Immunodetection reagent for blood virus screening and preparation method thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an immunodetection reagent for blood virus screening and a preparation method of the immunodetection reagent. The immunodetection reagent comprises the following raw materials in parts by weight: 100 parts of a capture reagent, 300 parts of a sealing agent, 300 parts of a cracking agent, 1 part of an HCV recombinant antigen, 1 part of an anti-HCVcAg monoclonal antibody, 0.2-0.6 part of hydroxymethylcoumarin, 10 parts of BSA, 2-6 parts of glutaric anhydride, 4-8 parts of TEOS and 100 parts of a PBS buffer solution. According to the present invention, the silica microsphere with the HCV recombinant antigen and the anti-HCVcAg monoclonal antibody coupled on the surface and the hydroxymethylcoumarin signal molecule loaded inside is constructed, and the HCV antigen and the HCV antibody in the serum are specifically recognized so as to achieve the efficient diagnosis and detection of the HCV infection in the whole cycle;

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to an immunological detection reagent for blood virus screening and a preparation method thereof. BACKGROUND

[0002] Hepatitis C is a blood-borne disease caused by HCV (Hepatitis C Virus), which is mainly transmitted and infected through the use of used syringes and needles, the reuse of medical equipment, blood transfusion and unprotected sexual contact. The disease has no obvious symptoms in the early stage of HCV infection, and is easily converted from acute to chronic, and from hepatitis to cirrhosis, and then to liver cancer, which poses a great threat to people's physical health and life safety. However, since HCV is an RNA virus with genetic heterogeneity and high variability, there is currently no effective HCV vaccine for the prevention and treatment of hepatitis C, and the current main method is to control the transmission route through large-scale screening and diagnosis. The current methods for detecting HCV include HCV antibody detection, HCV cAg (core antigen) detection, HCV RNA nucleic acid detection, imaging diagnosis and pathological diagnosis, among which the HCV antibody is an antibody protein produced by the immune cells of the human body after HCV infection. This detection method is relatively simple and fast, but the antibody appears late, and the diagnostic accuracy for early HCV infection is insufficient, and false positives are prone to occur; HCV cAg appears early and can be used as a serum marker for HCV replication, but when HCV antibody appears, it will combine with it to form an antigen-antibody complex, which interferes with the detection results of HCV cAg, so the detection sample needs to be pretreated to dissolve the immune complex, which reduces the sensitivity of HCV cAg.

[0003] Therefore, it is urgent to develop a detection reagent that can simultaneously capture antigen and antibody signals, has high sensitivity and simple operation, which is of great significance for improving hepatitis C screening and blocking HCV transmission. SUMMARY

[0004] In view of the above problems, the present application provides an immunological detection reagent for blood virus screening and a preparation method thereof, which aims to overcome the bottleneck problems of window period missed detection, insufficient sensitivity and complicated operation in the existing HCV detection technology, and to construct an integrated dual-function capture probe. The virus antigen and specific antibody are co-modified on the surface of the signal label microspheres, specifically bind to the free antigen and its antibody complex in the serum, so as to realize efficient coverage of the whole infection cycle. The signal molecules encapsulated in the microspheres and the lysis release mechanism are used to form a new type of HCV immunological detection solution with high sensitivity, strong specificity and convenient operation.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows: The application provides an immunological detection reagent for blood virus screening, which comprises the following raw materials in parts by weight: a capture reagent 100 parts, a blocking agent 300 parts, a lysis agent 300 parts, an HCV recombinant antigen 1 part, an anti-HCV cAg (HCV core antigen) monoclonal antibody 1 part, oxymethyl coumarin 0.2-0.6 parts, BSA (bovine serum albumin) 10 parts, glutaric anhydride 2-6 parts, TEOS (tetraethyl orthosilicate) 4-8 parts and PBS (phosphate) buffer 100 parts.

[0006] Further, the capture reagent is composed of the HCV recombinant antigen, the anti-HCV cAg monoclonal antibody and double distilled water in a mass ratio of 0.1:0.1:100.

[0007] Further, the blocking agent is selected from any one of sucrose solution, gelatin solution and skimmed milk.

