CN112255414A - Chemiluminescence kit for detecting anti-nonstructural protein antibody in serum of HCV (hepatitis C Virus) infected person and detection method - Google Patents
Chemiluminescence kit for detecting anti-nonstructural protein antibody in serum of HCV (hepatitis C Virus) infected person and detection method Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and discloses a chemiluminescence kit for detecting an anti-nonstructural protein antibody in serum of an HCV (hepatitis C virus) infected person, which comprises a microporous plate obtained by coating a recombinant HCV nonstructural 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 liquid B comprises carbamide peroxide and Phosphate Buffer (PB); the kit can perform in-vitro qualitative/quantitative determination on the antibodies against the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in the serum of HCV infectors, establishes the detection technology for the antibodies against the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in the serum of the HCV infectors and performs 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 field of biotechnology, relates to a chemiluminescence immunoassay qualitative/quantitative kit, and particularly relates to a detection technology and a chemical evaluation method for anti-nonstructural protein (NS2, NS3, NS 448, NS4B, NS5A and NS5B) antibodies in serum of HCV infectors by applying recombinant Hepatitis C Virus (HCV) nonstructural protein antigens (NS2, NS3, NS4A, NS4B, NS5A and NS5B) coated micro-plate and detecting generated coated antigen-antibody-enzyme labeled secondary antibody complex, so as to establish the detection technology and the chemical evaluation of the anti-nonstructural protein (NS2, NS3, NS4A, NS4B, NS5A and NS5B) antibodies in the serum of the HCV infectors.
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 age, the Direct use of recombinant protease inhibitors (DAAs) can greatly increase the proportion of patients that achieve sustained virological response (> 95%), which 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 HCV non-structural protein consists of NS2(2769-3419nt, 217aa), NS3(3420-5312nt, 631aa), NS4A (5313-5474nt, 54aa), NS4B (5475-6257nt, 261aa), NS5A (6258-7601nt, 448aa) and NS5B (7602-9371nt, 591aa)6 proteins, wherein the NS2 protein is a strong hydrophobic transmembrane protein and forms an NS2-3 protease complex with self-cleavage function together with the NS3 protein; the NS3 protein binds to the 3' end of the HCV genome, plays a regulatory role in viral RNA replication, and the NS3 gene is one of the important targets for the development of vaccines and anti-HCV drugs; the NS4A protein is used as a cofactor of the NS3 serine protease, forms a stable protein complex with the NS3 protein, and has important regulation effect on the activity of the serine protease; the NS4B protein is a highly hydrophobic membrane-associated localization protein, and can regulate the activity of RNA-dependent RNA polymerase of the NS5B protein; the NS5A protein is a phosphorylated, non-structural protein involved in the maturation and RNA replication of various HCV proteins; the activity of RNA-dependent RNA polymerase of NS5B protein is a key enzyme for HCV RNA replication, and RNA synthesis process guided by the activity of similar RNA-dependent RNA polymerase does not exist in mammalian cells, so RdRp and its coding gene NS5B become an ideal target for anti-HCV drug research. Nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) play an important role in viral proliferation and infection, chronic pathogenesis, hepatocellular carcinoma, immune dysfunction. Therefore, it is very important to evaluate the progress of the disease course and the therapeutic effect of the drugs of HCV-infected patients by detecting the antibodies produced by the stimulation of the HCV virus nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS 5B).
