CN116008540A - Method and kit for detecting residual quantity of total protein of baculovirus - Google Patents
Method and kit for detecting residual quantity of total protein of baculovirus Download PDFInfo
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Abstract
The invention discloses a double-antibody sandwich ELISA detection method and a kit for detecting the total protein residue of baculovirus. The detection method comprises the steps of immunizing animals by using 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 method. The detection method and the kit are simple and convenient to operate, have good specificity, accuracy, sensitivity, precision and reliability, good linear relation and wider linear range. The method can accurately and reliably detect the residual quantity of the total protein of the baculovirus in intermediate products and final products in the process, can be widely applied to quality control of the residual protein of the baculovirus in biological products such as vaccines, genes, proteins and the like produced by a baculovirus system, and has extremely important application value.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a method and a kit for detecting the residual quantity of total protein of baculovirus.
Background
Baculovirus is composed of baculovirus nucleocapsid, double-layered lipoprotein membrane coating it and single closed circular double-stranded DNA molecule, wherein the composition proteins include nucleocapsid proteins (VP 39, VP78/83, VP87, VP80, 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, VP40/41, etc.), inclusion body matrix proteins, etc. Among them, GP64 protein is a membrane fusion protein capable of recognizing and binding to a receptor on the surface of a host cell membrane, and mediating endocytosis to invade a virus into the host cell. Baculoviruses contain two virus particle forms, one is Budded Virus (BV), and during early replication of the virus, nucleocapsids are released from the cytoplasmic membrane by budding and then infect other cells, allowing the virus to spread in the insect. The other is an inclusion virus (OV), also known as polyhedron-modified virus (PDV), which is a virus that acquires a capsid in the nucleus and then coats it in a polyhedrin or granule envelope, and is not released outside the cell until after cell lysis, which primarily allows for transmission of infection between insects.
Baculoviruses are 80-180kb in genome size, which can be replicated and transcribed in the insect cell nucleus and then assembled in the nucleocapsid. The genome of baculovirus is able to accommodate large fragments of foreign DNA insertion. In addition, the culture operation of the insect cells is simple, the cost is low, and the insect cells can be adhered to and cultured in a suspension mode. Baculovirus-insect cell expression systems using baculovirus as vectors (hereinafter referred to as "baculovirus expression vector system" baculovirus expression vector system, BEVS) have been widely used for efficient expression of foreign proteins. The multi-core polyhedra virus (Autographa californica multiple nuclear polyhedrosis virus, acMNPV or AcNPV for short) of the noctuid has deeper research and most application, the genome size is 133894bp (NC_001623), and the encoding of the polyhedra virus is about 150 open reading frames, so that the polyhedra virus is an ideal carrier for expressing bioactive genes and proteins.
Various biological products such as vaccines, proteins, gene therapy products and the like are produced by adopting a baculovirus expression vector system, and part of baculovirus proteins in the system can remain in the final product as process-related impurities in the production process. Therefore, strict monitoring and control of these residual proteins is necessary. Although methods (for example, patent application No. 201611262295.5) are currently available for ELISA detection of baculovirus GP64 protein, these methods do not involve the residue of other baculovirus proteins in the product, and it is difficult to fully and accurately reflect the overall situation of residue of baculovirus proteins in the biological product. In order to more comprehensively evaluate the total residual amount of baculovirus proteins in biological products, it is desirable to develop a method capable of comprehensively and accurately detecting the total baculovirus proteins. Thus, the present invention developed an enzyme-linked immunosorbent assay (ELISA) assay and kit for baculovirus total protein. The detection method and the kit can be widely used for quality control of baculovirus protein residues in biological products such as vaccines, genes, proteins and the like produced by a baculovirus system, and have extremely important application values.
Disclosure of Invention
In order to solve the technical problems, the invention provides an ELISA double-antibody sandwich ELISA detection method and a kit for detecting baculovirus total proteins. The detection method and the kit can comprehensively, specifically, accurately and repeatedly detect the residual quantity of the total protein of the baculovirus in the sample.
Specifically, the first object of the invention is to provide a double-antibody sandwich ELISA detection method for detecting residual quantity of total protein of baculovirus in a sample. The method comprises the following steps:
(1) Coating an ELISA plate with a coating antibody, adding a sample to be detected, discarding liquid in a hole after incubation is completed, and cleaning;
(2) Adding a primary antibody, discarding liquid in the hole after incubation is completed, and cleaning;
(3) Adding a labeled secondary antibody, discarding liquid in the hole after incubation is completed, and cleaning;
(4) Carrying out a color reaction under proper conditions;
(5) Reading absorbance values (OD values);
(6) And calculating the total protein residual quantity of the baculovirus in the sample to be measured.
