CN112255411A - Chemiluminescence kit for detecting anti-nucleocapsid protein antibody in serum of HCV (hepatitis C Virus) infected person and detection method - Google Patents
Chemiluminescence kit for detecting anti-nucleocapsid protein antibody in serum of HCV (hepatitis C Virus) infected person and detection method Download PDFInfo
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- CN112255411A CN112255411A CN202010946264.1A CN202010946264A CN112255411A CN 112255411 A CN112255411 A CN 112255411A CN 202010946264 A CN202010946264 A CN 202010946264A CN 112255411 A CN112255411 A CN 112255411A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention relates to the technical field of biology, and discloses a chemiluminescence kit for detecting an anti-nucleocapsid protein (anti-Core) antibody in serum of an HCV (hepatitis C virus) infected person, which comprises a microporous plate obtained by coating recombinant HCV nucleocapsid protein antigen, and a sample diluent, a negative control, a positive control, an enzyme marker working solution, a substrate solution A, a substrate solution B and a washing solution which are respectively and independently packaged, wherein the substrate solution A comprises luminol, o-phenylphenol, 4-imidazole phenol and a carbonic acid buffer solution (CB); the substrate liquid B comprises carbamide peroxide and Phosphate Buffer (PB); the kit can perform in-vitro qualitative/quantitative determination on the anti-Core antibody in the serum of the HCV infected person, establish a detection technology of the anti-Core antibody in the serum of the HCV infected person and perform methodology evaluation, and can be applied to screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism and epidemiological research of the viral hepatitis C.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a chemiluminescence immunoassay qualitative/quantitative kit, in particular to a method for applying recombinant Hepatitis C Virus (HCV) nucleocapsid protein (Core) antigen coated microporous plate to detect the generated coated antigen-antibody to be detected-enzyme labeled secondary antibody compound, which can perform in-vitro qualitative/quantitative determination on the anti-Core antibody in the serum of HCV infected persons, establish the detection technology of the anti-Core antibody in the serum of HCV infected persons and perform methodology evaluation.
Background
Golafield M first reported non-A, non-B Hepatitis after transfusion in 1974, and Choo QL, Weiner AJ et al cloned the major pathogen of non-A, non-B Hepatitis using molecular biology techniques under the lead of Houghton M in 1989, followed by the gene sequence designated Hepatitis C Virus (HCV), and the resulting disease was called Hepatitis C (Hepatitis C). Hepatitis c has been shown to be a major blood-borne disease, with 50-85% of infected people becoming chronically infected and leading to chronic inflammatory necrosis and fibrosis of the liver, and 10-30% of patients developing cirrhosis of the liver, with an annual incidence of hepatocellular carcinoma (HCC) of about 1-7%, with great health and life risks to the patient, and has become a serious social and public health problem. The latest statistics of the world health organization in 2014 show that: more than about 1.85 million people worldwide are infected with HCV, with infection rates highest in east asia (3.8%), middle asia (3.7%), north/middle africa (3.6%), south asia (3.4%) and eastern europe (2.9%), with about 35 million people worldwide dying from hepatitis c-related diseases each year. The number of the hepatitis C morbidity of China is on the trend of increasing year by year, and the data of ' nationwide law definite infectious disease epidemic situation ' published by the national health and family planning committee of the people's republic of China all the year shows that: the number of patients in 2007 is less than 9.2 ten thousand, and the number of patients in 2014 is 20.3 ten thousand; according to the calculation, about 4000 ten thousand HCV infected persons exist in China, the infection rates of provinces and cities are different, the average infection rate is about 3.2 percent, and the HCV infection is a high-circulation region. With the progress of HCV infection treatment into the pan-genetic era, the Direct use of recombinant protease inhibitors (DAAs) can greatly increase the proportion (> 95%) of patients that achieve sustained virological response, and is expected to change the serious situation of HCV infection threat, but DAA treatment currently faces many problems to be solved: (1) the marginal population can not receive effective treatment, such as drug injection, prisoners, male sex, and the like, which aggravates the epidemic of diseases; (2) after treatment with the standardized DAA, there are still some patients with no sustained virologic response or treatment failure, and DAA treatment does not prevent HCV reinfection; (3) the medical insurance payment proportion is low, the treatment cost is high, and the treatment cost of a patient in each course of treatment is tens of thousands of yuan to hundreds of thousands of yuan RMB. It is thus known that chronic HCV infection remains a significant threat to human health and global public health safety for a long time in the future.
HCV belongs to the Flaviviridae family, the virion is spherical, the genome is single-strand positive-strand RNA, the full length is about 9.6kb, and has 7 genotypes (1-7) and 67 gene subtypes, which are common in China as 1b and 2a genotypes, the HCV genome consists of a 5' non-coding region (5' -NCR) at two ends, a 3' non-coding region (3' -NCR) and an Open Reading Frame (ORF) in the middle, the ORF is immediately downstream of the 5' -NCR, the genome arrangement sequence is 5' -C-E1-E2-NS1-NS2-NS3-NS4-NS5-3', a polyprotein precursor consisting of 3010-3030 amino acids can be encoded, as shown in FIG. 1, and 10 HCV proteins are produced by cleavage under the action of host cell signal peptidase and virus self-protease, as shown in FIG. 2, and comprise: 3 structural proteins, namely nucleocapsid protein (Core), envelope glycoprotein (E1, E2), transmembrane protein (P7); 6 non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS 5B). Core, E1, E2, P7 are assembled together into complete virus particles, and nonstructural proteins play an important role in the life cycle of the virus.