However, the research of chemiluminescence immunoassay kits for separately detecting antibodies against non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) at home and abroad and related technologies is still blank, and foreign scholars adopt recombinant Core, E1, E2, NS3, NS4A, NS4B, NS5A and NS5B proteins to establish an immunoblotting method to detect corresponding protein antibodies in the serum of HCV infected persons ((the method is applied to the detection of corresponding protein antibodies in the serum of HCV infected persons: (the method is applied to the detection of the HCV infected persons)
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 have employed HCV nucleocapsid region recombinant protein C-22, nonstructural protein NS3 region recombinant protein C-200 antigen, nonstructural protein NS4 region recombinant protein C-200 and NS5 as the fourth generation reagents mixed with coating antigen to detect antibodies (Warkad SD, Song KS, Pal D, et alGies Based on the Detection of antibiotics and antibodies.sensors (base). 4257.), the methods for detecting antibodies mainly include enzyme-linked immunosorbent assay (ELISA), colloidal gold method, and direct labeling of antigen by chemiluminescence method as disclosed in application publication No. CN103018455A to detect hepatitis c virus antigen antibody and detection kit, but these kits can only detect mixed antibodies in serum of HCV-infected person, cannot distinguish which protein component is antibody, and cannot fully reveal clinical significance of single HCV protein component in disease monitoring, prognosis evaluation, epidemiological research and other aspects of HCV-infected person; for example, the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) stimulate the body to produce antibodies against the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) and their circulating immune complexes, and do there are differences between HCV-infected patients in different genotypes and subtypes, different clinical stages (chronic, cirrhosis, liver cancer), before and after antiviral therapy? So far, no relevant report is found. Therefore, it is of great significance to establish a high-sensitivity detection method for detecting corresponding antibodies of serum nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) of HCV-infected persons or corresponding antibodies in circulating immune complexes for disease monitoring, prognosis evaluation and epidemiological research of HCV-infected persons. Therefore, a chemiluminescence immunoassay method for detecting the antibody of the anti-nonstructural protein (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in the serum of the HCV infected person is planned to be established, and the development of a kit is completed by carrying out methodology evaluation, so that technical support is provided for revealing the relation between the content change and different clinical typing, disease progression and DAA curative effect of the HCV infected person.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a chemiluminescence kit and a detection method for detecting antibodies against nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in serum of HCV-infected persons, the kit can perform in-vitro qualitative/quantitative determination on antibodies against nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in serum of HCV-infected persons, establish detection technology and methodology evaluation on antibodies against nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in serum of HCV-infected persons, and can be applied to screening diagnosis, disease monitoring, prognosis evaluation, disease mechanism and epidemiological research of viral hepatitis C.
In order to achieve the above purpose, the invention provides the following technical scheme:
a chemiluminescence kit for detecting an anti-nonstructural protein antibody in serum of an HCV (hepatitis C virus) infected person comprises a microporous plate obtained by coating a recombinant HCV nonstructural 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).
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-nonstructural protein antibodies), and the human immunodeficiency virus antibodies, treponema pallidum antibodies, hepatitis b virus surface antigen test is negative with ProClin300 preservative at a concentration of 0.2% by volume.
Preferably, the positive control is heat-inactivated HCV-infected (positive for anti-nonstructural protein antibodies) pooled serum or plasma, and the tests for human immunodeficiency virus antibodies, treponema pallidum antibodies, and hepatitis b virus surface antigen are negative, containing ProClin300 preservative at a concentration of 0.2% by volume.
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 Proclin300, 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%, Proclin300 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% Proclin300 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-nonstructural protein antibodies in serum of an HCV-infected person comprising the steps of:
(1) adding 100 mul of sample diluent into a recombinant HCV non-structural 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 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;
(5) measuring the relative luminescence intensity value RLIV of each hole by using a chemiluminescence immunoassay analyzer, and determining that the sample RLlV is positive when the ratio of the sample RLlV to the negative control RLIV mean value is more than or equal to 2.1; otherwise, it is negative.
Respectively adding a sample to be detected into a pre-coated recombinant HCV non-structural protein (NS2, NS3, NS4A, NS4B, NS5A and NS5B) antigen microplate, after incubation, washing to remove unbound components, adding an enzyme marker working solution for incubation, and washing to remove the unbound enzyme marker working solution; adding reaction substrate luminol, wherein the luminol is oxidized by horseradish peroxidase in an alkaline environment to be 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, and the Relative Luminescence Intensity Ratio (RLIR) is expressed by a relative luminescence intensity ratio (RLIR ═ sample RLIV/negative control RLIV ≥ 2.1 is positive) and is in direct proportion to the antibody concentration of the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in the serum of HCV infected persons.
