A reagent containing a swine pseudorabies virus gB protein magnetic microparticle coating, a preparation method and a kit

Abstract

The application discloses a reagent containing pig pseudorabies virus gB protein magnetic microparticle coating, a preparation method and a kit, and relates to the technical field of antibody detection. The preparation method of the reagent containing pig pseudorabies virus gB protein magnetic microparticle coating comprises the following steps: incubating magnetic microparticles and gB protein antibodies in a coating buffer solution; removing the reaction solution, and performing first sealing on the gB protein magnetic microparticle coating with a first sealing solution; after the first sealing is completed, the first sealing solution is washed away, and then the gB protein magnetic microparticle coating is sealed for the second time with a second sealing solution; after the second sealing is completed, the second sealing solution is washed away, and then resuspension is performed to obtain the reagent containing the gB protein magnetic microparticle coating. The two-step sealing method is adopted to prepare the reagent containing the pig pseudorabies virus gB protein magnetic microparticle coating, the sealing effect is good, and non-specific adsorption in the system is reduced. The kit provided by the application contains the reagent, and has the advantages of high analysis sensitivity, a wide linear range and high accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody detection technology, and particularly relates to a reagent containing a pig pseudorabies virus gB protein magnetic microparticle coating, a preparation method and a kit. BACKGROUND

[0002] Pseudorabies (PR) is a multi-animal co-infection disease caused by pseudorabies virus (PRV). Pseudorabies virus is a capsid double-stranded linear DNA virus, belonging to the Herpesviridae family, the alpha Herpesvirinae subfamily, and the Varicellovirus genus. PRV can infect a variety of mammals, including wild boars, pigs, bears, ruminants and rodents. Pigs are the source of infection and natural reservoir of pseudorabies, and pigs infected with PRV can cause fever, and breeding disorders in breeding pigs, and stillbirths, mummies and abortions in sows. The harm to piglets is the most serious, and piglets can show neurological symptoms and a mortality rate of up to 100%. Adult pigs are not seriously harmed, and are generally subclinical infections. Medium and large pigs show respiratory symptoms, and overall production performance is poor. At present, the disease is widespread in China, and it is difficult to eradicate once it occurs, causing huge economic losses to the pig industry. In the prevention and control of infectious diseases, immunization is an important measure to reduce the probability of disease. Large-scale pig farms recommend detecting the antibody level of the pig population 3-4 times per year, especially before and after immunization with a pseudorabies vaccine, to detect the antibody level of the pig population in a timely manner, and analyze whether the vaccine efficacy achieves the immune effect by comparison, so as to perform supplementary immunization. In addition, antibody detection can also be used for clinical diagnosis, for example, to check the condition after disease or treatment, or as a basis for the effectiveness of vaccination.

[0003] There are many methods for detecting pig pseudorabies antibodies at present, such as virus neutralization test, ELISA and immunochromatography. Virus neutralization test is an internationally recognized antibody detection method due to its strong specificity, but the method is time-consuming and cumbersome to operate, and requires professional personnel to operate. Latex agglutination test is simple to operate, highly sensitive, and suitable for rapid monitoring of clinical diseases, epidemiological investigation and initial screening of breeding herd quarantine and purification, but it has poor specificity and high false positive rate, and the final judgment needs to be made in combination with the clinical situation. Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used methods for detecting pseudorabies virus antibodies in the laboratory. Pseudorabies virus gB protein antibody blocking and competitive ELISA method is used to detect pig pseudorabies virus antibodies, which further improves the sensitivity of ELISA, but the test time still takes about one hour, and cannot be accurately quantified. Immunochromatographic test strip, this method takes less time, is convenient and fast, and is very suitable for clinical rapid detection in breeding farms, but the sensitivity and repeatability are slightly poor.

[0004] It is necessary to continue to develop new antibody detection methods based on the advantages and disadvantages of various detection methods, and to combine accurate quantification, differentiation between wild virus infection and immunity, and clinical rapid testing, which may be the development trend of future pseudorabies virus antibody detection. SUMMARY

[0005] The technical problem solved by the present application is based on the problems mentioned in the background art, and a reagent containing a pig pseudorabies virus gB protein magnetic microparticle coating, a preparation method and a kit are provided to improve the sensitivity and specificity of pig pseudorabies virus antibody detection.

