A method and kit for detecting the residual amount of baculovirus total protein

Abstract

The present invention discloses a double antibody sandwich enzyme-linked immunosorbent assay method and a kit for detecting the residual amount of baculovirus total protein. The detection method of the present invention comprises immunizing an animal with the prepared baculovirus total protein to obtain a coated antibody and a primary antibody, and detecting the baculovirus total protein by an enzyme-linked immunosorbent assay. The kit of the present invention comprises a coated antibody, a primary antibody, a labeled secondary antibody, a positive quantitative standard, and the like. The detection method and kit of the present invention are easy to operate, have good specificity, accuracy, sensitivity, precision and reliability, good linear relationship, and a wide linear range. The residual amount of baculovirus total protein in the intermediates and final products of the process can be accurately and reliably detected, and can be widely used in the quality control of baculovirus protein residues in vaccines, genes, proteins and other biological products produced by the baculovirus system, and has extremely important application value.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and more specifically, to a method and a kit for detecting the residual amount of total baculovirus protein. Background Art

[0002] Baculovirus is composed of a baculovirus nucleocapsid, a double-layer lipoprotein membrane that envelops it, and a single closed circular double-stranded DNA molecule, of which the constituent proteins include nucleocapsid proteins (VP39, VP78 / 83, VP87, VP80, and VP24, etc.), envelope proteins (PDV-E25, PDV-6e, PDV-E66, PDV-E56, PDV-E18, PDV-EC27, PDV-E35, PDV-EC27, BV / PDV-E26, PDV-43, GP64-67, and VP40 / 41, etc.), and inclusion body matrix proteins, etc. Among them, GP64 protein is a membrane fusion protein that can recognize and bind to receptors on the surface of host cell membranes, mediating endocytosis to allow the virus to invade host cells. Baculovirus contains two forms of virus particles, one is budded virus (BV), in the early stage of virus replication, the nucleocapsid obtains the capsule from the cytoplasmic membrane by budding and is released, and then infects other cells, allowing the virus to spread in the insect body. The other is occluded virus (OV), also known as polyhedron-derived virus (PDV), in the late stage of virus replication, the nucleocapsid obtains the capsule in the cell nucleus, and then is coated in the polyhedron protein or particle capsule, and is not released to the outside of the cell until the cell lyses, which mainly spreads infection between insects.

[0003] The genome size of baculovirus is 80-180kb, which can be replicated and transcribed in the nucleus of insect cells and then assembled in the nucleocapsid. The genome of baculovirus can accommodate large fragments of exogenous DNA insertion. In addition, the culture operation of insect cells is simple, low cost, and can be adhered to the wall and suspended. The baculovirus-insect cell expression system (referred to as "baculovirus expression vector system" baculovirus expression vector system, BEVS) with baculovirus as a vector has been widely used in the efficient expression of exogenous proteins. Among them, Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV or AcNPV) has been studied in depth and has the most applications. Its genome size is 133894bp (NC_001623), encoding about 150 open reading frames, and is an ideal vector for expressing biologically active genes and proteins.

[0004] At present, a variety of biological products such as vaccines, proteins, and gene therapy products are produced by using a baculovirus expression vector system. During the production process, some baculovirus proteins in the system will remain in the final product and become process-related impurities. Therefore, it is necessary to strictly monitor and control these residual proteins. Although there are methods (for example, patent application documents with application number 201611262295.5) for ELISA detection of baculovirus GP64 protein, these methods do not involve the residues of other baculovirus proteins in the product, and it is difficult to fully and accurately reflect the overall situation of baculovirus protein residues in biological products. In order to more comprehensively evaluate the total residual amount of baculovirus proteins in biological products, it is necessary to develop a method that can fully and accurately detect baculovirus total proteins. Therefore, the present invention develops an enzyme-linked immunosorbent (ELISA) detection method and kit for baculovirus total proteins. The detection method and kit of the present invention can be widely used in the quality control of baculovirus protein residues in biological products such as vaccines, genes, and proteins produced by baculovirus systems, and have extremely important application value. Summary of the invention

[0005] To solve the above technical problems, the present invention provides an ELISA double antibody sandwich enzyme-linked immunosorbent assay method and kit for detecting baculovirus total protein. The detection method and kit of the present invention can comprehensively, specifically, accurately and reproducibly detect the residual amount of baculovirus total protein in a sample.