Preferably, the antigen coated microplate is a recombinant HCV nonstructural protein antigen (NS2, NS3, NS4A, NS4B, NS5A, NS 5B); the method for coating the micro-porous plate comprises the following steps: diluting the recombinant HCV non-structural protein antigen to 0.5 mu g/ml by adopting a coating solution, adding the diluted HCV non-structural protein antigen into a microporous plate according to the amount of 100 mu l, standing overnight at 2-8 ℃ or 2h at 37 ℃, washing the plate to remove proteins which are not adsorbed on the plate, repeatedly washing for 3 times by using 300 mu l of washing solution per hole, and patting to 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 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 Proclin300, 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 needs to be checked repeatedly; the negative control value of the anti-nonstructural protein NS2 antibody is equal to or less than 115000, the negative control value of the NS3 antibody is equal to or less than 400000, the negative control value of the NS4A antibody is equal to or less than 400000, the negative control value of the NS4B antibody is equal to or less than 115000, the negative control value of the NS5A antibody is equal to or less than 400000, the negative control value of the NS5B antibody is equal to or less than 400000, and the difference between the reading of the two negative control wells and the average value of the negative controls is equal to or less than 10 percent, otherwise, the experiment is invalid and;
the positive control value of the anti-nonstructural protein NS2 antibody should be more than 241500, the positive control value of the NS3 antibody should be more than 840000, the positive control value of the NS4A antibody should be more than 840000, the positive control value of the NS4B antibody should be more than 241500, the positive control value of the NS5A antibody should be more than 840000, the positive control value of the NS5B antibody should be more than 840000, otherwise, the experiment is invalid and needs to be checked repeatedly.
Compared with the prior art, the invention has the beneficial effects that:
the kit of the present invention separates the antigen-antibody complex formed on the solid phase carrier from other substances in the liquid by washing; and then horseradish peroxidase-labeled secondary antibody is added to be combined on the 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, the RLIR (relative luminescence intensity ratio) is used for representing, and the concentration of the anti-non-structural protein 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 antibodies against the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in the serum of HCV infectors, establishes the detection technology for the antibodies against the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in the serum of the HCV infectors and performs 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 the anti-non-structural protein antibody detection kit
FIGS. 4(1) to (5) are sequencing charts of plasmid pGEx-4T-1-NS 2.
FIG. 5 is an electrophoretogram of NS2 protein.
FIG. 6 is a schematic diagram of an anti-nonstructural protein antibody chemiluminescence kit.
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 chemiluminescence immunoassay kits for separately detecting antibodies against non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) at home and abroad and related technologies is blank, foreign scholars adopt recombinant Core, E1, E2, NS3, NS4A, NS4B, NS5A and NS5B proteins to establish an immunoblotting method to detect corresponding protein antibodies in the serum of HCV infectors, 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, the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) stimulate the body to produce antibodies against the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) and their circulating immune complexes, and do there are differences between HCV-infected patients in different genotypes and subtypes, different clinical stages (chronic, cirrhosis, liver cancer), before and after antiviral therapy? So far, no relevant report is found. Therefore, it is of great significance to establish a high-sensitivity detection method for detecting corresponding antibodies of serum nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) of HCV-infected persons or corresponding antibodies in circulating immune complexes for disease monitoring, prognosis evaluation and epidemiological research of HCV-infected persons.
The embodiment provides a chemiluminescence kit for detecting an anti-nonstructural 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:
recombinant non-structural protein (NS2, NS3, NS4A, NS4B, NS5A and NS5B) antigens are pre-coated in the micropores, and anti-non-structural protein (NS2, NS3, NS4A, NS4B, NS5A and NS5B) antibodies to be detected in the blood serum of a detected sample are combined with corresponding antigens on the surface of the solid phase carrier. The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the liquid by washing. And then horseradish peroxidase-labeled secondary antibody is added to be combined on the solid phase carrier. And adding a reaction substrate of luminol, oxidizing the luminol by horseradish peroxidase in an alkaline environment to be in an excited state, radiating light with the maximum emission wavelength of 425nm when the luminol returns to a ground state from the excited state, performing signal conversion on a light signal generated by enzymatic luminescence through a photomultiplier to obtain a corresponding signal value, and expressing the corresponding signal value by using RLIR (relative luminescence intensity ratio), wherein the concentration of the antibody of the anti-non-structural protein to be detected (NS2, NS3, NS4A, NS4B, NS5A and NS5B) and the chemiluminescence intensity are in a linear quantitative relation 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 an anti-non-structural protein 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 microplates were coated with recombinant HCV non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) respectively, the NS2 recombinant proteins were obtained for autonomous recombinant expression (see appendix four for details), NS3 was obtained from ProSpec-Tany GeneTechnique, Inc., Israel (Spec: 100ug/100ul, batch No. 612 PHNS CV 3), NS4A was obtained from ABCAM, England (Spec: 1mg/ml, batch No. GR3249079-1), NS4B was obtained from ProSpec-Tany GeneTechnique, Israel (Spec-Tany GeneTechnique, Spec: 100ug/10ul, batch No. 407HCVNS401), NS5A was obtained from ProSpec-Tany GeneTechnique, Israel (Spec/10 ul, batch No. 612 ug/10ul, NS5B was obtained from ProSpec-Tany GeneTechnical, NyProSpec-Tany Technical, NyNt, Israya Spec-ProSpec-Tany GeneTechnical, Spec, Inc. (Spec, Spec: 100U;
(1) process for preparing NS2 antigen
The gene: plasmid PUC-NS2, which contains the HCV1b genotype NS2 nucleic acid sequence (Accession No: AJ238800), was synthesized by Ongzhou Hippon Biotechnology Inc.