[0006] To solve the above problems, the present application proposes the following technical solutions:

[0007] In a first aspect, the present application provides a preparation method of a reagent containing a pig pseudorabies virus gB protein magnetic microparticle coating, comprising the following steps:

[0008] Incubate the magnetic microparticles and the gB protein antibody in the coating buffer, the concentration of the magnetic microparticles is 3-5 mg / mL, and the concentration of the gB protein antibody is 10-40 μg / mL, to obtain a reaction solution containing a gB protein magnetic microparticle coating;

[0009] Remove the reaction solution, wash the gB protein magnetic microparticle coating, and perform a first blocking of the gB protein magnetic microparticle coating with a first blocking solution;

[0010] After blocking is completed, wash away the first blocking solution, and then perform a second blocking of the gB protein magnetic microparticle coating with a second blocking solution;

[0011] After blocking is completed, wash away the second blocking solution, resuspend the gB protein magnetic microparticle coating in a preservation solution to obtain a reagent containing the gB protein magnetic microparticle coating;

[0012] The first blocking solution is one or more of bovine serum albumin, ethanolamine, CE510, and glycine;

[0013] The second blocking solution is one or more of bovine serum albumin, ethanolamine, CE510, and glycine, and the second blocking solution is different from the first blocking solution.

[0014] Further, the first blocking solution is one or more of bovine serum albumin and CE510; and the second blocking solution is one or more of ethanolamine and glycine.

[0015] Further, the first blocking solution is CE510; and the second blocking solution is ethanolamine.

[0016] Further, the temperature of the first blocking is 24-38℃, and the blocking time is 4-8 hours.

[0017] Further, the temperature of the first sealing is 35-38℃, and the sealing time is 4-8 hours.

[0018] Further, the temperature of the second sealing is 2-45℃, and the sealing time is 15-24 hours.

[0019] Further, the temperature of the second sealing is 2-8℃, and the sealing time is 16-20 hours.

[0020] Further, the gB protein antibody is one of the full length, fragment or polypeptide of the porcine pseudorabies virus gB protein.

[0021] Further, the magnetic micro-particle has a particle size of 1000-3000nm.

[0022] Further, the coating buffer is any one of 0.02M-0.1M borate buffer, phosphate buffer, and has a pH of 8-10; the storage solution contains 0.02M-0.1M phosphate buffer, 0.5%-5% stabilizer, 0.05%-0.2% surfactant, and 0.01%-0.2% preservative.

[0023] Further, the stabilizer is one or more of the combination of bovine serum albumin, gelatin, trehalose, sucrose and casein; the surfactant is one or more of the combination of Tween 20, Tween 80 and S9; and the preservative is one or both of BND and Proclin 300.

[0024] In the second aspect, the application provides a reagent containing a porcine pseudorabies virus gB protein magnetic micro-particle coating, which is prepared by the preparation method of the first aspect.

[0025] In the third aspect, the application provides a kit for detecting porcine pseudorabies virus gB protein antibody, which comprises the reagent containing a porcine pseudorabies virus gB protein magnetic micro-particle coating prepared by the preparation method of the first aspect, or comprises the reagent containing a porcine pseudorabies virus gB protein magnetic micro-particle coating of the second aspect.

[0026] Further, the concentration of the reagent containing a porcine pseudorabies virus gB protein magnetic micro-particle coating is 0.08-0.4mg / mL.

[0027] Further, the kit further comprises a reagent containing a porcine pseudorabies virus gB protein aridin ester conjugate, a calibrator containing a porcine pseudorabies virus gB protein polyclonal antibody and a quality control product.

[0028] Further, the preparation method of the reagent containing a porcine pseudorabies virus gB protein aridin ester conjugate is as follows:

[0029] 1) Labeling, 100-300 μl of the arycridinester labeling buffer, 20-100 μg of the porcine pseudorabies virus gB protein and 0.00005-0.0005 mM of the arycridinester stock solution were sequentially added into a centrifuge tube to prepare 1 ml of the arycridinester conjugate of the porcine pseudorabies virus gB protein, the liquid was mixed by inverting the centrifuge tube, and the reaction was carried out at room temperature in the dark for 30 min;

[0030] 2) Termination, the arycridinester conjugate was obtained after the labeling reaction for 30 min, then 200 μl of the arycridinester termination buffer was added, and the mixture was mixed by hand inversion, and the reaction was carried out at room temperature in the dark for 30 min;