[0008] Further, the lysis agent is a 1 mol / L sodium hydroxide solution.

[0009] Further, the HCV recombinant antigen is an HCV NS3 recombinant antigen with a molecular weight of 23 kDa.

[0010] Further, the amino acid sequence of HCV cAg in the anti-HCV cAg monoclonal antibody is shown in SEQ ID NO. 1. Further, the base sequence of the encoding gene of HCV cAg is shown in SEQ ID NO. 2.

[0011] Further, the specific preparation method of the anti-HCV cAg monoclonal antibody is as follows: S1: selecting the amino acid site of 1-383 in the HCV viral protein sequence as HCV cAg, the amino acid sequence is SEQ ID NO. 1, determining the base sequence of the encoding HCV cAg and performing codon optimization to obtain the HCV cAg encoding gene as the target gene, i.e. SEQ ID NO. 2; S2: taking ScaⅠ / BsmⅠ as the enzyme cutting site and 6×His as the protein purification tag, connecting the target gene to the pET28a(+) expression vector, cutting and extracting to obtain the recombinant plasmid, transforming the recombinant plasmid into BL21 (DE3) competent cells to express the HCV cAg, centrifuging the BL21 (DE3) competent cells to obtain the precipitate, collecting the protein in the precipitate and purifying by column to obtain the HCV cAg; S3: preparing hybridoma cells by using the HCV cAg and performing positive cell screening and culture to obtain a monoclonal cell strain, injecting the monoclonal cell strain into the abdominal cavity of a mouse, centrifuging the ascites after extraction, collecting the supernatant, and the supernatant contains the anti-HCV cAg monoclonal antibody. S4: The supernatant was purified by chromatography, dialysis, and lyophilization to obtain anti-HCVcAg monoclonal antibody.

[0012] This invention also provides a method for preparing an immunoassay reagent for blood virus screening, comprising the following steps: Step 1: Mix anhydrous ethanol, deionized water and ammonia (mass ratio of 8:2:1) to prepare solution A. Disperse and dissolve 0.2-0.6 parts of hydroxymethylcoumarin and 4-8 parts of TEOS in anhydrous ethanol to obtain solution B. Slowly add solution B to solution A under magnetic stirring to obtain coumarin@SiO2 sol. Step 2: Centrifuge the coumarin@SiO2 sol, discard the supernatant to obtain the precipitate, resuspend the precipitate in anhydrous ethanol, add 2-6 parts of glutaric anhydride to it, heat the reaction for 3 h, centrifuge to collect the precipitate, and obtain carboxylated coumarin@SiO2 microspheres. Step 3: Disperse and dissolve one part of HCV recombinant antigen and carboxylated coumarin@SiO2 microspheres in MES buffer. Add EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to catalyze the covalent coupling of the amino groups in HCV recombinant antigen with the carboxyl groups in carboxylated coumarin@SiO2 microspheres. After the reaction is complete, centrifuge and collect the precipitate, which is the antigen-coupled microspheres. Disperse and dissolve one part of anti-HCVcAg monoclonal antibody and the precipitate in MES buffer. Add EDC and NHS and continue the reaction. Centrifuge and collect the antigen-antibody-coupled microspheres. Step 4: Take 10 portions of BSA and disperse and dissolve the antigen-antibody conjugated microspheres in deionized water to block the active sites on the antigen-antibody conjugated microspheres. Centrifuge and discard the supernatant to remove unbound BSA to obtain blocked microspheres. Resuspend the blocked microspheres in 100 portions of PBS buffer to obtain the binding reagent. Step 5: Take 100 portions of capture reagent, 300 portions of blocking agent, 300 portions of lysis agent and binding reagent and package them to obtain combined reagent, which is the immunoassay reagent for blood virus screening.

