CN104914244A - Kit for combined detection of hepatitis C virus antigen and antibody through chemiluminescence - Google Patents
Kit for combined detection of hepatitis C virus antigen and antibody through chemiluminescence Download PDFInfo
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- G—PHYSICS
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2333/186—Hepatitis C; Hepatitis NANB
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Abstract
The invention provides a kit for combined detection of hepatitis C virus antigen and antibody through chemiluminescence. According to the invention, the principles of chemiluminescence are utilized; anti-FITC-coated magnetic particles are bonded with an FITC-coated reagent and then undergo immunoreaction with a detected object; after immunoreaction of an AP-labeled substance, an immunoreaction chain (as shown in a figure 1 which is described in the specification) is constructed; luminous sensitivity of a substrate catalyzed by AP is far higher than enzyme immunoassay development, so reaction sensitivity is enhanced; meanwhile, FITC is used to simultaneously marking antigen and antibody, so hepatitis C virus antibody and core antigen are detected at the same time. The kit provided by the invention can more accurately detect hepatitis C, is free of leak detection, achieves the effect of early discovery and has a high application value in prevention of hepatitis C.
Description
Technical field
The invention belongs to hepatitis C detection technique field, be specifically related to a kind of C hepatitis virus antigen-antibody combined detection kit (chemoluminescence method).
Background technology
Hepatitis C is a kind of global infectious disease being infected by hepatitis C virus (Hepatitis C virus, HCV) and cause.There are 1. 7 hundred million hepatitis c virus infection persons in the whole world at present according to estimates, and the hepatitis C infections rate of China is approximately 30%, has 4000 ~ 6,000 ten thousand third hepatopaths at present at least.Wherein there is the infected of 80 ~ 85% to develop into chronic hepatitis C, wherein have again 20% can develop into liver fibrosis, finally have the patients with liver fibrosis generation hepatocellular carcinoma of 4 ~ 5%, endanger very serious.
At present, there is no the specific treatment medicine for hepatitis C virus, the development of hcv vaccine is also difficult because HCV makes a variation fast, and difficulty has breakthrough development in a short time.Therefore, the early diagnosis of HCV is of great importance for the examination HCV infection sources, guiding clinical treatment and Index for diagnosis.
Existing HCV detection mode clinically mainly contains PCR detection, antibody of HCV detects and core antigen of C type hepatitis virus detection kit, in three kinds of detection methods, PCR detection method is highly sensitive and result is accurate, but method complex operation, the personnel's technical capability needed is strong, is not easy to promote in each hospital; It is modal a kind of third liver detection method clinically that antibody of HCV detects reagent, but this detection exists one " window phase ", namely in the initial infection of the third liver, does not also occur immune antibody in patient body, just cannot obtain testing result in early days, cause the situation of failing to pinpoint a disease in diagnosis; Core antigen of C type hepatitis virus detect reagent recent years start gradually accept by hospital, market share amount also constantly increases, but is aimed at the later stage tracing detection of the third hepatopathy people, does not have good tracking effect.
Summary of the invention
Be directed to various detection method Problems existing, the reagent of antibody of HCV-antigen combined detection has just occurred, the invention provides a kind of chemoluminescence method joint-detection antibody of HCV-antigenic agents, this reagent utilizes chemiluminescence ratio juris, improve the sensitivity of detection, qualitative detection antigen and antibody simultaneously, can determine to detect the infection whether in sample with hepatitis C virus, have very high clinical value.
The present invention is achieved by the following measures:
A kind of chemoluminescence method joint-detection antibody of HCV-antigenic agents, its component is as follows:
component 1(R1) magnetic particle reagent:
Magnetic particle and sheep anti-FITC antibody connector 1%
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 2(R2) FITC labelled reagent:
The HCV-cAg antibody 1% of FITC mark
The NS3 albumen 1% of FITC mark
The NS4 albumen 1% of FITC mark
The NS5 albumen 1% of FITC mark
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 3(R3) AP labelled reagent:
The HCV-cAg antibody 1% of AP mark
The NS3 albumen 1% of AP mark
The NS4 albumen 1% of AP mark
The NS5 albumen 1% of AP mark
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 4(R4) Sample dilution:
Sodium hydrogen phosphate 1.974g/L
Potassium dihydrogen phosphate 0.2245g/L
BSA 0.5g/L
PC-300 0.1%
component 5(R5) luminous substrate:
Dioxane (AMPPD) 5mmol/L
component 6(R6) cleaning concentrate:
Sodium hydrogen phosphate 39.48g/L
Potassium dihydrogen phosphate 4.49g/L
Sodium chloride 18g/L
Tween-20 0.5%
All components all dissolves with deionized water, is mixed with solution state.
positive control:
Core antigen of C type hepatitis virus 1%
Antibody of HCV (anti-NS3) 1%
Antibody of HCV (anti-NS4) 1%
Antibody of HCV (anti-NS5) 1%
Dissolve with calf serum.
negative control:
BSA 4%
Dissolve with calf serum.
