Reagent containing porcine pseudorabies virus gB protein magnetic particle coating substance, preparation method and kit

Abstract

The invention discloses a reagent containing a porcine pseudorabies virus gB protein magnetic particle coating substance, a preparation method and a kit, and relates to the technical field of antibody detection. The preparation method of the reagent containing the porcine pseudorabies virus gB protein magnetic particle coating substance comprises the following steps: incubating magnetic particles and a gB protein antibody in a coating buffer solution; the reaction liquid is removed, and the gB protein magnetic particle coating and first confining liquid are subjected to first confining; after closing is completed, the first closing liquid is washed away, and the gB protein magnetic particle coating and second closing liquid are subjected to second-time closing; after closing is completed, the second closing liquid is washed away, resuspension is conducted, and the reagent containing the gB protein magnetic particle coating substance is obtained. The magnetic particle coating containing the porcine pseudorabies virus gB protein is prepared by adopting a two-step sealing method, the sealing effect is good, and non-specific adsorption in a system is reduced. The kit provided by the invention contains the reagents and has the advantages of high analysis sensitivity, wide linear range and high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody detection, and in particular to a reagent containing magnetic microparticle coating of porcine pseudorabies virus gB protein, a preparation method and a kit. Background Art

[0002] Porcine pseudorabies (PR) is a multi-animal co-infectious disease caused by pseudorabies virus (PRV). PRV is an enveloped double-stranded linear DNA virus belonging to the family Herpesviridae, subfamily Alphaherpesvirinae, and genus Varicellavirus. 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. Pigs infected with PRV can cause fever. Breeding pigs mainly show reproductive disorders, sows can produce stillbirths, mummies, and miscarriages. The most serious harm is to piglets, which can show neurological symptoms and a mortality rate of up to 100%. Adult pigs are not seriously harmed and are generally latently infected. Medium and large pigs will show respiratory symptoms and clinical manifestations such as poor overall production performance. At present, the disease is widely present in my country. Once it occurs, it is difficult to eradicate, causing huge economic losses to the pig industry. In the prevention and control of infectious diseases, vaccination is an important measure to reduce the probability of disease. Large-scale pig farms are recommended to regularly test the antibody level of pigs 3 to 4 times a year, especially before and after the pseudorabies vaccine is administered. The antibody level of the pigs needs to be tested in time to analyze whether the vaccine has achieved the immune effect through comparison, so as to carry out supplementary immunization. In addition, antibody testing can also be used for clinical diagnosis, for example, to check the condition after the disease or treatment, and can also be used as a basis for the effectiveness of vaccination.

[0003] There are many methods for detecting antibodies to pseudorabies in pigs, such as virus neutralization test, ELISA and immunochromatography. Virus neutralization test is an internationally used antibody detection method due to its strong specificity. The results of this method are accurate, but the time is long and the operation is cumbersome, and it needs to be operated by professionals. The latex agglutination test is easy to operate and has high sensitivity. It is suitable for rapid monitoring of clinical epidemics or epidemiological surveys and initial screening of quarantine and purification of breeding pigs, but its specificity is poor and the false positive rate is high. The final judgment should be made based on the clinical situation. Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used methods for detecting antibodies to pseudorabies virus in laboratories. Pseudorabies virus gB protein antibody blocking and competitive ELISA are used to detect antibodies to pseudorabies virus in pigs, which further improves the sensitivity of ELISA, but the test time still takes about 1 hour and cannot be accurately quantified. Immunochromatographic test strips, this method takes a short time, is convenient and fast, and is very suitable for rapid clinical detection in 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 current detection methods. The ability to accurately quantify and distinguish between wild virus infection and immunity and combine it with rapid clinical testing may be the future development trend of pseudorabies virus antibody detection. Summary of the invention

[0005] The technical problem to be solved by the present invention is based on the problem mentioned in the background technology, and provides a reagent, preparation method and kit containing magnetic microparticles coated with pseudorabies virus gB protein to improve the sensitivity and specificity of pseudorabies virus antibody detection.