[0013] The beneficial effects achieved by this invention are as follows: The immunological detection reagent for blood virus screening provided by the application constructs a multifunctional detection system integrating signal labeling, target capture and signal amplification, and solves the problem of decreased detection sensitivity caused by the formation of antigen-antibody complexes in traditional methods by covalently connecting virus-specific antigens and monoclonal antibodies to the surface of carboxyl-modified silica microspheres to form a microsphere that can synchronously and specifically bind HCV core antigens and HCV antibodies in serum, and realizes precise coverage of the whole cycle of hepatitis C virus infection. The hydroxymethyl coumarin dye with excellent optical properties and stability is used as a signal source and is wrapped inside the microspheres, which on the one hand avoids the weakening of the light absorption of hydroxymethyl coumarin, and on the other hand, hydroxymethyl coumarin has fluorescence properties and ultraviolet light absorption characteristics, and the characteristic absorption peak wavelength does not overlap with protein molecules, so when the silica microspheres are specifically combined with HCV markers, a large amount of hydroxymethyl coumarin is released by the action of a cleavage agent after the microspheres are broken, and the detection signal can be exponentially amplified without the need for coupling enzymes to realize the degradation of the substrate in traditional detection, thereby reducing the detection cost, and the detection process is simple, fast and easy to operate, has good stability, is easy to realize standardized production and quality control, and is conducive to promoting the development of HCV screening technology in the direction of being more precise, more convenient and more affordable. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 A schematic diagram of the principle mechanism of the immunological detection reagent of the application; Figure 2 A gel electrophoresis detection result of the HCV cAg protein prepared in Example 2; Figure 3 A gel electrophoresis detection result of the anti-HCV cAg monoclonal antibody prepared in Example 4. DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to one skilled in the art. In addition, any method and material similar or equivalent to those described can be used in the application. The preferred implementation methods and materials described herein are only used for demonstration, but cannot limit the content of the application.

[0017] In the following examples, unless otherwise specified, the conventional method is used; In the following examples and comparative examples, unless otherwise specified, the raw materials are new materials purchased on the market; The mechanism diagram of the immunological detection reagent for blood virus screening is shown in Figure 1 The capture reagent used is prepared from 1 part of HCV recombinant antigen, 1 part of anti-HCV cAg monoclonal antibody and 1000 parts of double distilled water; The HCV recombinant antigen used is HCV NS3 antigen; The mass fraction of the sucrose solution used is 2%; The mass fraction of the gelatin solution used is 0.25%; The mass fraction of the skim milk used is 5%; The volume fraction of the ammonia water used is 28%; The lysis solution used is 1 mol / L sodium hydroxide solution; The PBS buffer used is 0.1 M pH 7.4 PBS buffer.

[0018] Example 1: Plasmid construction A1: The sequence structure of the HCV viral protein is analyzed by using the GenBank database, the protein sequence length is 3011 aa, the structure is divided, and the order is core (1-191 aa), E1 (192-383 aa), E2 (384-746 aa), p7 (747-809 aa), NS2 (810-1026 aa), NS3 (1027-1657 aa), NS4A (1658-1711 aa), NS4B (1712-1972 aa), NS5A (1973-2420 aa), NS5B (2421-3011 aa). The core and E1 antigen sequences in the structural protein are constructed into plasmids, and the core antigen HCV cAg sequence is shown in SEQ ID NO. 1; A2: The codon analysis and optimization are carried out for the sequence shown in SEQ ID NO. 1, the corresponding target gene base sequence is shown in SEQ ID NO. 2, 6×His is used as a protein purification tag, ScaⅠ / BsmⅠ is added at both ends of the target gene as an enzyme digestion site, and the constructed gene is connected to the pET28a (+) expression vector to synthesize a recombinant plasmid; A3: The BL21 (DE3) competent cells are used as the recombinant plasmid transformation cells, 2 μL of the recombinant plasmid is added to the BL21 (DE3) competent cells under ice bath condition, mixed gently and incubated for 30 min, then heated to 42℃ to activate the cells for 120 s, and then quickly ice-bathed for 2 min to obtain a mixture; A4: The mixture is incubated in a non-resistant LB liquid medium at 37℃ and 120 rpm for 45 min, centrifuged at 5000 rpm for 5 min, the supernatant is discarded, and the precipitate is resuspended and plated on LB agar medium containing 50 μg / mL Kan +resistance plate, and incubated at 37℃ for 12 h. When colonies grew on the plate, single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL Kan + resistance plate, and incubated at 37℃ for 12 h. When colonies grew on the plate, single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL Kan A5: The plasmid synthesized by the recombinant strain was extracted by using a plasmid extraction kit, and sequencing was performed to confirm that the plasmid was the target plasmid.