In one specific embodiment of the present invention, the residual amount of the baculovirus total protein in the sample to be tested is calculated according to a standard curve, and the standard curve is drawn by using a positive quantitative standard.
In one embodiment of the invention, the coated antibody is a purified polyclonal antibody of rabbit anti-baculovirus total protein.
In one embodiment of the invention, the primary antibody is a positive serum of the total protein of the mouse anti-baculovirus.
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 invention, the secondary antibody is labeled with horseradish peroxidase (HRP).
In another embodiment of the invention, the positive quantitative standard is baculovirus total protein.
In a preferred embodiment of the present invention, the above-described purified polyclonal antibody against a total protein of baculovirus rabbit is obtained by the following preparation method: in the first immunization, antigen multipoint immunization is adopted, and baculovirus total protein is injected into rabbits; and (3) carrying out boosting on the obtained product with baculovirus total protein for 3-5 times after the primary immunization, taking serum after the final boosting, and purifying to obtain the purified polyclonal antibody of the rabbit anti-baculovirus total protein.
In a preferred embodiment of the present invention, the positive serum of the above-mentioned mouse anti-baculovirus total protein is obtained by the following preparation method: in the first immunization, antigen multipoint immunization is adopted, and baculovirus total protein is injected into mice; and (3) carrying out boosting on the baculovirus total protein for 3-5 times after the primary immunization, and taking serum after the final boosting, namely, obtaining the positive serum of the mouse anti-baculovirus total protein.
In a preferred embodiment of the invention, the baculovirus total protein is obtained by the following preparation method:
(1) Producing baculovirus;
(2) Harvesting and concentrating baculovirus;
(3) And (3) splitting the baculovirus to obtain the baculovirus total protein.
In a preferred embodiment of the invention, the positive quantitative standard concentration gradient is in the range of 0ng/ml to 200ng/ml. Preferably, the concentration gradient is 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml.
A second object of the present invention is to provide a double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting residual amount of total protein of baculovirus in a sample, the kit comprising: coating antibody, primary antibody, labeled secondary antibody, positive quantitative standard, etc.
In one embodiment of the invention, the coated antibody is a purified polyclonal antibody of rabbit anti-baculovirus total protein.
In another embodiment of the invention, the primary antibody is a positive serum of the total protein of the mouse anti-baculovirus.
In another embodiment of the invention, the secondary antibody is a goat anti-mouse secondary antibody or a horse anti-mouse secondary antibody, or the like. Preferably, the secondary antibody is a goat anti-mouse secondary antibody. In a preferred embodiment of the invention, the secondary antibody is labeled with horseradish peroxidase (HRP).
In another embodiment of the invention, the positive quantitative standard is baculovirus total protein.
In a preferred embodiment of the present invention, the above-described purified polyclonal antibody against a total protein of baculovirus rabbit is obtained by the following preparation method: in the first immunization, antigen multipoint immunization is adopted, and baculovirus total protein is injected into rabbits; and (3) carrying out boosting on the obtained product with baculovirus total protein for 3-5 times after the primary immunization, taking serum after the final boosting, and purifying to obtain the purified rabbit anti-baculovirus total protein polyclonal antibody.
In a preferred embodiment of the present invention, the positive serum of the above-mentioned mouse anti-baculovirus total protein is obtained by the following preparation method: in the first immunization, antigen multipoint immunization is adopted, and baculovirus total protein is injected into mice; and (3) carrying out boosting on the baculovirus total protein for 3-5 times after the primary immunization, and taking serum after the final boosting, namely, obtaining the positive serum of the mouse anti-baculovirus total protein.
In a preferred embodiment of the invention, the baculovirus total protein is obtained by the following preparation method:
(1) Producing baculovirus;
(2) Harvesting and concentrating baculovirus;
(3) And (3) splitting the baculovirus to obtain the baculovirus total protein.
The beneficial effects of the invention are as follows:
(1) In the prior art, detection is performed only by a single protein antibody, on one hand, since baculovirus particles contain a plurality of proteins, the whole residual quantity of baculovirus proteins cannot be comprehensively and objectively reflected by only using the single protein antibody. On the other hand, there may be a large difference in the composition and type of baculovirus protein remaining in different samples, as well as samples prepared by different processes, and it is difficult to accurately react the type, content, and variation of baculovirus remaining protein using only a single protein antibody. The technical scheme of the invention can more objectively, accurately and comprehensively detect and reflect the residual baculovirus proteins in various types of samples.