② strains: coli competent cells BL21(DE3) were stored in the laboratory.
Third, reagent: restriction enzymes Bam HI, Hind III were obtained from NEB (New England Biolabs) USA, KOD-PLUS series high-fidelity polymerase enzyme was obtained from TOYOBO Bio Japan, T4 DNA ligase was obtained from Biotech engineering (Shanghai) GmbH, protein Mark (14.4-116.0Kba) is Thermo scientific USA, and Ni-NTA-agarose affinity chromatography column is QIAGEN, Germany.
(iv) primers for amplifying the full-length NS2 gene (Hangzhou Ongke Biotechnology Co., Ltd.):
NS2-F：5’-ATCGGATCTGGTTCCGCGT GGATCC ATGGACCGGGAGATGGCAG-3’
NS2-R：5’-TTAGTGGTGGTGGTGGTG GTGGAGGAGTCGCCACCCCTG-3’
NS2-R1：
bam HI and HindIII sites (italic) were introduced into the primers, respectively, and 6 His (histidine) and the termination codon TAA (bold) were introduced into the downstream primer NS 2-R1.
(2) Preparation method of NS2 antigen
PCR amplification of HCV full-length NS 2:
the synthesized PUC-NS2 plasmid is used as a template, NS2-F and NS2-R are used as primers to carry out first round PCR amplification, and then the PCR product of the first round amplification is used as a template, and NS2-F and NS2-R1 are used as primers to carry out second round amplification. The PCR amplification parameters of both rounds were pre-denatured at 94 ℃ for 2min, followed by 30 cycles of 94 ℃ for 15s, 53 ℃ for 30s, and 68 ℃ for 30s, and finally extended at 72 ℃ for 7 min.
Secondly, PCR product identification and recombinant plasmid pGEx-4T-1-NS2 construction:
and (2) carrying out enzyme digestion on the PCR product and an expression vector pGEx-4T-1 by using restriction enzymes Bam HI and Hind III, wherein the enzyme digestion system is 50 mu l, and the enzyme digestion reaction system comprises: PCR product/pGEx-4T-143. mu.l, BamHI 1. mu.l, Hind III 1. mu.l, digestion buffer NEBuffer 2.15. mu.l (Tris-HCl buffer containing 1mol/L NaCl, 100mmol/L MgCl2, 10mmol/L DTT, pH7.9 and 500 mmol/L). And after uniformly mixing, placing the mixture in a water bath at 37 ℃ for digestion for 3-4h, and after the enzyme digestion is finished, recovering the required target fragment and the required vector fragment by agarose gel. After recovery, the PCR tube was removed, and 6 XLoading Buffer 9. mu.l (containing bromophenol blue at 0.05% by volume, xylene nitrile blue FF at 0.05% by volume, glycerol at 36% by volume, EDTA at 30mmol/L by mole, and NaOH at 2mol/L to adjust pH to 7.0) was added to the tube, and the mixture was mixed, and subjected to electrophoresis on agarose gel at 1.0% by volume at a voltage of 150V for 8min, and after the electrophoresis was completed, the gel was placed in a gel imaging system to observe whether the size of PCR amplified fragment was about 651 bp. Then, the gel containing the target fragment was cut out, transferred to 2 new sterile 1.5ml centrifuge tubes, and the gel pieces were cut up and DNA fragments were recovered using the AXYGEN gel recovery kit. The target fragment cut by restriction enzymes BamHI and HindIII and the expression vector pGEx-4T-1 are connected by T4 DNA ligase, the connection temperature is 16 ℃, and the connection time is 8-12 h. Finally sending the sequencing to Hangzhou Ongke biotechnology limited company for comparison with the sequence of the fragment of NS2 for consistency.
