Porcine pestivirus antibodies and detection kits

Abstract

The application discloses a swine fever antibody and a detection kit, the antibody comprises Fab, scFv and IgG, the IgG is anti-pig IgG-HRP secondary antibody, the variable region of the antibody is connected with a light chain constant region sequence and a heavy chain constant region downstream respectively, and the 293F cells are transfected, serum-free fermentation is carried out, the antibody is purified by using a protein G column and is quantified by using UV; the detection kit comprises a coated E2 enzyme label plate, a 2% BSA blocked blank plate, a PBST washing solution, anti-M13 HRP labeled secondary antibody, TMB color developing solution and sulfuric acid, the coated E2 enzyme label plate is a 96-hole enzyme label plate with 50 ng, E2 is coated in each hole, and the 2% BSA blocked blank plate is used as a control plate. The application adopts the coated E2 enzyme label plate as a substrate, provides a rapid and efficient antibody preparation method for specifically recognizing swine fever virus, simultaneously develops and applies a swine fever serum antibody detection kit by using the prepared specific antibody, obtains an Elisa kit for detecting swine fever virus infection, and uses the chessboard method to gradiently coat the antigen and gradiently add the sample coupling antibody.

Description

Technical Field

[0001] This invention relates to the field of phage display monoclonal antibody preparation technology, and particularly to classical swine fever antibodies and detection kits. Background Technology

[0002] Swine fever, also known as swine cholera, is a highly contagious disease in pigs characterized by multi-system hemorrhage. It can cause fever and death in pigs, impair their production performance, and cause economic losses. Whether in endemic areas, endemic areas, or areas where swine fever has been eradicated, it remains a significant disease that harms or threatens the pig farming industry.

[0003] After an outbreak of swine fever, the solutions include taking measures to completely cull sick and suspected pigs, using a combination of vaccination and culling, and implementing mandatory swine fever vaccination. Currently, there are two main types of swine fever vaccines widely used in production: one is the traditional live virus vaccine, and the other is the E2 enveloped glycoprotein subunit vaccine.

[0004] However, the E2 vaccine cannot provide strong protection against congenital infection, and the immune mechanism of this vaccine is not yet fully understood. Therefore, it is necessary to find a way to quickly and on a large scale detect swine fever infection and to test the effectiveness of the vaccine on a large scale. To this end, this invention proposes a swine fever antibody and a detection kit. Summary of the Invention

[0005] The purpose of this invention is to provide swine fever antibodies and detection kits to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a classical swine fever antibody, comprising Fab, scFv, and IgG, wherein the IgG is an anti-swine IgG-HRP secondary antibody, wherein the variable region of the antibody is coupled downstream of both the light chain constant region sequence and the heavy chain constant region, and after transfection into 293F cells and serum-free fermentation, the antibody is purified using a protein G column and quantified using UV. The antibody binds to six groups of E2 proteins, namely: MB001, MB016, MB021, MB0056, MB057, and MB113.

[0007] The CDR sequences of MB001 include CDR-L1: RASEN IYSYLA, CDR-L2: NAKTLAE, CDR-L3: QHHYGTPYT, CDR-H1: DYEMH, CDR-H2: AIYPETGGTAYNQKFKG and CDR-H3: QTGFYGGYSLDY;

[0008] The CDR sequence of MB016 includes CDR-L1: RASEN IYSYLS, CDR-L2: NAKTLPE, CDR-L3: QHHYGTPYT, CDR-H1: DYEMH, CDR-H2: AIYPETGGTAYNQKFKG and CDR-H3: QAGFYGTYSLDY;

[0009] The CDR sequence of MB021 includes CDR-L1: RSSQSLVHSNGNTYLH, CDR-L2: KVSNRFS, CDR-L3: SQSTHVPLT, CDR-H1: DYEMH, CDR-H2: AI DPETGGTAYNQKFKG and CDR-H3: GGNYVVYYAMDY;

