LB-A大平板上,于37℃培养箱培养12h;第二天用适量LB-A培养基洗脱平板上的全部菌落,加入终浓度为20%的甘油并混匀,放入-80℃超低温冰箱保存。
(5)筛选特异性纳米抗体
按照结合、洗涤、洗脱、扩增的方式,对上述噬菌体展示纳米抗体文库进行特异性筛选,抗原的包被浓度逐轮降低,依次为100、50、25、12.5μg/mL。具体步骤:向酶标板中加入相应浓度的抗原,100μL/孔,4℃环境包被过夜12h;弃上清,0.05%PBST洗板3次后加入封闭液(采用3%BSA和3%OVA交替封闭),300μL/孔,37℃环境封闭2h;将封闭液吸出,0.05%PBST洗板3次,加入噬菌体悬液,100μL/孔,37℃环境振荡孵育2h;弃上清,0.1%PBST洗板10~15次,加入0.1mol/LGly-HCl洗脱缓冲液(pH2.2)进行洗脱,100μL/孔,洗脱时间10min,加入1mol/LTris-HCl缓冲液(pH8.8)进行中和,15μL/孔,混匀,取10μL测定滴度,剩余的进行扩增后用于下一轮筛选。一共进行4轮筛选。
从测定滴度的平板上随机挑取单菌落进行噬菌体的拯救纯化,之后采用间接ELISA法鉴定阳性克隆。用PBS溶液稀释抗原至5μg/mL,加入酶标板,100μL/孔,4℃包被过夜12h;吸出包被液,PBST洗板3次,加入3%脱脂奶粉进行封闭,300μL/孔,37℃封闭2h;吸出封闭液,PBST洗板3次,加入扩增后的噬菌体悬液,100μL/孔,37℃孵育1h;吸出上清液,PBST洗板3次,加入HRP标anti-M13噬菌体单克隆抗体稀释液,100μL/孔,37℃孵育1h;吸出抗体溶液,PBST洗板3次,加入TMB显色液,100μL/孔,37℃孵育10min;加入2mol/L硫酸溶液终止反应,50μL/孔,立即用酶标仪测定OD450nm值。
(6)验证并表达特异性抗体
将筛选出的特异性抗体的重组质粒载体转入BL21大肠杆菌感受态细胞,涂布于含有50μg/mL卡那霉素的LB平板,37℃环境静置培养12h;挑取单菌落接种于5mL含50μg/mL卡那霉素的LB液体培养基,37℃、250r/min培养过夜12h;按1%的接种量接种于含50μg/mL卡那霉素的LB液体培养基,37℃、250r/min培养至OD600nm值为0.5,加入终浓度1mmol/L的IPTG,30℃、250r/min诱导表达8h;4℃、5000r/min离心8min,吸出上清液,收集菌体;加入PBS重悬菌体沉淀,冰浴超声波破碎菌体,破碎液于4℃、12000r/min离心15min,分离上清和沉淀。使用Ni2+-NTA亲和层析柱对重组蛋白进行纯化,使用SDS-PAGE分析纯化后的纳米抗体纯度,使用OctetRED96系统测定抗体的亲和力。
2、包被及封闭
配制包被液,配制方法为用包被稀释液(pH7.0,配方见表1)将IL-6包被特异性纳米抗体、PCT包被特异性纳米抗体、羊抗兔IgG稀释至1mg/mL;将硝酸纤维素膜贴于背衬卡后用划膜仪划膜,划膜参数为1μL/cm,划1条质控线(C线),划2条检测线(T线),划膜结束后37℃烘干24h。
表1包被稀释液配方
名称 | 含量 |
MOPSO | 10mM/L |
海藻糖 | 10mM/L |
硫酸铵 | 10mM/L |
封闭包被后的硝酸纤维素膜:配制封闭液(pH7.0,封闭液配方见表2),将封闭液倒入玻璃加样槽中,将烘干的包被板的硝酸纤维素膜的顶端贴上吸水纸,吸水纸与硝酸纤维素膜交叠1mm,随后将未贴吸水纸的一端放入盛装封闭液的加样槽中,20min后取出37℃烘干24h。
表2封闭液配方
名称 | 含量 |
MOPSO | 10mM/L |
海藻糖 | 10mM/L |
硫酸铵 | 10mM/L |
酪蛋白 | 0.2%(m/V) |
鱼明胶 | 0.2%(m/V) |
Tween20 | 0.2%(V/V) |
TritonX-100 | 0.2%(V/V) |
PVP40K | 0.2%(m/V) |
3、标记
取50μL荧光微球(固含量为1%),加入430μLMES缓冲液(50mM,pH6.0),最后加入20μLEDC(10mg/mL),室温反应10min,16000g离心弃上清,加入500μLPBS(10mM,pH7.4)超声分散荧光微球离心沉淀,分别加入IL-6标记纳米抗体、PCT标记纳米抗体及兔抗标记蛋白各200μg,室温反应2h,16000g离心弃上清,加入标记稀释液(pH7.0,标记稀释液配方见表3)500μL超声分散荧光微球离心沉淀,最后用喷金仪喷标记垫,标记垫为玻纤材料,37℃烘干24h。
表3标记稀释液配方
名称 | 含量 |
MOPSO | 10mM/L |
海藻糖 | 10mM/L |
硫酸铵 | 10mM/L |
甘氨酸 | 10mM/L |
酪蛋白 | 1%(m/V) |
鱼明胶 | 1%(m/V) |
Tween20 | 0.5%(V/V) |
TritonX-100 | 0.2%(V/V) |
