CN104407143A - Hepatitis c virus antigen-antibody joint detection kit - Google Patents
Hepatitis c virus antigen-antibody joint detection kit Download PDFInfo
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- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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Abstract
The invention belongs to the technical field of detection of hepatitis c, and particularly relates to a hepatitis c virus antigen-antibody joint detection kit. The kit comprises a protein coated plate, enzyme conjugates, control serum, sample diluent, washing concentrate, a color developing agent and a stop solution. According to the kit, anti-hepatitis c virus core antigen antibodies and other hepatitis c virus recombinant protein subunits are fixed on a solid-phase vector, so that hepatitis c virus core antigens and multiple subtype antibodies in a sample can be adsorbed onto the solid-phase vector through immune reaction, and missed detection is avoided; the enzyme conjugates in the kit are used for the enzyme labeling of corresponding antibodies and antigens paired with coated antibodies and antigens, and can be paired with the coated antibodies and antigens for use, so that the detection specificity is ensured; a protein structure-free macromolecular substance is adopted as a blocking solution, and meanwhile, a protein protectant is added into the blocking solution, so that the stability of the kit is effectively ensured.
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.
Background technology
Viral hepatitis type C, referred to as hepatitis C, the third liver, that one infects by hepatitis C virus (HCV) virus hepatitis caused, main through propagation such as blood transfusion, acupuncture, drug abuse, add up according to the World Health Organization (WHO), the infection rate of whole world HCV is about 3%, estimates that about 1.8 hundred million people have infected HCV, annual new hepatitis C case about 3.5 ten thousand example.Hepatitis C is global prevalence, and can cause the necrosis of liver chronic inflammation and fiberization, some patients can develop into cirrhosis even hepatocellular carcinoma (HCC).Continuation increases by mortality ratio (death that hepatic failure and hepatocellular carcinoma cause) relevant to HCV infection in following 20 years, very harmful to the health and lives of patient, has become serious society and public health problem.The the third liver epidemic situation number announced over the years according to the Ministry of Public Health as shown in Figure 1.
Hepatitis C virus (HCV) is addicted to after liver property slow virus HCV infection, and onset and the clinical symptoms pole of patient are not true to type, and is common, easily causes and fail to pinpoint a disease in diagnosis with subclinical infection.The chronicity incidence of HCV infection is apparently higher than hepatitis B, and comparatively hepatitis B easily occurs cirrhosis, liver cancer in early days, and mortality ratio is higher.Therefore the detection of HCV has great meaning to the early diagnosis of infection with hepatitis C virus and guiding clinical treatment.
Domestic also do not have hepatitis C (being called for short the third liver) to prevent vaccine appearance not have special effect medicine therapeutic so far yet, and early diagnosis is still the effective means preventing HCV from propagating.Mainly detect the RT-PCR method of HCVRNA at present for the reagent detecting the third liver, detect reagent for anti-HCV ELISA, and the ELISA being directed to HCV-cAg detects reagent.Wherein RT-PCR detection HCVRNA is expensive, and trivial operations, is unsuitable for extensive popularization; The c-hepatitis antibody that anti-HCV ELISA detects is after human body produces immune response to hepatitis C antigen, just can detect therefore have " window phase " that one longer, generally will infect latter 70 days ability and detect very well; Calendar year 2001 Ortho company is proposed detects HCV-cAg (HCV-cAg) ELISA reagent, evaluated by a large amount of clinical examination, proof detects Zao than anti-HCV reagent, but still exist certain undetected, cause in clinical practice, after reagent screening, after still having small part to transfuse blood, the third liver occurs.Have part blood station or hospital, application c-hepatitis antibody and HCV core antigen two kinds of kits detect simultaneously, but this not only increases the workload of medical personnel, and greatly increase testing cost.The defect of these detection methods constrains the clinical practice that the third liver detects reagent.
Summary of the invention
Be directed to now clinical to third liver detect exist undetected, and utilize the operation of plurality of reagents box to cause the problem that workload increases, cost increases, the invention provides the kit of a kind of C hepatitis virus antigen-antibody combined detection, this kit can sensitive, detect hepatitis C virus accurately.