[0006] Specifically, the first object of the present invention is to provide a double antibody sandwich enzyme-linked immunosorbent assay method for detecting the residual amount of total baculovirus protein in a sample. The method comprises the following steps:

[0007] (1) Coating the ELISA plate with the coating antibody, adding the sample to be tested, discarding the liquid in the well and washing after incubation;

[0008] (2) Add the primary antibody, discard the liquid in the wells and wash after incubation;

[0009] (3) Add the labeled secondary antibody, discard the liquid in the wells and wash after incubation;

[0010] (4) performing a color reaction under appropriate conditions;

[0011] (5) Read the absorbance value (OD value);

[0012] (6) Calculate the total amount of baculovirus protein remaining in the sample to be tested.

[0013] In a specific embodiment of the present invention, the residual amount of total baculovirus protein in the sample to be tested is calculated based on a standard curve, and the standard curve is drawn using a positive quantitative standard.

[0014] In a specific embodiment of the present invention, the coating antibody is a purified rabbit polyclonal antibody against baculovirus total protein.

[0015] In a specific embodiment of the present invention, the primary antibody is a positive serum of mouse against total baculovirus protein.

[0016] In a specific embodiment of the present invention, the secondary antibody is a goat anti-mouse secondary antibody or a horse anti-mouse secondary antibody, etc. Preferably, the secondary antibody is a goat anti-mouse secondary antibody. In a preferred embodiment of the present invention, the secondary antibody is labeled with horseradish peroxidase (HRP).

[0017] In another specific embodiment of the present invention, the positive quantitative standard is baculovirus total protein.

[0018] In a preferred embodiment of the present invention, the above-mentioned purified rabbit anti-baculovirus total protein polyclonal antibody is obtained by the following preparation method: during the first immunization, the rabbit is injected with baculovirus total protein using antigen multi-point immunization; after the first immunization, 3-5 booster immunizations are performed again with baculovirus total protein, and after the last booster immunization, serum is collected and purified to obtain purified rabbit anti-baculovirus total protein polyclonal antibody.

[0019] In a preferred embodiment of the present invention, the above-mentioned mouse anti-baculovirus total protein positive serum is obtained by the following preparation method: during the first immunization, the mouse is injected with baculovirus total protein using antigen multi-point immunization; after the first immunization, the mouse is boosted with baculovirus total protein for 3-5 times, and serum is collected after the last booster immunization, which is the mouse anti-baculovirus total protein positive serum.

[0020] In a preferred embodiment of the present invention, the baculovirus total protein is obtained by the following preparation method:

[0021] (1) Producing baculovirus;

[0022] (2) harvesting and concentrating baculovirus;

[0023] (3) Lysing the above baculovirus to obtain total baculovirus protein.

[0024] In a preferred embodiment of the present invention, the concentration gradient of the positive quantitative standard is in the range of 0 ng / ml-200 ng / ml. Preferably, the concentration gradient is 200 ng / ml, 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, and 6.25 ng / ml.

[0025] The second object of the present invention is to provide a double antibody sandwich enzyme-linked immunosorbent assay kit for detecting the residual amount of total baculovirus protein in a sample, the kit comprising: a coated antibody, a primary antibody, a labeled secondary antibody, a positive quantitative standard, and the like.

[0026] In a specific embodiment of the present invention, the coating antibody is a purified rabbit polyclonal antibody against baculovirus total protein.

