CN113189348A - Hepatitis C virus antibody detection kit and application thereof - Google Patents
Hepatitis C virus antibody detection kit and application thereof Download PDFInfo
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- 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
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- 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
- G01N33/576—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
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
Abstract
The invention discloses a hepatitis C virus antibody detection kit and application thereof, wherein the hepatitis C virus antibody detection kit comprises: a coated plate of hepatitis c virus antigen, an enzyme conjugate, and a chemiluminescent substrate. The kit prepared by the invention has the effects of strong specificity, good repeatability and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis and detection, and particularly relates to a hepatitis C virus antibody detection kit and application thereof.
Background
Viral hepatitis type c is caused by Hepatitis C Virus (HCV), mainly by infection through blood transfusion or blood products, hemodialysis, apheresis, kidney transplantation, intravenous injection of drugs, sexual transmission, mother-to-infant transmission, and the like. Hepatitis C is widely distributed and more likely to develop into chronic, cirrhosis and liver cancer. The transmission pathways for HCV mainly include: the transmission pathways of blood transmission, sexual transmission, maternal-fetal transmission, and some HCV-infected individuals are unclear.
So far, no effective vaccine for preventing hepatitis C has been successfully developed, so the effective way for preventing and treating hepatitis C is early diagnosis and early treatment. The current laboratory diagnostic methods for hepatitis c include: HCV serological tests, HCV RNA, genotype, and mutation tests. HCV RNA detection has good specificity, high sensitivity, high requirements on equipment and operators, and instability at the in vivo level in infected persons, thus the method has limitations. The detection results of HCV genotypes and variations are helpful for judging the treatment difficulty and making antiviral treatment schemes, but have high price, long time consumption and are not suitable for common screening. Commonly used serological tests for HCV employ enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA) and Recombinant Immunoblotting (RIBA), and anti-HCV antibodies are the hallmarks of serological detection. In ELISA, a porous polystyrene reaction plate is usually used as a solid phase carrier, an antigen or an antibody to be detected is fixed on the surface of the solid phase carrier, an enzyme-labeled antigen or antibody reacts with the specificity of the antigen or the antibody, a substrate is added, and the result is judged by a color reaction with an enzyme-labeling instrument. The Recombinant Immunoblotting (RIBA) is a confirmation test of HCV infection, mainly solves the false positive in an ELISA test, improves the specificity, but for the judgment of a positive result, RIBA cannot provide more information related to HCV infection and is not economical. The chemiluminescence immunoassay (CLIA) combines a chemiluminescence measuring technology with high sensitivity and a magnetic particle separation technology on the basis of enzyme immunoassay, adopts paramagnetic particles as a solid phase carrier, has small volume and large specific surface area, enlarges the reaction area, improves the detection sensitivity, needs to be matched with a full-automatic chemiluminescence instrument and a matched reagent during detection, and has high automation degree.
However, the chemiluminescence immunoassay method adopted at present is easy to have the phenomenon of false positive or false negative when detecting the hepatitis C antibody. There is still a need to develop a hepatitis c virus antibody detection kit with high detection sensitivity, strong specificity and good repeatability.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a hepatitis C virus antibody detection kit which can rapidly and accurately identify hepatitis C virus.
The invention also provides application of the hepatitis C virus antibody detection kit.
According to a first aspect of the invention, the hepatitis C virus antibody detection kit comprises a hepatitis C virus antigen coated plate, an enzyme conjugate and a chemiluminescent substrate.
In some embodiments of the invention, the kit further comprises a negative control, a positive control, a sample diluent, a concentrated wash solution, and a stop solution.
In some embodiments of the invention, the preparation of the hepatitis c virus antigen coated plate comprises the steps of: and diluting the hepatitis C virus antigen by adopting a coating buffer solution, and then coating the hepatitis C virus antigen to obtain the coated plate of the hepatitis C virus antigen.
In some embodiments of the invention, the volume ratio of the hepatitis c virus antigen to the coating buffer is 1: (500-8000).
In some embodiments of the invention, the coating buffer comprises 0.01-0.05% NaN3The pH of the 0.1% carbonate buffer solution is 7.5-9.0.
In some embodiments of the invention, the enzyme conjugate is an enzyme-labeled anti-human IgG monoclonal antibody.
