CN111474367A

CN111474367A - Kit for screening and detecting African swine fever virus P30 protein monoclonal antibody and preparation method thereof

Info

Publication number: CN111474367A
Application number: CN202010285019.0A
Authority: CN
Inventors: 孙统威; 周晶晶; 徐慧珍
Original assignee: Hangzhou Heng Ao Technology Co ltd
Current assignee: Hangzhou Heng Ao Technology Co ltd
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2020-07-31

Abstract

The invention discloses an E L ISA kit for identifying an African swine fever virus P30 protein monoclonal antibody, a preparation method and a use method of the kit, and the kit provided by the invention can be used for large-scale screening and detection of the African swine fever virus P30 protein monoclonal antibody on the premise of not using the African swine fever virus.

Description

Kit for screening and detecting African swine fever virus P30 protein monoclonal antibody and preparation method thereof

Technical Field

The invention relates to an E L ISA kit for identifying a monoclonal antibody of African swine fever virus P30 protein, a detection method and a preparation method thereof, belongs to the field of biomedicine, and particularly belongs to the field of virus epidemic disease diagnosis technology and animal quarantine.

Background

African Swine Fever (ASF) is an acute, hemorrhagic and virulent infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs and various wild pigs (such as African wild pigs, European wild pigs and the like). The world animal health Organization (OIE) classifies the animal epidemic disease as a legal report animal epidemic disease, and the disease is also a type of animal epidemic disease which is mainly prevented in China. The clinical symptoms of African swine fever are similar to swine fever symptoms, and the death rate of the most acute and acute infections is up to 100%, the clinical manifestations are fever (up to 40-42 ℃), fast heartbeat, dyspnea, partial cough, serous or mucoid purulent secretion of eyes and nose, cyanosis of skin, and obvious bleeding of lymph nodes, kidney and gastrointestinal mucosa.

The African Swine Fever virus is a large, double-stranded DNA virus with envelope, the genome of which is a single-molecule linear double-stranded DNA with covalently closed ends, the total length of the virus genome is 170kb-190kb, a conserved region in the center of about 125kb and variable regions at both ends, containing inverted terminal repeat sequences, the addition or deletion of which is the main cause of differences in the genome lengths of different isolates (Rafacl, Yancz, Javiarm, et al. analysis of the complex nucleotide Sequence of African Swine Virus [ J ]. Virology, 1995, 208: 249-279). ASFV is in the form of a regular icosahedron, about 200 nm in diameter, and is composed of multiple layers of proteins with nucleomimetic in the center, and a lipid envelope and a capsid protein, which are composed of 8280 major capsid proteins P72, P54 and P30, and in addition, at least three proteins are stabilized against adjacent proteins (20154. 7. III. F. 7. cell 3. cell)

The African swine fever virus belongs to a class of animal pathogenic microorganisms in the 'animal pathogenic microorganism classification catalogue' of China. Currently, only a few national-level research institutes can use african swine fever viruses for research and testing. In order to be able to study and prepare monoclonal antibodies for detecting African swine fever viruses, we used molecular biology techniques to clone and express the P30 protein and study monoclonal antibodies against the P30 protein on the basis of this, but no suitable method and kit were found in large-scale screening and evaluation of monoclonal antibodies against the P30 protein, since conventional methods require evaluation using African swine fever viruses.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a monoclonal antibody screening method which does not use the African swine fever virus and can screen specific proteins of the African swine fever virus in a large scale, preferably the African swine fever virus protein is P30 protein, and preferably the method is to establish an E L ISA kit aiming at the P30 protein of the African swine fever virus.

In order to realize the purpose of the invention, the invention provides an E L ISA kit for identifying the African swine fever virus P30 protein monoclonal antibody, which comprises an antigen coated plate, a sample diluent, a concentrated washing solution, a goat anti-mouse enzyme-labeled antibody, a positive control, a negative control, a chromogenic solution, a stop solution and an instruction book, wherein the antigen coated plate is an enzyme-labeled plate coated with P30 protein, the sample diluent is a 1 × PBST solution containing 1% BSA, the concentrated washing solution is a 25 × PBST solution and is diluted to a 1 × PBST solution before use, the goat anti-mouse enzyme-labeled antibody is obtained by diluting an enzyme-labeled secondary antibody by 10000 times by using the sample diluent, the positive control is positive serum with an OD450nm value of 0.9-1.2, the negative control is negative serum with an OD450nm value of less than 0.2, the chromogenic solution is TMB serum, and the single component of the stop solution is 2M H₂SO₄。

Preferably, the concentration of P30 protein coated on the antigen coated plate is 200 ng/well/100. mu.l.

Preferably, the positive control OD450nm value described herein is 1.1.

Preferably, the negative control OD450nm value described herein is < 0.1.

Preferably, the positive control OD450nm values are all more than or equal to 0.6, and the negative control OD450nm values are all less than 0.2, so that the test is established and the result is valid.