[0027] In another specific embodiment of the present invention, the primary antibody is a positive serum of mouse against baculovirus total protein.

[0028] In another specific embodiment of the present invention, the secondary antibody is a goat anti-mouse secondary antibody or a horse anti-mouse secondary antibody, etc. Preferably, the secondary antibody is a goat anti-mouse secondary antibody. In a preferred embodiment of the present invention, the secondary antibody is labeled with horseradish peroxidase (HRP).

[0029] In another specific embodiment of the present invention, the positive quantitative standard is baculovirus total protein.

[0030] In a preferred embodiment of the present invention, the above-mentioned purified rabbit anti-baculovirus total protein polyclonal antibody is obtained by the following preparation method: during the first immunization, the rabbit is injected with baculovirus total protein using antigen multi-point immunization; after the first immunization, the rabbit is again boosted with baculovirus total protein for 3-5 times, and after the last booster immunization, the serum is collected and purified to obtain the purified rabbit anti-baculovirus total protein polyclonal antibody.

[0031] In a preferred embodiment of the present invention, the above-mentioned mouse anti-baculovirus total protein positive serum is obtained by the following preparation method: during the first immunization, the mouse is injected with baculovirus total protein using antigen multi-point immunization; after the first immunization, the mouse is boosted with baculovirus total protein for 3-5 times, and serum is collected after the last booster immunization, which is the mouse anti-baculovirus total protein positive serum.

[0032] In a preferred embodiment of the present invention, the baculovirus total protein is obtained by the following preparation method:

[0033] (1) Producing baculovirus;

[0034] (2) harvesting and concentrating baculovirus;

[0035] (3) Lysing the above baculovirus to obtain total baculovirus protein.

[0036] The beneficial effects of the present invention are:

[0037] (1) In the prior art, only a single protein antibody is used for detection. On the one hand, since baculovirus particles contain multiple proteins, the use of a single protein antibody may not be able to fully and objectively reflect the overall residual amount of baculovirus protein. On the other hand, the composition and type of baculovirus protein remaining in different samples and samples prepared by different processes may vary greatly. It is difficult to accurately reflect the type, content and changes of baculovirus residual protein using only a single protein antibody. The technical solution of the present invention can more objectively, accurately and comprehensively detect and reflect the residual baculovirus protein in various types of samples.

[0038] (2) The technical solution of the present invention is easy to operate, has good specificity, accuracy, sensitivity, precision and reliability, good linear relationship and a wide linear range.

[0039] In conclusion, the detection method and kit of the present invention can accurately and reliably detect the residual amount of baculovirus total protein in the intermediates and final products of the process, and can be widely used in the quality control of baculovirus protein residues in biological products such as vaccines, genes, and proteins produced by the baculovirus system, and has extremely important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Standard curve drawn using positive quantitative standards. DETAILED DESCRIPTION

[0041] The following embodiments are only used to explain the present invention and are not used to limit the protection scope of the present invention.

[0042] Unless otherwise specified, the reagents and raw materials described in the following examples are all common commercially available raw materials, and the reagents are prepared using conventional methods. Methods not described in detail in the examples are all conventional operations in the art.

[0043] Example 1 Preparation of baculovirus total protein

[0044] 1. Production of Baculovirus

[0045] The bacmid DNA was transfected into Sf9 cells by liposome transfection and cultured in a cell culture incubator at 27°C for 72h-96h. The culture solution containing the bacmid was collected into a 15ml centrifuge tube, centrifuged (500×g, 5min) to remove the cell precipitate and impurities, and the supernatant was transferred into a new centrifuge tube as the P0 generation of the bacmid virus. Then, the P0 generation virus was used to infect new Sf9 cells to produce the virus. The cell culture supernatant was centrifuged (500×g, 5min), and the supernatant was transferred into a new tube as the P1 generation of the bacmid virus. In subsequent experiments, the P1 generation can be further amplified according to the demand for the bacmid virus.