[0019] Example 2: Expression and purification of HCVcAg protein B1: The recombinant strain with correct sequencing and capable of synthesizing the target plasmid was inoculated into a Kan + resistance plate, and incubated at 37℃ for 12 h. When colonies grew on the plate, single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL Kan + resistance plate, and incubated at 37℃ for 12 h. When colonies grew on the plate, single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL Kan B2: IPTG (isopropyl-β-D-thiogalactoside) was added to the amplification liquid to induce the expression of HCVcAg protein. The concentration of IPTG in the amplification liquid was 0.5 mmol / L. The amplification liquid was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded. The precipitated bacteria were collected, and cell lysis solution was added to the precipitated bacteria. After mixing by blowing, a lysis mixture was obtained. B3: The lysis mixture was ultrasonically broken in a cell disrupter, and centrifuged at 12000 rpm for 10 min. The protein precipitate was collected, resuspended, and passed through a Ni-NTA resin chromatography column. First, 20 times the column volume of Urea buffer was used for elution to remove impurity proteins, cell fragments and other substances that were not adsorbed on the chromatography column. Then, Urea buffer containing 500 mM imidazole was used to elute the target protein adsorbed on the chromatography column, and the eluate was collected. B4: TEV protease was added to the eluate to remove the 6×His tag. The eluate was passed through a Ni-NTA resin chromatography column again to obtain HCVcAg protein.

[0020] Example 3: Preparation of hybridoma cells C1: The HCVcAg protein was emulsified with Freund's complete adjuvant at a mass ratio of 1:1 to obtain an antigen solution. The antigen solution was injected subcutaneously into 8-week-old Balb / c mice at a dose of 100 μg per mouse for primary immunization. The mice were subcutaneously injected with the antigen solution again for booster immunization at 2 weeks and 4 weeks after the primary immunization, respectively. The antigen solution used in the booster immunization was emulsified with Freund's incomplete adjuvant, and the resistant mice were obtained. C2: Inject 100 μg of HCV cAg protein into the abdominal cavity of resistant mice, and after 3 days of feeding, remove the mouse spleen, grind to prepare a spleen cell suspension, mix the spleen cell suspension with Sp2 / 0 myeloma cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add PEG1500 to the precipitate and mix well, stand for 1 min, then add DMEM incomplete medium to terminate the fusion, continue to centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the precipitate with 20% FBS complete medium to obtain mixed cells; C3: Take 6-week-old healthy Balb / c mice, inject 0.5 mL of sterile paraffin oil into the abdominal cavity to stimulate macrophages, immerse the mice in 75% alcohol for 10 min for sterilization, cut open the abdominal skin to expose the abdominal cavity, inject 5 mL of DMEM incomplete medium into the peritoneum and aspirate, repeat 3 times, centrifuge the collected DMEM incomplete medium at 800 rpm for 5 min, discard the supernatant, resuspend the precipitate with 20% FBS complete medium, count, adjust the cell density to 1×10 5 cells / mL, and obtain feeder cells; C4: Mix the mixed cells and feeder cells in a volume ratio of 1:1, add 100 μL / well to a 96-well plate, culture in a cell incubator for 1 week, then change the medium, and when the cells grow to about 1 / 2 of the area at the bottom of the well under a microscope, use an HCV antibody detection kit to detect the positive results of the antibody in the cell supernatant, screen positive wells with good growth conditions and prepare a cell suspension, add to a new 96-well plate for culture, repeat the operation until the positive rate of the cell supernatant of the plate is 100%, and obtain hybridoma cells; C5: Proliferate the hybridoma cells to obtain monoclonal antibody cells, and adjust the cell density to 2×10 6 cells / mL.

