Monoclonal antibody and application thereof in preparation of cattle early pregnancy diagnostic reagent

Abstract

The invention relates to the technical field of cattle early pregnancy diagnosis, in particular to a monoclonal antibody and application thereof in preparation of a cattle early pregnancy diagnosis reagent. The monoclonal antibody provided by the invention comprises a monoclonal antibody B6. EPF is used as a research target, a hybridoma cell technology is applied, a specific antibody of EPF is obtained through cell fusion screening and subcloning and AKTA purification, an antigen competition ELISA detection method is established based on the principle that EPF-HRP and EPF in an actual bovine serum sample compete for a solid-phase EPF antibody, and when the cutoff value of bovine serum PI is set to be 0.5415, the specific antibody of EPF is detected. The early pregnancy detection method shows the best sensitivity (82.61%) and specificity (81.48%).

Description

Technical Field

[0001] The invention relates to the technical field of bovine early pregnancy diagnosis, in particular to a monoclonal antibody and application thereof in preparing a bovine early pregnancy diagnosis reagent. Background Art

[0002] Simple and effective early pregnancy diagnosis can improve the reproduction of animals such as cattle and sheep, shorten the interval between fetuses, and improve the economic benefits of breeding. It is a major problem that has long been urgently needed to be solved in intensive breeding production. At present, the main methods for early pregnancy diagnosis are observation, rectal examination, ultrasound examination, progesterone examination, and early pregnancy diagnostic markers (interferon tau induced genes (ISGs), miRNA in exosomes) detection. Observation is generally used as a supplementary method for early pregnancy diagnosis. Improper detection of rectal examination and ultrasound examination can easily lead to miscarriage. Progesterone detection technology has gradually matured, and many studies have reported on this. However, progesterone is a steroid hormone and a fat-soluble substance. The progesterone level varies greatly between individuals, and even high levels of progesterone cannot determine whether the embryo is alive. The sampling method and sampling time can also affect the progesterone content in milk fat. Therefore, the judgment criteria of this method are difficult to determine, and it has not yet been widely promoted and applied. ISGs detection is non-invasive, but changes in physiological states such as stress and inflammation can affect the expression of ISGs. These interfering factors may reduce the specificity of ISGs as a pregnancy diagnostic indicator. As a non-invasive and reproducible biomarker, exosomal miRNA has the potential to diagnose pregnancy loss in dairy cows. However, only a few studies have explored the application of exosomal miRNA in the diagnosis of pregnancy in dairy cows, and more clinical trials are needed to verify its feasibility as a pregnancy diagnostic marker. Compared with the above methods, immunological ELISA detection is promoted and applied because of its scientificity, high sensitivity, simple operation, and ability to detect changes in the content of pregnancy-related proteins in cattle blood. At present, commercial cattle early pregnancy detection ELISA kits are mainly produced by IDEX and Biotracking in the United States, but their import costs are high and they are rarely used in domestic breeding farms. Although there are commercial products of early pregnancy detection related kits in China, most of them are quantitative detection, and the test results between different products vary greatly, which cannot clearly distinguish pregnancy problems and cannot meet the actual needs of rapid early pregnancy diagnosis in the breeding industry. Therefore, it is urgent to develop a cattle early pregnancy detection method that is simple and fast to operate, can realize batch detection, and is highly efficient.

[0003] At present, the most popular pregnancy detection technology is to detect the content of pregnancy-specific protein B (PSP-B), pregnancy serum protein 60 (PSP60), pregnancy-associated glycoproteins (PAG) and early pregnancy factor (EPF) in the blood of pregnant cows through immunological methods. This immunological detection method of early pregnancy protein in blood is not only highly accurate and not affected by human factors, but also can avoid miscarriage caused by premature direct contact with the embryo. In addition, batch detection can be achieved with extremely high efficiency, which is particularly suitable for large-scale ranches. Among them, EPF is the earliest pregnancy-related protein detected. EPF can be detected from maternal serum 6 hours after mating of mice, 24 hours after fertilization of sheep, cattle, pigs and humans. Studies have shown that EPF can be used to determine whether the conceptus is aborted, detect whether embryo transplantation has failed, monitor the status of pregnant mothers and fetuses, etc., and has become a hot spot in the current research and development of early pregnancy detection. SmartYC et al. determined that EPF can persist throughout most of the pregnancy period by measuring the EPF activity in the serum collected from pregnant women every month. If the pregnancy is terminated, the EPF content in the blood will drop rapidly and drop to negative levels within 5-7 days. Sungwoo Park et al. prepared porcine EPF monoclonal antibodies to test the urine and blood of Duroc sows at 20 days of pregnancy with an accuracy rate of 70%; that is, the presence of EPF can be used as a pregnancy marker and an important indicator for early pregnancy diagnosis. Therefore, it is necessary to prepare monoclonal antibodies with excellent sensitivity and specificity for early pregnancy diagnosis in cattle. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a monoclonal antibody and its application in preparing a diagnostic reagent for early pregnancy in cattle. The monoclonal antibody provided by the present invention can be used to diagnose early pregnancy in cattle and has excellent sensitivity and specificity.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a monoclonal antibody, including monoclonal antibody B6;

[0007] The amino acid sequence of the heavy chain variable region of the monoclonal antibody B6 is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2.

[0008] The present invention provides a diagnostic reagent for early pregnancy in cattle, comprising the monoclonal antibody described in the above technical solution.

[0009] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a bovine early pregnancy diagnostic reagent.

[0010] The present invention also provides a method for detecting early pregnancy in cattle for a non-diagnostic purpose, comprising the following steps:

[0011] 1) coating the monoclonal antibody described in the above technical solution in a microplate to obtain a coated plate;

[0012] 2) washing, blocking and washing the coated plate obtained in step 1) to obtain a blocked plate;

[0013] 3) adding bovine serum solution and EPF-HRP solution to the blocking plate obtained in step 2) and incubating the plate, and washing the plate to obtain an incubation plate;

[0014] 4) Add 3,3′,5,5′-tetramethylbenzidine to the incubation plate obtained in step 3) and continue incubation. After terminating the reaction, detect OD 450 The inhibition rate PI value was calculated according to the formula: inhibition rate PI value = (blank control OD 450 -Bovine serum solution OD 450 ) / blank control OD 450 When the inhibition rate PI value is less than 0.5415, it is positive pregnant bovine serum; when the inhibition rate PI value is greater than 0.5415, it is negative pregnant bovine serum; when the inhibition rate PI value is equal to 0.5415, the final result is confirmed by repeated measurement.