The Core, E1, E2 and P7 proteins are main components of HCV coat protein skeleton, wherein the Core gene is located at 342-914nt site of HCV genome, encodes 191 amino acids, is a relatively conservative structural region in HCV genome, is an important component of virus nucleocapsid, and is used for assembling complete HCV virus particles under the action of glycoprotein, the amino terminal of the Core protein is rich in basic amino acid and highly conserved, and the carboxyl terminal of the Core protein has high hydrophobicity, is an important structural protein of HCV, and plays an important role in virus proliferation and infection, chronic pathogenesis, hepatocellular carcinoma and immune dysfunction. Therefore, it is very important to detect the antibody produced by the HCV virus protein Core to stimulate the body to evaluate the disease course progress and drug efficacy of HCV infected patients, and the research in this field is receiving wide attention from experts in molecular biology, infectious diseases and epidemiology.
However, the research of the chemiluminescence immunoassay kit for separately detecting the anti-nucleocapsid protein Core antibody at home and abroad and the related technology is still blank, and foreign scholars adopt recombinant Core, E1, E2, NS3, NS4A, NS4B and NS5A. NS5B protein immunoblotting method for detecting corresponding protein antibody in HCV infected person serum
M,Melén K,Porkka P,et al.Hepatitis C virus core,NS3,NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection[J]Virol J,2009,6:84.), the results show that: the antibody positivity rates for different proteins in HCV-infected subjects are different and we analyzed that may be related to the progression of the disease in HCV-infected subjects over different courses, different genotypes, before and after DAA treatment and the presence of circulating immune complexes in serum. Because of the low sensitivity of immunoblotting, the conventional kits for clinical diagnosis of hepatitis C adopt the recombinant protein C-22 in HCV nucleocapsid region, the recombinant protein C-200 antigen in non-structural protein NS3 region, the recombinant protein C-200 and NS5 in non-structural protein NS4 region, etc. as the fourth generation reagents mixed with coating antigen to detect antibody (Warkad SD, Song KS, Pal D, et al. development in the HCV Screening Technologies Based on the Detection of antibodies and antibodies. Sensors (base) 2019,19(19):4257.), the methods for detecting antibody mainly include enzyme linked immunosorbent assay (ELISA), colloidal gold method, and chemical luminescence method disclosed in application publication No. CN103018455A directly labeled antigen antibody Detection hepatitis C virus antigen antibody and Detection kit, but these kits can only detect mixed antibody in the serum of HCV infected person, antibodies which can not distinguish which protein components exist cannot comprehensively reveal the clinical significance of a single HCV protein component in the aspects of disease monitoring, prognosis evaluation, epidemiological research and the like of HCV infected persons; for example, does Core nucleocapsid protein stimulate the body to produce antibodies (anti-Core) and circulating immune complex (Core-IC), and there are differences before and after HCV infection, in different genotypes and subtypes, different clinical stages (chronic, liver cirrhosis, liver cancer), and antiviral treatment? So far, no relevant report is found. Therefore, the establishment of a high-sensitivity detection method for detecting the corresponding antibody of the Core protein in the serum of the HCV infected person or the corresponding antibody in the circulating immune complex is of great significance for disease monitoring, prognosis evaluation and epidemiological research of the HCV infected person. Therefore, we intend to establish a method for detecting HCVThe chemiluminescence immune analysis method of anti-HCV nucleocapsid protein antibody in the blood serum of an infected person, and the methodological evaluation are carried out to complete the development of the kit, thereby providing technical support for revealing the relationship between the content change and different clinical typing, disease progression and DAA curative effect of HCV infected persons.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a chemiluminescence kit and a detection method for detecting an anti-nucleocapsid protein Core antibody in the serum of an HCV (hepatitis C Virus) infected person; the kit can perform in-vitro qualitative/quantitative determination on the anti-Core antibody in the serum of the HCV infected person, establish a detection technology of the anti-Core antibody in the serum of the HCV infected person and perform methodology evaluation, and can be applied to screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism and epidemiological research of the viral hepatitis C.
In order to achieve the above purpose, the invention provides the following technical scheme:
a chemiluminescence kit for detecting an anti-nucleocapsid protein antibody in blood serum of an HCV (hepatitis C virus) infected person is characterized by comprising a microporous plate obtained by coating a recombinant HCV nucleocapsid protein antigen, and a sample diluent, an enzyme marker working solution, a substrate solution A, a substrate solution B and a washing solution which are respectively and independently packaged, wherein the substrate solution A comprises luminol, o-phenylphenol, 4-imidazole phenol and a carbonic acid buffer solution (CB); the substrate solution B comprises carbamide peroxide and Phosphate Buffer (PB).
Preferably, the substrate solution A comprises luminol with the mass concentration of 0.8g/L, o-phenylphenol with the mass concentration of 0.008g/L, 4-imidazolylphenol with the mass concentration of 0.025g/L and carbonic acid buffer solution (CB) with the pH value of 9.0 and the molar concentration of 0.1 mol/L; the substrate solution B contained urea peroxide at a mass concentration of 0.3g/L, and a phosphate buffer solution (PB) at a pH of 7.4 and a molar concentration of 0.2 mol/L.
Preferably, the sample diluent is Phosphate Buffered Saline (PBS) containing 20% bovine serum by volume, pH7.4 and 0.01mol/L molarity.
Preferably, the negative control is serum or plasma from a heat-inactivated non-HCV-infected person (negative for anti-nucleocapsid antibody), and the human immunodeficiency virus antibody, treponema pallidum antibody, hepatitis b virus surface antigen test is negative, containing ProClin 300 preservative at a concentration of 0.2% by volume.
Preferably, the positive control is heat-inactivated HCV-infected (anti-nucleocapsid antibody positive) pooled serum or plasma, and the tests for human immunodeficiency virus antibodies, treponema pallidum antibodies, hepatitis b virus surface antigen are negative, containing ProClin 300 preservative at a volume concentration of 0.2%.