In some embodiments of the invention, the enzyme-labeled anti-human IgG monoclonal antibody is a horseradish peroxidase-labeled anti-human IgG monoclonal antibody.
In some embodiments of the invention, the enzyme conjugate is diluted with an enzyme-labeled diluent, and the volume ratio of the enzyme conjugate to the enzyme-labeled diluent is 1: (500-10000).
In some embodiments of the invention, the volume ratio of enzyme conjugate to enzyme-labeled diluent is 1: 5000.
in some embodiments of the invention, each liter of enzyme labeling diluent comprises 1-5 g of sodium tetraborate, 3g/LCasein-Na 1-5 g of boric acid, 1-3 g of antipyrine, 1-5 g of liquid gelatin, 1000.1-0.5 g of Triton X, 1-3 g of glycerol, 8-12 g of calf serum, 3001-3 g of Proclin and 3-5 g of 1% carmine.
In some embodiments of the invention, each liter of enzyme-labeled diluent comprises 3g of sodium tetraborate, 3g/L, Casein-Na 2g of boric acid, 1g of antipyrine, 2g of liquid gelatin, Triton X-1000.3 g, 1g of glycerol, 10g of calf serum, Proclin 3001 g, and 5g of 1% carmine.
In some embodiments of the invention, the method for labeling the anti-human IgG monoclonal antibody with horseradish peroxidase is one of a sodium iodate-ethylene glycol method, a sodium periodate method and a glutaraldehyde two-step method.
In some embodiments of the invention, the anti-human IgG monoclonal antibodies are labeled with horseradish peroxidase using the sodium iodate-ethylene glycol method.
In some embodiments of the invention, the concentrated washing solution is 0.2-0.3 mmol/L phosphate buffer solution containing 150-250 g/L NaCl, 2-10 g/L KCl and 1-5% Tween-20, and the pH is 6.0-7.0.
In some embodiments of the invention, the concentrated wash solution is a 0.25M phosphate buffer solution containing 200g/L NaCl, 5g/L KCl and 3.0% Tween-20, with a pH of 6.2-6.4.
In some embodiments of the invention, the sample diluent comprises 1-2% calf serum, 0.1-0.4% glycerol, 0.05-0.2% Proclin300, and 0.1-0.4% fruit green in 1-6 mmol/L borate buffer solution
In some embodiments of the invention, the components of the sample diluent are phosphate buffered saline at ph7.4 containing 10% FBS (calf serum) by volume fraction.
In some embodiments of the present invention, the stop solution is 0.5-3 mol/L of H2SO4And (3) solution.
In some embodiments of the invention, the chemiluminescent substrate comprises substrate solution a and substrate solution B; the substrate A solution is a 5-20 mmol/L citric acid-sodium acetate buffer solution containing 0.5-0.8 g/L carbamide peroxide; the substrate B solution contains 0.3-0.5 g/L of TMB and 0.30 to 0.5 percent of acetone, 4 to 6 percent of absolute ethyl alcohol and 0.2 to 0.5g/L of EDTA-Na2And 8-15 mmol/L citric acid solution of 3-5% glycerin.
In some embodiments of the invention, the negative control is a negative control serum diluted with a control dilution, and the absorbance of the negative control is less than 0.05.
The positive control is positive control serum diluted by a reference substance diluent; the positive control has an absorbance greater than 1.5.
In some embodiments of the invention, the control dilution is a 2-7 mmol/L borate buffer solution comprising 1-3% calf serum, 0.05-0.2% glycerol, 0.05-0.2% Proclin300, and 0.1-0.5% fruit green.
In some embodiments of the invention, the control dilution is a 5mmol/L borate buffered solution comprising 2% calf serum, 0.1% glycerol, 0.1% Proclin300, and 0.2% fruit green.
The application of the hepatitis C virus antibody detection kit according to the second aspect of the invention is the application in preparing a hepatitis C virus detection reagent.
In some embodiments of the invention, the use is in the detection of hepatitis c virus.
A method for detecting antibodies to hepatitis c virus comprising the steps of: the hepatitis C virus antibody detection kit is used for detecting hepatitis C virus antibodies.