On the other hand, the invention also provides a method for detecting a sample to be detected by using the kit, which comprises the following steps: 1) sample dilution: diluting a sample to be detected by 1:100 times by using a sample diluent;

1) adding a sample: adding diluted sample, negative control and positive control, wherein the positive control and negative control are repeated for 2 times, 100 μ l/well, incubating at 37 deg.C for 1 hr,1 × PBST is washed 3 times, each hole is 300 mul, each time is 3 minutes, the last time is dried, 3) the goat anti-mouse enzyme-labeled antibody is added, each reaction hole is added with the goat anti-mouse enzyme-labeled antibody, 100 mul/hole is incubated for 1 hour at 37 ℃,1 × PBST is washed 5 times, each hole is 300 mul, each time is 3 minutes, the last time is dried, 4) the color developing solution is added, each reaction hole is added with 100 mul of color developing solution, the light-shielding color development is carried out for 10 minutes at 37 ℃, 5) the stopping solution is added, each reaction hole is added with 50 mul of stopping solution, and the result is measured within 10 minutes, 6) the test is carried out, the 450nm value of each hole is measured on an enzyme-labeling instrument, 7) the test effectiveness is judged, when the positive control OD450nm value is more than or equal to 0.6, the negative control OD450nm value is less than 0.2, the test is established and the result is effective, 8:

9) and (3) judging test results: and when the S/P value of the sample to be detected is more than or equal to 0.4, judging the sample to be detected as positive, and when the S/P value of the sample to be detected is less than 0.4, judging the sample to be detected as negative.

On the other hand, the invention also provides a method for preparing the kit, which comprises the steps of preparing an antigen coated plate, preparing and subpackaging a sample diluent, preparing and subpackaging a concentrated washing solution, preparing and subpackaging a goat anti-mouse enzyme-labeled antibody, preparing a positive control, preparing a negative control, subpackaging a developing solution, preparing a stopping solution and subpackaging.

Preferably, the preparation of the antigen coated plate comprises the following steps of 1) coating, diluting purified African swine fever virus P30 recombinant protein to 2 mu g/m L by using a coating buffer solution, uniformly mixing, adding an enzyme label plate with 100 mu l/hole, acting for 12-15 hours at 2-8 ℃, 2) washing by 1 × PBST for 3 times with 3 minutes each time and 300 mu l/hole, and finally patting dry, 3) sealing, adding 1% BSA-containing PBS with 200 mu l/hole, and sealing for 2 hours at 37 ℃, 4) washing by 1 × PBST for 3 times with 300 mu l/hole and 3 minutes each time and patting dry, 5) adding a protective agent, adding 10% trehalose, incubating for 2 hours at 37 ℃, patting dry, adding a drying agent, and vacuumizing for storage.

Preferably, the preparation of the positive control comprises the following steps of 1) immunization, namely taking 30 female BA L B/c mice with age of 6-8 weeks, taking purified P30 protein as immunogen (100 mu g/m L), carrying out subcutaneous multipoint injection immunization, wherein the immunization amount is 50 mu g/mouse, immunizing for 2 times at intervals of 2 weeks in the same method dose, carrying out primary immunization by using Freund's complete adjuvant, carrying out booster immunization by using Freund's incomplete adjuvant, 2) serum collection, wherein after 21 days of secondary immunization, blood is collected by using an eyeball extraction method, serum is separated, and the serum is stored at 20 ℃ for later use, 3) the collected serum is detected by using the method of claim 6, and the serum with OD450nm value of more than 0.9 and less than 1.2 is mixed as positive control and is subpackaged at 50 mu l/mouse and stored at 20 ℃.

Preferably, the negative control of the present invention is prepared by 1) serum collection of 30 SPF female BA L B/c mice of 6-8 weeks of age, body surface sterilization, orbital bleeding, centrifugation at 3000g for 30min, serum separation, storage at-20 ℃ for use, 2) detection of the collected serum using the method of claim 6, mixing the serum with OD450nm value less than 0.2 as negative control serum, dispensing 50 μ l/tube, and storage at-20 ℃.

The kit provided by the invention can be used for large-scale screening and detection of the African swine fever virus P30 protein monoclonal antibody on the premise of not using the African swine fever virus, can overcome the difficulty that no virus is used for screening and detection, and can obviously improve the screening efficiency of the African swine fever virus monoclonal antibody, especially the monoclonal antibody aiming at the P30 protein.

In addition, the method for establishing the kit and the detection method provided by the invention can also be used for screening and detecting monoclonal antibodies of other antigens of the African swine fever virus. Therefore, the method also belongs to the protection scope of the invention. On the other hand, the method can also be used for screening and detecting monoclonal antibodies of other antigens (not limited to African swine fever virus).

Drawings

FIG. 1PCR identification map, 1: DNA Marker, 2: Plasmid, 3: Plasmid Digested with XhoI-M L uI, 4: P30 Gene.

FIG. 2 shows the alignment of nucleotide sequence of African swine fever virus P30.

FIG. 3 is an SDS-PAGE electrophoresis of African swine fever virus P30 protein.

FIG. 4 positive cut-off values.

FIG. 5 negative cut-off values.

Detailed Description

The invention is further illustrated below with reference to specific examples. The various starting materials mentioned in the following examples are all commercially available unless otherwise specified.

The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention, the experimental procedures, for which specific conditions are not indicated in the examples, are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 preparation of African swine fever virus P30 protein

1 Material

1.1 Strain and vector Escherichia coli B L21 competent cells, purchased from Bomeidchi Ltd, and Gene Synthesis was synthesized by general biosystems (Anhui) Ltd (providing plasmid PET-30 a).

1.2 the main reagent DNAmarker is purchased from Thermo company, and the plasmid miniprep kit is purchased from AXYGEN company; other chemical reagents are all domestic analytical purifiers.

1.3 apparatus Centrif μ ge 5804R desk-top high speed centrifuge from Eppendorf, Germany; PCR instrument, nucleic acid electrophoresis instrument, VerSaDoc2000 gel imaging system, Bio-Rad, USA; HZQ-C air bath constant temperature double layer oscillator, purchased from Harbin Toming medical instruments and factories; SW-CJ-IF superclean bench, Sujing group Antai Co; -80 ℃ refrigerator, san ocean japan.