[0031] 3) Dialysis, the product obtained above was transferred into a dialysis bag, both ends of the bag were clamped with dialysis clamps, and the clamps were tightly clamped (fastened with an elastic band). 2 L of the arycridinester dialysis buffer was measured in a beaker, the dialysis bag containing the labeling agent was placed in the beaker, the beaker was placed on a stirrer, the stirrer was started, and the beaker was placed in a refrigerator at 2-8 °C, and the dialysis was carried out in the dark with stirring. The arycridinester dialysis buffer was replaced every 2 hours, and the dialysis was carried out for 3-5 times;

[0032] 4) The beaker was taken out, the dialysis bag was taken out, and all the labeling agent was transferred into a new centrifuge tube. 50% glycerol was added, 0.1% BSA was added to the centrifuge tube, and the mixture was gently mixed until homogeneous. The mixture was stored at -20 °C in the dark to obtain a reagent stock solution containing the arycridinester conjugate of the porcine pseudorabies virus gB protein;

[0033] 5) Dilution, the reagent stock solution containing the arycridinester conjugate of the porcine pseudorabies virus gB protein was added to the arycridinester diluent at a ratio of 1 / 1000-1 / 200 (v / v) to prepare an arycridinester working solution.

[0034] The arycridinester labeling buffer contains 0.1 M-0.3 M PB buffer solution or BB buffer solution, pH 7.0-9.0, 0.05-0.2 M NaCl or KCl, and 0.01%-0.2% preservative. The preservative is one or both of BND and Proclin 300.

[0035] The arycridinester termination buffer contains 0.1 M-0.3 M PB buffer solution or BB buffer solution, pH 7.0-9.0, 1%-5% lysine, glycine or alanine, and 0.01%-0.2% preservative. The preservative is one or both of BND and Proclin 300.

[0036] The arycridinester dialysis buffer is 20-40 mM PBS buffer solution or BB buffer solution.

[0037] The acridine ester diluent contains 0.02M-0.1M phosphate buffer, 0.5%-5% stabilizer, 0.05%-0.2% surfactant, 0.01%-0.2% preservative; the stabilizer is one or more combinations of bovine serum albumin, gelatin, trehalose, sucrose and casein; the surfactant is one or more combinations of Tween 20, Tween 80 and S9; and the preservative is one or both of BND and Proclin 300.

[0038] Further, the preparation method of the calibrator and the quality control is as follows:

[0039] 0.1-500U / mL porcine pseudorabies virus gB protein polyclonal antibody is added in the calibrator diluent to prepare the calibrator and the quality control with preset concentrations.

[0040] The calibrator diluent contains 0.01M-0.05M phosphate buffer, 0.5%-10% stabilizer, 0.05%-0.2% surfactant, 0.01%-0.2% preservative; the stabilizer is one or more combinations of bovine serum albumin, calf serum, goat serum, casein, sucrose and trehalose; the surfactant is one or more combinations of Tween 20, Tween 80, S9 and Triton X-100; and the preservative is one or both of BND and Proclin 300.

[0041] According to the technical scheme of the present application, the stabilizer in the above reagent mainly protects the activity of the protein and improves the stability of the reagent and prolongs the effective period of the product.

[0042] According to the technical scheme of the present application, the surfactant in the above reagent mainly promotes the uniform dispersion of each component in the reagent system and each substance in the detection sample and improves the detection precision and ensures the reproducibility of the results.

[0043] According to the technical scheme of the present application, the preservative prevents the adverse effects of microbial reproduction on the quality of the reagent, and any substance capable of inhibiting the growth of microorganisms can be used as the preservative of the present application.

[0044] Further, the kit of the present application further comprises an excitation solution A and an excitation solution B:

[0045] The excitation solution A contains 1% H2O2, 5% ethanol and 0.1% Tween 20.

[0046] The excitation solution B contains 0.1M sodium hydroxide, 5% ethanol and 0.1% Triton X-100.

[0047] The present application further provides a detection method for detecting porcine pseudorabies virus gB antibody by using the above kit, which comprises the following steps:

[0048] Test the working calibration sample, and make a master curve;

[0049] Calibrate the full-automatic chemical immune analyzer with the calibration sample, and obtain a calibration curve;

[0050] Test the quality control sample, and if the test value is within the specified target value range, the sample test can be performed;

[0051] The instrument takes the sample to be tested, adds the reagent containing the pig pseudorabies virus gB protein magnetic microparticle coating and the reagent containing the pig pseudorabies virus gB protein aziridine ester conjugate for immune reaction, adds the excitation liquid A and the excitation liquid B, enters the full-automatic immune detection system, measures the luminescence signal, and obtains the concentration of the pig pseudorabies virus gB protein antibody in the sample through the calibration curve.