A method for detecting the hepatitis C virus antibody by adopting the kit comprises the following steps:
s1, adding the sample solution to be tested, the negative control solution, the positive control solution and the blank control solution respectively until the envelope plate coated with the hepatitis C virus antigen is incubated, removing the liquid in the hole, washing and patting dry;
s2, adding an enzyme conjugate, removing liquid in the hole after incubation is finished, washing and patting dry;
s3, adding substrate liquid for color development;
s4, adding a stop solution;
s5, measuring the light absorption value of the reaction liquid, wherein the critical value cut-off =0.15+ the average light absorption value of the negative control, and when the average light absorption value of the negative control is less than 0.05, calculating according to 0.05; when the light absorption value of the sample to be detected is less than Cut-off, judging the sample to be detected to be negative; when the light absorption value of the sample to be detected is more than or equal to Cut-off, the sample is judged to be positive, and the method does not aim at the diagnosis or treatment of diseases.
The invention has the beneficial effects that: the invention provides a hepatitis C virus antibody detection kit and application thereof. The hepatitis C virus antibody detection kit specifically selects a coating plate coated with a hepatitis C virus antigen and a hepatitis C virus antigen marked by horseradish peroxidase, and is combined with a chemiluminescent substrate for color development, so that the detection kit has the functions of strong specificity, good repeatability, high sensitivity and rapid detection of the hepatitis C virus antibody. The detection kit disclosed by the invention also has the characteristics of simplicity and convenience in operation and strong practicability, and is suitable for being used by various basic medical units.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
The hepatitis C virus antigen used in this example was purchased from Beijing Hua Dagibei Biotech Co.
Example 1
The embodiment prepares a hepatitis C virus antibody detection kit, which comprises the following components:
(1) coated plate coated with hepatitis C virus antigen
After the hepatitis C virus antigen is purified by adopting affinity chromatography, a band is formed by detecting the hepatitis C virus antigen by using a polyacrylamide gel electrophoresis method, the molecular weight is about 40KD, the purity is more than or equal to 98 percent, and the purified antigen is stored at the temperature of minus 20 ℃ or below.
Coating: diluting the hepatitis C virus antigen with a coating buffer solution (the volume ratio of the hepatitis C virus antigen to the coating buffer solution is 1: 1000), adding the diluted hepatitis C virus antigen into the holes of a microporous plate according to the using amount of 100 mu L/hole, adsorbing for 48h at 37 ℃, and drying; and (3) sealing: adding phosphate buffer solution containing 0.8% of tryptone, 5% of sucrose and 0.02% of Proclin300 into the pores of the microporous plate at the dosage of 200 mu L/pore as a blocking solution, and blocking for 16h at 37 ℃; drying and sealing plates: removing the confining liquid, drying, and sealing to obtain the coated plate coated with the hepatitis C virus antigen.
The coating buffer solution contains 0.02% NaN30.1% carbonate buffer, pH 8.0.
(2) Chemiluminescent substrate
The chemiluminescent substrate comprises a substrate A solution and a substrate B solution; the substrate A solution is a 10mmol/L citric acid-sodium acetate buffer solution containing 0.6g/L carbamide peroxide; the substrate B solution contains 0.42g/L TMB (tetramethyl benzidine), 0.3% acetone, 4% absolute ethyl alcohol and 0.3g/L EDTA-Na210mmol/L citric acid solution of 3% glycerol.
(3) Enzyme conjugates
The enzyme conjugate is horseradish peroxidase labeled anti-human IgG monoclonal antibody.