[0021] Example 4: Preparation of anti-HCV cAg monoclonal antibody D1: Take 8-week-old healthy Balb / c mice, inject 0.5 mL of sterile paraffin oil into the abdominal cavity, feed for 7 days, then inject 0.5 mL of hybridoma cells into the abdominal cavity, and when the mouse abdomen is swollen, draw ascites, centrifuge at 5000 rpm to collect the upper liquid; D2: Under ice bath conditions, add an equal volume of saturated ammonium sulfate to the upper liquid, ice bath for 4 h, centrifuge at 4°C, 15000 rpm for 20 min, discard the supernatant, resuspend the precipitate with PBS to obtain a suspension, purify the suspension by Protein A / G column affinity chromatography and dialysis, the dialysis bag has a molecular weight of 10 kDa, collect the monoclonal antibody solution, and freeze-dry to obtain anti-HCV cAg monoclonal antibody.

[0022] Embodiment 5: The embodiment provides an immunological detection reagent for blood virus screening, which comprises the following raw materials in parts by weight: a capture reagent 100 parts, a sucrose solution 300 parts, a sodium hydroxide solution 300 parts, an HCV recombinant antigen 1 part, an anti-HCVcAg monoclonal antibody 1 part, hydroxymethyl coumarin 0.2 parts, BSA 10 parts, glutaric anhydride 2 parts, TEOS 8 parts and PBS buffer 100 parts, and the specific steps are as follows: Step 1: 40 parts of anhydrous ethanol, 10 parts of deionized water and 5 parts of ammonia water are mixed to prepare solution A, 0.2 parts of hydroxymethyl coumarin and 8 parts of TEOS are dispersed and dissolved in 20 parts of anhydrous ethanol to obtain solution B, solution B is slowly added to solution A under the condition of 30°C and 60 rpm magnetic stirring, and after the addition is completed, the stirring reaction is continued for 3 hours to obtain coumarin@SiO2 sol; Step 2: The coumarin@SiO2 sol is centrifuged at 12000 rpm for 15 min, the supernatant is discarded to obtain the precipitate, the precipitate is resuspended in 40 parts of anhydrous ethanol, and 2 parts of glutaric anhydride is added thereto, and the reaction is heated at 37°C for 3 hours, and the precipitate is collected by centrifugation to obtain carboxylated coumarin@SiO2 microspheres; Step 3: The HCV recombinant antigen 1 part and the carboxylated coumarin@SiO2 microspheres are dispersed and dissolved in 100 parts of 0.1 M pH 6.0 MES buffer, and 0.3 parts of EDC and 0.75 parts of NHS are added to catalyze the covalent coupling of the amino group in the HCV recombinant antigen and the carboxyl group in the carboxylated coumarin@SiO2 microspheres, and after the reaction is completed, the precipitate is collected by centrifugation, i.e. antigen-coupled microspheres, the anti-HCVcAg monoclonal antibody 1 part and the precipitate are further dispersed and dissolved in 100 parts of 0.1 M pH 6.0 MES buffer, EDC / NHS is added for continuous reaction, and centrifugation is performed to collect antigen-antibody coupled microspheres; Step 4: 10 parts of BSA and antigen-antibody coupled microspheres are dispersed and dissolved in deionized water to block the active sites on the antigen-antibody coupled microspheres, the supernatant is discarded by centrifugation to remove unbound BSA, and closed microspheres are obtained, and the closed microspheres are resuspended and dispersed in 100 parts of PBS buffer to obtain a binding reagent; Step 5: The capture reagent 100 parts, the sucrose solution 300 parts, the sodium hydroxide solution 300 parts and the binding reagent are dispensed to obtain a combined reagent, i.e. an immunological detection reagent for blood virus screening.