1.4 preparation of the principal solution

1.4.1L B Medium 5.0g yeast extract, 10g tryptone, 10g NaCl were weighed, dissolved in 1L purified water and autoclaved at 121 ℃ for 20 minutes.

1.4.2L B (Kan +) liquid medium L B medium was added kanamycin to a final concentration of 100. mu.g/m L, and stored at 2 to 8 ℃.

1.4.3L B (Kan +) solid culture medium 1.5g agar powder is added into a 100m L L B culture medium, the mixture is sterilized by high-pressure steam at 121 ℃ for 20 minutes, before use, the culture medium is melted in a microwave oven, the culture medium is cooled at room temperature, kanamycin is added when the temperature of the culture medium is reduced to about 50 ℃, the final concentration of kanamycin is 100 mug/m L, the culture medium is mixed evenly and then is turned over, a flat dish cover is opened, the culture medium is naturally cooled, and then the culture medium is stored at 2-8 ℃.

2 method

2.1 the structural protein coding gene p30 of the epidemic strain of African swine fever in China is selected by synthesizing the target fragment, the full length of the gene is 621bp, and the nucleotide sequence is shown in SEQ ID NO 1.

2.2 construction of recombinant Strain cloning sites NdeI (CATATG) -XhoI (CTCGAG), cloning vector pET-30a (+), the above plasmid construction was carried out by general biosystems (Anhui) Ltd.

2.3 transformation of competent cells frozen competent cells were dissolved on ice, 1. mu.l of plasmid DNA was added to 100. mu.l of competent cells and placed on ice for 30 minutes, followed by heat shock in a metal bath at 42 ℃ for 60 seconds and rapidly placed on ice for 5 minutes, the competent cells having taken up the foreign genetic material were added to a culture tube containing L B liquid medium preheated to 37 ℃ to allow a total volume of 1m L and 200r/min for 1 hour, the recovered inoculum was applied to a L B (Kan +) plate using an application gun, spread with a push rod until no liquid was visible on the surface of the medium, and after incubating the medium-containing surface downward for about 30 minutes, the plate was inverted and incubated at 37 ℃ overnight.

Selecting a single colony from the colony on the plate, inoculating the single colony in a 1m L L B (Kan +) liquid culture medium, culturing at 37 ℃ at 200r/min for 12-16 hours, collecting 1m L bacterial liquid, extracting recombinant plasmid, and verifying whether the recombinant plasmid contains the African swine fever p30 fragment by adopting PCR amplification reaction.

2.4 preservation of strains with correct sequencing are purely checked according to the appendix of the current Chinese veterinary pharmacopoeia, after the strains are checked to be qualified, the strains are taken and added with glycerol with the final concentration of 40 percent, and the mixture is placed at the temperature of minus 80 ℃ to be used as an original seed batch for proper preservation.

3P30 protein expression

3.1 screening and identifying positive strains, transforming the connecting products into competent cells, carrying out inverted culture at 37 ℃ for 12-16 hours, and then, obtaining a visible colony form which is smooth, single and scattered, neat in edge, glossy, moist and grey-white in surface and typical escherichia coli colony form, selecting 1 single colony from the competent cells for PCR identification and sequencing identification, wherein the PCR identification result (figure 1) is consistent with the expected size, the sequence comparison result has 100 percent homology with the P30 sequence of the African swine fever virus published by GenBank (figure 2), and the screened positive strains are named as B L-21-P30, carrying out pure inspection on the recombinant escherichia coli B L-21-P30, and the inspection results are pure and free of growth of infectious microbes or mold.

3.2B L-21-P30 strain, inoculating 1% of P30 protein expression to a liquid culture medium containing 100 μ g/M L kanamycin L B, culturing at 37 ℃ under the condition of 200r/min for 2-4 hours in a shaking manner until the OD600nm of the bacterial liquid is 0.6-0.8, adding 1.0mM IPTG (final concentration) for continuously inducing for 4 hours, centrifuging 5000g of the induced recombinant bacteria for 10 minutes, discarding the supernatant, dissolving the precipitate for 10 minutes by using PBS, crushing by using an ultrasonic cell crusher, centrifuging at 4 ℃ (12000r/min) for 30 minutes after crushing, discarding the supernatant, cracking the Tris inclusion body part by using a cracking solution (50mM, 8M Urea,0.5M NaCl) for 10 minutes, centrifuging at 12000r/min for 30 minutes, taking the supernatant, purifying by using a nickel column, washing the column by using 10 times of the cracking solution, adding the supernatant into a nickel column, slowly loading the protein solution, removing the impurity from the column, washing solution, storing the protein in a washing solution, eluting by using SDS, eluting with 300mM, eluting protein, and collecting the protein after detecting that the purity of 300. mu.8 mM SDS gel electrophoresis.

Example 2 establishment of a kit for identifying P30 protein monoclonal antibody of African swine fever virus

1 materials and methods

1.1 test materials

1.1.1 purified African Swine fever Virus P30 protein was prepared by Zhejiang university of Richardson university.

1.1.2 enzyme-labeled plates were purchased from Corning, USA.

1.1.3 goat anti-mouse enzyme-labeled antibodies were purchased from Huamei Bio Inc.

1.1.4 Single-component color developing solution of main reagent TMB was purchased from Beijing Solaibao Tech Co., Ltd; other chemical reagents are all domestic analytical purifiers.

1.1.5 Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma.

1.1.6 BA L B/c mice were purchased from Yangzhou university.

1.2 test methods

1.2.1 preparation and testing of Positive and negative sera

1.2.1.1 preparation of negative serum 30 SPF-grade female BA L B/c mice of 6-8 weeks of age were sterilized on the body surface, bled through the orbit, centrifuged at 3000g for 30min, and the serum, numbered N1-N30, was isolated and stored in a freezer at-20 deg.C for future use.