The testing process of the kit of a kind of chemoluminescence method of the present invention joint-detection hepatitis C virus antigen-antibody is as follows:
(1) experimentally need to prepare flat based tubes, to be placed on Magneto separate frame and to carry out numbering; By R6(cleaning concentrate) dilute with 20 times of deionized waters, for subsequent use;
(2) in test tube, add 15 μ l positive controls, negative control or sample, and add 15 μ l R4(Sample dilution);
(3) in test tube, 30 μ l R2(FITC labelled reagents are added with continuous liquid-moving machine);
(4) Magneto separate frame is covered with plastic foil, after being placed on oscillator concussion gently to thorough mixing, 37 DEG C of incubations 5 minutes;
(5) in test tube, 30 μ l R1(magnetic particle reagent are added with continuous liquid-moving machine), to ensure during application of sample that magnetic particle reagent fully mixes;
(6) Magneto separate frame is covered with plastic foil, after being placed on oscillator vibration gently to thorough mixing, 37 DEG C of incubations 5 minutes;
(7) be placed on magnetic separator by Magneto separate frame, guarantee often to prop up at the bottom of test tube and contact magnetic separator, leave standstill 2 minutes, then camber line topples over supernatant, inverted test tube is tipped upside down on thieving paper together with magnetic separator and pats dry;
(8) in test tube, add 300 μ l cleaning fluids with continuous liquid-moving machine, Magneto separate frame is taken off from magnetic separator, is placed in 1500 ~ 2000rpm on oscillator and shakes 20 seconds, to thoroughly mixing, repeat (7) operation 1 time;
(9) (8) operation 2 times are repeated;
(10) in test tube, 30 μ l R3 (AP labelled reagent) are added with continuous liquid-moving machine;
(11) Magneto separate frame is covered with plastic foil, after being placed on oscillator vibration gently to thorough mixing, 37 ± 1 DEG C of incubations 5 minutes;
(12) taken off from magnetic separator by Magneto separate frame, often pipe adds 300 μ l cleaning fluids, is placed in concussion extremely thoroughly mixing gently on oscillator, repeats (7) step and operate 1 time;
(13) repeat (8) step and operate 2 times;
(14) test tube rack is taken off from magnetic separator, each test tube adds 200 μ l luminous substrate, detects luminous intensity with Chemiluminescence Apparatus.
Critical value is determined: if during average RLU value≤100 of negative control average RLU value × 2.1(negative control, calculate by 100; During RLU>100, calculate by average RLU value × 2.1 of practical measurement negative control).The RLU value of test specimen is less than critical value then for HCV is negative.RLU value in test specimen is equal to or greater than critical value then for HCV is positive.
The reaction principle figure of kit of the present invention, as shown in Figure 1, by technical scheme of the present invention, a kind of Ag-Ab combined detection kit simultaneously detecting hepatitis C virus can be obtained, this kit is by being combined by the reagent of the magnetic particle of anti-FITC with FITC bag quilt by bag, immune response is being carried out with detected material, after the material immune response of AP mark, form immune response chain (as Fig. 1), the sensitivity of AP catalytic substrate luminescence exempts from colour developing higher than enzyme far away, enhance the sensitivity of reaction, utilize the mode of FITC simultaneously labelled antigen and antibody simultaneously, can detect antibody of HCV and cAg simultaneously.
The innovation of a kind of C hepatitis virus antigen of the present invention-antibody combined detection kit is:
(1) bag is utilized to be marked the antibody of anti-hepatitis c virus cAg and the mode of other hypotype recombinant proteins of hepatitis C virus by the magnetic particle of anti-FITC and FITC, by various third orgotein bag by magnetic particle, can ensure that the antibody of HCV core antigen in sample and multiple hypotype can be adsorbed onto magnetic particle surface by immune response, avoid undetected;
(2) the AP label in kit carries out AP enzyme labeling to coated antibody and the right corresponding antibody of psma ligand and antigen, can with coated antibody and antigen counterpart application, ensure that and the specificity that kit detects accurately detect hepatitis C virus;
(3) kit of the present invention, selects the principle of AP catalytic substrate luminescence, effectively ensure that the sensitivity of kit, even if also can detect under micro-state, reaches the effect early found.