Preferably, the enzyme label working solution is prepared by diluting an enzyme label stock solution to a mass concentration of 0.05 [ mu ] g/ml by using an enzyme label antibody diluent (the enzyme label stock solution is a horse radish peroxidase-labeled goat anti-human IgG label with a mass concentration of 1 mg/ml); the enzyme-labeled antibody diluent contained 5% by volume Bovine Serum Albumin (BSA), 0.5% by volume enzyme stabilizer, 0.1% by volume Tween-20, 5. mu.g/ml anti-mouse antibody blocking agent, 1. mu.g/L red pigment, 0.2% by volume Proclin 300, pH7.4, and 0.01mol/L Phosphate Buffered Saline (PBS).
Preferably, the washing solution is Phosphate Buffered Saline (PBS) containing Tween-20 at a volume concentration of 0.5%, Proclin 300 at a volume concentration of 0.2%, pH7.4 and a molarity of 0.01 mol/L; when in use, the washing solution is diluted by 20 times of concentrated washing solution according to the proportion of 1:19, and the 20 times of concentrated washing solution is Phosphate Buffered Saline (PBS) containing 10% Tween-20 by volume, 4% Proclin 300 by volume, pH7.4 and 0.2mol/L molar concentration.
The specific washing method comprises discarding the liquid in the hole, filling 300 μ l of diluted washing liquid into each hole, standing for no more than 60 s, discarding the washing liquid in the hole, washing for 3-5 times, and drying.
A chemiluminescent assay for detecting anti-nucleocapsid protein antibodies in the serum of an HCV-infected individual comprising the steps of:
(1) adding 100 mul of sample diluent into a recombinant HCV anti-nucleocapsid protein antigen coated microporous plate, adding 10 mul of sample to be detected, covering a sealing plate membrane, oscillating, uniformly mixing, incubating for 30min at the temperature of 37 +/-1 ℃, and washing to remove unbound components;
(2) setting antibody positive control 1 hole and negative control 2 holes, respectively adding negative control and positive control 100 mul each; setting a blank control hole 1 hole without adding any sample; covering a sealing plate film, oscillating and uniformly mixing, incubating for 30min at the temperature of 37 +/-1 ℃, and washing to remove unbound components;
(3) adding 100 mul of enzyme marker working solution into the rest holes except the blank control hole, covering a sealing plate film, oscillating and uniformly mixing, incubating for 30min at the temperature of 37 +/-1 ℃, and washing to remove the unbound enzyme marker working solution;
(4) sequentially adding 50 mul of substrate solution B and substrate solution A into each hole, oscillating, uniformly mixing, and placing for 15 minutes at room temperature in a dark place;
luminol is oxidized by horseradish peroxidase in an alkaline environment and is in an excited state, when the luminol returns to a ground state from the excited state, light with the maximum emission wavelength of 425nm is radiated, a corresponding signal value is obtained after signal conversion is carried out on a light signal generated by enzymatic luminescence through a photomultiplier, and the relative luminescence intensity ratio is in direct proportion to the concentration of an anti-Core antibody in the serum of an HCV infected person;
(5) measuring the relative luminescence intensity value RLIV of each hole by using a chemiluminescence immunoassay analyzer, and determining that the sample RLIV is positive when the ratio of the sample RLIV to the negative control RLIV mean value is more than or equal to 2.1; otherwise, it is negative.
A micro-porous plate for detecting anti-nucleocapsid protein antibody in HCV infected person serum, the antigen of the coating micro-porous plate is recombinant HCV nucleocapsid protein antigen (Core); the method for coating the micro-porous plate comprises the following steps: diluting the recombinant HCV anti-nucleocapsid protein antigen to 0.5 mu g/ml by adopting a coating solution, adding the diluted HCV anti-nucleocapsid protein antigen into a microporous plate according to the quantity of 100 mu l, keeping the temperature of 2-8 ℃ overnight or keeping the temperature of 37 ℃ for 2h, washing the protein which is not adsorbed on the plate by using a washing solution, repeatedly washing for 3 times by using 300 mu l of washing solution in each hole during use, and patting the solution dry;
after the coated microporous plate is washed, patted dry, immediately sealing; adding the sealing liquid into the coated microporous plate according to the amount of 200 mul per hole, sealing at 2-8 ℃ overnight or 37 ℃ for 2h, and throwing off the sealing liquid after sealing to facilitate the long-term storage of the coated microporous plate.
Preferably, the coating solution is Phosphate Buffer (PB) with pH7.4 and a molar concentration of 0.02 mol/L; the blocking solution contained 5% by mass/volume Bovine Serum Albumin (BSA), 1% by mass/volume sucrose, 4% by mass/volume gelatin, 0.2% by volume Proclin 300, and 0.01mol/L molarity Phosphate Buffered Saline (PBS), pH 7.4.
Preferably, when the RLIV value of the reagent blank is less than 30000, otherwise the experiment is invalid, and the test needs to be repeated; each negative control value is not more than 115000, and the difference between the reading of the two negative control wells and the average value of the negative control wells is not more than 10 percent, otherwise the experiment is invalid and needs to be checked repeatedly; each positive control value should be >241500, otherwise the experiment is invalid and the test is repeated.
Compared with the prior art, the invention has the beneficial effects that:
the micropore of the kit is pre-coated with recombinant Core protein antigen, and an anti-Core antibody to be detected in the serum of a detected sample is combined with a corresponding antigen on the surface of a solid phase carrier; separating the antigen-antibody complex formed on the solid phase carrier from other substances in the liquid by washing; then adding horseradish peroxidase-labeled secondary antibody to be combined on a solid phase carrier; adding a reaction substrate of luminol, wherein the luminol is oxidized by horseradish peroxidase in an alkaline environment and is in an excited state, when the luminol returns to a ground state from the excited state, light with the maximum emission wavelength of 425nm is radiated, a corresponding signal value is obtained after a light signal generated by enzymatic luminescence is subjected to signal conversion through a photomultiplier, RLIR (relative luminescence intensity ratio) is used for representing, and the concentration of the anti-Core antibody to be detected and the chemiluminescence intensity are in a linear quantitative relationship under a certain condition; the catalytic efficiency of the enzyme is high, so that the result of immune reaction is indirectly amplified, and the determination method has high sensitivity.