(2) The technical scheme of the invention has simple and convenient operation, good specificity, accuracy, sensitivity, precision and reliability, good linear relation and wider linear range.
In a word, the detection method and the kit can accurately and reliably detect the residual quantity of the total protein of the baculovirus in intermediate products and final products in the process, can be widely applied to quality control of the residual baculovirus protein in biological products such as vaccines, genes, proteins and the like produced by a baculovirus system, and have extremely important application values.
Drawings
Fig. 1: standard curves drawn with positive quantitative standards.
The specific embodiment is as follows:
the examples below are given for the purpose of illustration only and are not intended to limit the scope of the invention.
The reagent materials described in the examples below are commercially available common materials, except for the sources indicated, and the reagents were formulated in a conventional manner. Methods not described in detail in the examples are all routine in the art.
EXAMPLE 1 preparation of baculovirus Total protein
1. Production of baculoviruses
Sf9 cells were transfected with baculovirus plasmid DNA (Bacmid) by the liposome transfection method and cultured in a cell incubator at 27℃for 72h to 96h. The culture solution containing baculovirus was collected in a 15ml centrifuge tube, centrifuged (500×g,5 min) to remove cell pellet and impurities, and the supernatant was transferred to a new centrifuge tube as the baculovirus generation P0. Then, the P0 generation virus is used for infecting new Sf9 cells and generating virus. Cell culture supernatants were centrifuged (500 Xg, 5 min) and the supernatants were transferred into new tubes as baculoviruses for the P1 generation. In the subsequent experiments, the generation P1 can be amplified further according to the requirement of baculovirus.
2. Preparation of baculovirus concentrate
The supernatant containing baculovirus was harvested, filtered with a 0.22 μm filter membrane, transferred to a high-speed centrifuge tube, centrifuged (4 ℃,40000×g,1.5 h) to remove the supernatant, allowed to stand for 1min, and then the residual liquid at the bottom of the tube was aspirated with a pipette. Then adding a small amount of storage buffer solution, blowing and resuspending by using a pipette, and sucking the suspension into a new centrifuge tube. Mixing the precipitate heavy suspension in multiple separation tubes, and making into baculovirus concentrate, and preserving at 4deg.C or-80deg.C.
3. Preparation of baculovirus Total protein
Heating the baculovirus concentrate in 95 ℃ water bath for 30min or performing ultrasonic crushing to fully crack the baculovirus, thereby obtaining the baculovirus total protein. The total protein concentration of baculovirus was measured to be 0.78mg/ml by BCA method (BCA PROTEIN ASSAY KIT, thermo, cat# 22400-089), and then split-packed as positive quantitative standard in the subsequent ELISA detection experiments and antigen in the preparation of polyclonal antibodies and positive serum, stored at-80℃for later use.
EXAMPLE 2 preparation of polyclonal antibodies against Rabbit Total baculovirus proteins and Positive serum against mouse Total baculovirus proteins
Animal immunity, polyclonal antibody preparation method and polyclonal antibody purification method are adopted to prepare and/or purify the positive serum of the total protein of the anti-baculovirus and the polyclonal antibody. Firstly, the baculovirus total protein is used as an antigen to be mixed and emulsified with Freund's complete adjuvant, then 10-20 New Zealand white rabbits and mice are respectively immunized for the first time, and the baculovirus total protein is injected in a multipoint immunization mode. For New Zealand white rabbits, the injection dosage is 0.5 mg/animal; for mice, the injection dose was 0.1 mg/mouse. Boosting is carried out after 14 days of primary immunization, freund's incomplete adjuvant is adopted for antigen emulsification, other steps and dosages are the same as those of the primary immunization, the interval between each boosting is 10 days, and 3-5 times of boosting are carried out. Rabbit serum and mouse serum were harvested on day 7 after the last boost. The mouse serum is positive serum of the total protein of the mouse anti-baculovirus and is used as the first antibody in the subsequent ELISA detection method.