[0013] accompanying drawing explanation
The schematic diagram of Fig. 1 kit of the present invention.
For a better understanding of the present invention, further illustrate below in conjunction with specific embodiment.
embodiment 1
The component and the concentration that detect reagent are
component 1(R1) magnetic particle reagent:
Magnetic particle and sheep anti-FITC antibody connector 1%
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 2(R2) FITC labelled reagent:
The HCV-cAg antibody 1% of FITC mark
The NS3 albumen 1% of FITC mark
The NS4 albumen 1% of FITC mark
The NS5 albumen 1% of FITC mark
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 3(R3) AP labelled reagent:
The HCV-cAg antibody 1% of AP mark
The NS3 albumen 1% of AP mark
The NS4 albumen 1% of AP mark
The NS5 albumen 1% of AP mark
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 4(R4) Sample dilution:
Sodium hydrogen phosphate 1.974g/L
Potassium dihydrogen phosphate 0.2245g/L
BSA 0.5g/L
PC-300 0.1%
component 5(R5) luminous substrate:
Dioxane (AMPPD) 5mmol/L
component 6(R6) cleaning concentrate:
Sodium hydrogen phosphate 39.48g/L
Potassium dihydrogen phosphate 4.49g/L
Sodium chloride 18g/L
Tween-20 0.5%
All components all dissolves with deionized water, is mixed with solution state.
positive control:
Core antigen of C type hepatitis virus 1%
Antibody of HCV (anti-NS3) 1%
Antibody of HCV (anti-NS4) 1%
Antibody of HCV (anti-NS5) 1%
Dissolve with calf serum.
negative control:
BSA 4%
Dissolve with calf serum.
Testing process:
(1) experimentally need to prepare flat based tubes, to be placed on Magneto separate frame and to carry out numbering; By R6(cleaning concentrate) dilute with 20 times of deionized waters, for subsequent use;
(2) in test tube, add 15 μ l positive controls, negative control or sample, and add 15 μ l R4(Sample dilution);
(3) in test tube, 30 μ l R2(FITC labelled reagents are added with continuous liquid-moving machine);
(4) Magneto separate frame is covered with plastic foil, after being placed on oscillator concussion gently to thorough mixing, 37 DEG C of incubations 5 minutes;
(5) in test tube, 30 μ l R1(magnetic particle reagent are added with continuous liquid-moving machine), to ensure during application of sample that magnetic particle reagent fully mixes;
(6) Magneto separate frame is covered with plastic foil, after being placed on oscillator vibration gently to thorough mixing, 37 DEG C of incubations 5 minutes;
(7) be placed on magnetic separator by Magneto separate frame, guarantee often to prop up at the bottom of test tube and contact magnetic separator, leave standstill 2 minutes, then camber line topples over supernatant, inverted test tube is tipped upside down on thieving paper together with magnetic separator and pats dry;
(8) in test tube, add 300 μ l cleaning fluids with continuous liquid-moving machine, Magneto separate frame is taken off from magnetic separator, is placed in 1500 ~ 2000rpm on oscillator and shakes 20 seconds, to thoroughly mixing, repeat (7) operation 1 time;
(9) (8) operation 2 times are repeated;
(10) in test tube, 30 μ l R3 (AP labelled reagent) are added with continuous liquid-moving machine;
(11) Magneto separate frame is covered with plastic foil, after being placed on oscillator vibration gently to thorough mixing, 37 ± 1 DEG C of incubations 5 minutes;
(12) taken off from magnetic separator by Magneto separate frame, often pipe adds 300 μ l cleaning fluids, is placed in concussion extremely thoroughly mixing gently on oscillator, repeats (7) step and operate 1 time;
(13) repeat (8) step and operate 2 times
(14) test tube rack is taken off from magnetic separator, each test tube adds 200 μ l luminous substrate, detects luminous intensity with Chemiluminescence Apparatus.