[0046] 2. Preparation of Baculovirus Concentrate

[0047] Harvest the supernatant containing baculovirus, filter it with a 0.22μm filter membrane, transfer it to a high-speed centrifuge tube, centrifuge it (4℃, 40000×g, 1.5h), remove the supernatant, let it stand for 1min, and then use a pipette to suck out the residual liquid at the bottom of the tube. Add a small amount of storage buffer, pipette and resuspend it, and then suck the suspension into a new centrifuge tube. Mix the precipitate resuspension in multiple centrifuge tubes to make a baculovirus concentrate, and store it at 4℃ or -80℃ for later use.

[0048] 3. Preparation of Baculovirus Total Protein

[0049] The baculovirus concentrate was heated in a 95°C water bath for 30 min or ultrasonically disrupted to fully lyse the baculovirus and obtain the baculovirus total protein. The concentration of the baculovirus total protein was measured to be 0.78 mg / ml by the BCA method (BCA PROTEIN ASSAY KIT, Thermo, Catalog No.: 22400-089), and then aliquoted and used as a positive quantitative standard in subsequent ELISA detection experiments and as an antigen in the preparation of polyclonal antibodies and positive serum, and stored at -80°C for future use.

[0050] Example 2 Preparation of rabbit anti-baculovirus total protein polyclonal antibodies and mouse anti-baculovirus total protein positive serum

[0051] Animal immunization, polyclonal antibody preparation method and polyclonal antibody purification method are used to prepare and / or purify positive serum and polyclonal antibodies against total baculovirus protein. First, the total baculovirus protein is mixed and emulsified with Freund's complete adjuvant as an antigen, and then 10-20 New Zealand white rabbits and mice are first immunized, and the total baculovirus protein is injected in a multi-point immunization manner. For New Zealand white rabbits, the injection dose is 0.5 mg / rabbit; for mice, the injection dose is 0.1 mg / rabbit. 14 days after the first immunization, booster immunization is performed, and Freund's incomplete adjuvant is used for antigen emulsification. The other steps and doses are the same as the first immunization. The interval between each booster immunization is 10 days, and a total of 3-5 booster immunizations are performed. On the 7th day after the last booster immunization, rabbit serum and mouse serum are harvested respectively. Among them, the mouse serum is the positive serum of mouse anti-baculovirus total protein, which is used as the primary antibody in the subsequent ELISA detection method.

[0052] For rabbit serum, protein A affinity chromatography column was used for further purification. Take 100ml of rabbit serum, mix it with PBS buffer in a ratio of 1:1, and filter it with a 0.22μm filter membrane to obtain the treated sample. According to the operating instructions, the treated sample was purified using protein A resin. The specific operation is as follows: First, equilibrate the protein A column with 6 times the column volume of equilibrium solution (20mM / L PB buffer, 150mM / L NaCl, pH7.2) at a flow rate of 5ml / min. Adjust the pH of the sample (diluted rabbit serum) to 7.2, load it at room temperature, and flow rate 5ml / min. After complete equilibrium, add 20ml of eluent (0.1M / L Glycine, 150mM / LNaCl, pH3.2) for elution at a flow rate of 5ml / min, and collect the eluate. The purified polyclonal antibody against baculovirus total protein was obtained by concentrating and replacing the liquid with a 30KD ultrafiltration tube. The concentration was 7.6 mg / ml, which was packaged and stored at -80°C for use as a coating antibody in subsequent ELISA tests.

[0053] Example 3 Detection of baculovirus protein residues in samples using ELISA

[0054] 1. Coating antibody: Dilute the coating antibody (i.e., purified rabbit anti-baculovirus total protein polyclonal antibody) to a concentration of 160 μg / ml using PBS buffer, add 100 μl / well to the ELISA plate for coating, and let stand at 4°C overnight.