[0052] The kit for detecting the pig pseudorabies virus gB protein antibody provided by the application adopts the double-antigen sandwich method combined with the chemiluminescence detection technology, which not only improves the sensitivity and specificity of detection, but also can perform accurate quantification. Meanwhile, the raw materials of the calibration sample and the quality control sample matched in the kit adopt relatively stable polyclonal antibodies, and the stability is good. In combination with the chemiluminescence analyzer detection, the sample can be automatically batched, the pain point of manual operation of gB antibody detection is solved, and the clinical detection demand is met.

[0053] Compared with the prior art, the technical effects that can be achieved by the application include:

[0054] The preparation method of the reagent containing the pig pseudorabies virus gB protein magnetic microparticle coating provided by the application adopts a two-step blocking method to prepare the pig pseudorabies virus gB protein magnetic microparticle coating, and the blocking effect is good, the non-specific adsorption in the system is reduced, and the specificity of the detection system can be ensured. Further, due to the reduction of the background of the reagent, the detection rate of gB antibody positive serum can be improved, that is, the sensitivity of the detection system can be improved.

[0055] The preparation method of the reagent containing the pig pseudorabies virus gB protein magnetic microparticle coating provided by the application can ensure that the effect of the blocking agent is best by screening a suitable blocking agent and selecting different blocking times and blocking temperatures, so that the blocking is more complete, and the obtained reagent has good stability.

[0056] The detection kit provided by the application contains the above-mentioned reagent with low background, has the advantages of high analysis sensitivity, wide linear range and high accuracy, and has better detection performance; the calibration sample and the quality control sample prepared by using the polyclonal antibody have better stability than the calibration sample and the quality control sample prepared by using the antiserum, can replace the existing detection kit, and meet the clinical detection demand. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments will be clearly and completely described below. Obviously, the following described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application.

[0058] It should be understood that the terms "comprising" and "including" as used in the specification and the appended claims indicate the presence of the described features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0059] It should also be understood that the terms used in the specification of the embodiments of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0060] In the present application, unless otherwise specified, "%" refers to mass percentage.

[0061] Embodiment 1

[0062] The present embodiment provides a reagent containing a pig pseudorabies virus gB protein magnetic microparticle coating. The magnetic microparticles used are superparamagnetic nanometer microspheres, the surface modification functional group is toluenesulfonyl, and the particle size is 1000 nm.

[0063] The preparation method of the reagent is as follows:

[0064] 1) Magnetic microparticle cleaning, washing the magnetic microparticles with coating buffer, then enriching the magnetic microparticles with a magnetic stand, and aspirating the coating buffer, three times.

[0065] 2) Incubation, after washing the magnetic microparticles, the magnetic microparticles were placed in the coating buffer, and the final concentration of the magnetic microparticles was 4 mg / mL, then gB protein was added to a final concentration of 40 ug / mL, and incubated at 37°C for 18h.

[0066] 3) Blocking, using a magnetic stand to re-enrich the above coating, aspirating the reaction solution, washing three times with washing solution, adding an equal volume of CE510, and placing it at 2-8°C for 6h; after blocking, the coating was re-enriched with a magnetic stand, the reaction solution was aspirated, and the washing solution was washed three times, 50mM ethanolamine was added, and it was placed at 37°C for 18h.

[0067] 4) Preservation, after the end of the closing, the coated material is re-enriched by magnetic frame, the reaction solution is sucked, the washing solution is washed for three times, the magnetic micro-particle preservation solution is added, and the mother liquor containing the pig pseudorabies virus gB protein magnetic micro-particle coated material is obtained.

[0068] 5) Dilution, the above-mentioned mother liquor containing the pig pseudorabies virus gB protein magnetic micro-particle coated material is added into the magnetic micro-particle preservation solution, so that the final concentration is 0.2 mg / mL.

[0069] The coating buffer solution of the embodiment is 0.05 M boric acid buffer solution, pH 9.0.

[0070] The magnetic micro-particle preservation solution of the embodiment contains 0.05 M phosphate buffer solution, 2% bovine serum albumin, 2% trehalose, 0.1% Tween 20 and 0.1% Proclin 300.