[0010] The CDR sequence of MB056 includes CDR-L1: SASSS I SYMH, CDR-L2: DTSKLAS, CDR-L3: HQRSSYPT, CDR-H1: TYAMN, CDR-H2: RI RSKSNNYATYYADSVKD and CDR-H3: NLDY;

[0011] The CDR sequence of MB057 includes CDR-L1: KASQD I NSYLS, CDR-L2: RANRLVD, CDR-L3: LQYDEFPYT, CDR-H1: DYI ML, CDR-H2: NI NPYYGSTSYNLKFKG and CDR-H3: SGDGFSFAY;

[0012] The CDR sequence of MB113 includes CDR-L1: RASSSVSYMH, CDR-L2: ATSNLAS, CDR-L3: QQWSSNPYT, CDR-H1: DYEMH, CDR-H2: AI DPETGGTAYNQKFKG, and CDR-H3: QAGFYDGYSLDY.

[0013] The classical swine fever antibody detection kit includes a coated E2 ELISA plate, a blank plate blocked with 2% BSA, PBST washing solution, anti-M13 HRP-labeled secondary antibody, TMB chromogenic solution, and sulfuric acid. The coated E2 ELISA plate is a 96-well ELISA plate with 50 ng of E2 in each well. The blank plate blocked with 2% BSA serves as a control plate.

[0014] Preferably, the method for preparing the coated E2 microplate includes the following steps;

[0015] Step 1: Collect the spleens of mice. The spleens collected are from mice that have undergone multiple immunizations.

[0016] Step 2: Extract total RNA from the mouse spleen collected in Step 1;

[0017] Step 3: Construct antibody variable region PCR and phage vector. After quantifying the total RNA extracted in step 2 using a micro spectrophotometer, reverse transcription was performed using a 505L reverse transcription system. The antibody heavy and light chain splice fragments and pCom3 vector expression vector obtained by PCR were digested with enzymes and then ligated overnight according to the T4 ligase instructions to obtain the revived bacterial culture.

[0018] Step four: Library amplification and concentration. The revived bacterial culture obtained in step three is added to 2YT medium containing ampicillin, tetracycline and 2% glucose for amplification and concentration.

[0019] Step 5: Perform antigen coating and cell preparation. Dilute E2 protein to 10 μg / mL with carbonate buffer and add 100 μL to each well of a removable 96-well microplate. Passage and amplify as needed according to the number of cells required for the screening process.

[0020] Step six: Perform library screening and re-amplification. Take the concentrated library from step four and screen and re-amplify it to obtain the second-screened and amplified library.

[0021] Preferably, the specific steps for collecting the mouse spleen are as follows: using purified E2 protein as an immunogen, 6-8 week old healthy Balb / c mice are selected for immunization. For the first immunization, E2 protein is emulsified with an equal volume of Freund's complete adjuvant at a dose of 20 μg / mouse and then injected subcutaneously into the back of the mice at multiple points.

[0022] After the first immunization, mice were immunized every 14 days by subcutaneous injection of 20 μg / mouse of E2 protein mixed with an equal volume of Freund's incomplete adjuvant at multiple sites on the back. The mice were immunized a total of 4 times. The serum antibody titer of the mice was determined by indirect ELISA. When the titer reached a dilution of 243,000, the mice were boosted with 50 μg of E2 protein intraperitoneally for 4 days, and the spleen was collected.

[0023] Preferably, the specific steps for extracting total RNA are as follows: fresh spleen is collected and ground in a mortar with liquid nitrogen until it becomes powder. An appropriate amount is added to Trizol, quickly and thoroughly mixed, and then homogenized 3-5 times with a homogenizer.

[0024] After standing at room temperature for 5 minutes, take 1 ml of each sample and add it into two 1.5 ml EP tubes respectively. Add 200 μL of chloroform to the 1.5 ml EP tubes and tighten the sample tube caps.

[0025] Shake the test tube by hand for 1 minute, then let it stand for 5 minutes. Take about 3505 L of supernatant from each tube, and combine the supernatant from two tubes into one tube to obtain 600-7005 L of supernatant.