The technical scheme that the present invention solves the problems of the technologies described above employing is:
A kind of C hepatitis virus antigen-antibody combined detection kit, comprises albumen bag by plate and enzyme conjugates;
Described, albumen bag is prepared by following methods by plate: coating protein is diluted to 1:2000 concentration by the phosphate buffer of pH7.4 concentration 0.02mol/L, obtained coating buffer; PH7.4 concentration is that antigen is become finite concentration with antibody dilution by the phosphate buffer of 0.02mol/L; Add by 100 μ L/ holes the coating buffer diluted, wrap by 2 hours at 37 DEG C; Discard bag by the coating buffer in plate, bag is patted dry by plate, add bag by plate confining liquid by 100 μ L-300 μ L/holes, wrap at 2-8 DEG C by 16-20 hour; Discard bag by the confining liquid in plate, after bag is patted dry by plate, be positioned over humidity and be less than in the 35-39 DEG C of drying box of 30% and dry 4-6 hour, obtain albumen bag by plate.
The present invention's coating protein used is: a) antibody A 1 of anti-hepatitis c virus cAg; B) other hypotype recombinant proteins of hepatitis C virus are all other albumen of being expressed by HCV RNA such as NS3, NS4, NS5.
Described, enzyme conjugates prepares by the following method: 5mg horseradish peroxidase being dissolved in 1ml concentration is in 0.2mol/L, pH5.6 acetate buffer, then adds the ethanol solution 0.1ml that volumetric concentration is the DNF of 1%, stirred at ambient temperature 1h, obtains solution 1; The NaIO of the 0.1mol/L of fresh configuration is added in solution 1
40.5ml, places 30min, obtains solution 2 for 4 DEG C; In solution 2, add the ethylene glycol 1ml that volumetric concentration is 2.5%, stirred at ambient temperature 1h, obtains solution 3; In solution 3, add the albumen 5mg of horseradish peroxidase-labeled, regulate solution ph to 9.0 with the carbonate buffer solution that pH9.5 concentration is 1.0mol/L, mixing, 4 DEG C are spent the night, and obtain solution 4; In solution 4, add the sodium borohydride solution 0.1ml of 0.1mol/L, mixing, place 3h, obtain solution 5 for 4 DEG C; In solution 5, add concentration is 0.01mol/L, pH7.4 phosphate buffer, and 4 DEG C of dialysed overnight are changed liquid 3 times, obtained solution 6; By centrifugal for solution 6 30min, remove sediment, gained supernatant is enzyme conjugates.
The albumen of horseradish peroxidase-labeled of the present invention is: a) antibody A 2 of another avtive spot of core antigen of C type hepatitis virus; B) all other albumen of being expressed by HCV RNA such as NS3, NS4, NS5 of other hypotype recombinant proteins of hepatitis C virus pairing.
Described, wrap and be made up of following material by plate confining liquid: Tris-HCl damping fluid (pH=7.4) 0.1mol/L, macromolecular substances 0.5-1.0g/L, protein protective agent 1.5-10g/L, Sodium azide 0.5-1g/L;
Described, macromolecular substances is one or both the potpourri in PEG-20000, polyvinylpyrrolidone;
Described, protein protective agent is one or both the potpourri in trehalose, Tween-20.
Described, kit also comprises control serum, sample diluting liquid, concentrated cleaning solution, developer and stop buffer.