[0055] 2. Washing: Aspirate the liquid in the ELISA plate, add 200 μl of washing solution (Wash Concentrate (20×), CYGNUS, F004) to each well to wash each well, then discard the washing solution and pat dry on filter paper. Repeat the washing for 5 times.

[0056] 3. Blocking: Add blocking solution (prepared by dissolving 1.0 g skim milk powder in 20 ml PBS buffer), 200 μl / well, and incubate at 37°C for 2 h.

[0057] 4. Wash the plate: Same as step 2.

[0058] 5. Positive quantitative standard dilution: Use PBS buffer to dilute the positive quantitative standard (i.e., baculovirus total protein) in a gradient manner, with concentrations of 200 ng / ml, 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml and 6.25 ng / ml, for a total of 6 gradients.

[0059] 6. Sample addition: Take the coated ELISA plate, add 100 μl of sample or positive quantitative standard to each well, set up duplicate wells, incubate at 37°C for 2 hours, and use sample diluent as blank control.

[0060] 7. Wash the plate: Same as step 2.

[0061] 8. Add primary antibody: Add 100 μl of primary antibody diluted 4000 times with antibody diluent (i.e., mouse anti-baculovirus total protein positive serum) to each well and incubate at 37°C for 1.5 h.

[0062] 9. Wash the plate: Same as step 2.

[0063] 10. Add secondary antibody: Add 100 μl of secondary antibody (i.e., HRP-labeled goat anti-mouse secondary antibody) diluted 3000 times with antibody diluent to each well and incubate at 37°C for 1.5 h.

[0064] 11. Wash the plate: Same as step 2.

[0065] 12. Color development: Add 100 μl of TMB color development solution (TMB Substrate (ELISA), CYGNUS, F005) equilibrated at room temperature to each well and place at room temperature away from light for 10-30 minutes.

[0066] 13. Termination: Add 100 μl of stop solution (0.5 M sulfuric acid) to each well to terminate the color reaction.

[0067] 14. Detection: Use a multifunctional microplate reader to read the absorbance value of each well at 450nm.

[0068] 15. Data processing and sample determination:

[0069] (1) Drawing of standard curve: Data processing was performed using the software provided by the multifunctional microplate reader (SoftMax Pro 5.4.1), and four-parameter fitting was performed on the concentration values ​​of the positive quantitative standard and their corresponding absorbance values ​​to obtain the standard curve. The obtained standard curve is shown in Figure 1As shown, the results show that its R 2 Greater than 0.98, good linear relationship, quantitative range can be up to 6.25ng / ml-200ng / ml, detection limit can be as low as 6.25ng / ml.

[0070] (2) Sample test results: The concentration of the residual baculovirus protein in the sample is calculated based on the standard curve and the absorbance value of the measured sample. The test results of the intermediate sample and the finished sample are shown in Table 1, wherein intermediate sample 1 and intermediate sample 2 are intermediate samples at different stages of the sample purification process, respectively, and the finished sample is the finished sample obtained after the sample purification is completed.

[0071] Table 1 Detection results of the residual amount of total baculovirus protein in samples

[0072]

[0073] Example 4 Methodology Verification

[0074] 1. Exclusivity and specificity

[0075] Take the sample auxiliary material solution and the antibody diluent and dilute the positive quantitative standard with a concentration of 100 ng / ml in a ratio of 1:1 to obtain a spiked sample, and perform ELISA detection on the spiked sample (see Example 3 for specific steps). After obtaining the test results, calculate the spiked recovery rate: spiked recovery rate = [specified value of spiked sample / specified sample theoretical value] × 100%. If the recovery rate of the sample auxiliary material solution and the antibody diluent spiked sample is between 70% and 130%, it is considered that the method has good specificity and specificity.

[0076] The test results are shown in Table 2. The spiked recoveries of the sample auxiliary solution and sample diluent are between 80% and 120%, indicating that the method has good specificity and specificity.