[0006] In order to solve the above problems, the present invention proposes the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a reagent containing a magnetic particle coating of porcine pseudorabies virus gB protein, comprising the following steps:

[0008] Incubate the magnetic particles with gB protein antibodies in a coating buffer, wherein the concentration of the magnetic particles is 3 to 5 mg / mL, and the concentration of the gB protein antibodies is 10 to 40 μg / mL, to obtain a reaction solution containing gB protein magnetic particle coatings;

[0009] removing the reaction solution, washing the gB protein magnetic microparticle coating, and performing a first blocking on the gB protein magnetic microparticle coating with a first blocking solution;

[0010] After the blocking is completed, the first blocking solution is washed away, and the gB protein magnetic microparticle coating is then blocked for the second time with the second blocking solution;

[0011] After the blocking is completed, the second blocking solution is washed away, and the gB protein magnetic microparticle coating is added to the preservation solution and resuspended 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] Furthermore, the first blocking solution is one or more of bovine serum albumin and CE510; the second blocking solution is one or more of ethanolamine and glycine.

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

[0016] Furthermore, the temperature of the first sealing is 24 to 38° C., and the sealing time is 4 to 8 hours.

[0017] Furthermore, the temperature of the first sealing is 35-38° C., and the sealing time is 4-8 hours.

[0018] Furthermore, the temperature of the second sealing is 2 to 45° C., and the sealing time is 15 to 24 hours.

[0019] Furthermore, the temperature of the second sealing is 2 to 8° C., and the sealing time is 16 to 20 hours.

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

[0021] Furthermore, the particle size of the magnetic particles is 1000-3000 nm.

[0022] Furthermore, the coating buffer is any one of 0.02M-0.1M boric acid buffer and phosphate buffer, with a pH of 8-10; the preservation solution contains 0.02M-0.1M phosphate buffer, 0.5%-5% stabilizer, 0.05%-0.2% surfactant, and 0.01%-0.2% preservative.

[0023] Furthermore, 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, S9; and the preservative is one or two of BND and Proclin300.

[0024] In a second aspect, the present invention provides a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein, which is prepared by the preparation method described in the first aspect.

[0025] In a third aspect, the present invention provides a kit for detecting antibodies to the gB protein of pseudorabies virus of porcine, comprising a reagent containing magnetic microparticles coated with the gB protein of pseudorabies virus of porcine obtained by the preparation method described in the first aspect, or comprising a reagent containing magnetic microparticles coated with the gB protein of pseudorabies virus of porcine as described in the second aspect.

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

[0027] Furthermore, the kit also includes a reagent containing acridinium ester conjugate of pseudorabies virus gB protein, a calibrator containing polyclonal antibody of pseudorabies virus gB protein, and a quality control product.

[0028] Furthermore, the preparation method of the reagent containing the acridinium ester conjugate of pseudorabies virus gB protein is as follows:

[0029] 1) Labeling: prepare 1 ml of porcine pseudorabies virus gB protein acridinium ester conjugate, add 100-300 μl of acridinium ester labeling buffer, 20-100 μg of porcine pseudorabies virus gB protein, and 0.00005-0.0005 mM acridinium ester mother solution into a centrifuge tube, mix the mixture by inverting the liquid upside down, and react for 30 minutes at room temperature in the dark;

[0030] 2) Termination: After 30 minutes of labeling reaction, the acridinium ester conjugate is obtained, and then 200 μl of acridinium ester termination buffer is added, and the mixture is mixed by manual inversion, and the reaction is carried out at room temperature in the dark for 30 minutes;

[0031] 3) Dialysis, transfer all the above products to the dialysis bag, clamp the two ends with dialysis clips, clamp (tighten with rubber bands). Measure 2L of acridinium ester dialysis buffer into a beaker, put the dialysis bag with marker into the beaker, put the beaker on the stirrer, turn on the stirrer, put it in a 2-8℃ refrigerator, avoid light, stir, replace the acridinium ester dialysis buffer every 2 hours, and dialyze 3-5 times in total;

[0032] 4) Take out the beaker, take out the dialysis bag, transfer all the markers into a new centrifuge tube, add 50% glycerol, add 0.1% BSA to the centrifuge tube, gently mix until homogeneous, and store at -20°C in the dark to obtain a reagent stock solution containing acridinium ester conjugate of pseudorabies virus gB protein;

[0033] 5) Dilution: Add the reagent stock solution containing the acridinium ester conjugate of pseudorabies virus gB protein into the acridinium ester diluent at a ratio of 1 / 1000-1 / 200 (v / v) to prepare an acridinium ester working solution.

[0034] The acridinium ester labeling buffer contains 0.1M-0.3M PB buffer solution or BB buffer solution, pH 7.0-9.0, 0.05-0.2M NaCl or KCl, and 0.01%-0.2% preservative. The preservative is one or both of BND and Proclin300.

[0035] The acridinium ester termination buffer contains 0.1M-0.3M 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 Proclin300.