The kit can perform in-vitro qualitative/quantitative determination on the anti-Core antibody in the serum of the HCV infected person, establish the detection technology of the anti-Core antibody in the serum of the HCV infected person and perform methodology evaluation, and can be applied to screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism and epidemiological research of the viral hepatitis C.
Drawings
FIG. 1 shows the structural components of HCV genes and encoded polyprotein.
FIG. 2 is a 3D diagram of the structures of HCV structural and non-structural proteins.
FIG. 3 is a linear evaluation of chemiluminescence kit for anti-Core antibody
FIG. 4 is a schematic diagram of a chemiluminescence kit for an anti-Core antibody.
Detailed description of the preferred embodiments
The HCV genome is single-strand positive strand RNA, has about 9.6kb in total length, has 7 genotypes (1-7) and 67 gene subtypes, is common in 1b and 2a genotypes in China, consists of a 5' non-coding region (5' -NCR) and a 3' non-coding region (3' -NCR) at two ends and an Open Reading Frame (ORF) in the middle, is immediately downstream of the 5' -NCR, and has the genome arrangement sequence of 5' -C-E1-E2-NS1-NS2-NS3-NS4-NS5-3', and can encode a polyprotein precursor consisting of 3010-3030 amino acids, as shown in figure 1. Cleavage by host cell signal peptidase and viral autoproteases produces 10 HCV proteins, as shown in figure 2, including: 3 structural proteins, namely nucleocapsid protein (Core), envelope glycoprotein (E1, E2), transmembrane protein (P7); 6 non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS 5B).
At present, the research of the chemiluminescence immunoassay kit for separately detecting the anti-nucleocapsid protein Core antibody at home and abroad and the related technology is blank, foreign scholars adopt recombinant Core, E1, E2, NS3, NS4A, NS4B, NS5A and NS5B proteins to establish an immunoblotting method to detect the corresponding protein antibody in the serum of HCV infected persons, and the results show that: the antibody positivity rates for different proteins in HCV-infected subjects are different and we analyzed that may be related to the progression of the disease in HCV-infected subjects over different courses, different genotypes, before and after DAA treatment and the presence of circulating immune complexes in serum. Because the sensitivity of the immunoblotting method is not high, the conventional kits for clinically diagnosing hepatitis C adopt HCV nucleocapsid region recombinant protein C-22, non-structural protein NS3 region recombinant protein C-200 antigen, non-structural protein NS4 region recombinant protein C-200 and NS5 and the like as fourth generation reagents of mixed envelope antigens to detect antibodies, and the methods for detecting the antibodies mainly comprise enzyme-linked immunosorbent assay (ELISA), colloidal gold methods and the like, but the kits can only detect the mixed antibodies in the serum of HCV infectors and can not distinguish which protein component is the antibody, and can not fully reveal the clinical significance of a single HCV protein component in the aspects of disease monitoring, prognosis evaluation, epidemiological research and the like of the HCV infectors; for example, does Core nucleocapsid protein stimulate the body to produce antibodies (anti-Core) and circulating immune complex (Core-IC), and there are differences before and after HCV infection, in different genotypes and subtypes, different clinical stages (chronic, liver cirrhosis, liver cancer), and antiviral treatment? So far, no relevant report is found. Therefore, the establishment of a high-sensitivity detection method for detecting the corresponding antibody of the Core protein in the serum of the HCV infected person or the corresponding antibody in the circulating immune complex is of great significance for disease monitoring, prognosis evaluation and epidemiological research of the HCV infected person.
The embodiment provides a chemiluminescence kit for detecting an anti-nucleocapsid protein antibody in serum of an HCV (hepatitis C Virus) infected person, which is used for in-vitro auxiliary diagnosis or disease mechanism research, wherein the detection kit adopts a chemiluminescence immunoassay method and is based on the principle of an ELISA indirect method:
the recombinant Core protein antigen is coated in the micropores in advance, and an anti-Core antibody to be detected in the serum of a detected sample is combined with a corresponding antigen on the surface of the solid phase carrier; separating the antigen-antibody complex formed on the solid phase carrier from other substances in the liquid by washing; then adding horseradish peroxidase-labeled secondary antibody to be combined on a solid phase carrier; adding a reaction substrate of luminol, wherein the luminol is oxidized by horseradish peroxidase in an alkaline environment and is in an excited state, when the luminol returns to a ground state from the excited state, light with the maximum emission wavelength of 425nm is radiated, a corresponding signal value is obtained after a light signal generated by enzymatic luminescence is subjected to signal conversion through a photomultiplier, RLIR (relative luminescence intensity ratio) is used for representing, and the concentration of the anti-Core antibody to be detected and the chemiluminescence intensity are in a linear quantitative relationship under a certain condition; the catalytic efficiency of the enzyme is high, so that the result of immune reaction is indirectly amplified, and the determination method has high sensitivity.