[0077] Table 2 Specificity and exclusivity verification results

[0078]

[0079] 2. Accuracy

[0080] Take 100ng / ml (high concentration), 50ng / ml (medium concentration), 25ng / ml (low concentration) of positive quantitative standard respectively, and add them to the finished product sample or intermediate product sample at a ratio of 1:1 to obtain the spiked sample; take the sample diluent (PBS buffer), and add it to the finished product sample or intermediate product sample at a ratio of 1:1 to obtain the unspiked sample. Perform ELISA test on the spiked sample and the unspiked sample (refer to Example 3 for specific steps). Calculate the spiked recovery rate according to the test results: spiked recovery rate = (spiked sample measured value - unspiked sample measured value) / spiked concentration × 100%). If the spiked recovery rate is between 70% and 130%, it is considered that the accuracy of this method meets the requirements.

[0081] The test results of the accuracy of the spiked finished product samples and intermediate product samples are shown in Table 3-1 and Table 3-2, respectively. The recoveries of high, medium and low concentration positive quantitative standards are all between 70% and 120%, indicating that the accuracy of this method is good.

[0082] Table 3-1 Verification results of the accuracy of spiked finished product samples

[0083]

[0084] Table 3-2 Results of the verification of the accuracy of spiked intermediate samples

[0085]

[0086] 3. Repeatability

[0087] Take 50 ng / ml positive quantitative standard, perform ELISA test according to the operation steps of Example 3, repeat 5 replicates, calculate the average value and CV value of the test results. If the CV value of the measured value is less than 30%, it meets the requirements.

[0088] The test results are shown in Table 4. The CV value of the positive quantitative standard after repeated testing for 5 times was 2.8%, indicating that the method has good repeatability.

[0089] Table 4 Repeatability verification results

[0090]

[0091] 4. Intermediate precision

[0092] Two experimenters tested the 50 ng / ml positive quantitative standard respectively, and each experimenter repeated the test three times. The ELISA test was performed according to the operating steps of Example 3. The average value and CV value of the test results were calculated. If the CV value was less than 30%, it met the requirements.

[0093] The test results are shown in Table 5. Two experimenters each repeated the test three times, and the total CV value of 6 test results was 6.45%, indicating that the intermediate precision of this method is good.

[0094] Table 5 Intermediate precision verification results

[0095]

[0096] The present invention selects a purified rabbit anti-baculovirus total protein polyclonal antibody as a coating antibody and a mouse anti-baculovirus total protein positive serum as a primary antibody, and detects residual baculovirus protein in various samples by enzyme-linked immunosorbent assay (ELISA). The detection method of the present invention is methodologically validated, and the validation results show that the method has high specificity, strong specificity, high accuracy, good repeatability, and good precision. The detection method and kit of the present invention can be used to more comprehensively and sensitively detect the residual amount of baculovirus protein in samples produced by a baculovirus production system.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A double antibody sandwich enzyme-linked immunosorbent assay method for detecting the residual amount of baculovirus total protein in a sample, the method comprising the following steps: (1) Coat the ELISA plate with the coated antibody, add the sample to be tested, discard the liquid in the well and wash after incubation; (2) Add the primary antibody, discard the liquid in the well and wash after incubation; (3) Add the labeled secondary antibody, discard the liquid in the wells and wash after incubation; (4) Carrying out a color reaction under appropriate conditions; (5) Read the absorbance value; (6) Calculate the amount of total baculovirus protein remaining in the sample to be tested; in, The coating antibody is a purified polyclonal antibody of rabbit against baculovirus total protein; the purified polyclonal antibody of rabbit against baculovirus total protein is obtained by immunizing rabbit with baculovirus total protein as antigen; The primary antibody is a positive serum of mouse against total baculovirus protein.

2. The double antibody sandwich ELISA method according to claim 1, characterized in that: The residual amount of total baculovirus protein in the sample to be tested is calculated based on a standard curve, and the standard curve is drawn using a positive quantitative standard.