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

[0037] The acridinium ester diluent contains 0.02M-0.1M phosphate buffer, 0.5%-5% stabilizer, 0.05%-0.2% surfactant, and 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 two of BND and Proclin300.

[0038] Furthermore, the preparation method of the calibration product and the quality control product is as follows:

[0039] Add 0.1-500U / mL of polyclonal antibody against gB protein of pseudorabies virus to the calibrator diluent to prepare calibrators and quality control products with preset concentrations.

[0040] The calibration diluent contains 0.01M-0.05M phosphate buffer, 0.5%-10% stabilizer, 0.05%-0.2% surfactant, and 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 two of BND and Proclin300.

[0041] According to the technical solution of the present invention, the stabilizer in the above reagents mainly functions to protect the activity of the protein, while improving the stability of the reagents and extending the shelf life of the product.

[0042] According to the technical solution of the present invention, the surfactant in the above reagents mainly promotes the uniform dispersion of the components in the reagent system and the individual substances in the test sample and improves the detection precision to ensure the reproducibility of the results.

[0043] According to the technical solution of the present invention, the role of the preservative is to prevent the growth of microorganisms from adversely affecting the quality of the reagents. Any substance that can inhibit the growth of microorganisms can be used as the preservative of the present invention.

[0044] Furthermore, the kit of the present invention also includes an exciting solution A and an exciting solution B:

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

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

[0047] The present invention also provides a method for detecting porcine pseudorabies virus gB antibodies using the above kit, comprising the following steps:

[0048] Test working calibrants and create master curves;

[0049] Calibrate the fully automatic chemical immunoassay analyzer with the calibrator to obtain a calibration curve;

[0050] Test the quality control product. If the test value is within the specified target value range, the sample test can be carried out;

[0051] The instrument takes the sample to be tested, adds the above-mentioned reagent containing the magnetic microparticle coating of pseudorabies virus gB protein and the reagent containing the acridinium ester conjugate of pseudorabies virus gB protein to carry out immune reaction, adds stimulating solution A and stimulating solution B, enters the fully automatic immunoassay system, measures the luminescent signal, and obtains the concentration of pseudorabies virus gB protein antibodies in the sample through the calibration curve.

[0052] The kit for detecting antibodies to the gB protein of pseudorabies virus provided by the present invention adopts a double antigen sandwich method combined with chemiluminescence detection technology, which not only improves the sensitivity and specificity of the detection, but also can be accurately quantified. At the same time, the raw materials of the calibrators and quality control products in the kit adopt relatively stable polyclonal antibodies with good stability. With the detection of chemiluminescence analyzer, batch addition can be automatically performed, which solves the pain point of cumbersome manual operation of gB antibody detection and meets the needs of clinical detection.

[0053] Compared with the prior art, the technical effects achieved by the present invention include:

[0054] The preparation method of the reagent containing the porcine pseudorabies virus gB protein magnetic microparticle coating provided by the present invention adopts a two-step method to seal and prepare the porcine pseudorabies virus gB protein magnetic microparticle coating, has a good sealing effect, reduces nonspecific adsorption in the system, and can ensure good specificity of the detection system. Furthermore, due to the reduced background of the reagent, the detection rate of gB antibody positive serum can be increased, that is, the sensitivity of the detection system can be improved.

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

[0056] The detection kit provided by the present invention contains the above-mentioned reagent with low background, has the advantages of high analytical sensitivity, wide linear range and high accuracy, and has better detection performance; it is matched with calibrators and quality control products prepared with polyclonal antibodies, and has better stability than calibrators and quality control products prepared with antisera, and can replace existing detection kits, which meets clinical detection needs. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments are described clearly and completely below. Obviously, the embodiments described below are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

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

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

[0061] Example 1

[0062] This embodiment provides a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein. The magnetic microparticles used are superparamagnetic nanospheres, the surface modification functional groups are tosyl groups, and the particle size is 1000 nm.

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

[0064] 1) Washing of magnetic particles: Wash the magnetic particles with coating buffer, enrich the magnetic particles with a magnetic rack, remove the coating buffer, and wash three times.

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

[0066] 3) Blocking: re-enrich the coated material with a magnetic rack, remove the reaction solution, wash three times with washing solution, add an equal volume of CE510 and block at 2-8°C for 6 hours; after blocking, re-enrich the coated material with a magnetic rack, remove the reaction solution, wash three times with washing solution, add 50 mM ethanolamine and block at 37°C for 18 hours.