[0023] Embodiment 6: The embodiment provides an immunological detection reagent for blood virus screening, which comprises the following raw materials in parts by weight: a capture reagent 100 parts, a gelatin solution 300 parts, a sodium hydroxide solution 300 parts, an HCV recombinant antigen 1 part, an anti-HCVcAg monoclonal antibody 1 part, hydroxymethyl coumarin 0.4 parts, BSA 10 parts, glutaric anhydride 6 parts, TEOS 4 parts and PBS buffer 100 parts, and the specific steps are as follows: Step 1: 40 parts of anhydrous ethanol, 10 parts of deionized water and 5 parts of ammonia water were mixed to prepare solution A, 0.4 parts of hydroxymethyl coumarin and 4 parts of TEOS were dispersed and dissolved in 20 parts of anhydrous ethanol to obtain solution B, and solution B was slowly added to solution A under magnetic stirring at 30°C and 60 rpm, and the stirring was continued for 3 hours after the addition was completed to obtain coumarin@SiO2sol; Step 2: The coumarin@SiO2sol was centrifuged at 12000 rpm for 15 min, and the supernatant was discarded to obtain the precipitate, which was resuspended in 40 parts of anhydrous ethanol, and 6 parts of glutaric anhydride was added, and the reaction was heated at 37°C for 3 hours, and the precipitate was collected by centrifugation to obtain carboxylated coumarin@SiO2microspheres; Step 3: HCV recombinant antigen 1 part and carboxylated coumarin@SiO2microspheres were dispersed and dissolved in 100 parts of 0.1 M pH 6.0 MES buffer, and 0.2 parts of EDC and 0.5 parts of NHS were added to catalyze the covalent coupling of amino groups in HCV recombinant antigen and carboxyl groups in carboxylated coumarin@SiO2microspheres, and after the reaction was completed, the precipitate was collected by centrifugation, which was antigen-coupled microspheres, and 1 part of anti-HCVcAg monoclonal antibody was dispersed and dissolved in 100 parts of 0.1 M pH 6.0 MES buffer, and EDC / NHS was added for continuous reaction and centrifugation to obtain antigen-antibody coupled microspheres; Step 4: Take 10 parts of BSA and antigen-antibody coupled microspheres and disperse and dissolve in deionized water to block the active sites on the antigen-antibody coupled microspheres, centrifuge and discard the supernatant to remove unbound BSA, and obtain blocked microspheres, resuspend the blocked microspheres in 100 parts of PBS buffer to obtain a binding reagent; Step 5: Take 100 parts of capture reagent, 300 parts of gelatin solution, 300 parts of sodium hydroxide solution and binding reagent for dispensing to obtain a combination reagent, i.e. an immunological detection reagent for blood virus screening.

[0024] Example 7: The present embodiment provides an immunological detection reagent for blood virus screening, which comprises the following raw materials by weight: 100 parts of capture reagent, 300 parts of skimmed milk, 300 parts of sodium hydroxide solution, 1 part of HCV recombinant antigen, 1 part of anti-HCVcAg monoclonal antibody, 0.6 parts of hydroxymethyl coumarin, 10 parts of BSA, 4 parts of glutaric anhydride, 6 parts of TEOS and 100 parts of PBS buffer, and the specific steps are as follows: Step 1: 40 parts of anhydrous ethanol, 10 parts of deionized water and 5 parts of ammonia water were mixed to prepare solution A, 0.6 parts of hydroxymethyl coumarin and 6 parts of TEOS were dispersed and dissolved in 20 parts of anhydrous ethanol to obtain solution B, and solution B was slowly added to solution A under magnetic stirring at 30°C and 60 rpm, and the stirring was continued for 3 hours after the addition was completed to obtain coumarin@SiO2sol; Step 2: centrifuge the coumarin@SiO2sol 12000 rpm for 15 min, discard the supernatant to obtain the precipitate, resuspend the precipitate in 40 parts of anhydrous ethanol, and add glutaric anhydride 4 parts to it, heat the reaction at 37℃ for 3 h, centrifuge to collect the precipitate to obtain carboxylated coumarin@SiO2microspheres; Step 3: disperse and dissolve 1 part of HCV recombinant antigen and carboxylated coumarin@SiO2microspheres in 100 parts of 0.1 M pH 6.0 MES buffer, add 0.24 parts of EDC and 0.6 parts of NHS to catalyze the covalent coupling of amino groups in HCV recombinant antigen and carboxyl groups in carboxylated coumarin@SiO2microspheres, after the reaction is completed, centrifuge to collect the precipitate, which is the antigen-coupled microspheres, disperse and dissolve 1 part of anti-HCV cAg monoclonal antibody and the precipitate in 100 parts of 0.1 M pH 6.0 MES buffer, add EDC / NHS to continue the reaction, centrifuge to collect the antigen-antibody coupled microspheres; Step 4: disperse and dissolve 10 parts of BSA and antigen-antibody coupled microspheres in deionized water to block the active sites on the antigen-antibody coupled microspheres, centrifuge to discard the supernatant to remove unbound BSA, and obtain blocked microspheres, resuspend the blocked microspheres in 100 parts of PBS buffer to obtain the binding reagent; Step 5: take 100 parts of capture reagent, 300 parts of skimmed milk, 300 parts of sodium hydroxide solution, and the binding reagent to be dispensed to obtain a combination reagent, i.e. an immunological detection reagent for blood virus screening.