③ inducing expression of NS2 protein:
plasmid NS2 was transformed into E.coli competent cell BL21(DE3), cultured in 2 Xyeast extract peptone agar medium (YT) containing 100mg/L ampicillin (Amp) at 37 ℃ for 3-4hr, and when the absorbance OD600 was between 1.0-2.0, Isopropylthiogalactoside (IPTG) was added at a molar concentration of 1mmol/L, and the culture was induced for 3 hr.
Extraction and purification of NS2 protein:
the cells were collected by centrifugation and resuspended in buffer A (containing 200mmol/L NaCl, μ H8.0 and 50mmol/L Tris-HCl buffer solution) pH8.0 for ultrasonication; the centrifuged inclusion body precipitate was washed 1 time with buffer B (Tris-HCl buffer solution containing 150mmol/L NaCl, 1% Triton X-100 by volume, pH8.0 and 50 mmol/L); the centrifuged precipitate was washed, resuspended and dissolved in buffer C (containing NaCl 500mmol/L, Urea 8mol/L, Imidazole 10mmol/L, Tris-HCl buffer solution pH8.0 and 50 mmol/L) at pH8.0, then purified by Ni-NTA-agarose affinity column, and subjected to SDS-PAGE electrophoresis to collect the target protein.
Renaturation of NS2 protein:
and (4) treating the purified protein collected in the step (iv) and then dialyzing for renaturation. Calibrating the purified protein to be 1.0mg/ml, adding Dithiothreitol (DTT) with the molar concentration of 1mmol/L, and reacting for 30min at Room Temperature (RT); and then SDS with the mass volume concentration of 0.1 percent is added for RT reaction for 15 min. After the reaction, the sample was put into a dialysis bag 1: 100 and dialyzed against a dialysis buffer (pH7.5, phosphate buffer at a molar concentration of 0.01 mol/L).
(3) Results
Amplification of NS2 gene and construction of expression vector pGEX-4T-1:
the synthetic PUC-NS2 plasmid is used as a template, the full-length NS2 gene is amplified by PCR, electrophoresis identification after enzyme digestion shows that the insertion size of NS2 nucleic acid fragment is about 651bp and is consistent with the expected size, the PCR product is connected to pGEX-4T-1 vector, and the plasmid pGEx-4T-1-NS2 is obtained by correct sequencing (figure 4).
Expression, purification and renaturation of NS2 gene in escherichia coli competent cell BL21(DE 3):
the plasmid pGEx-4T-1-NS2 was transformed into E.coli competent cells BL21(DE3) to induce expression of GST (glutathione S-transferase) fusion protein with a molecular weight of 51kDa (FIG. 5), resulting in higher expression level. After the thalli are subjected to ultrasonic disruption, SDS-PAGE examination shows that the expressed protein forms inclusion bodies in precipitates. And dialyzing and renaturing the inclusion body by means of cracking and Ni-NTA-agarose affinity chromatography column chromatography to obtain the stable protein.
2. Diluting: diluting the recombinant HCV non-structural protein (NS2 or NS3 or NS4A or NS4B or NS5A or NS5B) antigen (the original nominal mass concentration is 1mg/ml) to 0.5 μ g/ml by using a coating solution [ Phosphate Buffer (PB) buffer solution with pH7.4 and the molar concentration of 0.02mol/L ];
3. coating: adding diluted HCV nonstructural protein (NS2 or NS3 or NS4A or NS4B or NS5A or NS5B) antigen into a microplate according to 100 μ l, and standing at 2-8 deg.C overnight (overnight for 20-24h), or at 37 deg.C for 2 h;
4. washing the plate after coating: washing off proteins which are not adsorbed on the plate, wherein each hole is 300 mu l, repeatedly washing for 3 times, and patting dry; wherein the concentrated washing solution (20X) is Phosphate Buffered Saline (PBS) containing 10% Tween-20 by volume, 4% Proclin300 by volume, pH7.4 and 0.2mol/L by mol concentration, 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% Proclin300 by volume, pH7.4 and 0.01mol/L by mol concentration 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 Proclin300 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 long-term storage of the coated plate in a sealing way.