The using method of kit of the present invention is as follows:
1) application of sample: take out detection kit, places 30 minutes for 15-30 DEG C; Plate is pre-seted each hole of blank, feminine gender and positive control at albumen bag, all the other every holes add 100 μ L sample diluting liquids, then add 100 μ L testing samples; Blank well adds 200 μ L sample diluting liquids; Positive and negative control wells adds 200 μ L feminine genders, positive control serum respectively, mixes rear 37 DEG C of water-baths and vibrates 90 minutes;
2) plate is washed: wash plate 4 times with the cleansing solution after 1:20 dilution, pat dry for the last time;
3) enzyme conjugates is added: every hole adds 200 μ L enzyme conjugates, 37 DEG C of water-baths 30 minutes, residual enzyme bond 4 DEG C preservation;
4) plate is washed: wash plate with step 2);
5) develop the color: first add developer A liquid 100 μ L, then add developer B liquid 100 μ L, 37 DEG C of water-bath lucifuge colour developing 10-15 minute;
6) stop: every hole adds 50 μ l stop buffers;
7) measure: in 10 minutes, measure each hole OD value with microplate reader 450nm wavelength;
Critical value is determined: if during negative control mean OD value+0.06(negative control mean OD value≤0.06, calculates by 0.06; During OD>0.06, calculate by practical measurement negative control mean OD value+0.06).The OD value of test specimen is less than critical value then for HCV-cAg is negative.OD value in test specimen is equal to or greater than critical value then for HCV-cAg is positive.
The Main Ingredients and Appearance of positive control serum used in kit of the present invention is the strong positive serum of HCV-cAg and subclass antibodies; Negative control sera is Main Ingredients and Appearance is not containing the normal human serum of HCV-cAg; The Main Ingredients and Appearance of sample diluting liquid is the PBS being added with lowlenthal serum; The Main Ingredients and Appearance of concentrated cleaning solution is NaCl, phosphate, Tween-20; The Main Ingredients and Appearance of developer A is carbamide peroxide; The Main Ingredients and Appearance of developer B is TMB.2HCL; The Main Ingredients and Appearance of stop buffer is 2M H
2sO
4.
The reaction principle figure of kit of the present invention, as shown in Figure 2.
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 wrapping by the mode of plate wrapping by hepatitis C virus core antibody and various subtype protein simultaneously, reduce the loss of detection, simultaneously by adding the confining liquid without protein macromolecule, ensure that the efficient, stable of kit, there is good potential clinical value.
The innovation of the kit of C hepatitis virus antigen of the present invention-antibody combined detection is:
(1) antibody of anti-hepatitis c virus cAg and other hypotype recombinant proteins of hepatitis C virus are fixed to solid phase carrier-albumen bag by plate, can ensure that the antibody of HCV core antigen in sample and multiple hypotype can be adsorbed onto on solid phase carrier by immune response, avoid undetected.
(2) enzyme conjugates in kit carries out 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 the specificity that kit detects.
(3) kit of the present invention, selects macromolecular substances as confining liquid, without protein structure, adds protein protective agent simultaneously, effectively ensure that the stability of kit in confining liquid.
Accompanying drawing explanation
Fig. 1 Ministry of Public Health announces the third liver epidemic situation statistical graph over the years;
The reaction principle figure of Fig. 2 kit of the present invention;
Fig. 3 embodiment 3 contrasts RT-PCR kit and detects correlativity.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
embodiment 1
A kind of C hepatitis virus antigen-antibody combined detection kit comprises control serum, sample diluting liquid, concentrated cleaning solution, developer, stop buffer, albumen bag by plate and enzyme conjugates.
Control serum used in kit comprises positive control serum and negative control sera, and the Main Ingredients and Appearance of positive control serum is the strong positive serum of HCV-cAg and subclass antibodies; Negative control sera is Main Ingredients and Appearance is not containing the normal human serum of HCV-cAg; The Main Ingredients and Appearance of sample diluting liquid is the PBS being added with lowlenthal serum; The Main Ingredients and Appearance of concentrated cleaning solution is NaCl, phosphate, Tween-20; The Main Ingredients and Appearance of developer A is carbamide peroxide; The Main Ingredients and Appearance of developer B is TMB.2HCL; The Main Ingredients and Appearance of stop buffer is 2M H
2sO
4.
Albumen bag is obtained by following steps by plate:
PH7.4 concentration is that antigen is become finite concentration with antibody dilution by the phosphate buffer of 0.02mol/L; Add by 100 μ L/ holes the coating buffer diluted, wrap by 2 hours at 37 DEG C; Discard bag by the coating buffer in plate, bag is patted dry by plate, add bag by plate confining liquid by 100 μ L-300 μ L/holes, wrap at 2-8 DEG C by 16-20 hour; Discard bag by the confining liquid in plate, after bag is patted dry by plate, be positioned over humidity and be less than in the 35-39 DEG C of drying box of 30% and dry 4-6 hour, obtain albumen bag by plate.