[0025] Example 8: The present embodiment provides a detection method of an immunological detection reagent for blood virus screening, which comprises the following materials: a high-binding enzyme-labeled plate, an immunological detection reagent, and a serum sample; The high-binding enzyme-labeled plate is a 96-well detachable high-binding enzyme-labeled plate purchased from Bi Yun Tian; The immunological detection reagent is an immunological detection reagent for blood virus screening prepared in Examples 5-7; The serum sample is a blood sample without anticoagulant collected in a clinical centrifugal separation; The detection method specifically comprises the following steps: Take high binding force enzyme labeled plate, add 0.5 parts of capture reagent in the immune detection reagent to the hole plate, incubate at 37℃ for 2h, adsorb and combine HCV recombinant antigen and anti-HCV cAg monoclonal antibody in the capture reagent at the bottom of the hole plate for capturing antigen or antibody material in the serum, after incubation, carefully suck out the capture reagent, add 1.5 parts of blocking agent (sucrose solution, gelatin solution or skim milk) to block the sites not combined with the capture reagent at the bottom of the hole plate, improve the detection accuracy; after carefully sucking out the blocking agent, add 1 part of serum sample and incubate for 15 min, promote the capture reagent to capture HCV antigen or HCV antibody in the serum sample, after incubation, carefully suck out the serum sample in the hole plate, add 0.5 parts of combination reagent and continue to incubate for 15 min, so that the microspheres modified with HCV recombinant antigen and anti-HCV cAg monoclonal antibody in the combination reagent specifically combine with the HCV antigen or HCV antibody captured by the capture reagent, after sucking out the combination reagent, invert the hole plate to drain the liquid, then add 1.5 parts of lysing agent, i.e. sodium hydroxide solution, and shake at 50 rpm for 5 min on a shaker to destroy the structure of the silica microspheres and expose the hydroxymethyl coumarin, detect at 325 nm wavelength by enzyme labeled instrument, and diagnose HCV infection negative / positive according to the standard curve.

[0026] SDS-PAGE gel electrophoresis investigation: the HCV cAg protein prepared in Example 2 was identified, and the molecular weight was investigated by SDS-PAGE gel electrophoresis method, using standard molecular weight Marker as a control, and the results are shown in Figure 2 ; the anti-HCV cAg monoclonal antibody prepared in Example 4 was identified by SDS-PAGE gel electrophoresis, and the results are shown in Figure 3 .

[0027] Specificity investigation: physiological saline, negative serum, negative whole blood, HCV antigen positive serum, HCV antibody positive serum and HCV antigen / antibody positive serum were used as detection samples, which were blank group, negative group 1, negative group 2, antigen group, antibody group and antigen antibody group respectively, the immune detection reagent prepared in Examples 5-7 was used, and the detection method described in Example 8 was used for HCV virus screening diagnosis, and the commercially available HCV core antigen detection kit and HCV antibody detection kit were used for reference, and the results are shown in Table 1.

[0028] Table 1 Immune reactivity of HCV

[0029] Figure 2 The results show that the molecular weight of HCV cAg protein is about 77 kDa, which is close to the target protein molecular weight of 76.67 kDa, and there is no impurity band, indicating that the HCV cAg protein has good purification degree.

[0030] Figure 3 The results show that the anti-HCV cAg monoclonal antibody appears two protein bands, about 25 kDa and 50 kDa, respectively, which are the light chain and heavy chain bands of the antibody, and no other obvious impurity bands, indicating that the anti-HCV cAg monoclonal antibody is well purified.