3. The double antibody sandwich ELISA method according to claim 1, characterized in that: The labeled secondary antibody is a labeled goat anti-mouse secondary antibody or a labeled horse anti-mouse secondary antibody.

4. The double antibody sandwich ELISA method according to claim 3, characterized in that: The label is horseradish peroxidase label.

5. The double antibody sandwich ELISA method according to claim 2, characterized in that: The positive quantitative standard is baculovirus total protein.

6. The double antibody sandwich ELISA method according to claim 1, characterized in that: The purified rabbit anti-baculovirus total protein polyclonal antibody is obtained by the following preparation method: during the first immunization, the rabbit is injected with baculovirus total protein by using antigen multi-point immunization; after the first immunization, the rabbit is again boosted with baculovirus total protein for 3-5 times, and serum is collected after the last booster immunization, and purified to obtain the purified rabbit anti-baculovirus total protein polyclonal antibody.

7. The double antibody sandwich ELISA method according to claim 1, characterized in that: The mouse anti-baculovirus total protein positive serum is obtained by the following preparation method: during the first immunization, the mouse is injected with baculovirus total protein by using antigen multi-point immunization; after the first immunization, the mouse is boosted with baculovirus total protein for 3-5 times, and serum is collected after the last booster immunization, which is the mouse anti-baculovirus total protein positive serum.

8. The double antibody sandwich ELISA method according to claim 5, characterized in that: The baculovirus total protein is obtained by the following preparation method: (1) Production of baculovirus; (2) harvesting and concentrating baculovirus; (3) Lysing the above baculovirus to obtain total baculovirus protein.

9. The double antibody sandwich ELISA method according to claim 2 or 5, characterized in that: The concentration gradient of the positive quantitative standard is in the range of 0 ng / ml-200 ng / ml.

10. A double antibody sandwich enzyme-linked immunosorbent assay kit for detecting the residual amount of baculovirus total protein in a sample, comprising a coating antibody, a primary antibody, a labeled secondary antibody and a positive quantitative standard; the coating antibody is a purified rabbit anti-baculovirus total protein polyclonal antibody, and the purified rabbit anti-baculovirus total protein polyclonal antibody is obtained by immunizing rabbits with baculovirus total protein as an antigen; the primary antibody is a positive serum of mouse anti-baculovirus total protein.

11. The double antibody sandwich ELISA kit according to claim 10, characterized in that: The labeled secondary antibody is a labeled goat anti-mouse secondary antibody or a labeled horse anti-mouse secondary antibody.

12. The double antibody sandwich ELISA kit according to claim 11, characterized in that: The label is horseradish peroxidase label.

13. The double antibody sandwich ELISA kit according to claim 10 or 11, characterized in that: The positive quantitative standard is baculovirus total protein.

14. The double antibody sandwich ELISA kit according to claim 10, characterized in that: The purified rabbit anti-baculovirus total protein polyclonal antibody is obtained by the following preparation method: during the first immunization, the rabbit is injected with baculovirus total protein by using antigen multi-point immunization; after the first immunization, the rabbit is again boosted with baculovirus total protein for 3-5 times, and serum is collected after the last booster immunization, and purified to obtain the purified rabbit anti-baculovirus total protein polyclonal antibody.

15. The double antibody sandwich ELISA kit according to claim 10, characterized in that: The mouse anti-baculovirus total protein positive serum is obtained by the following preparation method: during the first immunization, the mouse is injected with baculovirus total protein by using antigen multi-point immunization; after the first immunization, the mouse is boosted with baculovirus total protein for 3-5 times, and serum is collected after the last booster immunization, which is the mouse anti-baculovirus total protein positive serum.

16. The double antibody sandwich ELISA kit according to claim 13, characterized in that: The baculovirus total protein is obtained by the following preparation method: (1) Production of baculovirus; (2) harvesting and concentrating baculovirus; (3) Lysing the above baculovirus to obtain total baculovirus protein.

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