[0071] Example 2:

[0072] The embodiment provides a reagent containing pig pseudorabies virus gB protein magnetic micro-particle coated material, which is different from the embodiment 1 in that the closing of step 3) is different.

[0073] The closing of the embodiment 3) is specifically as follows: the above-mentioned coated material is re-enriched by magnetic frame, the reaction solution is sucked, the washing solution is washed for three times, an equal volume of CE510 is added, and is placed at 37°C for closing for 6 h; after the end of the closing, the coated material is re-enriched by magnetic frame, the reaction solution is sucked, the washing solution is washed for three times, 50 mM ethanolamine is added, and is placed at 2-8°C for closing for 18 h.

[0074] Example 3:

[0075] The embodiment provides a reagent containing pig pseudorabies virus gB protein magnetic micro-particle coated material, which is different from the embodiment 1 in that the closing of step 3) is different.

[0076] The closing of the embodiment 3) is specifically as follows: the above-mentioned coated material is re-enriched by magnetic frame, the reaction solution is sucked, the washing solution is washed for three times, an equal volume of CE510 is added, and is placed at 37°C for closing for 6 h; after the end of the closing, the coated material is re-enriched by magnetic frame, the reaction solution is sucked, the washing solution is washed for three times, 50 mM ethanolamine is added, and is placed at 2-8°C for closing for 18 h.

[0077] Example 4:

[0078] The embodiment provides a reagent containing pig pseudorabies virus gB protein magnetic micro-particle coated material, which is different from the embodiment 1 in that the closing of step 3) is different.

[0079] The 3) blocking of the present embodiment is specifically as follows: the above-mentioned coated material is re-enriched by a magnetic stand, the reaction solution is sucked off, and the washing solution is washed three times, an equal volume of CE510 is added, and the mixture is blocked at 37℃ for 6 hours; after the blocking is completed, the coated material is re-enriched by a magnetic stand, the reaction solution is sucked off, and the washing solution is washed three times, 50 mM ethanolamine is added, and the mixture is blocked at 45℃ for 18 hours.

[0080] Embodiment 5:

[0081] The present embodiment provides a reagent containing a pig pseudorabies virus gB protein magnetic microparticle coated material, which is different from the reagent of embodiment 1 in that the blocking of step 3) is different.

[0082] The 3) blocking of the present embodiment is specifically as follows: the above-mentioned coated material is re-enriched by a magnetic stand, the reaction solution is sucked off, and the washing solution is washed three times, an equal volume of CE510 is added, and the mixture is blocked at 37℃ for 6 hours; after the blocking is completed, the coated material is re-enriched by a magnetic stand, the reaction solution is sucked off, and the washing solution is washed three times, 50 mM ethanolamine is added, and the mixture is blocked at 37℃ for 18 hours.

[0083] Embodiment 6:

[0084] The present embodiment provides a reagent containing a pig pseudorabies virus gB protein magnetic microparticle coated material, which is different from the reagent of embodiment 1 in that the blocking of step 3) is different.

[0085] The 3) blocking of the present embodiment is specifically as follows: the above-mentioned coated material is re-enriched by a magnetic stand, the reaction solution is sucked off, and the washing solution is washed three times, an equal volume of CE510 is added, and the mixture is blocked at 37℃ for 6 hours; after the blocking is completed, the coated material is re-enriched by a magnetic stand, the reaction solution is sucked off, and the washing solution is washed three times, 50 mM ethanolamine is added, and the mixture is blocked at 37℃ for 18 hours.

[0086] Embodiment 7:

[0087] The present embodiment provides a reagent containing a pig pseudorabies virus gB protein magnetic microparticle coated material, which is different from the reagent of embodiment 1 in that the blocking of step 3) is different.

[0088] The 3) blocking of the present embodiment is specifically as follows: the above-mentioned coated material is re-enriched by a magnetic stand, the reaction solution is sucked off, and the washing solution is washed three times, an equal volume of CE510 is added, and the mixture is blocked at 37℃ for 6 hours; after the blocking is completed, the coated material is re-enriched by a magnetic stand, the reaction solution is sucked off, and the washing solution is washed three times, 50 mM ethanolamine is added, and the mixture is blocked at 37℃ for 18 hours.

[0089] The reagents containing pig pseudorabies virus gB protein magnetic microparticle coated materials provided in the above-mentioned embodiments 1-7 are made into reagent kits, which are corresponding to reagent kits 1-7, and the reagent containing a pig pseudorabies virus gB protein magnetic microparticle coated material is used as reagent 1.