[0026] Add an equal volume of isopropanol, mix well, let stand at room temperature for 20-30 min, centrifuge the sample at 11000 rpm and 4℃ for 10 min, discard the supernatant, add 7005 L of 75% ethanol, and wash the precipitate twice.

[0027] Centrifuge at 7000 rpm for 5 min at 4℃, discard the supernatant, air dry at room temperature, add an appropriate amount of DEPC water to completely dissolve, and obtain total RNA from the spleen.

[0028] Preferably, the PCR reaction conditions are: 94°C pre-denaturation for 2 min, 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, 72°C for 5 min extension, for 30 cycles;

[0029] The antibody heavy and light chain splicing conditions were as follows: 94℃ pre-denaturation for 5 min, 98℃ for 15 s, 58℃ for 30 s and 72℃ for 1 min for a total of 5 cycles; followed by 98℃ for 15 s, 68℃ for 1 min and 72℃ for 5 min.

[0030] Preferably, the culture time of the 2YT medium is 1 hour, then helper phage is added, and after static co-incubation at 37°C, it is cultured and recovered at 37°C and 250 rpm for 2 hours.

[0031] Subsequently, the supernatant was removed by centrifugation, and 2YT medium containing ampicillin, tetracycline, and kanamycin was added. The mixture was incubated overnight at 30°C and 250 rpm for amplification. The overnight culture supernatant was collected, and 4% PEG8000 and 3% NaCl were added. After mixing, the mixture was placed on ice for 1 hour, followed by centrifugation at 13000g for 40 minutes at 4°C. The supernatant was removed, and the mixture was dissolved in 5% of the total volume of sterile PBS buffer before concentration. Finally, 7% of the total volume of DMSO was added and mixed. The mixture was then stored at -80°C.

[0032] Preferably, the detection of the library involves diluting the 3rd amplified library by 1, 10, 100, and 1000 times, respectively, adding 100 μL per well to a coated E2 microplate and a blank plate blocked with 2% BSA, incubating at room temperature for 1 hour, washing twice with PBST buffer, wiping dry, adding ant i-M13 HRP-labeled secondary antibody, incubating at room temperature for 40 minutes, washing three times with PBST buffer, wiping dry, adding TMB chromogenic solution, developing at room temperature for 20 minutes, adding 2M sulfuric acid to stop the development, and reading the absorbance at OD450nm using a microplate reader.

[0033] The technical effects and advantages of this invention are as follows:

[0034] This invention uses coated E2 ELISA plates as substrates to provide a rapid and efficient method for preparing antibodies that specifically recognize classical swine fever virus. Furthermore, the prepared specific antibodies are used to develop and apply a classical swine fever serum antibody detection kit, resulting in an Elisa kit capable of detecting classical swine fever virus infection. The kit also utilizes a checkerboard gradient coating method for antigen and gradient loading of conjugated antibodies. Attached Figure Description

[0035] Figure 1 This is a flowchart illustrating the preparation process of the E2 microplate coated according to the present invention.

[0036] Figure 2 This is a bar chart showing the detection results of the binding of the labeled antibody of this invention to the E2 protein;

[0037] Figure 3 This is a line graph showing the results of detecting serum classical swine fever antibodies using the Elisa competitive method of this invention.

[0038] Figure 4 This is a bar chart showing the assembly and detection results of the kit of the present invention. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1: This invention provides a classical swine fever antibody, comprising Fab, scFv, and IgG. The IgG is an anti-swine IgG-HRP secondary antibody. The variable region of the antibody is linked downstream to the light chain constant region sequence and the heavy chain constant region, respectively. After transfection into 293F cells and serum-free fermentation, the antibody is purified using a protein G column and quantified using UV. Six groups of antibodies bind to the E2 protein, namely: MB001, MB016, MB021, MB0056, MB057, and MB113.