Tetronic(tm)1307 | 0.5%(m/V) |
PVP40K | 0.5%(m/V) |
PEG10000 | 0.5%(m/V) |
4、组装试剂卡
将干燥后的喷有标记的玻纤贴于包被板上,标记垫与硝酸纤维素膜交叠1mm,最后在带有背衬卡的包被板上贴上滤血膜,滤血膜与标记垫交叠1mm,切条机将贴好的大板斩切成4mm的小条,将小条装于塑料卡4即完成试剂卡制备,试剂卡放入铝箔袋并加干燥剂即完成检测试剂的制备。
本发明检测试纸条,在使用时,试纸条组装在由塑料上壳和塑料下壳插合而成的塑料外壳中(下壳上设置有供上壳插入的卡槽),即得到检测试纸卡,塑料上壳设有两个开孔,加样孔10和观察窗9,加样孔10对应于所述的检测IL-6/PCT试纸条的滤血膜样品垫1,塑料下壳上设置有固定检测试纸条的卡口,观察窗9对应于所述检测IL-6/PCT试纸条的第一检测线7、第二检测线8和质控线6,该检测IL-6/PCT试纸条可以从该塑料外壳中取出,如图3所示。
实施例3IL-6/PCT联合检测荧光免疫层析定量检测
本检测试剂可检测血清、血浆及全血,本发明试剂中,取血浆样本80μl加入检测缓冲液(PBS),混合10s后,将其加入到检测卡加样孔中,层析15min后使用干式荧光免疫分析仪读取浓度。
样本准备:不少于40例临床样本(肝素化抗凝血浆),样本IL-6和PCT含量尽可能在线性范围内均匀分布。
检测方法:使用实施例制备的检测试剂与对比检测试剂(CobasIL-6)分别检测同一临床样本,检测完成对检测结果进行统计分析,结果如图4和图5所示(说明:使用制备的检测试剂检测一个样本时同时检测IL-6和PCT两个标靶)。
图4横坐标为对比试剂(CobasIL-6)检测样本的IL-6含量,纵坐标为实施例制备试剂检测样本的IL-6含量,共检测IL-6含量为0-1000pg/mL的临床样本40例。利用所有样本双份测定值进行相关系数计算,试验结果为两组数据相关系数R2=0.9912(R2≥0.95),回归方程为y=0.9863x+1.8596。可以得出鼎润IL-6与CobasIL-6检测结果相关并具有统计学意义,即本发明制备的检测试剂与对比试剂相关性较好,在临床分析时可以获得基本一致的结果,结果符合临床分析要求,能够满足临床分析使用。
图5横坐标为对比试剂(CobasPCT)检测样本的PCT含量,纵坐标为实施例制备试剂检测样本的PCT含量,共检测PCT含量为0-100ng/mL的临床样本40例。利用所有样本双份测定值进行相关系数计算,试验结果为两组数据相关系数R2=0.9921(R2≥0.95),回归方程为y=0.9832x+0.3257。可以得出鼎润PCT与CobasPCT检测结果相关并具有统计学意义,即本发明制备的检测试剂与对比试剂相关性较好,在临床分析时可以获得基本一致的结果,结果符合临床分析要求,能够满足临床分析使用。
可见,本试剂盒检测一个样本只需要15min,检测结果迅速获得,帮助医生进一步采取干预措施;并且一个样本可同时检测IL-6、PCT两个标靶。
本发明的上述实施例仅仅是为说明本发明所作的举例,而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其他不同形式的变化和变动。这里无法对所有的实施方式予以穷举。凡是属于本发明的技术方案所引申出的显而易见的变化或变动仍处于本发明的保护范围之列。
Claims (10)
1. 一种PCT/IL-6荧光定量快速检测试纸条,其特征在于,所述检测试纸条由背衬卡以及粘贴在背衬卡上的滤血膜样品垫、荧光微球结合垫、硝酸纤维素膜检测垫和吸水垫组成,所述背衬卡两端分别设有滤血膜样品垫和吸水垫;在背衬卡中部设有硝酸纤维素膜检测垫,在硝酸纤维素膜检测垫与滤血膜样品垫之间设有荧光微球结合垫;所述荧光微球结合垫一端与滤血膜样品垫互相叠加,另一端与硝酸纤维素膜检测垫互相叠加;所述荧光微球结合垫上包括检测微球和质控微球,检测微球为表面包被有PCT标记纳米抗体和IL-6标记纳米抗体的荧光微球,质控微球为表面包被有兔抗标记蛋白的荧光微球;所述硝酸纤维素膜检测垫上沿长度方向依次平行间隔设置有第一检测线、第二检测线和质控线,所述第一检测线和第二检测线上分别包被有IL-6特异性纳米抗体和PCT特异性纳米抗体, 质控线上包被有羊抗兔IgG。
2.根据权利要求1所述PCT/IL-6荧光定量快速检测试纸条,其特征在于,所述IL-6或PCT特异性纳米抗体的制备方法包括如下步骤:
1)分别以IL-6抗原或PCT抗原免疫骆驼科动物,取免疫后骆驼科动物的外周血,提取外周血淋巴细胞RNA,然后将所述外周血淋巴细胞RNA反转录为cDNA,运用逆转录PCR扩增所述cDNA;
2)以步骤1)得到的cDNA为模板,采用巢式PCR扩增得到VHH基因片段;
3)将步骤2)得到的VHH基因片段连接表达载体,得到IL-6或PCT特异性抗体的重组表达载体,再将所述重组表达载体转导至宿主菌,获得表达IL-6或PCT特异性抗体的重组工程菌,然后诱导所述重组工程菌表达重组蛋白,对所述重组蛋白进行分离纯化处理,即得到所述IL-6或PCT特异性纳米抗体。