For rabbit serum, further purification was performed using protein a affinity chromatography columns. Rabbit serum was taken at 100ml and buffered with PBS according to 1:1, and filtering with a 0.22 μm filter membrane to obtain the treated sample. According to the instructions, the treated samples were purified using protein a resin. The specific operation is as follows: the protein A column was first equilibrated with 6 column volumes of equilibration solution (20 mM/L PB buffer, 150mM/L NaCl, pH 7.2) at a flow rate of 5ml/min. The pH of the sample (diluted rabbit serum) was adjusted to 7.2, and the sample was loaded at room temperature at a flow rate of 5ml/min. After complete equilibration, 20ml of eluent (0.1M/L Glycine,150mM/L NaCl, pH 3.2) was added for elution at a flow rate of 5ml/min and the eluent was collected. Concentrating and changing the solution by using a 30KD ultrafiltration tube to obtain the purified polyclonal antibody of the rabbit anti-baculovirus total protein, measuring the concentration to be 7.6mg/ml, subpackaging, preserving at-80 ℃, and using the polyclonal antibody as a coating antibody in the subsequent ELISA detection.
Example 3 detection of residual baculovirus protein in samples by ELISA method
1. Coating an antibody: the coated antibody (i.e., the purified polyclonal antibody against the total baculovirus protein) was diluted to a concentration of 160. Mu.g/ml with PBS buffer, and was added to the ELISA plate in an amount of 100. Mu.l/well for coating, and allowed to stand at 4℃overnight.
2. Washing the plate: the wells were rinsed by aspirating the liquid from the ELISA plate, adding 200. Mu.l of Wash solution (Wash Concentrate (20X), CYGNUS, F004) to each well, then removing the Wash solution and beating it dry on filter paper, and rinsing repeatedly 5 times.
3. Closing: blocking solution (prepared by dissolving 1.0g of skimmed milk powder in 20ml of PBS buffer) was added thereto, 200. Mu.l/well was incubated at 37℃for 2 hours.
4. Washing the plate: and the same as in step 2.
5. Positive quantitative standard dilution: positive quantification standards (i.e., baculovirus total protein) were diluted in gradient using PBS buffer at concentrations of 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml and 6.25ng/ml for a total of 6 gradients.
6. Sample adding: taking the coated ELISA plate, adding 100 μl of sample or positive quantitative standard into each corresponding well, respectively arranging multiple wells, and incubating at 37deg.C for 2 hr, wherein sample diluent is used as blank control.
7. Washing the plate: and the same as in step 2.
8. Adding primary antibody: mu.l of primary antibody (i.e.positive serum of mouse total anti-baculovirus proteins) diluted 4000-fold with antibody dilution was added to each well and incubated at 37℃for 1.5h.
9. Washing the plate: and the same as in step 2.
10. Adding a secondary antibody: mu.l of secondary antibody (i.e.HRP-labeled goat anti-mouse secondary antibody) diluted 3000-fold with antibody dilution was added to each well and incubated at 37℃for 1.5h.
11. Washing the plate: and the same as in step 2.
12. Color development: mu.l of TMB color development solution (TMB Substrate (ELISA), CYGNUS, F005) equilibrated at room temperature was added to each well, and the wells were left at room temperature for 10-30min in the absence of light.
13. And (3) terminating: 100 μl of stop solution (0.5M sulfuric acid) was added to each well to terminate the chromogenic reaction.
14. And (3) detection: the absorbance value of each well at 450nm was read using a multifunctional microplate reader.
15. Data processing and sample measurement:
(1) Drawing a standard curve: and (3) carrying out data processing by using self-contained software (softMax Pro 5.4.1) of a multifunctional enzyme-labeled instrument, and carrying out four-parameter fitting on the concentration value of the positive quantitative standard substance and the corresponding absorbance value thereof to obtain a standard curve. The standard curve obtained is shown in FIG. 1, and the result shows that R is 2 Above 0.98, the linear relation is good, the quantitative range can reach 6.25ng/ml-200ng/ml, and the detection limit can be as low as 6.25ng/ml.
(2) Sample detection results: the concentration of baculovirus protein residue in the sample is calculated from the standard curve and the absorbance value of the sample measured. The results of the detection of the intermediate sample and the final sample are shown in table 1, wherein the intermediate sample 1 and the intermediate sample 2 are intermediate samples of different stages of the sample in the purification process, respectively, and the final sample is a final sample obtained after the sample purification is completed.