[0015] Preferably, the microplate in step 1) is a transparent 96-well microplate;

[0016] The coating amount of the monoclonal antibody in the wells of the microplate is 0.1 μg / well.

[0017] Preferably, the coating conditions of the monoclonal antibody include: coating at 4° C. for 12 hours.

[0018] Preferably, the step 2) uses PBST buffer for washing and plate washing, the number of times is 3 times;

[0019] The cells were blocked with a 5% skimmed milk powder solution at 37°C for 1 h.

[0020] Preferably, in step 3), the amount of bovine serum solution added to the wells of the sealing plate is 50 μL / well, and the bovine serum solution is obtained by diluting the bovine serum 20 times;

[0021] The amount of EPF-HRP solution added to the wells of the closed plate is 50 μL / well. The EPF-HRP solution is obtained by srEPF protein after SUMO digestion and HPR labeling and then diluting 4000 times. The amino acid sequence of the srEPF protein is shown in SEQ ID No.3.

[0022] Preferably, the incubation conditions in step 3) include: time of 1 hour and temperature of 37°C.

[0023] Preferably, the amount of 3,3′,5,5′-tetramethylbenzidine added to the wells of the incubation plate in step 4) is 100 μL;

[0024] The conditions for continued incubation include: a temperature of 20 to 30° C. and a time of 15 min;

[0025] The reaction was terminated using 2 mol / L sulfuric acid solution.

[0026] Beneficial effects of the present invention:

[0027] The present invention takes EPF as the research target, applies hybridoma cell technology, screens through cell fusion and subcloning, obtains EPF-specific antibodies through AKTA purification, and obtains the antibody variable region sequence through sequencing, uses antibodies as detection probes, and establishes an antigen competition ELISA detection method based on the principle of EPF-HRP and EPF competition for solid-phase EPF antibodies in actual bovine serum samples. When the cutoff value of bovine serum PI is set to 0.5415, the early pregnancy detection method shows the best sensitivity (82.61%) and specificity (81.48%). The detection method established by the present invention has great potential for the detection of bovine serum samples, lays a foundation for subsequent basic research on EPF and early pregnancy detection based on EPF, and is crucial to reducing empty pregnancy cycles, maintaining the economic sustainability of dairy farms, and increasing the reproductive efficiency of cattle herds. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 Results of immunogen rEPF induction expression purification and Western-blot verification; a. EPF removal inhibition zone target gene amplification. b. Bacterial liquid PCR identification. c. rEPF protein expression and purification. M: protein marker. 1: whole bacteria after IPTG induction. 2: purified rEPF protein. d. Western blot analysis of rEPF protein, using anti-His tag antibody;

[0030] Figure 2Results of induced expression and purification of srEPF; a. Induced expression of srEPF protein. M: protein marker. 1: supernatant of bacterial lysate after IPTG induction. 2: precipitate of bacterial lysate after IPTG induction. 3: whole bacterial lysate 1 without IPTG induction. 4: whole bacterial lysate 2 without IPTG induction. b. srEPF protein affinity chromatography. M: protein marker. 1. Flow-through after srEPF binding to nickel column. 2. 40mM imidazole eluent. 3. 60mM imidazole eluent. 4. 80mM imidazole eluent. 5. 150mM imidazole eluent. 6. 200mM imidazole eluent. 7. 220mM imidazole eluent. 8. 250mM imidazole eluent. 9. 300mM imidazole eluent. c. Purified srEPF protein;

[0031] Figure 3 Purification and reactivity of EPF mAb; a. Purification of B6 using AKTA protein purification system. b. Identification of EPF mAb subtypes. c. Identification of EPF mAb affinity. d. Western-blot verification of the reactivity of srEPF recombinant protein with three EPF mAbs. e. Western-blot verification of the reactivity of natural EPF protein from pregnant bovine serum with three EPF mAbs;

[0032] Figure 4 To evaluate the application of EPF mAb in antigen competition ELISA; a. ELISA verified the specific binding of B6, F8, F6 and coupled EPF-HRP. b. Checkerboard method optimized B6 coating concentration and EPF-HRP dilution. c. Optimized ELISA coating conditions. d. Determined the OD of different dilutions of positive and negative serum samples of pregnant cows 450nm value;

[0033] Figure 5 The serum samples were tested by antigen competition ELISA based on B6. The ELISA method was used to detect 27 positive samples and 23 negative samples of pregnant bovine serum. a. ROC curve analysis of antigen competition ELISA. The AUC of antigen competition ELISA based on B6 was 0.833. b. The box plots of positive and negative serum of pregnant bovines tested by antigen competition ELISA based on B6 showed significant differences. c. There was no significant difference in the box plots of positive and negative serum of pregnant bovines tested by BovineEPF ELISAKIT kit.

[0034] Figure 6These are the predicted analysis results of the interaction between EPF mAb and EPF protein. A is the B6 monoclonal antibody structure predicted by AlphaFold, B is the EPF protein structure predicted by AlphaFold, C is the B6 monoclonal antibody structure predicted by PyRosetta, D is the scoring result of the complex formed by B6 and EPF predicted by AlphaFold, E is the visualization result of the complex between B6 and EPF predicted by AlphaFold, and F is the visualization result of the complex between B6 and EPF predicted by ClusPro. DETAILED DESCRIPTION

[0035] The present invention provides a monoclonal antibody, including monoclonal antibody B6;

[0036] The amino acid sequence of the heavy chain variable region of the monoclonal antibody B6 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2, which are specifically as follows:

[0037] SEQ ID No.1:

[0038] MECSWILPFILSVTSGVYSLVQLQQSGAELARPGASVKLSCKASGYTFTN YWMQWVKQRPGQGLEWIGAIYPGDFDTRYTQKFKGKATLTADKSSNTAYM QLSSLASEDSAVYYCVRWGWGQGAYWGQGTTLTVSS;

[0039] SEQ ID No.2:

[0040] MSVPTQVLGLLLLWLTGARCDIQMTQSSASQSASVGETVTITCRASENIY SYLAWFQQRQGKSPQLLIYNAETLAEGVPSRFSGSGSGTQFSLKINSLQPEDF GTYYCQHHYGSPWTFGGGTKLEIK.