embodiment 2
Conventional core antigen of C type hepatitis virus detection kit (euzymelinked immunosorbent assay (ELISA)) is selected from ortho company of the U.S., detects operation steps as follows:
(1) balance: from cold storage environment, take out kit and sample, place 30 minutes for 15-30 DEG C;
(2) dosing: all reagent shakes up before using, notes must not remaining crystallization, concentrated cleaning solution distilled water or deionized water 20 times dilution in bottle;
(3) application of sample: each detection all needs to arrange blank, feminine gender, each 2 holes of positive control.Blank well adds 200 μ l sample diluting liquids, and other each holes first add 100 μ l sample diluting liquids, and then the every hole of negative control hole adds 100 μ l negative controls, and the every hole of Positive control wells adds 100 μ l positive controls, and all the other each holes add 100 μ l testing samples;
(4) incubation: stick shrouding film after mixing, puts 37 DEG C of concussion incubations 90 minutes;
(5) wash plate: get rid of potpourri in hole, wash trigger washing lotion and wash plate 6 times (bottom irrigation), finally pat dry on thieving paper;
(6) enzyme-added: every hole adds enzyme conjugates 200 μ l respectively, sticks shrouding film, put 37 DEG C of incubations 30 minutes after mixing;
(7) plate is washed: with (5);
(8) develop the color: every hole adds each 100 μ l of developer A, B liquid, mixes a rearmounted 37 DEG C lucifuge and develops the color 10 minutes;
(9) measure: every hole adds 50 μ l stop buffers, mixing.Reaction terminating measured each hole OD value (need return to zero with blank when measuring with Single wavelength) with microplate reader 450nm or 450/630nm wavelength in 10 minutes.
embodiment 3
Accuracy analysis is carried out to the kit of chemoluminescence method joint-detection hepatitis C virus antigen-antibody, utilize the kit of ortho company of the U.S. as goldstandard, 40 routine clinical samples are detected, result is compared, detection method is as embodiment 1, embodiment 2, if difference appears in testing result, send third party inspection mechanism to utilize PCR detection method to verify, testing result is as shown in table 1:
Table 1 accuracy testing result
Detect through contrast, wherein No. 8 and No. 24 pattern detection results appearance exceptions, through third party's detection validation, embodiment 1(the present invention) result that detects is more accurate, occur that autoimmune antibody has mainly appearred in two abnormal routine samples, and the concentration of core antigen of C type hepatitis virus is lower, cause undetected.According to this testing result, illustrate that kit testing result of the present invention is more accurate, prevent undetected appearance.
embodiment 4
By the method for diluting positive sample, the sensitivity of checking kit.Embodiment is as follows:
Utilize normal negative sample to carry out gradient dilution in one hepatitis c virus-positive sample, utilize embodiment 1, embodiment 2 to detect the positive sample of each dilution gradient respectively, testing result is as shown in table 2.
Table 2 sensitivity technique result
| Dilution ratio | 1 | 1/2 | 1/4 | 1/8 | 1/16 | 1/32 |
| Embodiment 1 testing result | + | + | + | + | + | + |
| Embodiment 2 testing result | + | + | + | + | - | - |
Result according to table 2 shows, the sensitivity of embodiment 2 is 1/8, and the sensitivity of embodiment 1 can reach 1/32, this result illustrates that kit of the present invention has higher sensitivity, also can well detect even if the hepatitis C virus concentration in sample is very low, can reach and early find early preventive effect.
Traditional hepatitis c virus detection reagent box adopts euzymelinked immunosorbent assay (ELISA) to detect, sensitivity is lower, accuracy cannot ensure, the present invention utilizes chemiluminescence ratio juris, be combined by the reagent of the magnetic particle of anti-FITC with FITC bag quilt by bag, immune response is being carried out with detected material, after the material immune response of AP mark, form immune response chain (as accompanying drawing 1), the sensitivity of AP catalytic substrate luminescence exempts from colour developing higher than enzyme far away, enhance the sensitivity of reaction, utilize the mode of FITC simultaneously labelled antigen and antibody simultaneously, can detect antibody of HCV and cAg simultaneously, it is more accurate to detect for the third liver, can not be undetected, reach the effect early found, to the prevention of the third liver, there is very high using value.
Claims (2)
1. a kit for chemoluminescence method joint-detection hepatitis C virus antigen-antibody, its component is as follows:
component 1(R1) magnetic particle reagent:
Magnetic particle and sheep anti-FITC antibody connector 1%
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 2(R2) FITC labelled reagent:
The HCV-cAg antibody 1% of FITC mark
The NS3 albumen 1% of FITC mark
The NS4 albumen 1% of FITC mark
The NS5 albumen 1% of FITC mark
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 3(R3) AP labelled reagent:
The HCV-cAg antibody 1% of AP mark
The NS3 albumen 1% of AP mark
The NS4 albumen 1% of AP mark
The NS5 albumen 1% of AP mark
Tris 12.1g/L
BSA 0.5g/L
PC-300 0.1%
component 4(R4) Sample dilution:
Sodium hydrogen phosphate 1.974g/L
Potassium dihydrogen phosphate 0.2245g/L
BSA 0.5g/L
PC-300 0.1%
component 5(R5) luminous substrate:
Dioxane (AMPPD) 5mmol/L
component 6(R6) cleaning concentrate:
Sodium hydrogen phosphate 39.48g/L
Potassium dihydrogen phosphate 4.49g/L
Sodium chloride 18g/L
Tween-20 0.5%
All components all dissolves with deionized water, is mixed with solution state;
positive control:
Core antigen of C type hepatitis virus 1%
Antibody of HCV (anti-NS3) 1%
Antibody of HCV (anti-NS4) 1%
Antibody of HCV (anti-NS5) 1%
Dissolve with calf serum
negative control:
BSA 4%
Dissolve with calf serum.