Firstly, preparing a microporous plate for detecting anti-Core antibody
1. Micro-porous plate: 12 x 8 (detachable), a 96-hole microporous luminescent plate Nunc made of polypropylene used as a solid phase carrier; microplate Nunc is available from Thermo Fisher, USA. The microporous plate is coated with recombinant HCV Core protein antigen, and the Core recombinant protein is purchased from Israel ProSpec-Tany gene technology Co., Ltd, the specification is 100ug/100ul, and the batch number is 718PHCV 2211;
2. diluting: diluting the recombinant HCV Core protein antigen (the original calibration mass concentration is 1mg/ml) to 0.5 mu g/ml by adopting a coating solution [ Phosphate Buffer (PB) buffer solution with the pH value of 7.4 and the molar concentration of 0.02mol/L ];
3. coating: adding the diluted recombinant HCV Core protein antigen into a microplate according to the amount of 100 μ l, and standing overnight at 2-8 deg.C (overnight for 20-24h), or at 37 deg.C for 2 h;
4. washing the plate after coating: washing with washing solution to remove protein not adsorbed on the plate, washing with washing solution for 3 times repeatedly at a volume of 300 μ l per well, and patting to dry; wherein the concentrated washing solution (20X) is Phosphate Buffered Saline (PBS) containing 10% Tween-20 by volume, 4% Proclin 300 by volume and 0.2mol/L by mol concentration in pH7.4, and is diluted into washing solution (1X) by a ratio of 1:19 before use, and the washing solution (1X) is Phosphate Buffered Saline (PBS) containing 0.5% Tween-20 by volume, 0.2% Proclin 300 by volume and 0.01mol/L by mol concentration in pH7.4 for standby;
after the coated microporous plate is washed and patted dry, the coated microporous plate is immediately sealed, and if the drying time after plate washing exceeds half an hour, the activity of the coated antigen is obviously reduced;
5. and (3) sealing: adding a blocking solution (containing 5% by mass and volume Bovine Serum Albumin (BSA), 1% by mass and volume sucrose, 4% by mass and volume gelatin, 0.2% by volume Proclin 300 and 0.01mol/L Phosphate Buffered Saline (PBS) with the pH value of 7.4) into the coated microplate according to the amount of 200 mul per well, and blocking at the temperature of 2-8 ℃ for 20-24h or at the temperature of 37 ℃ for 2 h;
6. closing the back throwing plate: the sealing liquid is thrown away, which is beneficial to the sealing and long-term storage of the coated microporous plate.
7. And (3) drying: removing residual washing liquid on the microporous plate, sealing for long-term storage, if it is used in a short period, directly patting to dry, adding desiccant, and refrigerating at 2-8 deg.C for use.
Secondly, preparation of the kit
1. Preparation of sample diluent:
the sample diluent is Phosphate Buffered Saline (PBS) containing 20% bovine serum by volume, pH7.4 and 0.01mol/L molarity, and the preparation method is as follows: adding 200ml bovine serum into a 1L volumetric flask, and adding Na2HPO4·12H2O 2.9g，NaH2PO4·2H20.296g of O, 9g of NaCl and 3002ml of preservative Proclin, finally adding double distilled water to a constant volume of 1L, wherein the pH value of the solution is 7.4 +/-0.2, and refrigerating the prepared sample diluent in a refrigerator at 2-8 ℃ for later use.
2. Negative control preparation:
the negative control was serum or plasma from heat-inactivated non-HCV infected subjects (negative for anti-Core antibodies), and negative for the tests with hiv antibodies, treponema pallidum antibodies, hepatitis b virus surface antigen, containing ProClin 300 preservative at a concentration of 0.2% by volume.
3. Preparation of positive control:
the positive control is heat inactivated HCV infected person (positive anti-Core antibody) mixed serum or plasma, and human immunodeficiency virus antibody, treponema pallidum antibody and hepatitis B virus surface antigen test is negative, and contains 0.2% of ProClin 300 preservative by volume concentration. The preparation method comprises the following steps: collecting mixed serum or plasma of HCV infected person (human immunodeficiency virus antibody, treponema pallidum antibody, hepatitis B virus surface antigen test is negative), concentrating IgG antibody (50ml is concentrated to 10ml) by using U-Tube protein ultrafiltration concentration (30KD) Tube (purchased from Merck in Germany, model is: U-Tube 20-30), and adding ProClin 300 preservative with volume concentration of 0.2% into the concentrated mixed serum or plasma of HCV infected person.
4. Washing liquid:
the concentrated washing solution (20X) was phosphate buffered saline (pH7.4) containing Tween-20 at a volume concentration of 10%, Proclin 300 at a volume concentration of 4%, and a molar concentration of 0.2mol/LThe preparation method of the saline solution (PBS) comprises the following steps: adding Na into a 1L volumetric flask2HPO4·12H2O 58.0g，NaH2PO4·2H2O5.92 g, NaCl 180g, Tween-20100 ml and a preservative Proclin 30040 ml, finally adding double distilled water to a constant volume of 1L, wherein the pH value of the solution is 7.4 +/-0.2, and placing the prepared concentrated lotion (20 x) at normal temperature for later use.
5. Preparation of enzyme-labeled working solution
The working solution of the enzyme label is obtained by diluting an enzyme label stock solution to a mass concentration of 0.05 mu g/ml by using an enzyme label antibody diluent (the enzyme label stock solution is a horse radish peroxidase-labeled goat anti-human IgG label with a mass concentration of 1mg/ml, and the enzyme label stock solution is purchased from Hangzhou Longji Biotech Co., Ltd.); the enzyme-labeled antibody diluent is prepared from 5% by mass of Bovine Serum Albumin (BSA), 0.5% by mass of an enzyme stabilizer, 0.1% by volume of Tween-20, 5 μ g/ml by mass of an anti-mouse antibody blocking agent, 1 μ g/L by mass of a red pigment, 0.2% by volume of Proclin 300, pH7.4 and 0.01mol/L by mole of Phosphate Buffered Saline (PBS) by a preparation method comprising the following steps: adding Na into a 1L volumetric flask2HPO4·12H2O 2.9g，NaH2PO4·2H20.296g of O, 9g of NaCl, 3002ml of preservative Proclin, 50g of Bovine Serum Albumin (BSA), 5ml of enzyme stabilizer, 201 ml of Tween-201, 5mg of anti-mouse antibody blocking agent and 1 mu g of red pigment, finally adding double distilled water to a constant volume of 1L, setting the pH value of the solution to be 7.4 +/-0.2, and refrigerating the prepared enzyme-labeled antibody diluent at 2-8 ℃ for later use.