[0090] Each reagent kit further includes reagent 2 (a reagent containing a pig pseudorabies virus gB protein aridinium ester conjugate), a calibrator, a quality control, an excitation solution A, and an excitation solution B of the same composition. The following is a specific description:

[0091] ①Reagent 2 (reagent containing porcine pseudorabies virus gB protein aziridine ester conjugate) and its preparation method, the steps are as follows:

[0092] 1) Labeling, for every 1ml porcine pseudorabies virus gB protein aziridine ester conjugate prepared, 300μl of aziridine ester labeling buffer, 100μg of gB protein, and 0.000025mM of aziridine ester stock solution were sequentially added into a centrifuge tube, mixed, and reacted at room temperature in the dark for 30min.

[0093] 2) Termination, after 30min of labeling reaction, aziridine ester conjugate was obtained, aziridine ester termination buffer (0.1 times the reaction volume) was added into the aziridine ester conjugate, and mixed by hand, and reacted at room temperature in the dark for 30min.

[0094] 3) Dialysis, the product obtained above was transferred into a dialysis bag, both ends of the dialysis bag were clamped, and the bag was clamped tightly (fastened with an elastic band), and it was confirmed that there was no liquid leakage. 2L of aziridine ester dialysis buffer was measured in a beaker, the dialysis bag containing the labeling product was placed in the beaker, the beaker was placed on a stirrer, the stirrer was started, and the beaker was placed in a 2-8℃ refrigerator, and the dialysis was carried out in the dark with stirring. The aziridine ester dialysis buffer was replaced every 2h, and the dialysis was carried out for 4 times.

[0095] 4) The beaker was taken out, the dialysis bag was taken out, and all the labeling product was transferred into a new centrifuge tube. 50% glycerol was added, 0.1% BSA was added into the centrifuge tube, and mixed gently until homogeneous. The mixture was stored at -20℃ in the dark to obtain a reagent stock solution containing porcine pseudorabies virus gB protein aziridine ester conjugate.

[0096] 5) Dilution, the reagent stock solution containing porcine pseudorabies virus gB protein aziridine ester conjugate was taken, and 1 / 500 (v / v) was added into aziridine ester diluent to prepare aziridine ester working solution.

[0097] The aziridine ester labeling buffer contains 0.1M PB buffer solution, pH 8.0, 0.15M NaCl, and 0.1% Proclin300.

[0098] The aziridine ester termination buffer contains 0.1M PB buffer solution, pH 8.0, 1% glycine, and 0.1% Proclin300.

[0099] The aziridine ester dialysis buffer is 20mM PBS buffer solution.

[0100] The aziridine ester diluent contains 0.05M phosphate buffer, 0.5% casein, 2% sucrose, 0.1% S9, and 0.1% Proclin300.

[0101] ② Preparation of calibrators and quality control products

[0102] 0.1-500U / mL porcine pseudorabies virus gB protein polyclonal antibody was added in the calibrator diluent, and the calibrator and quality control were configured with preset concentration, wherein:

[0103] The concentration of calibrator 1 was 0U / mL;

[0104] The concentration of calibrator 2 was 2±0.4U / mL;

[0105] The concentration of calibrator 3 was 200±40U / mL;

[0106] The concentration of quality control 1 was 2±0.4U / mL;

[0107] The concentration of quality control 2 was 50±25U / mL;

[0108] The calibrator diluent contains 0.05M phosphate buffer, 1% casein, 2% sucrose, 0.1% Tween 80, 0.05% Proclin 300, and 0.05% BND.

[0109] ③Preparation of the stimulating solution

[0110] The stimulating solution A contains 1% H2O2, 5% ethanol, and 0.1% Tween 20;

[0111] The stimulating solution B contains 0.1M sodium hydroxide, 5% ethanol, and 0.1% triton X-100.

[0112] Performance test experiment 1

[0113] The above kits 1-7 were respectively used to detect 300 cases of porcine pseudorabies virus antibody negative serum and 188 cases of porcine pseudorabies virus antibody positive serum, and the specificity and sensitivity of each kit were investigated. Among them, the sensitivity = the number of positive samples detected by the kit / the number of clinically diagnosed positive samples × 100%, and the specificity = the number of negative samples detected by the kit / the number of clinically diagnosed negative samples × 100%. The detection results are shown in Table 1.