[0041] The present invention also provides a diagnostic reagent for early pregnancy in cattle, comprising the monoclonal antibody described in the above technical solution. The present invention has no special limitation on other components in the diagnostic reagent, and those skilled in the art can routinely set them according to the monoclonal antibody.

[0042] The present invention also provides the use of the monoclonal antibody described in the above technical solution in the preparation of a bovine early pregnancy diagnostic reagent.

[0043] The present invention provides a method for detecting early pregnancy in cattle with a non-diagnostic destination, comprising the following steps:

[0044] 1) coating the monoclonal antibody described in the above technical solution in a microplate to obtain a coated plate;

[0045] 2) washing, blocking and washing the coated plate obtained in step 1) to obtain a blocked plate;

[0046] 3) adding bovine serum solution and EPF-HRP solution to the blocking plate obtained in step 2) and incubating the plate, and washing the plate to obtain an incubation plate;

[0047] 4) Add 3,3′,5,5′-tetramethylbenzidine to the incubation plate obtained in step 3) and continue incubation. After terminating the reaction, detect OD 450 The inhibition rate PI value was calculated according to the formula: inhibition rate PI value = (blank control OD 450 -Bovine serum solution OD 450 ) / blank control OD 450 When the inhibition rate PI value is less than 0.5415, it is positive pregnant bovine serum; when the inhibition rate PI value is greater than 0.5415, it is negative pregnant bovine serum; when the inhibition rate PI value is equal to 0.5415, repeated measurements are required to confirm the final result.

[0048] The present invention coats the monoclonal antibody described in the above technical solution in a microplate to obtain a coated plate. In the present invention, the microplate is preferably a transparent 96-well microplate. In the present invention, the coating amount of the monoclonal antibody in the wells of the microplate is preferably 0.1 μg / well. The present invention does not specifically limit the source of the transparent 96-well microplate, and conventional commercially available products can be used, such as those purchased from Guangzhou Jie Te Biofiltration Co., Ltd. In the present invention, the coating conditions of the monoclonal antibody preferably include: coating at 4°C for 12 hours. The present invention preferably dissolves the monoclonal antibody in a CBS solution before coating, and the concentration of the CBS solution is preferably 0.05 mol / L, and the pH value is preferably 9.6.

[0049] The present invention washes, blocks and washes the obtained coated plate to obtain a blocked plate. The present invention preferably uses PBST buffer for washing and washing, and the number of times is 3 times. The present invention preferably uses a skimmed milk powder solution with a mass percentage of 5% for blocking at 37°C for 1 hour.

[0050] The invention adds bovine serum solution and EPF-HRP solution to the obtained closed plate, incubates the plate, and washes the plate to obtain the incubation plate.

[0051] In the present invention, the amount of bovine serum solution added to the wells of the closed plate is preferably 50 μL / well, and the bovine serum solution is preferably obtained by diluting the bovine serum 20 times. In the present invention, the amount of EPF-HRP solution added to the wells of the closed plate is preferably 50 μL / well, and the EPF-HRP solution is preferably obtained by diluting srEPF protein 4000 times after SUMO digestion and HPR labeling, and the srEPF amino acid sequence after removing the immunosuppressive peptide (FRDGDILGKYV) is as shown in SEQ ID No. 3, specifically as follows:

[0052] MAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVVAV GSGSKGKGGEIQPVSVKVGDKVLLPEYGGTKVVLDDKDYFL.

[0053] The method adds 3,3',5,5'-tetramethylbenzidine to the obtained incubation plate and continues incubation. After terminating the reaction, OD is detected. 450 The inhibition rate PI value was calculated according to the formula: inhibition rate PI value = (blank control OD 450 -Bovine serum solution OD 450 ) / blank control OD 450 When the inhibition rate PI value is less than 0.5415, it is positive pregnant bovine serum; when the inhibition rate PI value is greater than 0.5415, it is negative pregnant bovine serum; when the inhibition rate PI value is equal to 0.5415, it is necessary to confirm the final result through repeated measurements, that is, use other methods for auxiliary verification.

[0054] In the present invention, the amount of 3,3',5,5'-tetramethylbenzidine added to the well of the incubation plate is preferably 100 μL. In the present invention, the conditions for continued incubation preferably include: a temperature of 20-30°C and a time of 15 minutes. In the present invention, a 2 mol / L sulfuric acid solution is preferably used to terminate the reaction.

[0055] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] 1. Materials and Methods

[0058] 1.1 Materials

[0059] 1.1.1 Experimental animals

[0060] BALB / c female mice, 6-8 weeks old, were purchased from Liaoning Changsheng Biotechnology Company.

[0061] 1.1.2 Materials and reagents

[0062] IPTG (isopropylthiogalactosyl) was purchased from China Solebao Technology Co., Ltd.; Rainbow pre-stained protein marker was purchased from Beijing Jumei Biotechnology Co., Ltd.; M5 HiPer ECL Western HRP Substrate (ultrasensitive ECL luminescent liquid) was purchased from Beijing Jumei Biotechnology Co., Ltd.; TMB colorimetric solution A and solution B were purchased from Beijing Meikewande Biotechnology Co., Ltd.; goat anti-mouse IgG secondary antibody was purchased from Immunoway; skim milk powder was purchased from Sangon Biotechnology Co., Ltd.; 0.22μM PPVDF membrane and 0.5mL ultrafiltration tube were purchased from Merck Millipore; 96-well ELISA plate was purchased from Guangzhou Jiete Biofiltration Co., Ltd.; HRP coupling kit was purchased from Beijing Meikewande Biotechnology Co., Ltd.; ELISA kit for mouse monoclonal antibody subclass identification was purchased from Luoyang Bio-Tong Experimental Materials Center; Bovine EPF ELISAKIT was purchased from Shanghai Lanpai Biotechnology Co., Ltd.; bovine serum was collected from Jiutai Cattle Farm in Changchun, Jilin Province and processed by conventional centrifugation.