[0041] The CDR sequence of MB001 includes CDR-L1: RASEN IYSYLA, CDR-L2: NAKTLAE, CDR-L3: QHHYGTPYT, CDR-H1: DYEMH, CDR-H2: AIYPETGGTAYNQKFKG and CDR-H3: QTGFYGGYSLDY;

[0042] The CDR sequences of MB016 include CDR-L1: RASEN IYSYLS, CDR-L2: NAKTLPE, CDR-L3: QHHYGTPYT, CDR-H1: DYEMH, CDR-H2: AIYPETGGTAYNQKFKG and CDR-H3: QAGFYGTYSLDY;

[0043] The CDR sequence of MB021 includes CDR-L1: RSSQSLVHSNGNTYLH, CDR-L2: KVSNRFS, CDR-L3: SQSTHVPLT, CDR-H1: DYEMH, CDR-H2: AI DPETGGTAYNQKFKG and CDR-H3: GGNYVVYYAMDY;

[0044] The CDR sequences of MB056 include CDR-L1: SASSSI SYMH, CDR-L2: DTSKLAS, CDR-L3: HQRSSYPT, CDR-H1: TYAMN, CDR-H2: RI RSKSNNYATYYADSVKD and CDR-H3: NLDY;

[0045] The CDR sequence of MB057 includes CDR-L1: KASQDI NSYLS, CDR-L2: RANRLVD, CDR-L3: LQYDEFPYT, CDR-H1: DYI ML, CDR-H2: NI NPYYGSTSYNLKFKG and CDR-H3: SGDGFSFAY;

[0046] The CDR sequence of MB113 includes CDR-L1: RASSSVSYMH, CDR-L2: ATSNLAS, CDR-L3: QQWSSNPYT, CDR-H1: DYEMH, CDR-H2: AI DPETGGTAYNQKFKG and CDR-H3: QAGFYDGYSLDY;

[0047] Recombinant antibodies targeting different CDR differences were constructed and expressed. Light chain constant region sequences and heavy chain constant regions were ligated downstream of the variable region of the antibody, respectively. After transfection into 293F cells and serum-free fermentation, the antibodies were purified using a protein G column and quantified using UV. The antibodies were then conjugated with horseradish peroxidase at a molar ratio of 1:1, and the binding of the conjugation product to E2 was detected. The results showed that all six antibodies had excellent binding to the E2 protein.

[0048] Referring to the classical swine fever E2 gene sequence published in GenBank, the main antigenic region of the classical swine fever antigen E2 protein was analyzed. Non-immunologically irrelevant regions such as transmembrane and intracellular regions were removed. After introducing and optimizing the DNA sequence with mammalian cell codons, the E2 protein expression gene fragment was synthesized and cloned into the pCDNA3.1 eukaryotic expression vector. A 6*hi s tag was added to the C-terminus of the protein for purification. The vector, after being correctly sequenced, underwent endotoxin-free medium-volume plasmid extraction.

[0049] The extracted endotoxin-free plasmids were transfected into HEK293F cells using PEI transfection reagent (purchased from Anolun Biotechnology) at an appropriate plasmid ratio. The transfected cells were cultured in serum-free 293 cells to a density of 2*106. After transfection, the cells were fermented at 37°C, 165 RPM, and 5% CO2 for 7 days, and the supernatant was collected.

[0050] The supernatant was purified using Ni column affinity elution. The target protein was eluted in 500 mM imidazole in PBS buffer and dialyzed into PBS (pH 7.4). The purity and yield of the purified protein were verified by SDS-PAGE. The purified E2 recombinant protein was coated onto an ELISA plate at 0.5 μg / mL overnight at 4°C. After blocking and washing with 3% skim milk powder, the plate was incubated with swine fever positive and negative serum for 40 minutes and then washed again. Anti-swine IgG-HRP secondary antibody was added and incubated. The plate was then developed with TMB chromogenic solution for 20 minutes, and the absorbance was measured at 450 nm using an ELISA reader.