Enzyme conjugates prepares by the following method:
5mg horseradish peroxidase being dissolved in 1ml concentration is in 0.2mol/L, pH5.6 acetate buffer, then adds the ethanol solution 0.1ml that volumetric concentration is the DNF of 1%, and stirred at ambient temperature 1h, obtains solution 1; The NaIO of the 0.1mol/L of fresh configuration is added in solution 1
40.5ml, places 30min, obtains solution 2 for 4 DEG C; In solution 2, add the ethylene glycol 1ml that volumetric concentration is 2.5%, stirred at ambient temperature 1h, obtains solution 3; In solution 3, add the albumen 5mg of horseradish peroxidase-labeled, regulate solution ph to 9.0 with the carbonate buffer solution that pH9.5 concentration is 1.0mol/L, mixing, 4 DEG C are spent the night, and obtain solution 4; In solution 4, add the sodium borohydride solution 0.1ml of 0.1mol/L, mixing, place 3h, obtain solution 5 for 4 DEG C; In solution 5, add concentration is 0.01mol/L, pH7.4 phosphate buffer, and 4 DEG C of dialysed overnight are changed liquid 3 times, obtained solution 6; By centrifugal for solution 6 30min, remove sediment, gained supernatant is enzyme conjugates.
The bag of the present embodiment is made up of following material by plate confining liquid
Tris-HCl damping fluid (pH=7.4) 0.1mol/L; PEG-20000 1g/L; Tween-20 5g/L; Trehalose 10g/L; Sodium azide 1g/L.
2. kit application and detect running program
1) application of sample: take out detection kit, places 30 minutes for 15-30 DEG C; Plate is pre-seted blank, feminine gender, each hole of positive control at albumen bag, all the other every holes add 100 μ L sample diluting liquids, then add 100 μ L testing samples; Blank well adds 200 μ L sample diluting liquids; Positive and negative control wells respectively adds 200 μ L yin, yang control serums, mixes rear 37 DEG C of water-baths and vibrates 90 minutes;
2) plate is washed: wash plate 4 times with the cleansing solution after 1:20 dilution, pat dry for the last time;
3) enzyme conjugates is added: every hole adds 200 μ L enzyme conjugates, 37 DEG C of water-baths 30 minutes, residual enzyme bond 4 DEG C preservation;
4) plate is washed: wash plate with step 2);
5) develop the color: first add developer A liquid 100 μ L, then add developer B liquid 100 μ L, 37 DEG C of water-bath lucifuge colour developing 10-15 minute;
6) stop: every hole adds 50 μ l stop buffers;
7) measure: in 10 minutes, measure each hole OD value with microplate reader 450nm wavelength;
Critical value is determined: if during negative control mean OD value+0.06(negative control mean OD value≤0.06, calculates by 0.06; During OD>0.06, calculate by practical measurement negative control mean OD value+0.06).The OD value of test specimen is less than critical value then for HCV-cAg is negative.OD value in test specimen is equal to or greater than critical value then for HCV-cAg is positive.
embodiment 2
A kind of C hepatitis virus antigen-antibody combined detection kit comprises control serum, sample diluting liquid, concentrated cleaning solution, developer, stop buffer, albumen bag by plate and enzyme conjugates.
Control serum used in kit comprises positive control serum and negative control sera, and the Main Ingredients and Appearance of positive control serum is the strong positive serum of HCV-cAg and subclass antibodies; Negative control sera is Main Ingredients and Appearance is not containing the normal human serum of HCV-cAg; The Main Ingredients and Appearance of sample diluting liquid is the PBS being added with lowlenthal serum; The Main Ingredients and Appearance of concentrated cleaning solution is NaCl, phosphate, Tween-20; The Main Ingredients and Appearance of developer A is carbamide peroxide; The Main Ingredients and Appearance of developer B is TMB.2HCL; The Main Ingredients and Appearance of stop buffer is 2M H
2sO
4.