[0063] 1.2 Methods

[0064] 1.2.1 Preparation of immunogen rEPF

[0065] The bovine EPF gene sequence (NM_002157.3) was obtained from NCBI, and the CDS region of the sequence without the inhibition region was selected as the target gene. Polyhistidine (6×His) was added to the N-terminus of the sequence. The upstream and downstream primers were designed using Primer Primer5.0, and the upstream Nde I and downstream Xho I restriction sites were added, respectively, and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The target gene was amplified by PCR, connected to the pMD18-T vector, and transformed into Escherichia coli DH5α competent cells. After plating and culture, a single colony was picked. After the bacterial solution was correctly identified, the recombinant plasmid was double-digested, the target gene was connected to the pET-22b vector, and transformed into Escherichia coli BL21 (DE3) competent cells, inoculated in liquid LB medium containing ampicillin, and cultured at 37°C until the logarithmic growth phase. The OD was measured using a spectrophotometer. 450nmWhen the value is about 0.5, add 1mM IPTG to induce for 5h, collect the bacterial solution, perform SDS-PAGE gel electrophoresis and stain. Then add 0.25mol / LKCl solution to the protein gel until it is completely immersed, react at 4℃ for 5min, cut the target band and put it into the dialysis bag, add conventional electrophoresis solution until the target band is completely immersed, seal it, place it in the electrophoresis tank with ice-water mixture, and act at 80V for 2h to obtain purified rEPF protein. The purified rEPF protein (SEQ ID No.3) (this part only provides the EPF sequence without the inhibition zone) is identified by SDS-PAGE and Western blot.

[0066] Target fragment amplification:

[0067] Table 1 PCR system

[0068] Reagents Addition volume (μL) 10×PCR buffer 5 2.5 mM dNTP 4 22bEPF-276-F 2 22bEPF-276-R 2 Synthetic plasmid template 1 EXTaq enzyme 0.5 <![CDATA[ddH2O]]> 35.5 total 50

[0069] PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 45 s, annealing at 53°C for 30 s, extension at 72°C for 45 s, for a total of 33 cycles; extension at 72°C for 10 min.

[0070] Connect the purified target fragment into the pMD18-T vector. Connect pMD18-T system 10μL: PCR gel recovery product 4μL, pMD18-T vector 1μL, SolutionⅠ5μL. Connect pMD18-T conditions: 16℃, overnight. Transform the ligation product into E. coli DH5α competent cells, plate and pick the monoclonal strain to amplify in LB liquid culture medium, and send the correct monoclonal strain verified by bacterial liquid PCR to Shanghai Biotech sequencing for further verification.

[0071] The positive pMD18-T-DH5a with the correct verification result was extracted, and the target fragment was recovered by restriction gel digestion. pET22b target gene and expression restriction digestion system and conditions: positive EPF-pMD18-T / empty vector pET22b25ul; Nde I 1ul; EcoR I1ul; 10×QuickCut Buffer 3ul; 37℃, 60min. And transformed into Escherichia coli BL21 (DE3) competent cells, inoculated in liquid LB medium containing ampicillin, and cultured at 37℃ until the logarithmic growth phase.

[0072] The nucleotide sequence of the target gene is shown in SEQ ID No.4, and is as follows:

[0073] CATATGGCAGGACAGGCATTTAGAAAGTTTCTTCCCCTCTTTGACCGAGTATTAGTTGAAAGAAGTGCGGCCGAAACTGTAACCAAAGGAGGGATTATGCTTCCAGAAAAATCACAAGGAAAAGTATTGCAAGCAACGGTGGTAGCTGTTGGATCAGGCTCTAAAGGA AAGGGTGGAGAGATTCAACCAGTTAGTGTGAAAGTTGGAGATAAAGTTCTTCTCCCAGAATATGGAGGCACCAAAGTAGTTCTAGACGACAAGGATTATTTCTTATTTAGAGATGGTGACATTCTTGGGAAATATGTCGACCTCGAGCACCACCACCACCACCACTGA.

[0074] Upstream and downstream primer sequences: Based on the primers with upstream Nde I and downstream Xho I restriction sites, the fragment after the inhibition region (276 bp) was removed (connected to pET-22b), and the primer sequences are as follows:

[0075] 22bEPF-276-F (SEQ ID No. 5):

[0076] GGAATTCCATATGGCAGGACAGGCATTTA;

[0077] 22bEPF-276-R (SEQ ID No. 6): CCGCTCGAGAAATAAGAAATAATCCT.

[0078] 1.2.2 Preparation of original srEPF for detection

[0079] The research team has successfully constructed the pET-28a-srEPF positive plasmid (Wang Cong, Wang Han, Zhang Hailing, et al. Prokaryotic expression of recombinant early pregnancy factor and preparation of polyclonal antibodies [J]. Hubei Agricultural Sciences, 2023, 62(05): 129-34.), and transferred it into BL21 (DE3) competent cells, revived the correctly identified positive bacterial solution, and prepared and purified srEPF according to the literature method. The amino acid sequence is shown in SEQ ID No. 3.

[0080] 1.2.3 Preparation of EPF mAbs

[0081] The immunogen rEPF was used to subcutaneously immunize 6-8 week old female Balb / c mice three times on the back, with an interval of 14 days and an immunization dose of 50 μg / mouse. The serum antibody titer was detected by indirect ELISA, and mice with higher titers were selected for booster immunization. At the end of the immunization, the immunized mice were euthanized, and the spleen cell suspension was fused with the SP2 / 0 cells that had been resuscitated in advance, and the fused cell suspension was inoculated into a 96-well plate with feeder cells. After 8-10 days of culture, the cell supernatant was taken for indirect ELISA detection, and the positive hybridoma cells were picked. The screened positive hybridoma cells were subcloned by the limiting dilution method. After three subclones, the screened hybridoma cells were picked for expansion culture. Subsequently, 10-week-old Balb / c mice were pretreated by intraperitoneal injection of 0.5 mL of liquid paraffin, and one week later, 1x10 6 hybridoma cells, observe the swelling of the mouse abdomen, extract ascites when the abdomen is swollen, centrifuge at 8000r / min for 10min and freeze.

[0082] 1.2.4EPF Ascites Antibody Purification

[0083] The collected mouse ascites was mixed with the binding buffer at a ratio of 1:1. After the mixture was filtered through a 0.22 μm filter, the mouse ascites was purified by affinity chromatography using the AKTA protein purification system.