[0051]

[0052] Table 1

[0053] The test results are shown in Table 1 above. Positive serum showed obvious binding, while negative serum did not bind.

[0054] Example 2: This invention provides a classical swine fever antibody detection kit, comprising a coated E2 ELISA plate, a 2% BSA-blocked blank plate, PBST washing buffer, ant i-M13 HRP-labeled secondary antibody, TMB chromogenic solution, and sulfuric acid. The coated E2 ELISA plate is a 96-well ELISA plate with 50 ng of E2 in each well, and the 2% BSA-blocked blank plate serves as a control plate.

[0055] The preparation method of coated E2 microplates includes the following steps;

[0056] Step 1: Collect the spleens of mice. The spleens collected are from mice that have undergone multiple immunizations.

[0057] The specific steps for collecting mouse spleens are as follows: using purified E2 protein as the immunogen, healthy 6-8 week old Ba lb / c mice are selected for immunization. For the first immunization, E2 protein is emulsified with an equal volume of Freund's complete adjuvant at a dose of 20 μg / mouse and then injected subcutaneously into the back of the mice at multiple points.

[0058] After the first immunization, mice were immunized every 14 days by subcutaneous injection of 20 μg / mouse of E2 protein mixed with an equal volume of Freund's incomplete adjuvant at multiple sites on the back. The mice were immunized a total of 4 times. The serum antibody titer of the mice was determined by indirect ELISA. When the titer reached a dilution of 243,000, the mice were boosted with 50 μg of E2 protein intraperitoneally for 4 days, and the spleen was collected.

[0059] Step 2: Extract total RNA from the mouse spleen collected in Step 1;

[0060] The specific steps for extracting total RNA are as follows: fresh spleen is collected and ground in a mortar with liquid nitrogen until it becomes powder. An appropriate amount is added to Trizol, quickly and thoroughly mixed, and then homogenized 3-5 times with a homogenizer.

[0061] After standing at room temperature for 5 minutes, take 1 ml of each sample and add it into two 1.5 ml EP tubes respectively. Add 200 μL of chloroform to the 1.5 ml EP tubes and tighten the caps on the sample tubes.

[0062] Shake the test tube by hand for 1 minute, then let it stand for 5 minutes. Take about 3505 L of supernatant from each tube, and combine the supernatant from two tubes into one tube to obtain 600-7005 L of supernatant.

[0063] Add an equal volume of isopropanol, mix well, let stand at room temperature for 20-30 min, centrifuge the sample at 11000 rpm and 4℃ for 10 min, discard the supernatant, add 7005 L of 75% ethanol, and wash the precipitate twice.

[0064] Centrifuge at 7000 rpm for 5 min at 4℃, discard the supernatant, air dry at room temperature, add an appropriate amount of DEPC water to completely dissolve, and obtain total RNA from the spleen.

[0065] Step 3: Construct antibody variable region PCR and phage vector. After quantifying the total RNA extracted in step 2 using a micro spectrophotometer, reverse transcription was performed using a 505L reverse transcription system. The antibody heavy and light chain splice fragments and pCom3 vector expression vector obtained by PCR were digested with enzymes and then ligated overnight according to the T4 ligase instructions to obtain the revived bacterial culture.

[0066] The PCR reaction conditions were: 94℃ pre-denaturation for 2 min, 94℃ for 30 s, 55℃ for 30 s and 72℃ for 30 s, 72℃ for 5 min extension, for 30 cycles.

[0067] The conditions for splicing the antibody heavy and light chains were as follows: 94℃ pre-denaturation for 5 min, 98℃ for 15 s, 58℃ for 30 s and 72℃ for 1 min for a total of 5 cycles; followed by 98℃ for 15 s, 68℃ for 1 min and 72℃ for 5 min.