The horseradish peroxidase labeled anti-human IgG monoclonal antibody is labeled by adopting a sodium iodate-ethylene glycol method to label the anti-human IgG monoclonal antibody by horseradish peroxidase, and the specific method comprises the following steps:
a. weighing 4.0mg of horseradish peroxidase (HRP) in a brown glass bottle, adding 0.4mL of 0.2mol/LDE1 acetate buffer solution for dissolving, and putting the solution into a rotor and putting the solution into the bottle;
b. placing the magnetic stirrer in an environment of 2-8 ℃ for working, then placing the solution bottle in the step a on the magnetic stirrer, adjusting the rotating speed to be slowest, and adding 0.1mol/L NaIO under the condition that a rotor in the solution bottle continuously rotates40.2mL of solution is mixed evenly, then the whole bottle body is wrapped by tinfoil, and the reaction is carried out for 30min in a dark place;
c. selecting a dialysis bag with a proper specification, cutting to a proper length, placing in a beaker filled with a proper amount of purified water, heating and boiling for 1-2min (the dialysis bag with the proper specification is selected according to the total marked liquid amount, so that the bag opening of the dialysis bag can be completely opened for adding liquid), and reserving for later use;
d. after the reaction in the step c is finished, taking out the magnetic stirrer and the solution bottle from the environment of 2-8 ℃, standing at room temperature, adding 0.4mL of 2.5% glycol aqueous solution under the condition of keeping the rotor in the solution bottle to continuously rotate, uniformly mixing, and reacting for 30min at room temperature in a dark place;
e. after the reaction in step d is finished, adding 2mL of ice-cooled absolute ethyl alcohol into every 0.5mL of solution, uniformly mixing, centrifuging for 5min by 1120g of centrifugal force (the rotating speed of a ZY0500 centrifugal machine is regulated to 2500 r/min), fully sucking supernatant by using a pipette, and adding 0.5mL of purified water to redissolve and precipitate;
f. folding one end of the dialysis bag, clamping and sealing, measuring the re-solution, the antibody or the antigen in the step e according to actual requirements, respectively adding the re-solution, the antibody or the antigen into the dialysis bag (calculating according to the proportion that 4mg of antigen is added into 1mL of re-solution and 1mg of antigen is added into 1mL of re-solution), uniformly mixing, slowly extruding bubbles in the bag upwards to separate the solution, folding the other end of the dialysis bag, clamping and sealing, and marking the name of the antigen and the antibody on a clamp. A3L beaker was filled with the appropriate amount of CB solution (carbonate buffer) and placed on a large rotor, which was then placed in a magnetic stirrer and stirred overnight at 2-8 deg.C.
g. Adding 5mg/mL of NaHB4Solution 0.08mL was terminated (NaHB)4The solution dosage calculation method comprises the following steps: 5mg/mL NaHB is added according to the requirement of 1mgHRP420 mu L), mixing, standing at 2-8 ℃ for 2h, and mixing once after 1 hour;
h, adding glycerol with the same volume, mixing evenly, and then adding 1mol/L NaH2PO4Adjusting pH to about 7.0, and storing at-20 deg.C or below.
(4) Negative control solution
The negative control is negative control serum diluted by a control diluent.
The control dilution is a 5mmol/L borate buffer solution containing 2% calf serum, 0.1% glycerol, 0.1% Proclin300, and 0.2% fruit green; the absorbance of the negative control was less than 0.05.
(5) Positive control solution
The positive control is positive control serum diluted by a control product diluent.
The method for detecting HCV antibodies by using the kit of the embodiment comprises the following steps:
s1, preparing a washing solution: using distilled water or deionized water as a solvent for 1: 25, diluting;
s2, taking out the coated plate, adding 100 mu L of sample to be detected into each hole, setting 2 holes for negative control, positive control and blank control, respectively adding 100 mu L of corresponding control serum into the negative control hole and the positive control hole, adding 100 mu L of sample diluent into the blank control hole, and uniformly mixing; covering with sealing plate glue, and incubating at 37 deg.C in dark for 60 min;
s3, washing plates: washing the plate with washing solution for 5 times, standing for 1min each time, and drying;
s4, addition of enzyme conjugate: 50. mu.l/well of enzyme conjugate was added. After the plate is covered with the glue, the plate is placed at 37 ℃ and incubated for 30 minutes in a dark place;
s5, color development and termination: adding substrate solution, developing at 37 deg.C in dark for 10min, and adding stop solution 50 μ L/well;
s6, washing plates: washing the plate with washing solution for 5 times, standing for 1min each time, and drying;
s7, measurement: zero calibration is carried out by a blank control hole with 450nm (single wavelength) or 450nm/630nm (double wavelength) of an enzyme-labeling instrument, and the light absorption value of each hole is measured;
s8, analyzing and judging results: cutoff-off =0.15+ mean absorbance of negative control, calculated as 0.05 when mean absorbance of negative control is < 0.05; when the light absorption value of the sample to be detected is less than Cut-off, judging the sample to be detected to be negative; and when the light absorption value of the sample to be detected is more than or equal to Cut-off, judging the sample to be detected to be positive.