[0084] 1.2.5 Identification of immunological properties of EPF mAb

[0085] Titer determination: 1 μg / mL recombinant protein was coated on the ELISA plate, 100 μL / well, overnight at 4°C; 5% skim milk powder was used for blocking for 1 hour; EPF mAb was diluted at a ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, 1:1024000, 100 μL / well for incubation for 1 hour; HRP-labeled goat anti-mouse IgG antibody was added, 100 μL / well; 1 hour later, substrate was added for color development for 10-15 minutes; the reaction was terminated with stop solution, and OD was determined 450nm The absorbance value was set at the absorbance value, and the absorbance ratio of the monoclonal antibody and the negative control (P / N) was detected. If P / N>2.1, it was judged as positive, and the positive result with the largest dilution ratio was taken as the measured antibody titer.

[0086] Subtype identification: refer to the instructions of the antibody subtype kit for determination.

[0087] Identification of affinity constant (Ka): The affinity constant Ka of the monoclonal antibody was determined by referring to the Beatty saturation method. The microplate was coated with srEPF at mass concentrations of 0.5 and 1 mg / L, and the EPF mAb was diluted in multiples for indirect ELISA detection. The reaction curve was drawn and calculated according to the formula:

[0088]

[0089] Where n = [Ag]t / [Ag′]t; [Ag]t and [Ag′]t represent the molar concentrations of the two antigen coatings; [Ab]t and [Ab′]t represent the maximum OD at two different coating concentrations. 450nm The antibody molar concentration corresponding to half the value.

[0090] Western-blot identification: Prokaryotically expressed srEPF and natural pregnant bovine serum were mixed with loading buffer to prepare samples. After 15% SDS-PAGE electrophoresis, the protein was transferred from the gel to a 0.22μM PVDF membrane using the reaction conditions of wet transfer at 200mA and 1h30min. Blocked with 5% skim milk at room temperature for 2h, then the EPF monoclonal antibody was diluted 1:1000 and incubated at 4℃ for 12h. The membrane was washed 3 times with TBST, 10min each time. HRP-labeled goat anti-mouse IgG was diluted 1:5000 and incubated at 37℃ for 1h. The membrane was washed four times with TBST and developed using M5 HiPer ECLWestern HRP Substrate (ultrasensitive ECL luminescent liquid).

[0091] 1.2.6 Analysis of the performance of early pregnancy diagnosis based on EPF and mAb antigen competition ELISA

[0092] In order to evaluate the potential use of EPF mAbs as diagnostic reagents in early pregnancy detection in cattle, an antigen competition ELISA based on EPFmAb was designed. The EPF purified by SUMO digestion of srEPF was labeled with HRP (Beijing Meikewande Biotechnology Co., Ltd.). After successful labeling, the free EPF antigen in the serum competed with EPF-HRP for solid-phase EPF mAb for antigen competition ELISA. Eight positive and five negative serum samples from pregnant cattle were used, and antigen competition ELISA was performed after serial dilution of 1:10-1:80 with PBS. The OD 450nmValue. After the conditions were optimized, the specific detection was as follows: B6 was fixed in a transparent 96-well microplate (Guangzhou Jie Te Biofiltration Co., Ltd.) at 4°C for 12 hours at 0.1 μg per well in CBS (0.05 mol / L, pH 9.6). After washing three times with PBST, 5% skim milk powder was blocked at 37°C for 1 hour. After washing the plate three times, 50 μL of a mixed solution of EPF-HRP (1:4000, volume multiple) and bovine serum (1:20, volume multiple) were added to each well. Incubate at 37°C for 1 hour. After washing three times with PBST, 100 μL of the enzyme substrate 3,3′,5,5′-tetramethylbenzidine was added to each well and incubated at room temperature for 15 minutes. 50 μL of 2 mol / L sulfuric acid solution was added to each well to terminate the reaction, and Epoch TM Ultra-microplate spectrophotometer ( The optical density (OD) at 450 nm was measured and analyzed by the US Department of Energy's Vermont Laboratory.

[0093] 1.2.7 Repeatability evaluation of competitive ELISA

[0094] Five positive and five negative serum samples from pregnant cows were tested using ELISA plates coated with the same batch. Each sample was tested three times, and the average of the three repeated tests was calculated. The results were statistically analyzed, and the relative standard deviation (RSD) within the plate was calculated, RSD (%) = (standard deviation / average) × 100%. Three different batches of ELISA plates were used for inter-plate repetitions. The above samples were tested in the same way, and the inter-plate RSD was calculated to evaluate the repeatability of this method.

[0095] 1.2.8 Evaluation of the practicality of competitive ELISA

[0096] 27 positive and 23 negative serum samples from pregnant cows were collected and tested simultaneously using this competitive method and a standardized kit. The tests were repeated three times in parallel to evaluate the practicability of this method.

[0097] 2. Results and Analysis

[0098] 2.1 Results of immunogen rEPF-induced expression

[0099] The synthetic plasmid with the target fragment was used as a template for PCR amplification, and the product was identified by agarose gel electrophoresis. Figure 1 As shown in a, there is a bright band at 276 bp. After the target fragment was recovered and purified by an agarose gel recovery box, it was connected to the pMD18-T vector and transformed into E. coli DH5α competent cells. The bacterial solution was identified by PCR ( Figure 1b), there is a bright band at 276 bp, which is consistent with the expected size, indicating that the recombinant plasmid pMD18-T-rEPF was successfully constructed. After double restriction digestion of pMD18-T-rEPF, it was transformed into BL21 competent cells together with the vector pET-22b. After PCR identification of the bacterial solution, the pET-22b-rEPF positive bacterial solution was induced by IPTG to express, and the target protein was expressed at about 11 kDa. After gel purification, SDS-PAGE verified that the purification was successful ( Figure 1 Since the rEPF protein contains a HIS tag, it was verified by Western blot using an anti-HIS tag antibody. The results showed that ( Figure 1 In middle d), a specific single band of rEPF protein was observed at about 11 kDa, which was consistent with the relative molecular mass of the expected protein, indicating that the rEPF protein was successfully expressed and purified and could be used for the next step of monoclonal antibody preparation.