3.根据权利要求2所述PCT/IL-6荧光定量快速检测试纸条,其特征在于,所述骆驼科动物为羊驼,所述表达载体为噬菌体展示载体pHEN1,所述宿主菌为大肠杆菌。
4.一种炎症检测剂盒,其特征在于,包括权利要求1~3任一项所述PCT/IL-6荧光定量快速检测试纸条。
5.一种如权利1所述PCT/IL-6荧光定量快速检测试纸条的制备方法,其特征在于,包括以下步骤:
S1:制备权利要求2所述IL-6特异性纳米抗体和PCT特异性纳米抗体;
S2:用包被稀释液将IL-6特异性纳米抗体、PCT特异性纳米抗体和羊抗兔IgG稀释成1mg/mL的包被液;然后分别于贴在背衬卡的硝酸纤维素膜上划膜间隔包被,分别得到第一检测线、第二检测线和质控线,划膜参数为1μL/cm,划膜结束后,置于37~40℃条件下干燥30~60min;
S3:将封闭液倒入玻璃加样槽中,将步骤S2烘干的包被板的硝酸纤维素膜的顶端贴上吸水垫,吸水垫与硝酸纤维素膜交叠1mm,随后将未贴吸水垫的一端放入盛装封闭液的加样槽中,10~30min后取出37~40℃烘干24~36h;
S4:取荧光微球加入MES缓冲液,再加入 10mg/mL的EDC,室温反应10min,16000g离心弃上清;然后加入 PBS缓冲溶液,超声分散荧光微球离心沉淀,再依次加入IL-6标记纳米抗体、PCT标记纳米抗体及兔抗标记蛋白,室温反应2h,16000g离心弃上清,加入标记稀释液超声分散荧光微球离心沉淀,最后用喷金仪喷标记垫,标记垫为玻纤材料,置于37~40℃条件下烘干24~36h,即得到荧光微球结合垫;
S5:将步骤S4得到的荧光微球结合垫贴于包被板上,荧光微球结合垫与硝酸纤维素膜交叠1mm,最后在带有背衬卡的包被板上贴上滤血膜样品垫,滤血膜样品垫与荧光微球结合垫交叠1mm,即得到所述检测试纸条。
6.根据权利要求5所述PCT/IL-6荧光定量快速检测试纸条的制备方法,其特征在于,所述包被稀释液为MOPSO、海藻糖和硫酸铵的混合溶液,所述包被液缓冲液的pH为7.0。
7.根据权利要求5所述PCT/IL-6荧光定量快速检测试纸条的制备方法,其特征在于,所述封闭液由MOPSO、海藻糖、硫酸铵、酪蛋白、鱼明胶、Tween20、Triton X-100和PVP 40K组成,所述封闭液的pH为7.0。
8.根据权利要求5所述PCT/IL-6荧光定量快速检测试纸条的制备方法,其特征在于,所述标记稀释液的配方为:10mM/L的MOPSO、10mM/L的海藻糖、10mM/L的硫酸铵、0.2%(m/V)的酪蛋白、0.2%(m/V)的鱼明胶、0.2%(V/V)的Tween20、0.2%(V/V)的Triton X-100和0.2%(m/V)的PVP 40K,所述标记稀释液的pH为7.0。
9.一种PCT/IL-6荧光定量快速检测的方法,其特征在于,取待测样本加入检测缓冲液,混合10s后,加到权利要求1所述检测试纸条的样本垫上,将检测试纸条插入荧光分析仪的检测孔,放置15min,以检测线和质控线的荧光强度比值为纵坐标,PCT和IL-6标准溶液浓度为横坐标计算样品中PCT和IL-6的浓度值。
10.根据权利要求9所述的检测方法,所述待测样本为全血、血清或血浆。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010688922.1A CN111856041A (zh) | 2020-07-17 | 2020-07-17 | 一种pct/il-6荧光定量快速检测试纸条及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010688922.1A CN111856041A (zh) | 2020-07-17 | 2020-07-17 | 一种pct/il-6荧光定量快速检测试纸条及其制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111856041A true CN111856041A (zh) | 2020-10-30 |
Family
ID=72983931
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010688922.1A Pending CN111856041A (zh) | 2020-07-17 | 2020-07-17 | 一种pct/il-6荧光定量快速检测试纸条及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111856041A (zh) |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005037056A2 (en) * | 2003-10-06 | 2005-04-28 | University Of Maryland, Baltimore | Method for the detection of a biomolecule in a test sample |