6. Substrate solution B:
the substrate solution B is a phosphate buffer solution (PB) containing carbamide peroxide with a mass concentration of 0.3g/L, a pH value of 7.4 and a molar concentration of 0.2mol/L, and the preparation method comprises the following steps: adding Na into a 1L volumetric flask2HPO4·12H2O 58.0g，NaH2PO4·2H2O5.92 g and carbamide peroxide 0.3g, and finally adding double distilled water to a constant volume of 1L, wherein the pH value of the solution is 7.4 +/-0.2; and storing the prepared substrate solution B at room temperature for later use.
7. Substrate solution A:
the substrate solution A was a carbonate buffer solution (CB) containing 0.8g/L luminol, 0.008g/L orthophenylphenol, 0.025 g/L4-imidazolylphenol, pH9.0, and 0.1mol/L molarity, and prepared by the following method: adding 0.56g of anhydrous sodium carbonate, 7.96g of sodium bicarbonate, 0.8g of luminol, 0.008g of o-phenylphenol and 0.025g of 4-imidazole phenol into a 1L constant volume bottle, and finally adding double distilled water to constant volume to 1L, wherein the pH value of the solution is 9.0 +/-0.1; and (4) preparing the substrate solution A, and refrigerating the substrate solution A at the temperature of 2-8 ℃ in a dark place for later use.
8. A sealing plate film and a self-sealing bag (containing a drying agent).
9. Each kit of reagent is composed of sample diluent, negative control, positive control, enzyme marker working solution, substrate solution A, substrate solution B, washing solution, a microporous plate (96 holes) for detecting anti-Core antibody, a sealing plate film, a self-sealing bag (containing drying agent) and the like which are respectively and independently packaged as shown in figure 4.
Thirdly, collecting serum: collecting venous blood to collect serum or collecting plasma by using ethylene diamine tetraacetic acid disodium salt or potassium salt (EDTA) anticoagulation, and storing the serum or plasma sample detected within one week at 2-8 ℃, wherein if the serum or plasma sample needs to be stored for a long time, the serum or plasma sample needs to be placed at-20 ℃ to avoid repeated freezing and thawing and cross contamination.
Fourth, detection method
1. Preparation of
(1) The kit components were removed from the box and allowed to equilibrate to room temperature.
(2) Adding the concentrated washing solution into deionized water or distilled water according to the proportion of 1:19, and uniformly mixing for later use.
2. Detection of
(1) Sample adding: fixing the recombinant HCV anti-nucleocapsid protein antigen coated microporous plate on a plate frame, setting 2 holes of negative control and 1 hole of positive control in each test, and respectively adding 100 mul of negative control and positive control; setting a blank control hole 1 hole without adding any sample;
adding 100 mul of sample diluent into the rest holes, adding 10 mul of samples to be detected, covering a sealing plate membrane, oscillating, uniformly mixing, incubating for 30min at the temperature of 37 +/-1 ℃, and washing to remove the unbound components;
(2) adding an enzyme marker working solution: adding no enzyme marker working solution into the blank control holes, and adding 100 mu l of enzyme marker working solution into the other holes;
(3) and (3) incubation: covering a sealing plate film, oscillating and uniformly mixing, and incubating for 30min at the temperature of 37 +/-1 ℃;
(4) washing: discarding liquid in the holes, filling the diluted washing liquid into each hole, standing for no more than 60 seconds, discarding the washing liquid in the holes, repeatedly washing for 5 times, and then drying by patting;
(5) color development: sequentially adding 50 mul of substrate solution B and substrate solution A into each hole, oscillating, uniformly mixing, and placing for 15 minutes at room temperature in a dark place;
(6) and (3) detection: measuring Relative Luminescence Intensity Value (RLIV) of each hole by using a chemiluminescence immunoassay analyzer (TZD-CL-200S), calculating relative luminescence intensity ratio (RLIR ═ sample RLIV/negative control RLIV mean value) of each hole, and determining that the sample RLIV is positive when the ratio of the sample RLIV to the negative control RLIV mean value is more than or equal to 2.1; otherwise, it is negative.
(7) Quality control
The blank value of the reagent is less than 30000, otherwise, the experiment is invalid and needs to be checked repeatedly.
And secondly, each negative control value is not more than 115000, the difference between the reading of the two negative control holes and the average value of the negative control is not more than 10 percent, otherwise, the experiment is invalid and needs to be checked repeatedly.
③ every positive control should be >241500, otherwise the experiment is invalid and needs to be checked repeatedly.
Fifth, detecting instrument
Semi-automatic chemiluminescence immunoassay analyzer (TZD-CL-200S, Xiamen Tianzhongda Biotech Co., Ltd.)
Sixth, experimental data
1. Precision test
Imprecision within the batch: the screened samples with high and low concentrations are taken, the detection is repeated for 20 times (holes), the RLIR mean value and the SD are calculated, and the CV percent in the batch is calculated.
Batch imprecision: the screened samples with high and low concentrations are taken and respectively detected 1 time in the morning and afternoon every day, 20 batches of detection is carried out for 10 days, the RLIR mean value and SD are calculated, the CV percent between batches is calculated, and the result is shown in Table 1.
Table 1 precision experimental statistics and performance determination (n ═ 20)
As can be seen from Table 1, the intra-batch coefficient of variation CV% was less than 10% and the inter-batch coefficient of variation CV% was less than 15%.
2. Margin and detection limits
(1) Blank limit: selecting 5 uninfected HCV examinee serums, measuring 2 batches of the uninfected HCV examinees each day for 6 days, and recording RLIR values to obtain 60 results in total; class I errors (α ═ 0.05), LoB, were 5% likely to contain the test substance; the results of the blank samples were sorted from small to large by non-parametric tests, and the 95 th percentile value was the blank limit LoB, and the results are shown in table 2.
TABLE 2 blank sample assay results (RLIR)
As can be seen from Table 2, the 95 th percentile of the blank sample measurements was estimated by a nonparametric method, i.e., the 95 th percentile was found to be 1.13 RLIR.