[0067] 4) Preservation: After the sealing is completed, the coated material is re-enriched using a magnetic rack, the reaction solution is aspirated, the washing solution is washed three times, and a magnetic particle preservation solution is added to obtain a mother solution containing the magnetic particle coated material of the pseudorabies virus gB protein.

[0068] 5) Dilution: the mother solution containing the magnetic microparticle coating of the pseudorabies virus gB protein is added to the magnetic microparticle storage solution to make the final concentration be 0.2 mg / mL.

[0069] The coating buffer in this example is 0.05 M boric acid buffer, pH 9.0.

[0070] The magnetic particle storage solution of this example contains 0.05M phosphate buffer, 2% bovine serum albumin, 2% trehalose, 0.1% Tween 20 and 0.1% Proclin300.

[0071] Embodiment 2:

[0072] This embodiment provides a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein, which is different from Embodiment 1 in that the blocking in step 3) is different.

[0073] 3) Blocking in this embodiment specifically comprises: re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding an equal volume of CE510 and blocking at 37° C. for 6 h; after blocking, re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding 50 mM ethanolamine and blocking at 2-8° C. for 18 h.

[0074] Embodiment 3:

[0075] This embodiment provides a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein, which is different from Embodiment 1 in that the blocking in step 3) is different.

[0076] 3) Blocking in this embodiment specifically comprises: re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding an equal volume of CE510 and blocking at 37° C. for 6 h; after blocking, re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding 50 mM ethanolamine and blocking at 25° C. for 18 h.

[0077] Embodiment 4:

[0078] This embodiment provides a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein, which is different from Embodiment 1 in that the blocking in step 3) is different.

[0079] 3) Blocking in this embodiment specifically comprises: re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding an equal volume of CE510 and blocking at 37° C. for 6 h; after blocking, re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding 50 mM ethanolamine and blocking at 45° C. for 18 h.

[0080] Embodiment 5:

[0081] This embodiment provides a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein, which is different from Embodiment 1 in that the blocking in step 3) is different.

[0082] 3) Blocking in this embodiment specifically comprises: re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding an equal volume of CE510 and blocking at 37° C. for 6 h; after blocking, re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding 50 mM ethanolamine and blocking at 37° C. for 18 h.

[0083] Embodiment 6:

[0084] This embodiment provides a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein, which is different from Embodiment 1 in that the blocking in step 3) is different.

[0085] 3) Blocking in this embodiment is specifically as follows: re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding an equal volume of 1wt% BSA and blocking at 37°C for 6h; after blocking, re-enriching the coated material with a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding 50mM ethanolamine and blocking at 2-8°C for 18h.

[0086] Embodiment 7:

[0087] This embodiment provides a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein, which is different from Embodiment 1 in that the blocking in step 3) is different.

[0088] 3) Blocking in this embodiment specifically comprises: re-enriching the coated material using a magnetic rack, removing the reaction solution, washing three times with a washing solution, adding an equal volume of CE510 and blocking at 37° C. for 6 hours.

[0089] The reagent containing the magnetic microparticle coating of the gB protein of pseudorabies virus provided in the above Examples 1-7 is prepared into a kit. Corresponding to kit 1-7, the reagent containing the magnetic microparticle coating of the gB protein of pseudorabies virus is used as reagent 1.

[0090] Each kit also includes reagent 2 (a reagent containing acridinium ester conjugate of pseudorabies virus gB protein), calibrators, quality control products, stimulating solution A and stimulating solution B of the same composition. The following is a detailed description:

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

[0092] 1) Labeling: for each 1 ml of pseudorabies virus gB protein acridinium ester conjugate prepared, 300 μl of acridinium ester labeling buffer, 100 μg of gB protein, and 0.000025 mM acridinium ester stock solution were added to a centrifuge tube, the mixture was mixed by inverting the liquid upside down, and the reaction was carried out for 30 min at room temperature in the dark.

[0093] 2) Termination: After 30 minutes of labeling reaction, an acridinium ester conjugate is obtained. Acridinium ester termination buffer (reaction volume × 0.1) is added to the acridinium ester conjugate, and the mixture is mixed by manual inversion. The reaction is allowed to react for 30 minutes at room temperature in the dark.

[0094] 3) Dialysis, transfer all the above products to the dialysis bag, clamp the two ends with dialysis clips, clamp (tighten with rubber bands), and make sure there is no leakage. Measure 2L of acridinium ester dialysis buffer into a beaker, put the dialysis bag containing the marker into the beaker, put the beaker on the stirrer, turn on the stirrer, put it in a 2-8℃ refrigerator, avoid light, stir, replace the acridinium ester dialysis buffer every 2 hours, and dialyze 4 times in total.