1.2.1.2 preparation of Positive serum 30 female BA L B/c mice of 6-8 weeks old were also selected, immunized with purified P30 protein as immunogen (100. mu.g/m L) by subcutaneous multipoint injection at an amount of 50. mu.g/mouse 2 times at 2 weeks intervals, the dose of the method was the same as above, Freund's complete adjuvant was used for the first immunization, Freund's incomplete adjuvant was used for the second boosting immunization, blood was collected by an eye-ball extraction method 21 days after the second immunization, serum was isolated and numbered P1-P30, and stored at-20 ℃ for further use.

1.2.2 establishment of Indirect E L ISA method

1.2.2.1 determination of the coating concentration of the recombinant P30 protein and determination of the optimal dilution multiple of the serum the optimal working concentration of the recombinant protein and the optimal dilution multiple of the serum to be tested are determined by matrix titration, the purified recombinant P30 protein is diluted with a coating solution (0.05M carbonate buffer pH9.6) to concentrations of 4. mu.g/M L, 2. mu.g/M L, 1. mu.g/M L and 0.5. mu.g/M L, an ELISA plate is coated at 100. mu. L/well, a negative (N1) and a positive serum (P1) are diluted at a rate of 1:100, 1:200, 1:400 and 1:800, respectively, each serum dilution corresponds to 8 antigen coating concentrations, a sample diluent (1 × PBST solution containing 1% of BSA-labeled anti-rat-peroxidase is diluted 10000 times, an indirect immunoassay procedure is performed according to the ISA method, a determination is performed with an enzyme reader, the optimal OD of 450 of each well is calculated as the optimal dilution value of the positive serum P nm and the optimal dilution of the serum OD P nm, and the optimal dilution value of the optimal dilution of the goat serum dilution of the same concentration of the goat peroxidase-goat serum is selected as the optimal dilution.

1.2.2.2 optimization of the working concentration of goat anti-mouse enzyme-labeled antibody E L ISA plate is coated with the optimal working concentration of recombinant protein, closed, added with negative (N1) and positive serum (P1) with the optimal dilution times, the goat anti-mouse enzyme-labeled antibody is diluted according to the proportion multiple ratio of 1:5000, 1:10000 and 1:20000, the known negative and positive serum is detected according to the operation procedure of the indirect E L ISA method, an enzyme-labeling instrument is used for measuring the OD450nm value of each hole, and the P/N value is compared to determine the optimal dilution of the enzyme-labeled antibody.

1.2.2.3 selection of blocking conditions blocking solutions were selected from 1 × PBST solutions containing 0.5% BSA, 1% BSA, 5% skim milk, blocking times were 1 hour incubation at 37 deg.C, 2 hours incubation at 37 deg.C, 2.5 hours incubation at 37 deg.C, respectively, E L ISA detection was performed, P/N values were compared, and optimal blocking conditions were determined.

1.2.2.4 antigen-antibody reaction conditions and time the reaction conditions and time of the antigen-antibody were determined by matrix titration. And (3) diluting the negative serum and the positive serum according to the optimal dilution times respectively, and detecting the negative serum and the positive serum respectively, wherein the antigen-antibody reaction temperature is 25 ℃, and the antigen-antibody reaction time is 37 ℃ and is 1 hour, 1.5 hours and 2 hours. The OD450nm value of each well was measured by a microplate reader, and the P/N (positive/negative) value was calculated. The conditions for the maximum P/N value are selected as the reaction conditions and time of the antigen-antibody.

1.2.2.5 goat anti-mouse enzyme-labeled antibody action condition and time and determining enzyme-labeled antibody working condition and time by a matrix titration method. Diluting the negative serum and the positive serum according to the optimal dilution times respectively, and detecting respectively; action temperature: 4 ℃, 37 ℃, action time: the reaction is carried out for 14 hours at 4 ℃, for 45 minutes, 60 minutes and 90 minutes at 37 ℃, and an OD450nm value of each hole is measured by a microplate reader to calculate a P/N (positive/negative) value. The dilution factor of the pore with the largest P/N value is selected as the optimal action temperature and time of the enzyme-labeled antibody.

1.2.2.6 the concentration and reaction time of the substrate are respectively detected, the reaction time is respectively 8 minutes, 10 minutes and 15 minutes, the OD450nm value of each hole is measured by a microplate reader, and the P/N (positive/negative) value is calculated. The dilution factor of the pore with the largest P/N value was chosen as the optimal substrate concentration and reaction time.

1.2.3 preparation of Positive and negative controls

1.2.3.1 preparation of negative control 30 parts of negative sera total from N1 to N30 were tested according to the method of 1.2.2 and the conditions determined, and sera with OD450nm values less than 0.2 were mixed as negative control sera and dispensed at 50. mu.l/tube and stored at-20 ℃.

1.2.3.2 preparation of Positive control 30 parts of positive sera from P1 to P30 were tested according to the method of 1.2.2 and the conditions determined, and sera with OD450nm values greater than 0.9 and less than 1.2 were mixed as positive control sera and were aliquoted at 50. mu.l/tube and stored at-20 ℃.

1.2.4 operating the negative and positive controls according to the determined diagnostic method, performing 50 times of repeatability tests, and calculating

Positive control

And determining the establishment conditions of the negative control and the positive control in the kit.

1.2.5 determination of negative and positive cut-off values 203 known negative sera and 200 known positive sera were tested according to the diagnostic method defined above, and all the results were statistically analyzed (ROC analysis) using MedCalc software, and the optimal cut-off point was determined based on sensitivity (Sn95), specificity (Sp95) and area of the curve (AUCs) at 95% CIs.