(2) Detection limit
Selecting 5 parts of a serum sample with the expected concentration of 1-5 times LoB as a low-concentration sample, detecting for 2 times every day, continuously measuring for 6 days, and calculating LoD by adopting a nonparametric analysis method; the results of 12 consecutive measurements of 5 low concentration samples are shown in table 3, the results are in a non-normal distribution, and the LOD is estimated using a non-parametric program, namely: LOD ═ LOB + Ds. β, Ds. β is the difference between the median (M) and the 5 th percentile values determined for the low concentration samples.
TABLE 3 dilution series sample assay results (RLIR)
As shown in Table 3, the median of the values obtained in the serial dilution samples was (30 th order value + 31 th order value)Rank/2 ═ 3.48RLIR, DS-βThe median result, 5 th percentile (1.91), LoD absorbance LoB + DS-βSince 1.13+1.91 is 3.04, the detection limit LoD concentration of the kit is 3.04 RLIR.
3. Linearity
Analytical Measurement Range (AMR): according to the design of the file requirement of EP6-A2, 6 samples are prepared according to the proportion of 5L, 4L +1H, 3L +2H, 2L +3H, 1L +4H and 5H by taking the average value of 2 detections of each sample and taking the expected average value and the measured average value as the abscissa and the ordinate respectively to obtain a regression equation (shown in figure 3), wherein the anti-Core antibody y is 0.991X-0.141, the b is between 0.97 and 1.03, the correlation coefficient r is not less than 390.975 (r2 is not less than 0.95), and the analysis measurement range of the anti-Core antibody is respectively 1.64-17.06 IR, which can be seen in figure 3.
4. Interference experiment
The serum specimen positive to rheumatoid factor, hepatitis B virus surface antibody, hepatitis B virus e antibody, treponema pallidum antibody, human immunodeficiency virus I and/or II antibody is mixed with the positive serum of anti-Core (8.53RLIR) antibody in the same amount, and the sample diluent is used as the reference, so that no cross reaction occurs in the result.
5. Example applications
The developed anti-HCV nucleocapsid protein Core antibody detection reagent is adopted to detect corresponding antibodies in the serum of HCV infected persons (45 cases of chronic HCV infected persons are randomly selected), and the positive rate of the anti-Core antibody in the chronic HCV infected persons is counted, and the result is shown in table 4.
TABLE 4 survey of serum anti-Core antibody positivity of chronic HCV-infected patients
Injecting: an anti-HCV mixed antibody screening kit produced by Xiamen creative company is adopted.
As is clear from Table 4, the anti-Core antibody positivity (positive for RLIR. gtoreq.2.1) was 100% (45/45).
6. Selection of optimum enzyme-labeled antibody concentration and antigen working concentration
(1) 50. mu.l, 100. mu.l and 150. mu.l of enzyme-labeled antibody were added to the coated wells, incubated, washed, and after addition of a luminescent substrate, RLIV was read, and the results are shown in Table 5.
TABLE 5 selection of enzyme-labeled antibody work (RLIV)
As can be seen from Table 5, the sample addition amounts of 50. mu.l and 100. mu.l had a greater effect on the sensitivity and a lesser effect on the negative samples; the sample addition amount of 100. mu.l had a smaller effect on sensitivity and a larger effect on background than that of 150. mu.l. The sample adding amount of 50uI and 100 mul enzyme conjugate both act in the effective space of the coating, so the sensitivity is in a better linear relation, and the background is not influenced, while the sample adding amount of 150 mul enzyme conjugate is outside the effective space of the coating plate, so the sensitivity influence is not large, and the enzyme conjugate has non-specific adsorption of a blank plate which is partially or not completely sealed, so the background is much higher. From this, it was found that the optimum amount of enzyme conjugate to be added to the anti-Core antibody detection kit was 100. mu.l.
(2) Chessboard titration method for selecting working concentration of coating antigen
Firstly, serially diluting an antigen by using a coating solution (after the antigen concentration is 1mg/ml, the antigen concentration is 0.25 mug/ml, 0.5 mug/ml, 1 mug/ml, 2ug/ml and 4 mug/ml, coating is carried out according to the row at the temperature of 2-8 ℃ for overnight, and washing is carried out;
adding sample for positive control and negative control, keeping temperature and washing;
thirdly, adding an enzyme-labeled antibody diluted according to the working concentration, and performing heat preservation and washing;
adding substrate for color development, and reading RLIV value.
TABLE 6 Core protein antigen coating concentration comparison (RLIV)
As can be seen from table 6, the coating concentration of the antigen has a large influence on the coating result: within 2-0.5ug/ml, the antigens adsorbed on the coated plate are all in a saturated state, and the influence on the result is large; further dilution down significantly affected the sensitivity, demonstrating that 0.5ug/ml (1:2000) was at the critical point for saturation of antigen coating, and that the coating concentration was most economical.
Claims (10)
1. A chemiluminescence kit for detecting an anti-nucleocapsid protein antibody in blood serum of an HCV (hepatitis C virus) infected person is characterized by comprising a microporous plate obtained by coating a recombinant HCV nucleocapsid protein antigen, and a sample diluent, a negative control, a positive control, an enzyme marker working solution, a substrate solution A, a substrate solution B and a washing solution which are independently packaged respectively, wherein the substrate solution A comprises luminol, o-phenylphenol, 4-imidazole phenol and a carbonic acid buffer solution (CB); the substrate solution B comprises carbamide peroxide and Phosphate Buffer (PB).
2. The chemiluminescent kit for detecting the anti-nucleocapsid protein antibody in the serum of an HCV-infected person according to claim 1, wherein the substrate solution A comprises luminol with a mass concentration of 0.8g/L, o-phenylphenol with a mass concentration of 0.008g/L, 4-imidazolylphenol with a mass concentration of 0.025g/L, a carbonic acid buffer (CB) with a pH of 9.0 and a molar concentration of 0.1 mol/L; the substrate solution B contained urea peroxide at a mass concentration of 0.3g/L, and a phosphate buffer solution (PB) at a pH of 7.4 and a molar concentration of 0.2 mol/L.