[0095] 4) Take out the beaker, take out the dialysis bag, transfer all the markers into a new centrifuge tube, add 50% glycerol, add 0.1% BSA to the centrifuge tube, gently mix until homogeneous, and store at -20°C away from light to obtain a reagent stock solution containing acridinium ester conjugate of pseudorabies virus gB protein.

[0096] 5) Dilution: Take the reagent mother solution containing the acridinium ester conjugate of pseudorabies virus gB protein and add it to the acridinium ester diluent at a ratio of 1 / 500 (v / v) to prepare the acridinium ester working solution.

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

[0098] The acridinium ester stop buffer contained 0.1 M PB buffer solution, pH 8.0, 1% glycine, and 0.1% Proclin300.

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

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

[0101] ②Preparation of calibration products and quality control products

[0102] Add 0.1-500U / mL of polyclonal antibody to the gB protein of pseudorabies virus to the calibrator diluent to prepare calibrators and quality control products with preset concentrations, where:

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

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

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

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

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

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

[0109] ③ Preparation of excitation solution

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

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

[0112] Performance test experiment 1

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

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

[0115]

[0116]

[0117] From the results in Table 1, it can be seen that compared with kit 7, the reagent containing the magnetic microparticle coating of the gB protein of pseudorabies virus in kit 1-6 is blocked by a two-step method. The detection sensitivity of kit 1-6 is higher than that of kit 7, which indicates that the reagent containing the magnetic microparticle coating of the gB protein of pseudorabies virus of porcine is blocked by a two-step method better than the one-step method. This is because in the present invention, the first step is blocked for 6 hours by a conventional magnetic bead blocking method, but the second step is added, and the blocking is performed for 18 hours by replacing the blocking agent. The superposition of the two blocking agents prolongs the blocking time, which is conducive to completely blocking the blank sites on the magnetic beads that are not bound to the target protein.

[0118] The above results also show that the temperature of the two blockings has a significant effect on the blocking effect. Different blocking agents have different optimal reaction temperatures. The secondary blocking step of the present invention uses different temperatures. The blocking agents of macromolecules such as CE510 and BSA are blocked at 37°C for 6 hours, which can occupy the blank sites on the magnetic beads in a short time. The remaining unblocked sites are blocked for a longer time at 2-45°C. From the comparison between the various embodiments, it can be seen that the blocking effect at 2-8°C is the best. The sensitivity and specificity of kit 2 are the best because the macromolecular CE510 is first used for blocking at 37°C. CE510 is a compound synthesized from PEG and oligoamine, which is covalently coupled to the surface groups of the magnetic beads without specific adsorption, and has a better blocking effect than bovine serum albumin; and then the small molecule ethanolamine is used for blocking. Due to the small molecular weight of ethanolamine, it is easier to disperse to the remaining blank sites at 2-8°C to react with the magnetic bead groups, promote the blocking effect, and further improve the sensitivity of the reagent.

[0119] Performance test experiment 2

[0120] The stability tests were performed on the reagent kits 1-7 consisting of the reagent 1 containing the magnetic microparticle coating of the pseudorabies virus gB protein and the reagent 2 containing the acridinium ester marker, respectively, to examine the storage stability at 2-8°C for 12 months. 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 in Table 2, it can be seen that in the kits 1-7 of the present invention, the reagents 2 are the same, and only the reagents 1 are derived from the examples 1-7 respectively. From the perspective of stability, the reagents containing the magnetic microparticles coated with the pseudorabies virus gB protein in the two-step method of the example 1-6 are significantly better than the example 7 in terms of stability. At the same time, different sealing temperatures also have different effects on the stability of the sealing. The deviations of the kits 2-3 are less than 10% when stored at 2-8°C for 12 months, and they have good stability. The main reason is that the more thoroughly the reagent 1 is sealed, the less likely it is to have further sealing phenomenon and background fluctuations when the kit is stored at 2-8°C for a long time. At the same time, in the reagent 1, two sealing agents are used to superimpose and seal at a specific temperature, and the interaction greatly improves the stability of the reagent.

[0128] Performance test experiment 3

[0129] The positive controls of the existing ELISA kits are all prepared with antiserum. This experiment sets up a comparison of calibrators and quality controls (the porcine pseudorabies virus antiserum is diluted to 0.1-500U / mL with calibrator diluent) to compare the accelerated stability with the calibrators and quality controls in this kit. The experimental results are shown in Table 3.