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 3002 ml 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 heat-inactivated non-HCV-infected [ anti-nonstructural-protein (NS2, NS3, NS4A, NS4B, NS5A, NS5B) antibody negative ] serum or plasma, and the tests for human immunodeficiency virus antibodies, treponema pallidum antibodies, hepatitis b virus surface antigen were negative, containing ProClin300 preservative at a concentration of 0.2% by volume.
3. Preparation of positive control: the positive control was heat-inactivated HCV-infected [ anti-nonstructural protein (NS2, NS3, NS4A, NS4B, NS5A, NS5B) antibody positive ] pooled serum or plasma, and human immunodeficiency virus antibodies, treponema pallidum antibodies, hepatitis b virus surface antigen test negative, containing ProClin300 preservative at a concentration of 0.2% by volume. 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 ProClin300 preservative with volume concentration of 0.2% into the concentrated mixed serum or plasma of HCV infected person.
4. Washing liquid: the concentrated cleaning solution (20X) is Phosphate Buffered Saline (PBS) containing 10% by volume of Tween-20, 4% by volume of Proclin300, pH7.4 and 0.2mol/L of molarity, and is prepared by the following method: 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 cleaning solution (20 x) at normal temperature for later use.
5. Preparation of enzyme marker 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 contains 5% by mass volume of Bovine Serum Albumin (BSA), 0.5% by volume of enzyme stabilizer, 0.1% by volume of Tween-20, 5. mu.g/ml by mass of anti-mouse antibody blocking agent, 1. mu.g/L by mass of red pigment, and 0 by volume.2% Proclin300, Phosphate Buffered Saline (PBS) with pH7.4 and a molarity of 0.01mol/L, prepared by the following method: adding Na into a 1L volumetric flask2HPO4·12H2O 2.9g，NaH2PO4·2H20.296g of O, 9g of NaCl, 3002 ml 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 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-imidazolylphenol into a 1L volumetric flask, finally adding double distilled water to achieve a constant volume of 1L, enabling the pH value of the solution to be 9.0 +/-0.1, preparing a substrate solution A, and refrigerating the substrate solution A at the temperature of 2-8 ℃ in a dark place for standby.
8. A sealing plate film and a self-sealing bag (containing a drying agent).
9. Finally, the reagent is matched and subpackaged according to 96 tests of each kit (as shown in figure 6), and the reagent kit comprises a sample diluent, a negative control, a positive control, an enzyme marker working solution, a substrate solution A, a substrate solution B, a washing solution, a microporous plate (96 holes) for detecting antibodies of anti-non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), a sealing plate film, a self-sealing bag (containing a drying agent) and the like which are independently packaged respectively
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 lotion into deionized water or distilled water according to the ratio of 1: 19, and mixing uniformly for later use.
2. Detection of
(1) Sample adding: and (3) fixing the recombinant HCV non-structural protein antigen coating 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.
② each negative control value of the kit: the antibody of the anti-nonstructural protein NS2 should be less than or equal to 115000, the antibody of NS3 should be less than or equal to 400000, the antibody of NS4A should be less than or equal to 400000, the antibody of NS4B should be less than or equal to 115000, the antibody of NS5A should be less than or equal to 400000, the antibody of NS5B should be less than or equal to 400000, and the difference between the reading of two negative control wells and the average value of the negative control wells is less than or equal to 10%, otherwise.
③ every positive control value of the kit: the antibody of the anti-non-structural protein NS2 should be more than 241500, the antibody of NS3 should be more than 840000, the antibody of NS4A should be more than 840000, the antibody of NS4B should be more than 241500, the antibody of NS5A should be more than 840000, and the antibody of NS5B should be more than 840000, otherwise, the experiment is invalid and needs to be tested repeatedly.
Fifthly, detecting the 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 non-parametric method is used to estimate the 95 th percentile of the blank sample measurement result, i.e. the result of the 95 th percentile is: NS 2: 1.57RLIR, NS 3: 1.72RLIR, NS 4A: 1.34RLIR, NS 4B: 1.37RLIR, NS 5A: 1.62RLIR, NS 5B: 1.49 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 can be seen from table 3, the median of the values detected in the serially diluted samples (30 th rank + 31 th rank)/2,DS-βmedian result-5 th percentile result, LoD absorbance LoB + DS-βTherefore, the detection limit LoD concentrations of the kit are respectively: NS2 ═ 4.8RLIR, NS3 ═ 5.31RLIR, NS4A ═ 4.02RLIR, NS4B ═ 4.21RLIR, NS5A ═ 5.08RLIR, and NS5B ═ 4.63 RLIR.