US20060148097A1 (en) * | 2005-01-06 | 2006-07-06 | Sysmex Corporation | Test strip for immunochromatography |
JP2007248373A (ja) * | 2006-03-17 | 2007-09-27 | Univ Of Fukui | 免疫測定用ブロッキング剤組成物およびそれを用いたブロッキング方法 |
US20150024414A1 (en) * | 2013-07-22 | 2015-01-22 | Sandia Corporation | Amplification of biological targets via on-chip culture for biosensing |
RU2603269C1 (ru) * | 2015-10-26 | 2016-11-27 | Федеральное Государственное Бюджетное Учреждение Науки Институт Молекулярной Биологии Им. В.А. Энгельгардта Российской Академии Наук (Имб Ран) | Рекомбинантные однодоменные антитела, специфически связывающие интерлейкин-6 человека, способ их получения и использования для детекции этого белка |
CN108383907A (zh) * | 2018-03-21 | 2018-08-10 | 北京科卫临床诊断试剂有限公司 | 针对降钙素原的纳米抗体及其应用 |
US20180292394A1 (en) * | 2016-04-11 | 2018-10-11 | Joshua Caine Soldo | Sample depletion and enrichment to improve the quality of diagnostic test results |
CN109975557A (zh) * | 2019-04-29 | 2019-07-05 | 厦门稀土材料研究所 | Il-6/pct联合检测时间分辨检测试剂盒及方法 |
CN110133283A (zh) * | 2019-05-06 | 2019-08-16 | 中生北控生物科技股份有限公司 | Pct和il-6联合检测试剂盒及其制备方法 |
-
2020
- 2020-07-17 CN CN202010688922.1A patent/CN111856041A/zh active Pending
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005037056A2 (en) * | 2003-10-06 | 2005-04-28 | University Of Maryland, Baltimore | Method for the detection of a biomolecule in a test sample |
US20060148097A1 (en) * | 2005-01-06 | 2006-07-06 | Sysmex Corporation | Test strip for immunochromatography |
JP2007248373A (ja) * | 2006-03-17 | 2007-09-27 | Univ Of Fukui | 免疫測定用ブロッキング剤組成物およびそれを用いたブロッキング方法 |
US20150024414A1 (en) * | 2013-07-22 | 2015-01-22 | Sandia Corporation | Amplification of biological targets via on-chip culture for biosensing |
RU2603269C1 (ru) * | 2015-10-26 | 2016-11-27 | Федеральное Государственное Бюджетное Учреждение Науки Институт Молекулярной Биологии Им. В.А. Энгельгардта Российской Академии Наук (Имб Ран) | Рекомбинантные однодоменные антитела, специфически связывающие интерлейкин-6 человека, способ их получения и использования для детекции этого белка |