2 results

2.1 preparation and detection of Positive and negative sera

2.1.1 preparation 30 parts of positive serum and 30 parts of negative serum are respectively prepared according to the test method, each part of serum is more than or equal to 1m L, and the serum is stored at the temperature of minus 80 ℃ for standby.

2.1.2 IFA detection results show that 30 positive serums (P1-P30) are all positive, and 30 negative serums (N1-N30) are all negative.

2.2 establishment of Indirect E L ISA method

2.2.1 determination of coating concentration of recombinant P30 protein and determination of optimal dilution factor of serum, the matrix titration method showed that when the amount of coated antigen was 200 ng/well/100. mu. L (i.e., 2. mu.g/m L) and the optimal dilution factor of serum was 1:100, the highest P/N was detected (Table 1), which was the best detection result, therefore, the optimal coating amount of antigen was 200 ng/well/100. mu.l (i.e., 2. mu.g/m L) and the optimal dilution factor of serum was 1: 100.

TABLE 1 optimal coating concentration of P30 protein antigen and optimal dilution of positive serum

Note: (+) denotes positive serum, and (-) denotes negative serum

2.2.2 optimization of the working concentration of goat anti-mouse enzyme-labeled antibody when the dilution of the enzyme-labeled antibody is 1:10000, the detected P/N value reaches the highest value (Table 2); therefore, the optimal working concentration of the enzyme-labeled antibody was determined to be 1: 10000.

TABLE 2 determination of optimal dilution factor of enzyme-labeled antibody

2.2.3 selection of blocking conditions the highest P/N values were determined when the blocking solution was 1 × PBST containing 1% BSA at 37 ℃ for 2 hours (Table 3), and therefore the optimal blocking conditions were determined to be 1 × PBST containing 1% BSA at 37 ℃ for 2 hours.

TABLE 3 determination of optimal blocking conditions (Positive sample P/N values)

2.2.4 antigen antibody reaction conditions and time the antigen antibody reaction conditions were tested by the matrix titration method and the results showed that: the antigen-antibody reaction had the highest P/N value at 37 ℃ for 1 hour (Table 4), which was not different from the P/N value at 25 ℃ for 2 hours. Considering that the general veterinary laboratories are equipped with biochemical incubators, in order to shorten the operation time and improve the working efficiency, we determine the optimal reaction conditions for antigen and antibody as follows: the reaction was carried out at 37 ℃ for 1 hour.

TABLE 4 antigen antibody reaction conditions and time

2.2.5 the action condition and time of the goat anti-mouse enzyme-labeled antibody are tested on the action temperature and the reaction time of the goat anti-mouse enzyme-labeled antibody under the condition that the working concentration of the goat anti-mouse enzyme-labeled antibody is 1:10000, and the results show that: the maximum P/N value was reached at 37 ℃ for 60 minutes (Table 5). Therefore, the optimal action conditions of the goat anti-mouse enzyme-labeled antibody are confirmed as follows: and the reaction is carried out for 60 minutes at 37 ℃.

TABLE 5 goat anti-mouse enzyme-labeled antibody action conditions and time P/N results

2.2.6 substrate concentration and reaction time after substrate addition E L ISA detection at 37 deg.C for 8 min, 10 min, 15 min, substrate optimum action time was selected, OD per well was measured with microplate reader_450nmValues, P/N values for the respective action times were calculated, and the results showed that the P/N value was the highest at 10 minutes of substrate addition. Therefore, the substrate reaction time at 37 ℃ was selected to be 10 minutes as the optimum substrate concentration and reaction time. The specific results are shown in Table 6.

TABLE 6 results of substrate reaction time P/N

2.3 preparation of Positive and negative controls P1-P30 and N1-N30 were tested separately according to the established E L ISA protocol, wherein the OD450 values of P1-P5, P11-P14, P18-P20 were all greater than 0.9 and less than 1.2, preferably, the OD450nm value is 1.1, the OD450 values of N1-N12, N15-N25, N28-N30 are all less than 0.2, preferably, the OD450nm value is less than 0.1, these serums meeting the requirements of the positive and negative controls were mixed and then stored at-20 ℃ according to 50. mu.l/branch.

2.4 Exception of Condition

The negative and positive controls were operated according to the established diagnostic method and subjected to 50 replicates with the following results:

TABLE 7 test results of the repeatability test of negative and positive controls

Considering the difference in the operation of the operators, the conditions are determined to be: positive control well OD_450nmThe reading should be greater than or equal to 0.6; negative control wells OD per well_450nmThe reading should be less than 0.2.

2.5 determination of Positive and negative cutoff values

203 portions of known negative sera (prepared according to the method of 1.2.1, sera from 203 BA L B/c blank mice), 200 portions of known positive sera (prepared according to the method of 1.2.1, sera from 200 BA L B/c mice immunized with P30 protein) were tested according to the diagnostic method determined above, all the test results were subjected to statistical analysis (ROC analysis) using MedCalc software, and determined from sensitivity under 95% CIs conditions (Sn95), specificity (Sp95) and area of the curve under 95% CIs (AUCs). The analysis results showed (FIGS. 4, 5), positive cut-off value (S/P value) was 0.399, and negative cut-off value (S/P value) was 0.399, which is the optimum cut point for the system analysis.