Comparative example 1
The comparative example employed a hepatitis C virus antibody detection kit (enzyme-linked immunosorbent assay) purchased from Shanghai Kehua bioengineering, Inc.
Comparative example 2
The difference between the comparative example and the hepatitis C virus antibody detection kit prepared in example 1 is that the chemiluminescent substrate adopts the color developing agents of hydrogen peroxide solution A and tetramethyl benzidine solution B, and the volume ratio of the hydrogen peroxide solution A to the tetramethyl benzidine solution B is 1: 1.
Test examples
1. Concentration selection of coated hepatitis C Virus antigens
Coating buffer (containing 0.02% NaN) was used3The pH = 8.0) of the 0.1% carbonate buffer solution is prepared by diluting hepatitis C virus antigen (purchased from Beijing Hua Daji bie Biotechnology Co., Ltd.) according to the volume ratio of 1:500, 1:1000, 1:2000, 1:4000 and 1:8000 respectively to prepare coating solution, and coating the coating plate; and selecting a kit produced by Shanghai Kehua bioengineering GmbH as a control kit and an in-plant quality control substance (2 NCU/mL quality control substance in Guangdong province is diluted by HBV/HCV/HIV/TP negative plasma in a progressive manner to 5 titers which are 0.125-2 NCU/mL respectively), selecting the concentration of the enveloped hepatitis C virus antigen by adopting a matrix titration method, and detecting the absorbance values of the positive sample and the negative sample under different enveloped hepatitis C virus antigen concentrations, thereby selecting the optimal concentration range of the enveloped hepatitis C virus antigen. The protocol for the selection of the concentration of hepatitis C virus-coated antigen by the square matrix titration method is designed as shown in Table 1.
TABLE 1
Note: S1-S5 are confirmed positive samples, and S6-S10 are confirmed negative samples.
The experimental results of the matrix titration method for selecting the concentration of the antigen coated with the hepatitis C virus are shown in Table 2, and it can be seen from the table that the detection effect is adversely affected when the dilution ratio of the antigen coated with the hepatitis C virus is too high or too low, and through comprehensive comparison, when the dilution ratio of the antigen coated with the hepatitis C virus is 1: (500-2000) can achieve better detection effect, and the dilution ratio of the coated hepatitis C virus antigen is 1:1000, the effect is best, the light absorption value is stable and saturated, the detection sensitivity is high, and the specificity is good.
TABLE 2
2. Working concentration selection of enzyme-labeled hepatitis C Virus antigen
The enzyme-labeled antibody is obtained by labeling an anti-human IgG monoclonal antibody (purchased from SIGMA-ALDRICH) by a sodium iodate-ethylene glycol method. Diluting an enzyme-labeled antibody by using an enzyme-labeled diluent (0.2% Casein-Na, 0.1% antipyrine, 0.2% liquid gelatin, 0.003% Triton X-100, 0.1% glycerol, 1% calf serum, 0.1% Proclin300 and 0.5% carmine in a 5mmol/L borate buffer solution) according to the volume ratio of 1:500 to 1:1000 to 1:2000 to 1:5000 to 1:10000 to prepare a horseradish peroxidase-labeled anti-human IgG monoclonal antibody solution; and a kit produced by Shanghai Kehua bioengineering GmbH is selected as a control kit and quality control products (N, A1, A2, A3, A4, A5 and A6) produced by Beijing Congthenstein biotechnology, and the light absorption values of the positive sample and the negative sample under different enzyme-labeled antibody working concentrations are detected, so that the optimal working concentration range of the enzyme-labeled antibody is selected. The protocol design in which the square matrix titration method selects the working concentration of enzyme-labeled antibody is shown in table 3.
TABLE 3
Note: S1-S5 are confirmed positive samples, and S6-S10 are confirmed negative samples.