[0068] After digesting the antibody heavy and light chain splice fragments and pCom3 vector expression vector obtained by PCR, ligation was performed overnight according to the T4 ligase instructions. The competent XL1-blue bacteria were taken out and thawed at 4°C. At the same time, the electroporation cuvette was pre-cooled at 4°C for 10 minutes. The voltage of the electroporator was adjusted to 1800V / mm. The cuvette containing competent plasmids was added to the electroporation cuvette, and the cells were incubated on ice for half a minute. Then, electroporation was performed for 5ms, and SOC medium was quickly added. The competent cells were then removed and electroporated. This process was repeated several times. The competent cells were then combined and collected. After recovery, 2YT medium was added and the cells were incubated at 37°C and 250rpm for several minutes. After recovery, 100uL of bacterial culture was taken, diluted, plated, and the library volume was calculated.

[0069] Step four: Library amplification and concentration. The revived bacterial culture obtained in step three is added to 2YT medium containing ampicillin, tetracycline and 2% glucose for amplification and concentration.

[0070] The culture time in 2YT medium was 1 hour, then helper phage was added, and after static co-incubation at 37°C, it was cultured at 37°C and 250 rpm for 2 hours for recovery.

[0071] Subsequently, the supernatant was removed by centrifugation, and 2YT medium containing ampicillin, tetracycline, and kanamycin was added. The mixture was incubated overnight at 30°C and 250 rpm for amplification. The overnight culture supernatant was collected, and 4% PEG8000 and 3% NaCl were added. After mixing, the mixture was placed on ice for 1 hour, followed by centrifugation at 13000g for 40 minutes at 4°C. The supernatant was removed, and the mixture was dissolved in 5% of the total volume of sterile PBS buffer before concentration. Finally, 7% of the total volume of DMSO was added and mixed. The mixture was then stored at -80°C.

[0072] Step 5: Perform antigen coating and cell preparation. Dilute E2 protein to 10 μg / mL with carbonate buffer and add 100 μL to each well of a removable 96-well microplate. Passage and amplify as needed according to the number of cells required for the screening process.

[0073] E2 protein was diluted to 10 μg / mL with carbonate buffer (pH 9.6), and 100 μL was added to each well of a removable 96-well microplate. The plate was incubated overnight at 4°C. The next day, the plate was removed, the liquid in the wells was drained, and the plate was washed twice with PBST solution and drained. Then, 2% BSA solution was added for blocking at room temperature for 1 hour. The plate was washed twice more with PBST solution, drained, and stored at -20°C for later use. HeLa cells were revived and cultured in DMEM medium, and passaged as needed according to the number of cells required for the screening process.

[0074] Step six: Perform library screening and re-amplification. Take the concentrated library from step four and screen and re-amplify it to obtain the second screened and amplified library.

[0075] For library detection, the 3rd amplified library was diluted 1-fold, 10-fold, 100-fold, and 1000-fold, respectively. 100 μL was added to each well of an E2 microplate coated with ELISA and a blank plate blocked with 2% BSA. After incubation at room temperature for 1 hour, the plates were washed twice with PBST buffer and dried. ANT i-M13 HRP-labeled secondary antibody was added and incubated at room temperature for 40 minutes. The plates were washed three times with PBST buffer and dried. TMB chromogenic solution was added and the plates were incubated at room temperature for 20 minutes. Finally, 2M sulfuric acid was added to stop the incubation. The absorbance was read at OD450 nm using a microplate reader.

[0076] Example 3: Monoclonal expression, detection, and sequence analysis;

[0077] After appropriate dilution of the positive library for the second round of testing, it was used to infect *E. coli* XL1-blue and plated. The next day, single colonies were picked from the plates and cultured. One copy of the culture was kept as a sequencing backup. The remaining culture was incubated with helper phages, then added to 2YT medium containing ampicillin, tetracycline, and kanamycin, and incubated overnight at 30°C and 250 rpm for amplification. 50 ng of E2 was coated per well in a 96-well microplate, for a total of one plate. A blank plate blocked with 2% BSA was used as a control. After blocking, washing, and drying, a 10-fold diluted expression sample was added, and the plate was incubated at room temperature for 1 hour. After washing twice with PBST buffer and drying, ant was added. i-M13HRP-labeled secondary antibody was incubated at room temperature for 40 minutes, washed three times with PBST solution, dried, and TMB chromogenic solution was added. After chromogenic development at room temperature for 20 minutes, 2M sulfuric acid was added to stop the chromogenic development. The absorbance value was read at OD450nm using a microplate reader. Positive clones with an OD value greater than 0.4 were selected, numbered, and sequenced for analysis. The results of positive clone detection are shown in Table 2 below.