3. Linearity
Analytical Measurement Range (AMR): designed as required in the EP6-A2 file, 6 samples were prepared from serum of an uninfected HCV subject (low value L: anti-NS 2 antibody 1.641.63RLIR, anti-NS 3 antibody 1.86RLIR, anti-NS 4A antibody 1.65RLIR, anti-NS 4B antibody 1.58RLIR, anti-NS 5A antibody 1.94RLIR, anti-NS 5B antibody 1.81RLIR) and serum of an HCV infected person (high value H: anti-NS 2 antibody 17.0644.5RLIR, anti-NS 3 antibody 34.17RLIR, anti-NS 4A antibody 29.66RLIR, anti-NS 4B antibody 17.95RLIR, anti-NS 5A antibody 17.14RLIR, anti-NS 5B antibody 33.09RLIR) in proportions of 5L, 4L + IH, 3L +2H, 2L +3H, 1L +4H, 5H, as determined by mean value test of each sample, as expected mean value and as expected coordinate regression (see the equation for the horizontal regression): anti-NS 2 antibody y-0.9894X-0.5756, anti-NS 3 antibody y-1.0079X-0.2776, anti-NS 4A antibody y-1.042X-0.1212, anti-NS 4B antibody y-0.9887X +0.0041, anti-NS 5A antibody y-1.0231X-0.3606, anti-NS 5B antibody y-0.9597X +0.4362, b is between 0.95 and 1.05, correlation coefficient r is not less than 0.975(r2 not less than 0.95), then the analytical measurement ranges of the HCV nonstructural protein antibodies are NS 2: 1.63-44.5 RLIR; NS 3: 1.86 to 34.17 RLIR; NS 4A: 1.65 to 29.66 RLIR; NS 4B: 1.58 to 17.95 RLIR; NS 5A: 1.94-17.14 RLIR; NS 5B: 1.81 to 33.09RLIR, see FIG. 3.
4. Interference experiment
Serum samples positive for rheumatoid factor, hepatitis B virus surface antibody, hepatitis B virus e antibody, treponema pallidum antibody, human immunodeficiency virus type I and/or type II antibody were mixed with anti-NS 2(30.53RLIR), NS3(14.28RLIR), NS4A (12.75RLIR), NS4B (10.33RLIR), NS5A (14.91RLIR), NS5B (24.11RLIR) positive sera, respectively, and no cross reaction was observed with the sample diluent as a control.
5. Example applications
The developed anti-non-structural protein 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-non-structural protein antibody in the chronic HCV infected persons is counted, and the result is shown in tables 4 and 5.
TABLE 4 survey of the positivity of serum anti-nonstructural protein antibodies (except NS2, RLIR) of chronic HCV-infected patients
Injecting: an anti-HCV mixed antibody screening kit produced by Xiamen creative company is adopted.
TABLE 5 survey of the antibody positivity of chronic HCV-infected persons to NS2 protein
Injecting: an anti-HCV mixed antibody screening kit produced by Xiamen creative company is adopted.
As can be seen from tables 4 and 5, the positive rates of the anti-non-structural protein antibodies (positive RLIR ≧ 2.1) were respectively calculated for the patients with chronic HCV infection: anti-NS 2 antibody 20% (9/45), anti-NS 3 antibody 71.11% (32/45), anti-NS 4A antibody 35.56% (16/45), anti-NS 4B antibody 84.44% (38/45), anti-NS 5A antibody 55.56% (25/45), anti-NS 5B antibody 6.67% (3/45).
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 6.