TABLE 1 results of detection of residual amount of Total protein of baculovirus in samples
Example 4 methodological verification
1. Specificity and specificity
Taking a sample auxiliary material solution and an antibody diluent according to the following steps of 1:1 to a concentration of 100ng/ml to obtain a labeled sample, and performing ELISA detection on the labeled sample (see example 3 for specific steps). After the detection result is obtained, calculating the labeling recovery rate: the labeling recovery rate is = [ actual measured value of labeling sample/theoretical value of labeling sample ] ×100%. If the recovery rate of the sample auxiliary material solution and the antibody diluent added with the standard sample is between 70% and 130%, the specificity and the specificity of the method are considered to be good.
The detection results are shown in Table 2, the standard recovery rate of the sample auxiliary material solution and the sample diluent is 80% -120%, and the specificity of the method are good.
TABLE 2 specificity and specificity verification results
2. Accuracy of
Positive quantitative standards of 100ng/ml (high concentration), 50ng/ml (medium concentration), 25ng/ml (low concentration) were taken respectively, and were prepared according to 1:1, adding the mixture into a finished product sample or an intermediate product sample to obtain a labeled sample; sample dilutions (PBS buffer) were taken according to 1:1 is added into a finished product sample or an intermediate product sample to obtain an unlabeled sample. ELISA assays were performed on both the labeled and unlabeled samples (see example 3 for specific steps). Calculating the labeling recovery rate according to the detection result: the accuracy of the method is considered to meet the requirement if the labeling recovery rate is between 70% and 130%.
The detection results of the labeling accuracy of the finished product sample and the intermediate product sample are shown in the table 3-1 and the table 3-2 respectively, and the recovery rates of the high, medium and low concentration positive quantitative standard products are all between 70% and 120%, which indicates that the accuracy of the method is good.
TABLE 3-1 end product sample labeling accuracy verification results
Table 3-2 results of sample labeling accuracy verification of intermediate
3. Repeatability of
ELISA was performed using 50ng/ml positive quantitative standard, and 5 replicates were performed according to the procedure of example 3, and the average and CV values were calculated from the results of the detection. If the CV value of the measured value is less than 30%, the measurement is satisfactory.
The results of the tests are shown in Table 4, and the CV value of the positive quantitative standard for repeated 5 times is 2.8%, which shows that the method has good repeatability.
TABLE 4 repeatability verification results
4. Intermediate precision
Two test persons respectively test 50ng/ml positive quantitative standard, each person is repeated for 3 times, ELISA test is carried out according to the operation steps of the example 3, the average value and CV value of the test results are calculated, and if the CV value is less than 30%, the test results meet the requirements.
The results of the tests are shown in Table 5, and the two laboratory workers each repeated 3 times and 6 times, and the total CV value is 6.45%, which shows that the intermediate precision of the method is good.
TABLE 5 results of intermediate precision validation
The invention selects the purified polyclonal antibody of the rabbit anti-baculovirus total protein as a coating antibody and the positive serum of the mouse anti-baculovirus total protein as a primary antibody, and detects baculovirus residual proteins in various samples by enzyme-linked immunosorbent assay (ELISA). The detection method of the invention is subjected to methodological verification, and the verification result shows that: the method has the advantages of high specificity, strong specificity, high accuracy, good repeatability and good precision. The detection method and the kit can be used for detecting the residual quantity of the baculovirus protein in the sample produced by the baculovirus production system more comprehensively and sensitively.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (18)
1. A double-antibody sandwich enzyme-linked immunosorbent assay method for detecting residual amounts of total proteins of baculovirus in a sample, the method comprising the steps of:
(1) Coating an ELISA plate with a coating antibody, adding a sample to be detected, discarding liquid in a hole after incubation is completed, and cleaning;
(2) Adding a primary antibody, discarding liquid in the hole after incubation is completed, and cleaning;
(3) Adding a labeled secondary antibody, discarding liquid in the hole after incubation is completed, and cleaning;
(4) Carrying out a color reaction under proper conditions;
(5) Reading the absorbance value;
(6) And calculating the total protein residual quantity of the baculovirus in the sample to be measured.
2. The method for detecting the double-antibody sandwich enzyme-linked immunosorbent assay according to claim 1, wherein the residual quantity of the baculovirus total protein in the sample to be detected is calculated according to a standard curve, and the standard curve is drawn by using a positive quantitative standard.
3. The method for double-antibody sandwich enzyme-linked immunosorbent assay according to claim 1 or 2, wherein: the coated antibody is a polyclonal antibody of purified rabbit anti-baculovirus total protein.
4. A double antibody sandwich enzyme-linked immunosorbent assay according to any of claims 1 to 3, wherein: the primary antibody is a positive serum of the total protein of the mouse anti-baculovirus.