Albumen bag is obtained by following steps by plate:
PH7.4 concentration is that antigen is become finite concentration with antibody dilution by the phosphate buffer of 0.02mol/L; Add by 100 μ L/ holes the coating buffer diluted, wrap by 2 hours at 37 DEG C; Discard bag by the coating buffer in plate, bag is patted dry by plate, add bag by plate confining liquid by 100 μ L-300 μ L/holes, wrap at 2-8 DEG C by 16-20 hour; Discard bag by the confining liquid in plate, after bag is patted dry by plate, be positioned over humidity and be less than in the 35-39 DEG C of drying box of 30% and dry 4-6 hour, obtain albumen bag by plate.
Enzyme conjugates prepares by the following method:
5mg horseradish peroxidase being dissolved in 1ml concentration is in 0.2mol/L, pH5.6 acetate buffer, then adds the ethanol solution 0.1ml that volumetric concentration is the DNF of 1%, and stirred at ambient temperature 1h, obtains solution 1; The NaIO of the 0.1mol/L of fresh configuration is added in solution 1
40.5ml, places 30min, obtains solution 2 for 4 DEG C; In solution 2, add the ethylene glycol 1ml that volumetric concentration is 2.5%, stirred at ambient temperature 1h, obtains solution 3; In solution 3, add the albumen 5mg of horseradish peroxidase-labeled, regulate solution ph to 9.0 with the carbonate buffer solution that pH9.5 concentration is 1.0mol/L, mixing, 4 DEG C are spent the night, and obtain solution 4; In solution 4, add the sodium borohydride solution 0.1ml of 0.1mol/L, mixing, place 3h, obtain solution 5 for 4 DEG C; In solution 5, add concentration is 0.01mol/L, pH7.4 phosphate buffer, and 4 DEG C of dialysed overnight are changed liquid 3 times, obtained solution 6; By centrifugal for solution 6 30min, remove sediment, gained supernatant is enzyme conjugates.
The bag of the present embodiment is made up of following material by plate confining liquid:
Tris-HCl damping fluid (pH=7.4) 0.01mol/L; Polyvinylpyrrolidone 0.5g/L; Tween-20 0.5g/L; Trehalose 1g/L; Sodium azide 0.1g/L.
2. kit application and detect running program with embodiment 1.
embodiment 3
A kind of C hepatitis virus antigen-antibody combined detection kit comprises control serum, sample diluting liquid, concentrated cleaning solution, developer, stop buffer, albumen bag by plate and enzyme conjugates.
Control serum used in kit comprises positive control serum and negative control sera, and the Main Ingredients and Appearance of positive control serum is the strong positive serum of HCV-cAg and subclass antibodies; Negative control sera is Main Ingredients and Appearance is not containing the normal human serum of HCV-cAg; The Main Ingredients and Appearance of sample diluting liquid is the PBS being added with lowlenthal serum; The Main Ingredients and Appearance of concentrated cleaning solution is NaCl, phosphate, Tween-20; The Main Ingredients and Appearance of developer A is carbamide peroxide; The Main Ingredients and Appearance of developer B is TMB.2HCL; The Main Ingredients and Appearance of stop buffer is 2M H
2sO
4.
Albumen bag is obtained by following steps by plate:
PH7.4 concentration is that antigen is become finite concentration with antibody dilution by the phosphate buffer of 0.02mol/L; Add by 100 μ L/ holes the coating buffer diluted, wrap by 2 hours at 37 DEG C; Discard bag by the coating buffer in plate, bag is patted dry by plate, add bag by plate confining liquid by 100 μ L-300 μ L/holes, wrap at 2-8 DEG C by 16-20 hour; Discard bag by the confining liquid in plate, after bag is patted dry by plate, be positioned over humidity and be less than in the 35-39 DEG C of drying box of 30% and dry 4-6 hour, obtain albumen bag by plate.