The results of the experiments of the matrix titration method for selecting the working concentration of the enzyme-labeled antibody are shown in Table 4, and it can be seen from the table that when the working concentration of the enzyme-labeled hepatitis C virus antigen is more than 1:5000 hours, the sensitivity is high, but the specificity is poor; when the working concentration of the enzyme-labeled hepatitis C virus antigen is less than 1: at 5000, the specificity was good, but the sensitivity was low. In summary, when the dilution ratio of the coated hepatitis c virus antigen is 1: (2000-10000) can achieve better detection effect, when the working concentration of the enzyme-labeled hepatitis C virus antigen is 1:5000, the effect is best, the light absorption value is stable and saturated, and the advantages of high sensitivity and good specificity can be achieved.
TABLE 4
3. Selection of chemiluminescent substrates
The absorbance values of the positive sample and the negative sample were measured using the hepatitis c virus antibody detection kit prepared in example 1 and the hepatitis c virus antibody detection kit prepared in comparative example 2, and the experimental protocol is shown in table 5.
TABLE 5
Note: S1-S5 are confirmed positive samples, and S6-S10 are confirmed negative samples.
As shown in table 6, it can be seen that the hepatitis c virus antibody detection kit prepared in example 1 and the hepatitis c virus antibody detection kit prepared in comparative example 2 both achieve relatively good detection effects, but the hepatitis c virus antibody detection kit prepared in example 1 has higher sensitivity, stable and saturated light absorption value, and has the advantages of high sensitivity and good specificity.
TABLE 6
4. Product performance testing
The test comparison in the laboratory was carried out using the examination reagent (kit prepared in example 1) and the control reagent (kit prepared in comparative example 1) using 100 confirmed positive clinical specimens and 100 confirmed negative clinical specimens, and a2 × 2 table (table 7) was established and calculated using the following formula:
positive coincidence = a/(a + C) × 100%;
negative coincidence = D/(B + D) × 100%;
total coincidence rate = (a + D)/(a + B + C + D);
TABLE 7
Control reagent: the hepatitis C virus antibody detection kit (ELISA) obtained from Shanghai Kowa bioengineering, Inc. in comparative example 1 was used.
The assessment reagent: the hepatitis C virus antibody detection kit prepared in the embodiment 1 of the invention.
Evaluation criteria:
1. positive and negative match rates: the value range is between 0 and 100 percent, and the closer the value is to 100 percent, the greater the authenticity is.
2. The total coincidence rate refers to the proportion of samples with consistent detection results of the assessment reagent and the contrast reagent in the total detection samples, the value range is between 0 and 100 percent, the closer the value is to 100 percent, the higher the coincidence degree with the contrast method is.
It makes sense to judge the consistency between 0 and +1 for Kappa values. The larger the Kappa number, the better the consistency. It is generally considered that the Kappa value is 0.75 or more, indicating that a considerably satisfactory degree of conformity has been achieved. If the Kappa value is < 0.4, the degree of conformity is not satisfactory.
The test results of 200 clinical samples were shown in table 8 and the statistical results are shown in table 9 using the kits provided in example 1 and comparative example 1.
TABLE 8
TABLE 9
As can be seen from tables 8-9, the contrast reagent detects 99 positive samples and 101 negative samples from 200 clinically-provided clinical comparison samples; the assessment reagent detects 99 positive cases and 101 negative cases.
Positive agreement rate =99/99 × 100% = 100%;
negative coincidence =101/101 × 100% = 100%;
total coincidence =200/200 × 100% = 100%;
Kappa=1.00。
kappa =1.00 as tested by the SPSS15.0 software Kappa test,Pless than 0.01, the consistency of the detection results of the assessment reagent and the contrast reagent has statistical significance, and the detection consistency is excellent. The hepatitis C virus antibody detection kit prepared by the invention has good detection effect.
5. And (3) specificity test:
the hepatitis C virus antibody detection kit prepared in embodiment 1 of the application is used for detecting hepatitis B virus, hepatitis B virus antibody, hepatitis A virus antibody and hepatitis C virus standard positive serum, except that the light absorption value of the standard positive serum of the hepatitis A virus antibody is larger than the critical value cut-off, the light absorption values of other serum are smaller than the critical value cut-off, and the method accords with the judgment standard of negative serum, and shows that the specificity of the method is good.
6. Sensitivity detection
The kit prepared in example 1 and the kit of comparative example 1 are used for simultaneously detecting the serum containing the hepatitis C virus with different dilution times, and the table shows that when the serum is diluted by 5-60 times, the detection antibodies of the two kits are positive, and when the serum is diluted by 80 times, the kit of comparative example 1 has false negative, which indicates that the self-made kit has higher sensitivity.