5. The method for double antibody sandwich enzyme-linked immunosorbent assay according to any one of claims 1 to 4, wherein: the labeled secondary antibody is labeled goat anti-mouse secondary antibody or labeled horse anti-mouse secondary antibody, etc., preferably the label is horseradish peroxidase label.
6. The method for double antibody sandwich enzyme-linked immunosorbent assay according to any one of claims 2 to 5, wherein: the positive quantitative standard is baculovirus total protein.
7. The method for double-antibody sandwich enzyme-linked immunosorbent assay according to claim 3, wherein: the purified polyclonal antibody of the rabbit anti-baculovirus total protein is obtained by the following preparation method: in the first immunization, antigen multipoint immunization is adopted, and baculovirus total protein is injected into rabbits; and (3) carrying out boosting on the obtained product with baculovirus total protein for 3-5 times after the primary immunization, taking serum after the final boosting, and purifying to obtain the purified polyclonal antibody of rabbit anti-baculovirus total protein.
8. The method for double-antibody sandwich enzyme-linked immunosorbent assay according to claim 4, wherein the method comprises the following steps: the positive serum of the mouse anti-baculovirus total protein is obtained by the following preparation method: in the first immunization, antigen multipoint immunization is adopted, and baculovirus total protein is injected into mice; and (3) carrying out boosting on the baculovirus total protein for 3-5 times after the primary immunization, and taking serum after the final boosting, namely, obtaining the positive serum of the mouse anti-baculovirus total protein.
9. The method for double-antibody sandwich enzyme-linked immunosorbent assay according to claim 6, wherein: the baculovirus total protein is obtained by the following preparation method:
(1) Producing baculovirus;
(2) Harvesting and concentrating baculovirus;
(3) And (3) splitting the baculovirus to obtain the baculovirus total protein.
10. The method of double antibody sandwich enzyme-linked immunosorbent assay according to any of claims 2 to 9, wherein: the concentration gradient range of the positive quantitative standard is 0ng/ml-200ng/ml.
11. A double-antibody sandwich ELISA kit for detecting the residual quantity of the total protein of baculovirus in a sample comprises a coated antibody, a primary antibody, a labeled secondary antibody, a positive quantitative standard and the like.
12. The double-antibody sandwich enzyme-linked immunosorbent assay kit according to claim 11, wherein: the coated antibody is a polyclonal antibody of purified rabbit anti-baculovirus total protein.
13. The double antibody sandwich enzyme-linked immunosorbent assay kit according to claim 11 or 12, wherein: the primary antibody is a positive serum of the total protein of the mouse anti-baculovirus.
14. The double antibody sandwich enzyme-linked immunosorbent assay kit according to any one of claims 11 to 13, wherein: the labeled secondary antibody is labeled goat anti-mouse secondary antibody or labeled horse anti-mouse secondary antibody, etc., preferably the label is horseradish peroxidase label.
15. The double antibody sandwich enzyme-linked immunosorbent assay kit according to any of claims 11 to 14, wherein: the positive quantitative standard is baculovirus total protein.
16. The double-antibody sandwich enzyme-linked immunosorbent assay kit according to claim 12, wherein: the purified polyclonal antibody of the rabbit anti-baculovirus total protein is obtained by the following preparation method: in the first immunization, antigen multipoint immunization is adopted, and baculovirus total protein is injected into rabbits; and (3) carrying out boosting on the obtained product with baculovirus total protein for 3-5 times after the primary immunization, taking serum after the final boosting, and purifying to obtain the purified polyclonal antibody of rabbit anti-baculovirus total protein.
17. The double-antibody sandwich enzyme-linked immunosorbent assay kit according to claim 13, wherein: the positive serum of the mouse anti-baculovirus total protein is obtained by the following preparation method: in the first immunization, antigen multipoint immunization is adopted, and baculovirus total protein is injected into mice; and (3) carrying out boosting on the baculovirus total protein for 3-5 times after the primary immunization, and taking serum after the final boosting, namely, obtaining the positive serum of the mouse anti-baculovirus total protein.
18. The double-antibody sandwich enzyme-linked immunosorbent assay kit according to claim 15, wherein: the baculovirus total protein is obtained by the following preparation method:
(1) Producing baculovirus;
(2) Harvesting and concentrating baculovirus;
(3) And (3) splitting the baculovirus to obtain the baculovirus total protein.
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