[0031] In the results of Table 1, the immunological detection reagent prepared in Examples 5-7 has high accuracy in the detection results of HCV antigen and HCV antibody, and the interfering substances in the negative serum or blood sample do not affect the HCV negative / positive diagnosis results, and the HCV recombinant antigen and the anti-HCV cAg monoclonal antibody contained in the immunological detection reagent do not produce specific binding, mainly because the modified HCV protein regions are different, which are core, E1 and NS3 regions, so the immunological detection reagent provided by the present application has good whole-cycle HCV detection effect.

[0032] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0033] The above describes the present application and its embodiments, which is not restrictive, and the drawings shown are only one of the embodiments of the present application, and the actual application is not limited thereto. In summary, if a person skilled in the art is inspired by it, without departing from the purpose of the present application, without creative design, similar ways and embodiments of the technical solution should belong to the protection scope of the present application.

Claims

1. An immunoassay reagent for blood viral screening, characterized by, The immunological detection reagent comprises the following raw materials by weight: 100 parts of a capture reagent, 300 parts of a blocking agent, 300 parts of a lysis agent, 1 part of an HCV recombinant antigen, 1 part of an anti-HCV cAg monoclonal antibody, 0.2-0.6 parts of hydroxymethyl coumarin, 10 parts of BSA, 2-6 parts of glutaric anhydride, 4-8 parts of TEOS and 100 parts of PBS buffer; The specific preparation method of the anti-HCV cAg monoclonal antibody is as follows: S1: Analyzing the HCV viral protein sequence and selecting the HCV cAg amino acid sequence, determining and optimizing the gene sequence encoding the HCV cAg as the target gene; S2: Constructing a recombinant plasmid for the target gene, transforming, expressing and purifying the recombinant plasmid to obtain the HCV cAg; S3: Preparing hybridoma cells using the HCV cAg and screening to obtain a monoclonal cell strain, injecting the monoclonal cell strain into the abdominal cavity of a mouse for culture, extracting ascites, centrifuging and collecting the supernatant; S4: Purifying and freeze-drying the supernatant to obtain the anti-HCV cAg monoclonal antibody.

2. The immunoassay test reagent for viral screening of blood according to claim 1, characterized by, The HCV recombinant antigen is an HCV NS3 recombinant antigen; The HCV cAg amino acid sequence is shown in SEQ ID NO. 1; The gene sequence encoding the HCV cAg is shown in SEQ ID NO.

2.

3. The immunoassay test reagent for viral screening of blood according to claim 2, characterized by, The blocking agent is selected from any one of sucrose solution, gelatin solution and skimmed milk; The lysis agent is a 1 mol / L sodium hydroxide solution.

4. A method for the preparation of an immunoassay reagent for viral screening of blood according to any one of claims 1 to 3, characterized in that, Specifically comprises the following steps: Step 1: mixing anhydrous ethanol, deionized water and ammonia water to prepare solution A, dispersing and dissolving hydroxymethyl coumarin and TEOS to obtain solution B, and adding solution B dropwise into solution A to obtain coumarin@SiO2 sol; Step 2: treating the coumarin@SiO2 sol with glutaric anhydride to obtain carboxylated coumarin@SiO2 microspheres; Step 3: covalently coupling the HCV recombinant antigen and the anti-HCV cAg monoclonal antibody with the carboxylated coumarin@SiO2 microspheres in sequence to obtain antigen-antibody coupled microspheres; Step 4: dispersing and centrifuging BSA and the antigen-antibody coupled microspheres to discard the supernatant to obtain blocked microspheres, and resuspending the blocked microspheres in PBS buffer to obtain a combined reagent; Step 5: dispensing the capture reagent, the blocking agent, the lysis agent and the combined reagent to obtain a combined reagent, i.e. an immunological detection reagent for blood virus screening.

5. The method for preparing an immunoassay reagent for blood virus screening according to claim 4, characterized by, In step 1, the mass ratio of the anhydrous ethanol, deionized water and ammonia water is 8:2:1; In step 3, EDC and NHS are used in the covalent coupling process.

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