[0130] Table 3 Stability test results of calibrators and quality control products

[0131]

[0132] From the results in Table 3, it can be seen that the accelerated stability of calibrators and quality controls made with polyclonal antibodies is better than that of calibrators and quality controls made with antisera.

[0133] Performance test experiment 4

[0134] This experiment uses kit 2 as an example to analyze the performance of the kit of the present invention:

[0135] (1) Analytical sensitivity (detection limit)

[0136] Five samples with concentrations close to the detection limit of the kit were selected (numbered: LoD1-LoD5), and each sample was tested 5 times. A sample with a concentration of 0 was also tested. The results are shown in Table 4-5. The number of test results of this reagent that were less than the blank limit was no more than 3, which met the requirements.

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

[0138]

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

[0140] Number of tests 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 showed that the analytical sensitivity (detection limit) of the kit provided by the present invention was no more than 0.1 U / mL, which met the requirements.

[0142] (2) Linear range

[0143] The kit provided by the present invention has a linear correlation coefficient r value 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) Repeatability

[0147] The test was repeated 10 times with sample 1 and sample 2 respectively, and the results are shown in Table 7.

[0148] Table 7 Repeatability results

[0149]

[0150]

[0151] The repeatability of the kit provided by the invention is within 5%.

[0152] (4) Accuracy

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

[0154] Table 8 Accuracy test results

[0155]

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

[0157] According to the results of Tables 4 to 8 above, it can be seen that the detection kit provided by the present invention has the advantages of high analytical sensitivity, wide linear range, good repeatability, high accuracy, and low cost, which can meet the needs of current clinical testing.

[0158] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0159] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing a reagent containing magnetic microparticles coated with porcine pseudorabies virus gB protein, characterized in that: The following steps are involved: Incubate the magnetic particles with gB protein antibodies in a coating buffer, wherein the concentration of the magnetic particles is 3 to 5 mg / mL, and the concentration of the gB protein antibodies is 10 to 40 μg / mL, to obtain a reaction solution containing gB protein magnetic particle coatings; removing the reaction solution, washing the gB protein magnetic microparticle coating, and performing a first blocking on the gB protein magnetic microparticle coating with a first blocking solution; After the blocking is completed, the first blocking solution is washed away, and the gB protein magnetic microparticle coating is then blocked for the second time with the second blocking solution; After the blocking is completed, the second blocking solution is washed away, and the gB protein magnetic microparticle coating is added to the preservation solution and resuspended to obtain a reagent containing the gB protein magnetic microparticle coating; The first blocking solution is one or more of bovine serum albumin, ethanolamine, CE510, and glycine; 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.

2. The preparation method according to claim 1, characterized in that The temperature of the first sealing is 24-38° C., and the sealing time is 4-8 hours.

3. The preparation method according to claim 2, characterized in that: The temperature of the second sealing is 2 to 45° C., and the sealing time is 15 to 24 hours.

4. The preparation method according to claim 1, characterized in that: The gB protein antibody is one of the full-length, fragment or polypeptide of the gB protein of pseudorabies virus.

5. The preparation method according to claim 1, characterized in that: The particle size of the magnetic particles is 1000-3000nm.

6. The preparation method according to claim 1, characterized in that: The coating buffer is any one of 0.02M-0.1M boric acid buffer and phosphate buffer, with a pH of 8-10; the preservation solution contains 0.02M-0.1M phosphate buffer, 0.5%-5% stabilizer, 0.05%-0.2% surfactant, and 0.01%-0.2% preservative.

7. A reagent containing magnetic microparticles coated with pseudorabies virus gB protein, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6.

8. A kit for detecting antibodies to the gB protein of pseudorabies virus, characterized in that: A reagent comprising the magnetic microparticle coating of the gB protein of pseudorabies virus obtained by the preparation method according to any one of claims 1 to 6, or a reagent comprising the magnetic microparticle coating of the gB protein of pseudorabies virus according to claim 7.

9. The kit according to claim 8, characterized in that The concentration of the reagent containing the magnetic microparticle coating of the porcine pseudorabies virus gB protein is 0.08-0.4 mg / mL.

10. The kit according to claim 8, characterized in that It also includes reagents containing acridinium ester conjugates of pseudorabies virus gB protein, calibrators and quality control products containing polyclonal antibodies of pseudorabies virus gB protein.

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