TABLE 10 detection results of different dilution times of the infected sera
7. Repeatability detection
The hepatitis c virus antibody detection kit prepared in example 1 of the present invention was used to repeatedly test the same sample to be tested (2 NCU/mL quality control substance in Guangdong province was diluted to 1NCU/mL with HBV/HCV/HIV/TP negative plasma) for 10 wells to obtain a set of S/CO data, and the average value (X) and standard deviation (S) were calculated to obtain the relative standard deviation (coefficient of variation, CV) values, which were strictly performed according to the operating protocol during the operation, and the test results are shown in table 11.
TABLE 11
As can be seen from Table 11, the coefficient of variation CV is 6.02%, which meets the requirement that the coefficient of variation CV is less than or equal to 15%, and thus the hepatitis C virus antibody detection kit prepared by the present invention has good repeatability when used for actual detection.
8. Shelf life test of kit
The storage condition of the kit is 2-8 ℃, the maximum absorbance value (zero addition) of the kit and the actual measurement value of the addition of the hepatitis C virus antibody are within the normal range after the kit is stored for 13 months, and the stability of the test result is good. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 1 week under the storage condition of 37 ℃, and the result shows that all indexes of the kit completely meet the requirements. And (3) taking the freezing condition of the kit into consideration, putting the kit into a refrigerator at the temperature of-20 ℃ for freezing for 2 weeks, and determining results show that all indexes of the kit are completely normal. From the above results, it was found that the kit could be stored at 2-8 ℃ for 13 months or more.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A hepatitis C virus antibody detection kit, characterized in that the kit comprises: a coated plate of hepatitis c virus antigen, an enzyme conjugate, and a chemiluminescent substrate; the preparation of the hepatitis C virus antigen coated plate comprises the following steps: diluting the hepatitis C virus antigen by adopting a coating buffer solution, uniformly mixing and coating to obtain a coated plate of the hepatitis C virus antigen; the volume ratio of the hepatitis C virus antigen to the coating buffer solution is 1: (500-8000).
2. The hepatitis C virus antibody detection kit according to claim 1, wherein the enzyme conjugate is an enzyme-labeled anti-human IgG monoclonal antibody.
3. The hepatitis C virus antibody detection kit according to claim 2, wherein the enzyme-labeled anti-human IgG monoclonal antibody is a horseradish peroxidase-labeled anti-human IgG monoclonal antibody.
4. The hepatitis C virus antibody detection kit according to claim 1, wherein the enzyme conjugate is diluted with an enzyme-labeled diluent, and the volume ratio of the enzyme conjugate to the enzyme-labeled diluent is 1: (500-10000).
5. The hepatitis C virus antibody detection kit according to claim 1, wherein the chemiluminescent substrate comprises a substrate solution A and a substrate solution B; the substrate A solution is a 5-20 mmol/L citric acid-sodium acetate buffer solution containing 0.5-0.8 g/L carbamide peroxide; the substrate B solution contains 0.3-0.5 g/L of LTMB, 0.3-0.5% of acetone, 4-6% of absolute ethyl alcohol and 0.2-0.5 g/L of EDTA-Na2And 8-15 mmol/L citric acid solution of 3-5% glycerin.
6. The hepatitis C virus antibody detection kit according to claim 1, characterized in that the kit further comprises a negative control, a positive control, a sample diluent, a concentrated washing solution and a stop solution.
7. The hepatitis C virus antibody detection kit according to claim 6, wherein the negative control is a negative control serum diluted with a control diluent.
8. The hepatitis C virus antibody detection kit according to claim 6, wherein the positive control is a positive control serum diluted with a control diluent.
9. The hepatitis C virus antibody detection kit according to claim 6, wherein the concentrated washing solution is 0.2 to 0.3mmol/L phosphate buffer solution containing 150 to 250g/L NaCl, 2 to 10g/L KCl and 1 to 5% Tween-20, and has a pH of 6.0 to 7.0.
10. Use of the hepatitis c virus antibody detection kit according to any one of claims 1 to 9 in the preparation of a hepatitis c virus detection reagent.
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