TABLE 6 selection of enzyme-labeled antibody work (RLlV)
As can be seen from Table 6, 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 50ul and 100 mul enzyme conjugate acts 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 optimal enzyme conjugate loading amount of the anti-nonstructural protein (NS2, NS3, NS4A, NS4B, NS5A, NS5B) 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 7 comparison of the coating concentration of the nonstructural protein antigens (RLIV)
As can be seen from table 7, 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-nonstructural protein antibody in serum of an HCV (hepatitis C virus) infected person is characterized by comprising a microporous plate obtained by coating a recombinant HCV nonstructural 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 chemiluminescence kit for detecting an anti-nonstructural protein antibody in serum of an HCV-infected person in accordance with claim 1, wherein the substrate solution A comprises luminol at a mass concentration of 0.8g/L, o-phenylphenol at a mass concentration of 0.008g/L, 4-imidazolylphenol at a mass concentration of 0.025g/L, a carbonic acid buffer (CB) at pH9.0 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 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-nonstructural protein antibodies in serum of an HCV-infected person according to claim 1 wherein the negative control is heat-inactivated (anti-nonstructural protein antibody negative) serum or plasma of a non-HCV-infected person and tests negative for human immunodeficiency virus antibodies, treponema pallidum antibodies, hepatitis b virus surface antigen, ProClin300 preservative at a volume concentration of 0.2%;
the positive control is heat inactivated HCV infected person (positive anti-non-structural protein antibody) mixed serum or plasma, and the tests of human immunodeficiency virus antibody, treponema pallidum antibody and hepatitis B virus surface antigen are negative, and the positive control contains 0.2% of ProClin300 preservative by volume concentration.
5. The chemiluminescence kit for detecting the anti-nonstructural protein antibody in the serum of an HCV (hepatitis C Virus) infected person, as claimed in claim 1, wherein the working solution of the enzyme marker is prepared by diluting a stock solution of the enzyme marker with a diluent of the enzyme marker antibody to a mass concentration of 0.05 μ g/ml, and the stock solution of the enzyme marker is a goat anti-human IgG marker labeled with horseradish peroxidase to 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 Proclin300, pH7.4, and 0.01mol/L Phosphate Buffered Saline (PBS).
6. The chemiluminescent kit of claim 1 wherein the washing solution comprises tween-20 at a concentration of 0.5% by volume, Proclin300 at a concentration of 0.2% by volume, Phosphate Buffered Saline (PBS) at ph7.4 at a molarity of 0.01 mol/L.
7. A chemiluminescent assay for detecting an anti-nonstructural protein antibody in serum of an HCV-infected subject comprising the steps of:
(1) adding 100 mul of sample diluent into a recombinant HCV non-structural 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 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); the substrate liquid B comprises carbamide 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 nonstructural protein antigen (NS2, NS3, NS4A, NS4B, NS5A, NS 5B);
the method for coating the micro-porous plate comprises the following steps: diluting the recombinant HCV non-structural protein antigen to 0.5 mu g/ml by adopting a coating solution, adding the diluted HCV non-structural protein antigen into a microporous plate according to the amount of 100 mu l, standing overnight at 2-8 ℃ or 2h at 37 ℃, washing the plate to remove proteins which are not adsorbed on the plate, repeatedly washing for 3 times by using 300 mu l of washing solution per hole, and patting to 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 plate.
9. The chemiluminescent assay for the detection of antibodies to nonstructural proteins in the serum of an HCV infected person of claim 8 wherein the coating solution is Phosphate Buffer (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 Proclin300, and 0.01mol/L molarity Phosphate Buffered Saline (PBS), pH 7.4.
10. The chemiluminescent assay for the detection of antibodies to nonstructural proteins in the serum of an HCV-infected person of any one of claims 7 to 9 wherein the test is repeated when the reagent blank RLIV value is <30000 and when the test is otherwise invalid; the negative control value of the anti-nonstructural protein NS2 antibody is equal to or less than 115000, the negative control value of the NS3 antibody is equal to or less than 400000, the negative control value of the NS4A antibody is equal to or less than 400000, the negative control value of the NS4B antibody is equal to or less than 115000, the negative control value of the NS5A antibody is equal to or less than 400000, the negative control value of the NS5B antibody is equal to or less than 400000, and the difference between the reading of the two negative control wells and the average value of the negative controls is equal to or less than 10 percent, otherwise, the experiment is invalid and;
the positive control value of the anti-nonstructural protein NS2 antibody should be more than 241500, the positive control value of the NS3 antibody should be more than 840000, the positive control value of the NS4A antibody should be more than 840000, the positive control value of the NS4B antibody should be more than 241500, the positive control value of the NS5A antibody should be more than 840000, the positive control value of the NS5B antibody should be more than 840000, otherwise, the experiment is invalid and needs to be checked repeatedly.
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