[0100] 2.2 Detection of original srEPF induced expression results

[0101] After the pET-28a-srEPF positive bacterial solution was activated, the expression was induced by IPTG, and the SDS-PAGE analysis was performed after ultrasonic disruption. The results are as follows: Figure 2 As shown in a, the srEPF protein supernatant was expressed, which was in line with expectations. The supernatant after fragmentation was purified by affinity chromatography, and the results were as follows: Figure 2 As shown in b, imidazole can be eluted at a concentration of 80-300mmol / L with high purity. The eluate is collected, srEPF is concentrated by ultrafiltration using a 10kDa ultrafiltration tube, and imidazole is replaced with PBS. Figure 2 In middle c, there is a clear band at 26 kDa, and the result is consistent with expectations.

[0102] 2.3 Preparation and characterization of EPF mAb

[0103] The present invention screened and obtained three positive hybridoma cell lines that can stably secrete specific monoclonal antibodies against EPF protein, which were named 8E6BB6, 8E6BF8, and 11E3GF6, respectively. A portion of the positive cell lines were frozen for future use, and a portion was expanded for culture, and EPF mAb was prepared by inducing ascites in mice. The collected mouse ascites was mixed with an equal volume of binding buffer and purified using the AKTA protein purification system. The results are as follows: Figure 3 As shown in a, the ascites fluid passes through the affinity chromatography column, the antibody is captured, and the impurity protein peak flows through. After elution, a single antibody peak appears as the target peak. The purified EPF mAbs B6, F8, and F6 were taken and their titers were measured respectively. The results are shown in Table 2. The titers of the three antibodies all reached 10 -6 above.

[0104] Table 2 EPF mAb titer determination

[0105]

[0106]

[0107] The mouse monoclonal antibody subclass kit was used to identify the subclasses of the three prepared EPF monoclonal antibodies. The results are as follows Figure 3 Middle b shows that the OD of the three EPF monoclonal antibodies B6, F8, and F6 is IgG1. 450nm Therefore, it was determined that the three monoclonal antibodies were all of IgG1 subclass.

[0108] EPF mAb affinity curves Figure 3 As shown in c, taking EPF mAb B6 as an example, when the coating antigen concentration is 0.5 μg / mL and 1.0 μg / mL, [Ab]t and [Ab′]t are calculated according to the formula to be 1.35×10 -10 mol / L and 9.22×10 -11 mol / L, calculated Ka = 1.4 × 10 9 L / mol, and the affinity constants of F8 and F6 were 1.2×10 9 L / mol, 8.7×10 8 L / mol, indicating that the present invention has prepared EPF mAb with good affinity.

[0109] To detect the specificity of the reaction between EPF mAb and EPF natural protein, SDS polyacrylamide electrophoresis was performed using srEPF recombinant protein and pregnant bovine serum as antigens. After transfer to the membrane, the obtained EPF mAb was used for Western-blot detection. The results are shown in Figure 3 Figure d shows that the purified monoclonal antibody specifically binds to the srEPF recombinant protein at 26 kDa. Figure 3 Figure e shows that the three monoclonal antibodies can specifically bind to pregnant bovine serum near 25 kDa and 45 kDa, indicating that the purified EPFmAb has good specificity and can be used to establish subsequent detection methods.

[0110] 2.4 Evaluation of the potential application of EPF mAbs in antigen competition ELISA

[0111] Appropriate concentrations of B6, F8, and F6 were coated respectively, and EPF-HRP at different dilutions was added to verify successful coupling by ELISA. Figure 4 As shown in a, coupling OD 450nmThe absorbance value decreased with the increase of EPF-HRP dilution. EPF was successfully coupled to HRP, and the specific binding effect of B6 with EPF-HRP was better than that of F8 and F6. Therefore, B6 was selected for subsequent experiments. Using the chessboard method, the coating concentrations of EPF mAb B6 were 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, and 0.125μg / mL, and the dilutions of EPF-HRP were 500 times, 1000 times, 2000 times, 4000 times, and 8000 times, respectively, for ELISA determination. The results are as follows Figure 4 As shown in b, when OD 450nm When the absorbance value is around 1, the detection is more sensitive, so the OD value of 1 is selected. Considering the principle of saving antibodies, the coating concentration of EPF mAb B6 is finally determined to be 1μg / mL, and the corresponding EPF-HRP dilution factor is 4000 times. According to the optimized EPF mAb coating dosage and the optimal dilution of EPF-HRP, four coating conditions are set: 4℃12h, 37℃1h, 37℃2h, 37℃4h, and a negative control group is set for antigen competition ELISA experiment, with coating conditions as the horizontal axis and OD as the horizontal axis. 450 value and inhibition rate PI = (blank OD 450nm - Sample OD 450nm ) / Blank OD 450nm Draw a graph for the vertical axis respectively, and the results are as follows Figure 4 As shown in c, when coated at 4℃ for 12h, the PI value is the highest, so 4℃ for 12h is selected as the coating condition. 8 positive and 5 negative serum samples of pregnant cows were diluted with PBS in a continuous volume ratio of 1:10-1:80 for testing, and the positive and negative serum at each dilution was analyzed at OD 450nm The absorbance under . The test results are shown in Figure 4 As shown in Figure d, when the dilution ratio is 1:10, 1:40, and 1:80, the OD of positive and negative serum 450nm There is basically no difference in the values. When the dilution ratio is 1:20, the OD 450nm The values ​​overlap significantly, indicating that the difference is significant at this dilution and can distinguish between positive and negative sera.

[0112] 2.5 Evaluation of Repeatability and Reproducibility of Competition ELISA

[0113] 5 positive and 5 negative serum samples of pregnant cows were measured using ELISA plates coated with the same batch and three different batches, and the intra-batch and inter-batch CVs were calculated to evaluate the precision of the method. When the intra-batch CV was less than 10%, the test was considered to have good repeatability, and when the inter-batch CV was less than 15%, the test was considered to have good reproducibility. The results of this test are shown in Table 3. The intra-batch CV was 0.51%-6.42%, and the inter-batch CV was 2.45%-10.24%, which can fully meet the detection of EPF in the sample, indicating that the antigen competition ELISA method initially established in this study has good repeatability and reproducibility.