TABLE 8203 known negative samples (S/P values)

TABLE 9200 known Positive samples (S/P values)

1.335	1.345	1.665	1.229	0.707	0.399	0.416
							0.432	1.132	0.416	0.616	0.752	1.445	0.518
0.406	0.983	1.392	0.626	0.964	0.673	0.596
							0.625	1.381	0.41	2.167	1.888	1.943	1.709
0.67	0.462	0.412	1.493	0.472	1.023	1.971
							0.458	0.93	0.427	0.561	1.647	0.565	0.992
1.032	1.035	0.454	0.903	0.488	1.809	0.492
							0.654	2.161	0.497	1.394	1.883	0.891	1.275
0.678	1.386	0.588	1.944	0.499	2.066	1.049
							0.448	1.41	0.612	0.99	0.49	0.451	0.425
1.033	0.802	0.644	0.557	2.165	2.197	0.425
							1.503	0.514	0.715	1.671	0.447	1.372	1.903
2.148	2.107	0.752	1.676	0.4	1.373	0.461
							1.031	0.48	0.778	0.685	2.018	0.603	1.6
1.038	2.075	0.816	0.503	0.989	1.505	0.477
							1.04	1.736	0.944	0.44	0.827	0.681	0.773
1.519	2.097	0.981	2.097	1.906	2.129	1.798
							1.539	1.338	1.137	0.81	0.496	1.747	0.79
1.942	0.86	1.148	1.964	0.481	0.839	0.888
							1.471	2.083	1.178	0.4	0.432	1.223	0.407
0.588	1.393	1.183	2.007	0.688	0.482	0.588
							0.753	1.926	1.212	1.715	0.506	0.631	0.834
1.757	1.092	1.397	2.077	0.516	2.017	1.294
							1.914	1.92	1.463	1.165	0.549	0.566	1.542
0.619	0.91	1.738	1.493	0.464	1.776	1.384
							1.19	0.462	1.745	0.706	1.028	0.499	1.907
1.894	0.836	1.754	0.82	1.328	0.853	0.957
							0.438	0.501	1.79	1.03	0.821	0.896	1.726
0.614	2.175	1.919	0.468	/	/	/

Example 3 detection method of kit for identifying P30 protein monoclonal antibody of African swine fever virus

1. Sample dilution: diluting a sample to be detected by 1:100 times by using a sample diluent;

2. adding samples, namely adding diluted samples, negative controls and positive controls, wherein the positive controls and the negative controls are repeated for 2 times, incubating for 1 hour at 37 ℃ in each hole, washing for 3 times by 1 × PBST, washing for 3 minutes in each hole by 300 mu l, and finally drying by patting;

3. adding goat anti-mouse enzyme-labeled antibody, namely adding the goat anti-mouse enzyme-labeled antibody into each reaction hole, incubating for 1 hour at 37 ℃, washing for 5 times by 1 × PBST (Perkin Elmer-Partriker) at 300 mu l of each hole for 3 minutes, and finally drying by patting;

4. adding a color development liquid: adding 100 mul of color development liquid into each reaction hole, and developing for 10 minutes at 37 ℃ in a dark place;

5. adding a stop solution: mu.l of stop buffer was added to each reaction well, and the results were measured over 10 minutes.

6. And (3) detection: measuring the OD450nm value of each hole on a microplate reader;

7. and (3) testing validity judgment: when the positive control OD450nm values are all more than or equal to 0.6, and the negative control OD450nm values are all less than 0.2, the test is established, and the result is effective;

S/P value calculation:

9. and (3) judging test results: and when the S/P value of the sample to be detected is more than or equal to 0.4, judging the sample to be detected as positive, and when the S/P value of the sample to be detected is less than 0.4, judging the sample to be detected as negative.

Example 4 African swine fever virus P30 protein monoclonal antibody identification kit preparation and Assembly

1. Preparation of the kit

1) Preparing an antigen coated plate:

① coating, diluting the purified African swine fever virus P30 recombinant protein to 2 μ g/M L with coating buffer solution (0.05M carbonate buffer solution pH9.6), mixing, adding enzyme label plate with 100 μ l/hole, and acting at 2-8 deg.C for 12-15 hr;

② washing with 1 × PBST for 3 times (3 min each time) and 300 μ l per well, and drying in the last time;

③ blocking, adding PBS containing 1% BSA, 200 μ l/well, blocking at 37 deg.C for 2 hours;

④ washing with 1 × PBST for 3 times (300 μ l/well) for 3 min, and drying;

⑤ adding protective agent, adding 10% trehalose, incubating at 37 deg.C for 2 hr, drying, adding desiccant, and vacuum preserving.

2) The sample diluent is 1 × PBST solution containing 1% BSA, and the formula is as follows, 0.27g potassium dihydrogen phosphate, 0.2g potassium chloride, 3.58g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride are weighed and dissolved in 800m L double distilled water, 0.5m L Tween-20, ProClin300 with the final concentration of 0.01% (m/V) and BSA with the final concentration of 1% (m/V) are added, the pH value is adjusted to 7.2 after dissolution, then the double distilled water is added to 1L, the mixture is mixed evenly, the filter membrane with 0.22 μm is used for filtration and sterilization, and the quantitative split charging is carried out.

3) The concentrated washing solution is 25 × PBST solution, diluted to 1 × PBST solution before use, and its formulation is that 6.8g potassium dihydrogen phosphate, 5g potassium chloride, 89.5g disodium hydrogen phosphate dodecahydrate, 198.7g sodium chloride are weighed and dissolved in 800m L double distilled water, 1.25% Tween-20 (V/V) and preservative ProClin300 (V/V) with final concentration of 0.1% are added, the double distilled water is added to 1L, mixed evenly, filtered and sterilized by 0.22 μm filter membrane, and quantitatively packaged.

4) The goat anti-mouse enzyme-labeled antibody is obtained by diluting an enzyme-labeled secondary antibody by 10000 times by using a sample diluent.