[0078]

[0079] Table 2

[0080] Example 4: Monoclonal antibody expression and antibody enzyme labeling;

[0081] After sequence alignment, we constructed and expressed recombinant antibodies targeting different CDR differences. Light chain constant region and heavy chain constant region sequences were ligated downstream of the variable region of the antibody, respectively. After transfection into 293F cells and serum-free fermentation, the antibodies were purified using a Protein G column and quantified using UV spectroscopy. The antibodies were then conjugated with horseradish peroxidase at a 1:1 molar ratio, and the conjugation products were tested for binding to E2. The results showed that all six antibodies exhibited excellent binding to the E2 protein. The results are shown in Table 3 below. Figure 2 As shown;

[0082]

[0083] Table 3

[0084] Example 5: Detection of serum classical swine fever antibodies using the Elisa competitive assay;

[0085] To screen for the optimal antigen coating amount and the best amount of conjugated antibody, a checkerboard gradient coating method was used for antigen coating and gradient loading of conjugated antibody. The E2 antigen protein coating amount was tested from 0.02ug / mL to 2ug / mL, and the amount of conjugated antibody was tested from 0.05ug / mL to 0.5ug / mL. To maximize the detection of classical swine fever antibodies, we selected two methods: diluting and undiluted loading of the swine serum for testing. Known classical swine fever positive serum was used as a positive control, and classical swine fever negative serum was used as a negative control. The loading volume ranged from 50uL to 100uL.

[0086] First, the serum to be tested was added to enzyme-linked immunosorbent assay (ELISA) plates coated with antigens of different concentrations. After pre-binding for 30-120 minutes, the serum sample was washed off, and the conjugate antibody was added for further incubation for 30-60 minutes. The plates were then washed 2-3 times, and TMB chromogenic buffer was added for 20-30 minutes. The readings were taken at 450 nm using an ELISA reader. Competitive inhibition was clearly detected in positive serum when the coating concentration was in the range of 0.02-2 μg / mL and the conjugate antibody concentration was in the range of 0.05-0.5 μg / mL. MB001 antibody showed the most significant inhibition, with the most pronounced inhibition observed at a coating concentration of 0.5 μg / mL, a doubled serum sample, and a conjugate antibody concentration of 0.2 μg / mL. The test results are as follows: Figure 3 As shown.

[0087] Example 6: Reagent kit assembly and testing;

[0088] M001 antibody was selected as the preferred antibody. The key conditions for the detection method were a coating concentration of 0.5 μg / mL, double the original serum sample, and a conjugated antibody concentration of 0.2 μg / mL. A kit was assembled for further testing of classical swine fever serum. During the testing process, 8 positive sera and 4 negative sera were selected for detection, with a blank buffer used as a control. The detection method described in Example 5 was followed. The results showed that all 10 positive sera samples exhibited significant competition with a competition rate greater than 40%, while the 10 negative sera samples showed no competition or less than 20% competition background. The results are as follows. Figure 4 As shown.

[0089] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A classical swine fever antibody, characterized in that, The antibody binds to the E2 protein, and the antibody is MB001. The CDR sequences of MB001 are CDR-L1: RASENIYSYLA, CDR-L2: NAKTLAE, CDR-L3: QHHYGTPYT, CDR-H1: DYEMH, CDR-H2: AIYPETGGTAYNQKFKG and CDR-H3: QTGFYGGYSLDY.

2. A classical swine fever antibody detection kit, comprising the classical swine fever antibody as described in claim 1.

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