[0114] Table 3 Intra- and inter-batch reproducibility of competitive ELISA based on B6

[0115]

[0116] 2.6 Analysis of the practical application of B6-based competitive ELISA in the diagnosis of early pregnancy in cattle

[0117] The established antigen competition ELISA and a similar commercial kit (Bovine EPF ELISA KIT) were used to parallelly test a total of 50 serum samples, including 27 positive serum samples from pregnant cows and 23 negative serum samples (serum samples were determined to be positive or negative based on follow-up) to evaluate the practicality of the detection method. Figure 5 The area under the ROC curve AUC of a,dc-ELISA was 0.833, the standard error SE = 0.06, and the 95% confidence interval was 0.714-0.951. According to the ROC curve analysis, when the PI cutoff value of the competitive ELISA was 0.5415 (PI < 0.5415, positive pregnant bovine serum), the specificity and sensitivity were the best, which were 82.61% and 81.48%, respectively, both greater than 70%, and the test was considered to perform well. Figure 5 As shown in b, the DC-ELISA test showed significant differences in positive and negative pregnant bovine serum. The OD 450nm The value was significantly higher than that of positive pregnant bovine serum. 450nm There was no significant difference in the values ​​( Figure 5 (c).

[0118] 2.7 Amino Acid Sequence Composition of Monoclonal Antibody Variable Region

[0119] In view of the good reaction characteristics between B6 antibody and EPF, the present invention sequenced the variable region of B6 antibody and used bioinformatics software to perform structure prediction and interaction analysis to reserve cell and gene resources for subsequent antibody applications.

[0120] Cell culture and RNA extraction

[0121] Take the cells out of the liquid nitrogen tank, resuscitate and expand the culture, and collect the cultured cells; Cell / Tissue Total RNA Isolation Kit V2 kit instructions, extract cell RNA. Reverse transcription to obtain cDNA. Using the universal primers for the variable region of the IgG subtype mouse monoclonal antibody, the above cDNA was used as a template to perform PCR amplification on the variable region of the light chain and heavy chain, and the amplified products were identified by DNA gel, and the PCR products were recovered by gel cutting using the kit of TIANGEN. The above light chain and heavy chain PCR products were connected with 5minTA / Blunt-Zero Cloning Kit (C601-02), transformed into Top10 competent cells, and colony PCR was performed with M13F(-47) / M13R(-48) primers, and positive clones were selected for sequencing.

[0122] Antibody variable region sequence composition:

[0123] According to sequencing analysis, the sequence of the heavy chain variable region (VH) of the B6 monoclonal antibody is shown in SEQ ID No. 1, and the sequence of the light chain variable region (VL) is shown in SEQ ID No. 2;

[0124] B6 heavy chain amino acid sequence (136aa)

[0125] MECSWILPFILSVTSGVYSLVQLQQSGAELARPGASVKLSCKASGYTFTN YWMQWVKQRPGQGLEWIGAIYPGDFDTRYTQKFKGKATLTADKSSNTAYM QLSSLASEDSAVYYCVRWGWGQGAYWGQGTTLTVSS;

[0126] B6 light chain amino acid sequence (127aa)

[0127] MSVPTQVLGLLLLWLTGARCDIQMTQSSASQSASVGETVTITCRASENIY SYLAWFQQRQGKSPQLLIYNAETLAEGVPSRFSGSGSGTQFSLKINSLQPEDF GTYYCQHHYGSPWTFGGGTKLEIK;

[0128] Analysis of interaction between B6 monoclonal antibody and target EPF:

[0129] This embodiment uses three advanced computational biology methods to predict the structure and interaction of B6 monoclonal antibody and EPF protein for modeling and analysis. AlphaFold is a protein structure prediction method based on deep learning, which can generate high-precision three-dimensional structure prediction results based on the local and global features of protein sequence information. PyRosetta is a molecular modeling tool based on the Rosetta algorithm that can perform protein structure optimization, docking and energy evaluation. By gradually optimizing the structure, it tends to a more stable low-energy state, thereby improving the accuracy and biological rationality of the model. ClusPro is a protein-protein docking tool that uses rigid docking and energy-driven clustering analysis to predict the binding conformation of protein complexes. It clusters the different conformations obtained by docking and gives corresponding energy scores to evaluate the stability and reliability of the complex.

[0130] In this example, the three-dimensional structures of B6 monoclonal antibody and EPF protein were predicted based on AlphaFold (see Figure 6 Middle A, Figure 6 B), and predicted the scoring results of the complex formed by the two. The key parameters of their interaction are analyzed as follows: most of the binding interface residues have pLDDT>90 ( Figure 6 The blue area in D shows the prediction error of the main chain (Cα atoms). The accuracy is close to the crystallographic resolution, indicating that the prediction confidence of this region is high and the structure is stable; ipTM = 0.64: ​​the interface topology matching degree is above the medium level (ipTM>0.5 is considered reliable), the spatial arrangement of interface residues is reasonable, but the local conformation such as side chain orientation still has room for optimization; pTM = 0.72: the overall folding has a high similarity with the natural conformation (pTM>0.5 is considered a credible model), indicating that the overall structural topology accuracy of the B6 and EPF protein complex is high, which can support subsequent functional studies (such as mutation verification, molecular docking); in the EPE heat map ( Figure 6 In D (right side), the prediction error in the green area is less than This indicates that the relative spatial positioning deviation of the bonding interface is low and the interface structure is highly correlated (see Figure 6 (middle D).

[0131] Based on the above scoring results, the complex of B6 and EPF was visualized ( Figure 6 E), showing that the two have formed a reasonable three-dimensional protein conformation, the confidence of key interface residues is high, and the overall folding is compact and stable. Among them, the heavy chain of B6 monoclonal antibody is marked in green, the light chain is marked in yellow, and the EPF protein is marked in purple.

[0132] PyRosetta was further used to perform overall structure prediction and energy evaluation on the B6 monoclonal antibody (heavy chain and light chain sequences), and the structure of the B6 monoclonal antibody predicted by PyRosetta was obtained (see Figure 6 C: Green represents heavy chain, yellow represents light chain). The results show that its total energy is about -467.61REU (Rosetta energy unit), indicating that the B6 monoclonal antibody is in a relatively stable low-energy state as a whole. The structure generally presents a typical "Y"-shaped antibody appearance, which is consistent with the expected immunoglobulin folding characteristics. ClusPro was used to predict the interaction between the B6 monoclonal antibody predicted by PyRosetta and the EPF protein predicted by AlphaFold. The complex model shown in the results (see Figure 6 F in the middle: green is B6 heavy chain, yellow is light chain, and blue is EPF protein). Combined with the data analysis results: the structural energy of the complex is about -373.3, indicating that this complex is the most stable and shows stable binding, indicating that the B6 monoclonal antibody and EPF protein have a high binding credibility and can be used as the focus of subsequent research.