3. The chemiluminescent kit for detecting antibodies to the nucleocapsid protein in the serum of an HCV-infected person of claim 1 wherein the sample diluent is Phosphate Buffered Saline (PBS) containing 20% by volume bovine serum, pH7.4 and 0.01 mol/L.
4. The chemiluminescent kit for detecting anti-nucleocapsid protein antibodies in serum of an HCV infected person according to claim 1 wherein the negative control is heat inactivated non-HCV infected person (anti-nucleocapsid antibody negative) serum or plasma and tests negative for human immunodeficiency virus antibodies, treponema pallidum antibodies, hepatitis b virus surface antigen, ProClin 300 preservative at a concentration of 0.2% by volume;
the positive control is heat inactivated HCV infected person (anti-nucleocapsid protein antibody positive) mixed serum or plasma, and human immunodeficiency virus antibody, treponema pallidum antibody and hepatitis B virus surface antigen test is negative, and contains ProClin 300 preservative with volume concentration of 0.2%.
5. The chemiluminescent kit for detecting the anti-nucleocapsid protein antibody in the serum of the HCV infected person according to claim 1, wherein the enzyme label working solution is prepared by diluting an enzyme label stock solution with an enzyme label antibody diluent to a mass concentration of 0.05 μ g/ml, and the enzyme label stock solution is a horse radish peroxidase-labeled goat anti-human IgG label with a mass concentration of 1 mg/ml; the enzyme-labeled antibody diluent contained 5% by volume Bovine Serum Albumin (BSA), 0.5% by volume enzyme stabilizer, 0.1% by volume Tween-20, 5. mu.g/ml anti-mouse antibody blocking agent, 1. mu.g/L red pigment, 0.2% by volume Proclin 300, pH7.4, and 0.01mol/L Phosphate Buffered Saline (PBS).
6. The chemiluminescent kit for detecting the anti-nucleocapsid protein antibody in the serum of an HCV-infected person of claim 1 wherein the washing solution comprises Tween-20 at a volume concentration of 0.5%, Proclin 300 at a volume concentration of 0.2%, Phosphate Buffered Saline (PBS) at pH7.4 and a molar concentration of 0.01 mol/L.
7. A chemiluminescent assay for detecting an anti-nucleocapsid protein antibody in the serum of an HCV-infected person comprising the steps of:
(1) adding 100 mul of sample diluent into a recombinant HCV anti-nucleocapsid protein antigen coated microporous plate, adding 10 mul of sample to be detected, covering a sealing plate membrane, oscillating, uniformly mixing, incubating for 30min at the temperature of 37 +/-1 ℃, and washing to remove unbound components;
(2) setting antibody positive control 1 hole and negative control 2 holes, respectively adding negative control and positive control 100 mul each; setting a blank control hole 1 hole without adding any sample; covering a sealing plate film, oscillating and uniformly mixing, incubating for 30min at the temperature of 37 +/-1 ℃, and washing to remove unbound components;
(3) adding 100 mul of enzyme marker working solution into the rest holes except the blank control hole, covering a sealing plate film, oscillating and uniformly mixing, incubating for 30min at the temperature of 37 +/-1 ℃, and washing to remove the unbound enzyme marker working solution;
(4) sequentially adding 50 mul of substrate solution B and substrate solution A into each hole, oscillating, uniformly mixing, and placing for 15 minutes at room temperature in a dark place; wherein the substrate solution A comprises luminol, o-phenylphenol, 4-imidazole phenol and carbonic acid buffer solution (CB); substrate solution B includes urea peroxide and phosphate buffer (PB;
(5) measuring the relative luminescence intensity value RLIV of each hole by using a chemiluminescence immunoassay analyzer, and determining that the sample RLIV is positive when the ratio of the sample RLIV to the negative control RLIV mean value is more than or equal to 2.1; otherwise, it is negative.
8. The chemiluminescent detection method of claim 7 wherein the antigen coated microwell plate is recombinant HCV transmembrane protein antigen (P7);
the preparation method of the coated microporous plate comprises the following steps: diluting the recombinant HCV anti-nucleocapsid protein antigen to 0.5 mu g/ml by adopting a coating solution, adding the diluted HCV anti-nucleocapsid protein antigen into a microporous plate according to the quantity of 100 mu l, keeping the temperature of 2-8 ℃ overnight or keeping the temperature of 37 ℃ for 2h, washing the protein which is not adsorbed on the plate by using a washing solution, repeatedly washing for 3 times by using 300 mu l of washing solution in each hole during use, and patting the solution dry; after the coated microporous plate is washed, patted dry, immediately sealing;
adding the sealing liquid into the coated microporous plate according to the amount of 200 mul per hole, sealing at 2-8 ℃ overnight or 37 ℃ for 2h, and throwing off the sealing liquid after sealing to facilitate the long-term storage of the coated microporous plate.
9. The chemiluminescent assay for the detection of anti-nucleocapsid protein antibodies in the serum of an HCV-infected person of claim 8 wherein the coating solution is phosphate buffered saline (PB) at pH7.4 and a molarity of 0.02 mol/L; the blocking solution contained 5% by mass/volume Bovine Serum Albumin (BSA), 1% by mass/volume sucrose, 4% by mass/volume gelatin, 0.2% by volume Proclin 300, and 0.01mol/L molarity Phosphate Buffered Saline (PBS), pH 7.4.
10. The chemiluminescent assay for the detection of anti-nucleocapsid protein antibodies in serum of an HCV infected person of any one of claims 7 to 9 wherein the test is repeated when the RLIV value of the reagent blank is <30000, otherwise the test is invalid; each negative control value is not more than 115000, and the difference between the reading of the two negative control wells and the average value of the negative control wells is not more than 10 percent, otherwise the experiment is invalid and needs to be checked repeatedly; each positive control value should be >241500, otherwise the experiment is invalid and the test is repeated.
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