5) The positive control is positive serum with an OD450nm value of 0.9-1.2, and the specific preparation method is shown in example 2.

6) The negative control is negative serum with OD450nm value less than 0.2, and the specific preparation method is shown in example 2.

7) The color developing solution is TMB single-component color developing solution or other general color developing solutions, and is quantitatively packaged.

8) The stop solution is 2M H₂SO₄The formula is that 891ml of ultrapure water is taken, 109ml of concentrated sulfuric acid is slowly added, and the mixture is uniformly mixed, and the preparation is safe.

2. Kit assembly (192 wells/box or 480 wells/box), kits were assembled according to the following table:

example 5 screening of monoclonal antibodies against African swine fever virus P30 protein

5.1 preparation of ASFV immunogen recombinant Escherichia coli B L-21-P30 basic seed batch strain is inoculated with liquid culture medium containing 100 mug/M L kanamycin L B in a proportion of 1%, the liquid culture medium is subjected to shaking culture at 37 ℃ and 200r/min for 2-4 hours, when bacterial liquid OD600nm is 0.6-0.8, 1.0mM IPTG is added to continue to induce for 4 hours, 5000g of the induced recombinant bacteria is centrifuged for 10 minutes, supernatant is discarded, a precipitate is dissolved by PBS for 10 minutes, an ultrasonic cell crusher is used for crushing, after crushing, 4 ℃ centrifugation (12000r/min) for 30 minutes, supernatant is discarded, the precipitate inclusion body part is lysed by lysate (50mM Tris, 8M Urea,0.5M NaCl) for 10 minutes, the supernatant is centrifuged for 30 minutes by 12000r/min, a nickel column is used for purification, a Tris column is used for cleaning a column, the supernatant is added into a nickel column, protein liquid is slowly loaded, the protein liquid is loaded, the column is used for preservation, the 10 times of the lysate, the protein liquid is used for later use, the protein liquid is washed for 10 times of SDS, the supernatant, the protein liquid is centrifuged 30 minutes, the supernatant is collected by SDS, the gel, the supernatant is used for removing, the protein is used for electrophoresis, the protein is used for purification, the protein is used for removing, the protein is used for purification, the protein is used for detecting the protein, the protein.

5.2 animal immunization Balb/c mice were immunized with the prepared ASFV antigen. The immunization method comprises subcutaneously immunizing 100 μ g/antigen emulsified in Freund's complete adjuvant, injecting 50 μ g/antigen emulsified in Freund's incomplete adjuvant into abdominal cavity after 3 weeks, and 3 days before fusion, performing booster immunization on 100 μ g/antigen without adjuvant.

5.3 cell fusion feeder cells were first plated in 96-well cell plates at 37 ℃ with 5% CO₂Aseptically taking spleens of immunized mice, preparing splenocytes suspension, mixing SP2/0 with the splenocytes suspension (the ratio is 1: 5-1: 10) in a centrifuge tube of 50m L, evenly mixing the spleens, centrifuging the mixture for 10 minutes at 1500r/min, removing supernatant, putting the centrifuge tube filled with the cells mixture in a water bath of 37 ℃, slowly dripping 50% PEG 1m L preheated to 37 ℃ within 1 minute, gently stirring the mixture while adding, then slowly adding 1640 liquid 10m L preheated to 37 ℃, finally adding 30m L1640 liquid, centrifuging the mixture for 5 minutes at 1000r/min, collecting precipitates, suspending the fused cells by HAT culture medium, inoculating the cells in a 96-well culture plate paved with feeder cells, and suspending the cells at 100 mu l/well and 5% CO at 37 ℃ and 5%₂Culturing in an incubator. Observing on the second day after fusion, supplementing 1 drop HAT culture medium into each hole on the fourth day, and sucking 100 mul culture medium to replace 100 mul HT culture medium on 8-10 days. Antibody detection was performed when the fused cell colonies grew to culture well 1/4 and the medium turned slightly yellow.

5.4 the media of 5.3 was assayed using the kit prepared and assembled in example 4 and using the assay method of example 3.

5.5 screening results of the hybridoma cell positive clone strains are fused and then screened to 4 positive cell strains, three positive cell strains with higher titer are selected for subcloning to obtain a monoclonal antibody hybridoma cell strain 2 strain which stably secretes the African swine fever virus, the monoclonal cell strain with the highest E L ISA detection value is selected for expansion and cryopreservation and is respectively named as 7D5 and 4C11, and specific results are shown in the following table.

TABLE 10 screening results of hybridoma cell-positive clones

The above description is not intended to limit the invention, nor is the invention limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the spirit of the invention.

Sequence listing

<110> Hangzhou Hengolo Tech Co Ltd

<120> kit for screening and detecting African swine fever virus P30 protein monoclonal antibody and preparation method thereof