[0114] Table 1 Clinical detection results of kits 1-7

[0115]

[0116]

[0117] From the results of Table 1, it can be seen that the reagents containing the pig pseudorabies virus gB protein magnetic microparticle coating in kits 1-6 are blocked by two-step method compared with kit 7. The detection sensitivity of kits 1-6 is higher than that of kit 7, which shows that the reagent containing the pig pseudorabies virus gB protein magnetic microparticle coating blocked by two-step method is superior to that blocked by one-step method. This is because in the present application, the first step adopts the conventional magnetic bead blocking method for blocking for 6 hours, but a second step is added to block for 18 hours by changing the blocking agent, and the effects of the two blocking agents are superimposed while the blocking time is prolonged, which is conducive to completely blocking the blank sites on the magnetic beads that are not combined with the target protein.

[0118] The above results also show that the temperature of the two blocking steps has a significant effect on the blocking effect, and the optimal reaction temperature of different blocking agents is different. The two blocking steps in the present application adopt different temperatures. The blocking agent of macromolecules such as CE510 and BSA is blocked at 37°C for 6 hours, which can occupy the blank sites on the magnetic beads in a relatively short time, and the remaining unblocked sites are blocked for a long time under the condition of 2-45°C. It can be seen from the comparison between the various examples that the effect of blocking at 2-8°C is the best. The sensitivity and specificity of kit 2 are the best, because CE510, a macromolecule, is first used for blocking at 37°C. CE510 is a compound synthesized by PEG and oligoamine, which is covalently coupled with the magnetic bead surface group and has no specific adsorption, and the blocking effect is better than that of bovine serum albumin. Then, small molecule ethanolamine is used for blocking. Since ethanolamine has a small molecular weight, it is easier to disperse into the remaining blank sites and react with the magnetic bead group at 2-8°C, which promotes the blocking effect and further improves the sensitivity of the reagent.

[0119] Performance test experiment 2

[0120] The stability of kits 1-7 composed of the above-mentioned reagent 1 containing the pig pseudorabies virus gB protein magnetic microparticle coating and reagent 2 containing the acridinium ester label was tested respectively, and the 2-8°C storage stability for 12 months was investigated. The results are shown in Table 2.

[0121] Table 2 Stability results of kits 1-7

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] From the results of Table 2, in the kits 1-7 of the present application, reagent 2 is the same, and only reagent 1 is derived from Examples 1-7, respectively. From the stability, the reagent containing the pig pseudorabies virus gB protein magnetic microparticle coating material blocked by the two-step method in Examples 1-6 is obviously superior to Example 7 in stability, and at the same time, the blocking temperature also has different effects on the stability of blocking. The deviation of kits 2-3 stored at 2-8℃ for 12 months is less than 10%, which has good stability. The main reason is that the more complete the blocking of reagent 1 is, the less the further blocking phenomenon occurs under long-term storage of the kit at 2-8℃, which leads to the fluctuation of the background. At the same time, in reagent 1, two blocking agents are used to stack blocking at a specific temperature, and the interaction makes the stability of the reagent greatly improved.

[0128] Performance test experiment 3

[0129] The existing positive control of enzyme immunoassay kit is prepared by antiserum. In the present experiment, the comparative calibration and quality control (pig pseudorabies virus antiserum is diluted to 0.1-500 U / mL by using calibration diluent) are set to compare the accelerated stability with the calibration and quality control in the present kit, and the experimental results are shown in Table 3.

[0130] Table 3 Stability experimental results of calibration and quality control

[0131]

[0132] From the results of Table 3, the accelerated stability of the calibration and quality control prepared by using polyclonal antibody is better than that of the calibration and quality control prepared by using antiserum.

[0133] Performance test experiment 4

[0134] In the present experiment, the kit 2 is taken as an example to analyze the performance of the kit of the present application.

[0135] (1) Analysis of sensitivity (detection limit)

[0136] Five samples (number: LoD1-LoD5) with approximate detection limit concentration of the kit are selected respectively, each sample is detected for 5 times, and the 0 concentration sample is detected at the same time, and the results are shown in Tables 4-5. The value of the detection result of the present kit is not more than 3 less than the blank limit, which meets the requirements.