Enzyme conjugates prepares by the following method:
5mg horseradish peroxidase being dissolved in 1ml concentration is in 0.2mol/L, pH5.6 acetate buffer, then adds the ethanol solution 0.1ml that volumetric concentration is the DNF of 1%, and stirred at ambient temperature 1h, obtains solution 1; The NaIO of the 0.1mol/L of fresh configuration is added in solution 1
40.5ml, places 30min, obtains solution 2 for 4 DEG C; In solution 2, add the ethylene glycol 1ml that volumetric concentration is 2.5%, stirred at ambient temperature 1h, obtains solution 3; In solution 3, add the albumen 5mg of horseradish peroxidase-labeled, regulate solution ph to 9.0 with the carbonate buffer solution that pH9.5 concentration is 1.0mol/L, mixing, 4 DEG C are spent the night, and obtain solution 4; In solution 4, add the sodium borohydride solution 0.1ml of 0.1mol/L, mixing, place 3h, obtain solution 5 for 4 DEG C; In solution 5, add concentration is 0.01mol/L, pH7.4 phosphate buffer, and 4 DEG C of dialysed overnight are changed liquid 3 times, obtained solution 6; By centrifugal for solution 6 30min, remove sediment, gained supernatant is enzyme conjugates.
The bag of the present embodiment is made up of following material by plate confining liquid:
Tris-HCl damping fluid (pH=7.4) 0.05mol/L; PEG-20000 0.5g/L; Polyvinylpyrrolidone 0.5g/L; Tween-20 (Tween-20) 1g/L; Trehalose 5g/L; Sodium azide 0.5g/L.
2. kit application and detect running program with embodiment 1.
measure of merit 1
Selected 100 routine clinical samples detect, and the kit prepared using embodiment 1-3 is as experimental example 1-3, and the core antigen of C type hepatitis virus kit of ortho company of the U.S. as a comparison case, detects clinical sample respectively.Testing result is as shown in table 1.
The testing result of table 1 100 sample application two kinds of reagent
| Positive | Negative | Accuracy/% | |
| Experimental example 1 | 36 | 64 | 100 |
| Experimental example 2 | 36 | 64 | 100 |
| Experimental example 3 | 36 | 64 | 100 |
| Comparative example | 35 | 65 | 98 |
As shown in Table 1, it is consistent that kit prepared by embodiment 1-3 detects sample yin and yang attribute result, and the kit detection of ortho company of the U.S. has an example to be detected as false negative, and the later stage carries out tracing detection to this sample, and this patient's pattern detection is hepatitis C virus infection.Therefore the kit of this experiment preparation, detecting yin and yang attribute coincidence rate is 100%, not undetected.
measure of merit 2
Utilize kit prepared by embodiment 3, the goldstandard real-time quantitative PCR detection method that contrast hepatitis C virus detects, the correlativity of checking two kits, contrast agent is chosen as hepatitis C virus (HCV) kit for detecting nucleic acid (PCR-fluorescence probe method) of Da'an Gene Company, Zhongshan University, detect 40 routine clinical samples (covering high low value), result as shown in Figure 3 simultaneously.
Through the comparison with goldstandard, kit prepared by embodiment 3 has the testing result of the high correlativity with goldstandard, detects 40 sample correlation coefficient r=0.9977, illustrates that the result that two kits are detecting clinical sample is basically identical.
Known by above-mentioned measure of merit, the invention provides a kind of C hepatitis virus antigen-antibody combined detection kit, can detect the antigen of infection with hepatitis C virus generation and the antibody of each subtype protein simultaneously, testing result is accurate, yin and yang attribute coincidence rate can reach 100%, comparison goldstandard simultaneously (RT-PCR detection kit) has high consistency, and the present invention has very high potential value for clinical detection hepatitis C virus.