US20180292394A1 (en) * | 2016-04-11 | 2018-10-11 | Joshua Caine Soldo | Sample depletion and enrichment to improve the quality of diagnostic test results |
CN108383907A (zh) * | 2018-03-21 | 2018-08-10 | 北京科卫临床诊断试剂有限公司 | 针对降钙素原的纳米抗体及其应用 |
CN109975557A (zh) * | 2019-04-29 | 2019-07-05 | 厦门稀土材料研究所 | Il-6/pct联合检测时间分辨检测试剂盒及方法 |
CN110133283A (zh) * | 2019-05-06 | 2019-08-16 | 中生北控生物科技股份有限公司 | Pct和il-6联合检测试剂盒及其制备方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11635438B2 (en) | IL-6 detection based early diagnosis and prediction of systemic inflammatory response syndrome and sepsis in asymptomatic patients | |
WO2017107974A1 (zh) | 血清psmd4蛋白的检测试剂盒及其检测方法与应用 | |
CN103477229B (zh) | 用于预测败血症患者中呼吸衰竭、肾衰竭或血小板减少症的风险的方法和试剂盒 | |
US10126312B2 (en) | Diagnostic method for urinary tract infection | |
CN110361547B (zh) | 一种化学发光定量检测粪便潜血的试剂及其检测方法和其在检测下消化道健康的用途 | |
WO2008151808A1 (en) | Diagnostic method | |
US20240019433A1 (en) | Assays and methods for the diagnosis of post-streptococcal disorders | |
WO2017125007A1 (zh) | 用于诊断活动性结核的方法和试剂盒 | |
CN109633163B (zh) | 降钙素原/c反应蛋白二合一检测试剂盒 | |
CN114058593A (zh) | SARS-CoV-2的N抗原的单克隆抗体、检测方法及其用途 | |
CN111856041A (zh) | 一种pct/il-6荧光定量快速检测试纸条及其制备方法和应用 | |
US20170097352A1 (en) | Immunoglobulin-bound extracellular vesicles and uses thereof | |
CN107505459B (zh) | 定量检测人h-fabp的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法 | |
CN112979815B (zh) | 一种抗he4的纳米抗体1g8及其应用 | |
US20150369820A1 (en) | Novel disease-marker | |
CN113045663A (zh) | 一种抗he4的纳米抗体1a8及其应用 | |
CN105242049A (zh) | 一种鉴别诊断急性支原体、细菌和病毒感染的试剂盒及其应用 | |
CN117069857B (zh) | 猫NT-proBNP蛋白的双特异性抗体及其用途 | |
CN104459134A (zh) | 脑损伤早期诊断s100胶体金免疫测试盒及其制备方法 | |
CN113831401B (zh) | 一种sle抗原表位多肽及其在sle诊断中的作用 | |
CN117285635B (zh) | 抗肝素结合蛋白的单克隆抗体组合物及应用 | |
CN113817025B (zh) | Sle抗原表位多肽在鉴别sle和其他自身免疫疾病中的作用 | |
WO2023061388A1 (zh) | 半乳糖凝集素-3的免疫测定 | |
WO2022263648A1 (en) | A method for predicting sepsis and septic shock | |
CN104459133A (zh) | 急性肾损伤诊断ngal胶体金免疫测试盒及其制备方法 |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201030 |
|
RJ01 | Rejection of invention patent application after publication |