[0137] Table 4 Blank limit test results (concentration: U / mL)

[0138]

[0139] Table 5 Detection limit test results (concentration: U / mL)

[0140] Test number LOD1 LOD2 LOD3 LOD4 LOD5 1 0.07 0.07 0.07 0.08 0.07 2 0.07 0.08 0.07 0.06 0.07 3 0.07 0.07 0.08 0.07 0.07 4 0.08 0.07 0.07 0.07 0.07 5 0.07 0.07 0.07 0.07 0.07

[0141] The results show that the analysis sensitivity (detection limit) of the kit provided by the application is not more than 0.1 U / mL, which meets the requirements.

[0142] (2) Linear range

[0143] The linear correlation coefficient r of the kit provided by the application is greater than 0.9990 in the range of 0.1-500 U / mL, and the test results are shown in Table 6.

[0144] Table 6 Linear range test results

[0145]

[0146] (3) Reproducibility

[0147] The sample 1 and sample 2 are respectively tested for 10 times, and the results are shown in Table 7.

[0148] Table 7 Reproducibility results

[0149]

[0150]

[0151] The reproducibility of the kit provided by the application is within 5%.

[0152] (4) Accuracy

[0153] The accuracy measurement refers to adding a known amount of antibody to the sample diluent, comparing the concentration value after adding with the theoretical concentration value, and calculating the deviation, and the test results are shown in Table 8.

[0154] Table 8 Accuracy test results

[0155]

[0156] The accuracy deviation of the kit is within ±6%, which meets the requirements.

[0157] According to the results of Tables 4-8 above, the detection kit provided by the application has the advantages of high analysis sensitivity, wide linear range, good reproducibility, high accuracy, low cost of the kit, and can meet the needs of clinical detection at present.

[0158] In the above embodiments, the description of each embodiment has its own emphasis, and the parts not described in detail in a certain embodiment can be referred to the related description of other embodiments.

[0159] The above is a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present application, and these modifications or replacements should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing a reagent containing a magnetic microparticle coating of a porcine pseudorabies virus gB protein, characterized in that, The method comprises the following steps: incubating magnetic microparticles with gB protein antibodies in a coating buffer, the concentration of the magnetic microparticles being 3-5 mg / mL, and the concentration of the gB protein antibodies being 10-40 μg / mL, to obtain a reaction solution containing gB protein magnetic microparticle coating; removing the reaction solution, washing the gB protein magnetic microparticle coating, and performing first blocking of the gB protein magnetic microparticle coating with a first blocking solution; after the first blocking, washing away the first blocking solution, and performing second blocking of the gB protein magnetic microparticle coating with a second blocking solution; after the second blocking, washing away the second blocking solution, resuspending the gB protein magnetic microparticle coating in a storage solution, and obtaining a reagent containing the gB protein magnetic microparticle coating; the first blocking solution is CE510, and the second blocking solution is ethanolamine; the temperature of the first blocking is 35-38℃, and the blocking time is 4-8 hours; the temperature of the second blocking is 2-8℃, and the blocking time is 15-24 hours.

2. The production method according to claim 1, wherein The gB protein antibodies are one of a full-length, a fragment, or a polypeptide of a porcine pseudorabies virus gB protein.

3. The production method according to claim 1, wherein The magnetic microparticles have a particle size of 1000-3000 nm.

4. The production method according to claim 1, wherein The coating buffer is any one of a 0.02M-0.1M borate buffer or a 0.02M-0.1M phosphate buffer, and has a pH of 8-10; and the storage solution contains a 0.02M-0.1M phosphate buffer, 0.5%-5% stabilizer, 0.05%-0.2% surfactant, and 0.01%-0.2% preservative.

5. A reagent comprising a magnetic microparticle coating of a porcine pseudorabies virus gB protein, characterized in that, The reagent containing the gB protein magnetic microparticle coating is prepared by the preparation method of any one of claims 1-4.

6. A kit for detecting antibodies to gB protein of porcine pseudorabies virus, characterized by comprising: The reagent containing the gB protein magnetic microparticle coating is prepared by the preparation method of any one of claims 1-4.

7. The kit of claim 6, wherein The concentration of the reagent containing the gB protein magnetic microparticle coating is 0.08-0.4 mg / mL.

8. The kit of claim 6, wherein The reagent containing the gB protein magnetic microparticle coating is prepared by the preparation method of any one of claims 1-4. The reagent containing the gB protein magnetic microparticle coating is prepared by the preparation method of any one of claims 1-4. The concentration of the reagent containing the gB protein magnetic microparticle coating is 0.08-0.4 mg / mL. The reagent containing the gB protein magnetic microparticle coating is prepared by the preparation method of any one of claims 1-4.

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