<160>5

<170>PatentIn version 3.5

<210>1

<211>621

<212>DNA

<213> African swine fever virus P30 nucleotide sequence

<400>1

catatggatt tcatcctgaa tatcagtatg aaaatggaag ttatcttcaa gaccgatctg 60

cgtagtagca gtcaggttgt ttttcatgca ggcagtctgt ataattggtt tagtgtggaa 120

attatcaaca gtggccgcat tgttaccacc gccattaaga ccctgctgag taccgtgaaa 180

tatgatattg ttaaaagcgc ccatatctat gcaggccagg gttataccga acatcaggca 240

caggaagaat ggaatatgat tctgcatgtt ctgtttgaag aagaaaccga aagcagtgcc 300

agcagcgaaa gcattcatga aaagaatgat aatgagacca acgaatgcac cagcagtttt 360

gaaaccctgt ttgaacagga accgagcagt gaagaaccga aagatagcaa actgtatatg 420

ctggcccaga aaaccgttca gcatattgaa cagtatggta aagccccgga ttttaataag 480

gttattcgcg cccataattt tattcagacc attcatggca ccccgctgaa agaagaagaa 540

aaagaagttg tgcgtctgat ggttattaag ctgctgaaaa agaataagct gctgagccat 600

ctgcatctga tgtttctcga g 621

Claims

1. The E L ISA kit for identifying the African swine fever virus P30 protein monoclonal antibody is characterized by comprising an antigen coated plate, a sample diluent, a concentrated washing solution, a goat anti-mouse enzyme-labeled antibody, a positive control, a negative control, a developing solution, a stopping solution and an instruction book, wherein:

the antigen coating plate is an ELISA plate coated with P30 protein;

the sample diluent is 1 × PBST solution containing 1% BSA;

the concentrated washing solution is 25 × PBST solution, and is diluted to 1 × PBST solution before use;

the goat anti-mouse enzyme-labeled antibody is obtained by diluting an enzyme-labeled secondary antibody by 10000 times by using a sample diluent;

the positive control is positive serum with an OD450nm value of 0.9-1.2;

the negative control is negative serum with an OD450nm value less than 0.2;

the color developing liquid is TMB single-component color developing liquid;

the stop solution is 2M H₂SO₄。

2. The kit according to claim 1, wherein the concentration of P30 protein coated on the antigen-coated plate is 200 ng/well/100. mu.l.

3. The kit of claim 1, wherein the positive control OD450nm value is 1.1.

4. The kit of claim 1, wherein the negative control OD450nm value is < 0.1.

5. The kit of claim 1, wherein the positive controls OD450nm are all greater than or equal to 0.6, and the negative controls OD450nm are all less than 0.2, the test is established and the result is valid.

6. A method for detecting a sample to be tested by using the kit of claim 1, comprising the steps of:

1) sample dilution: diluting a sample to be detected by 1:100 times by using a sample diluent;

2) adding samples, namely adding diluted samples, negative controls and positive controls, wherein the positive controls and the negative controls are repeated for 2 times, incubating for 1 hour at 37 ℃ in each hole, washing for 3 times by 1 × PBST, washing for 3 minutes in each hole by 300 mu l, and finally drying by patting;

3) adding goat anti-mouse enzyme-labeled antibody, namely adding the goat anti-mouse enzyme-labeled antibody into each reaction hole, incubating for 1 hour at 37 ℃, washing for 5 times by 1 × PBST (Perkin Elmer-Partriker) at 300 mu l of each hole for 3 minutes, and finally drying by patting;

4) adding a color development liquid: adding 100 mul of color development liquid into each reaction hole, and developing for 10 minutes at 37 ℃ in a dark place;

5) adding a stop solution: mu.l of stop buffer was added to each reaction well, and the results were measured over 10 minutes.

6) And (3) detection: measuring the OD450nm value of each hole on a microplate reader;

7) and (3) testing validity judgment: when the positive control OD450nm values are all more than or equal to 0.6, and the negative control OD450nm values are all less than 0.2, the test is established, and the result is effective;

8) and (3) calculating an S/P value:

7. The method for preparing the kit of claim 1, wherein the method comprises the steps of preparing an antigen coated plate, preparing and subpackaging a sample diluent, preparing and subpackaging a concentrated washing solution, preparing and subpackaging a goat anti-mouse enzyme-labeled antibody, preparing a positive control, preparing a negative control, subpackaging a developing solution, preparing and subpackaging a stopping solution.

8. The method of claim 7, wherein the antigen coated plate is prepared by the steps of:

1) coating, namely diluting the purified African swine fever virus P30 recombinant protein to 2 mu g/m L by using a coating buffer solution, uniformly mixing, adding an enzyme label plate with 100 mu l/hole, and acting for 12-15 hours at the temperature of 2-8 ℃;

2) washing with 1 × PBST for 3 times (3 min each time) with 300 μ l per well, and drying for the last time;

3) and (3) sealing: adding PBS containing 1% BSA, 200. mu.l/well, blocking at 37 ℃ for 2 hours;

4) washing with 1 × PBST for 3 times (300 μ l per well for 3 min) and drying;

5) adding a protective agent: adding 10% trehalose, incubating at 37 deg.C for 2 hr, drying, adding desiccant, and vacuum preserving.

9. The method of claim 7, wherein the preparation of the positive control comprises the steps of:

1) immunizing 30 female BA L B/c mice of 6-8 weeks old with purified P30 protein as immunogen (100 μ g/m L), injecting subcutaneously at multiple points with the immunization amount of 50 μ g/mouse, immunizing for 2 times at intervals of 2 weeks, and using Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the second boosting immunization;

2) collecting serum: collecting blood by eyeball picking method 21 days after the second immunization, separating serum, and storing at-20 deg.C for use;

3) the method of claim 6, wherein the collected sera are tested by mixing sera with OD450nm values greater than 0.9 and less than 1.2 as positive control, and storing at-20 deg.C in 50 μ l/tube.

10. The method of claim 7, wherein the negative control is prepared by the steps of:

1) collecting serum, namely collecting 30 SPF female BA L B/c mice of 6-8 weeks old, sterilizing the body surface, collecting blood through eye sockets, centrifuging for 30min at 3000g, separating serum, and storing at-20 ℃ for later use;

2) the collected sera were tested by the method of claim 6 and pooled as negative control sera with OD450nm values less than 0.2 and dispensed at 50 μ l/tube and stored at-20 ℃.