2. the testing process of the kit of a kind of chemoluminescence method joint-detection hepatitis C virus antigen-antibody according to claim 1 is as follows:
(1) experimentally need to prepare flat based tubes, to be placed on Magneto separate frame and to carry out numbering; By R6(cleaning concentrate) dilute with 20 times of deionized waters, for subsequent use;
(2) in test tube, add 15 μ l positive controls, negative control or sample, and add 15 μ l R4(Sample dilution);
(3) in test tube, 30 μ l R2(FITC labelled reagents are added with continuous liquid-moving machine);
(4) Magneto separate frame is covered with plastic foil, after being placed on oscillator concussion gently to thorough mixing, 37 DEG C of incubations 5 minutes;
(5) in test tube, 30 μ l R1(magnetic particle reagent are added with continuous liquid-moving machine), to ensure during application of sample that magnetic particle reagent fully mixes;
(6) Magneto separate frame is covered with plastic foil, after being placed on oscillator vibration gently to thorough mixing, 37 DEG C of incubations 5 minutes;
(7) be placed on magnetic separator by Magneto separate frame, guarantee often to prop up at the bottom of test tube and contact magnetic separator, leave standstill 2 minutes, then camber line topples over supernatant, inverted test tube is tipped upside down on thieving paper together with magnetic separator and pats dry;
(8) in test tube, add 300 μ l cleaning fluids with continuous liquid-moving machine, Magneto separate frame is taken off from magnetic separator, is placed in 1500 ~ 2000rpm on oscillator and shakes 20 seconds, to thoroughly mixing, repeat (7) operation 1 time;
(9) (8) operation 2 times are repeated;
(10) in test tube, 30 μ l R3 (AP labelled reagent) are added with continuous liquid-moving machine;
(11) Magneto separate frame is covered with plastic foil, after being placed on oscillator vibration gently to thorough mixing, 37 ± 1 DEG C of incubations 5 minutes;
(12) taken off from magnetic separator by Magneto separate frame, often pipe adds 300 μ l cleaning fluids, is placed in concussion extremely thoroughly mixing gently on oscillator, repeats (7) step and operate 1 time;
(13) repeat (8) step and operate 2 times;
(14) test tube rack is taken off from magnetic separator, each test tube adds 200 μ l luminous substrate, detects luminous intensity with Chemiluminescence Apparatus.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510276415.6A CN104914244A (en) | 2015-05-27 | 2015-05-27 | Kit for combined detection of hepatitis C virus antigen and antibody through chemiluminescence |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510276415.6A CN104914244A (en) | 2015-05-27 | 2015-05-27 | Kit for combined detection of hepatitis C virus antigen and antibody through chemiluminescence |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108896380A (en) * | 2018-05-21 | 2018-11-27 | 苏州佑君环境科技有限公司 | A kind of Chemiluminescence Apparatus is in machine serum dilution and preparation method thereof |
| CN109406796A (en) * | 2018-12-11 | 2019-03-01 | 迈克生物股份有限公司 | Rheumatoid factor detection reagent box and its detection method |
| CN109917124A (en) * | 2018-10-30 | 2019-06-21 | 郑州安图生物工程股份有限公司 | A kind of hepatitis C virus antigen-antibody combined detection kit |
-
2015
- 2015-05-27 CN CN201510276415.6A patent/CN104914244A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108896380A (en) * | 2018-05-21 | 2018-11-27 | 苏州佑君环境科技有限公司 | A kind of Chemiluminescence Apparatus is in machine serum dilution and preparation method thereof |
| CN109917124A (en) * | 2018-10-30 | 2019-06-21 | 郑州安图生物工程股份有限公司 | A kind of hepatitis C virus antigen-antibody combined detection kit |
| CN109406796A (en) * | 2018-12-11 | 2019-03-01 | 迈克生物股份有限公司 | Rheumatoid factor detection reagent box and its detection method |
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