Claims (5)
1. C hepatitis virus antigen-antibody combined detection kit, is characterized in that, kit comprises albumen bag by plate and enzyme conjugates;
Described albumen bag is prepared by following methods by plate: antigen is become 1:2000 concentration with antibody dilution by the phosphate buffer of pH7.4 concentration 0.02mol/L, obtained coating buffer; Add by 100 μ L/ holes the coating buffer diluted, wrap by 2 hours at 37 DEG C; Discard bag by the coating buffer in plate, bag is patted dry by plate, add bag by plate confining liquid by 100 μ L-300 μ L/holes, wrap at 2-8 DEG C by 16-20 hour; Discard bag by the confining liquid in plate, after bag is patted dry by plate, be positioned over humidity and be less than in the 35-39 DEG C of drying box of 30% and dry 4-6 hour, obtain albumen bag by plate;
Described enzyme conjugates prepares by the following method: 5mg horseradish peroxidase being dissolved in 1ml concentration is in 0.2mol/L, pH5.6 acetate buffer, add that volumetric concentration is 1% again 2, the ethanol solution 0.1ml of 4-dinitrofluorobenzene, stirred at ambient temperature 1h, obtain solution 1; The NaIO of the 0.1mol/L of fresh configuration is added in solution 1
40.5ml, places 30min, obtains solution 2 for 4 DEG C; In solution 2, add the ethylene glycol 1ml that volumetric concentration is 2.5%, stirred at ambient temperature 1h, obtains solution 3; In solution 3, add the albumen 5mg of horseradish peroxidase-labeled, regulate solution ph to 9.0 with the carbonate buffer solution that pH9.5 concentration is 1.0mol/L, mixing, 4 DEG C are spent the night, and obtain solution 4; In solution 4, add the sodium borohydride solution 0.1ml of 0.1mol/L, mixing, place 3h, obtain solution 5 for 4 DEG C; In solution 5, add concentration is 0.01mol/L, pH7.4 phosphate buffer, and 4 DEG C of dialysed overnight are changed liquid 3 times, obtained solution 6; By centrifugal for solution 6 30min, remove sediment, gained supernatant is enzyme conjugates.
2. kit according to claim 1; it is characterized in that, described bag is made up of following material by plate confining liquid: the Tris-HCl damping fluid 0.1mol/L of pH=7.4, macromolecular substances 0.5-1.0g/L, protein protective agent 1.5-10g/L, Sodium azide 0.5-1g/L.
3. kit according to claim 2, is characterized in that, described macromolecular substances is one or both the potpourri in PEG-20000, polyvinylpyrrolidone.
4. kit according to claim 2, is characterized in that, described protein protective agent is one or both the potpourri in trehalose, Tween-20.
5. kit according to claim 1, is characterized in that, described kit also comprises control serum, sample diluting liquid, concentrated cleaning solution, developer and stop buffer.
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|---|---|---|---|
| CN201410698139.8A CN104407143B (en) | 2014-11-28 | 2014-11-28 | A kind of C hepatitis virus antigen-antibody combined detection kit |
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|---|---|---|---|
| CN201410698139.8A CN104407143B (en) | 2014-11-28 | 2014-11-28 | A kind of C hepatitis virus antigen-antibody combined detection kit |
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| CN104407143A true CN104407143A (en) | 2015-03-11 |
| CN104407143B CN104407143B (en) | 2016-04-27 |
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| WO2017008179A1 (en) * | 2015-07-06 | 2017-01-19 | 山东博科生物产业有限公司 | Reagent for high throughput combined detection of hepatitis c virus antigen and antibody |
| CN106353502A (en) * | 2016-08-25 | 2017-01-25 | 山西医科大学 | ELISA kit for detecting human cytomegalovirus (HCMV) IgG and IgM antibodies |
| CN108931635A (en) * | 2018-05-04 | 2018-12-04 | 上海交通大学 | A kind of standard antigen dilution for superparamagnetism immunochromatography chip |
| 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 |
| CN110261616A (en) * | 2019-04-30 | 2019-09-20 | 广东菲鹏生物有限公司 | A kind of hepatitis c virus detection reagent box |
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| CN110261616B (en) * | 2019-04-30 | 2021-07-20 | 广东菲鹏生物有限公司 | Hepatitis C virus detection kit |
| CN115575631A (en) * | 2022-09-30 | 2023-01-06 | 深圳盛源生物技术有限公司 | Kit for detecting content of inhibin A in blood plasma |
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