[0133] AlphaFold predicts that B6 and EPF complex ( Figure 6 Middle E) and ClusPro predicted complex of B6 and EPF ( Figure 6 Compared with (F), the complex presents a relatively loose conformation in some areas, which may be due to the sampling algorithm limitation of ClusPro or insufficient treatment of binding site flexibility. However, from the perspective of overall folding and energy distribution, the complex still has a reasonable three-dimensional structure and good stability, which is in line with expectations.

[0134] In summary, the interaction between B6 monoclonal antibody and EPF protein was predicted by various computational modeling and analysis strategies (AlphaFold, PyRosetta, ClusPro). The results showed that the two had good binding characteristics, and all scores and energy analyses supported their stable binding. The prediction results can provide a reliable theoretical basis for antibody affinity maturation and the optimization of corresponding detection methods.

[0135] 3. Discussion

[0136] Early and ultra-early pregnancy diagnosis in cattle is an important part of preserving pregnancy, reducing empty pregnancy, increasing production performance and improving reproduction rate. The ideal early pregnancy diagnosis technology in cattle should have the characteristics of showing diagnostic effect within one estrus cycle after mating, with an accuracy rate of more than 85% for pregnancy and non-pregnancy diagnosis, safety for mother and fetus, simple method, economical and practical. In the past, traditional diagnosis used ultrasonic detection and rectal detection methods, which would cause invasive damage to cattle. Improper operation would also lead to embryo death, causing great economic losses. In recent years, with the development of analytical technology, it has been found that animal embryos can produce progesterone, testosterone, EPF, pregnancy-specific protein, pregnancy-related protein, pregnancy serum protein, etc. EPF is the earliest pregnancy-related factor in maternal serum in early pregnancy. It can appear several hours after pregnancy and disappear within a few hours after embryo abortion or death. Therefore, it can not only be used for early pregnancy diagnosis, but also as a sign of embryo status in late pregnancy. The research on EPF is of great significance to the dairy farming industry.

[0137] EPF is an immunosuppressive active protein that appears in the serum of the mother after fertilization. In 1998, Clara et al. characterized and identified EPF and found that EPF is an immunomodulatory active peptide with Fc receptors. Studies have shown that EPF affects the function of lymphocytes by inhibiting the synthesis of lymphocyte DNA, thereby downregulating T cell-mediated immune function, so that the fetus will not be cleared as a foreign body in the mother's body. Therefore, in order to prevent the interference of immunosuppression in the preparation of EPF monoclonal antibodies and enhance their immunogenicity, this study used the conventional molecular cloning technology of the research group to express the protein of EPF with the gene of the inhibitory region removed in prokaryotes as an immunogen, and prepared EPF monoclonal antibodies through hybridoma technology. The titer analysis showed that the expressed recombinant antigen had good immunogenicity and could stimulate the body to produce a good immune response, providing a good experimental basis for the preparation of hybridomas and the acquisition of antibodies.

[0138] It is reported that the basic activity of EPF is a polypeptide containing 101 amino acids. However, due to the complexity of serum itself, different laboratories use different methods to separate and purify EPF from different species at different gestational periods, and the molecular weights obtained are also different. Wilsons et al. purified two polypeptides with EPF activity from the serum of ewes at 3 to 8 weeks of pregnancy, with molecular weights of 20 kDa and 67 kDa, respectively. Metha et al. separated and purified EPF active polypeptides from the serum of pregnant women at 5-12 weeks of pregnancy. The molecular weight of the purified EPF was about 21 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The analysis by gel permeation chromatography (HPLC) showed that its molecular weight was 28 kDa. Xiang Jiyao et al. separated and purified the blood of pregnant cows and extracted the EPF polypeptide with a high activity molecular weight of 44 kDa. In this study, a high-titer and high-affinity EPF ascites mouse monoclonal antibody was prepared. Western-blot identification showed that the three antibodies could specifically bind to the serum of pregnant cows near 25 kDa and 45 kDa. The molecular weight of EPF varies. It is speculated that it may be that it carries certain carriers, or the protein itself forms self-aggregates such as dimers and trimers. Its molecular existence form is relatively complex, but the specific way that leads to the difference in its molecular weight has not been reported in detail yet, and further in-depth research is needed.

[0139] AlphaFold uses a deep learning strategy to predict the three-dimensional structure of proteins based on their sequence information, and has a high prediction accuracy for monomeric proteins or multi-protein complexes. PyRosetta is a molecular modeling tool based on the Rosetta algorithm that can perform structural optimization, docking, and energy evaluation on proteins. The core idea is to gradually optimize the structure so that the system tends to a more stable low-energy state. Generally speaking, the lower the energy, the more stable the structure. ClusPro is a protein-protein docking tool that uses rigid docking and energy-driven clustering analysis to predict the binding conformation of protein complexes. The tool clusters the different conformations generated by docking and evaluates the stability and reliability of the conformation based on the energy score. Using the above three modeling and docking strategies to analyze the structure and interaction of B6 monoclonal antibody and EPF protein, it can be found that: AlphaFold showed high confidence and accuracy in both monomeric protein prediction and complex docking; PyRosetta further predicted and optimized the energy of B6 monoclonal antibody to obtain a more stable antibody structure;

[0140] ClusPro provides multiple possible complex conformations by docking and clustering B6 monoclonal antibody and EPF protein, and evaluates its stability with energy score to obtain a stable complex conformation. The comprehensive analysis of the three methods shows that B6 monoclonal antibody and EPF protein have good binding characteristics, and the prediction results can provide a reliable theoretical basis for antibody affinity maturation and corresponding detection method optimization.

[0141] ELISA is a sensitive, specific and simple method for detecting bacteria, proteins and viruses. It is widely used in the fields of biology and medicine because of its accuracy and repeatability, stable reagents and cheap equipment. Some studies have reported on the detection of EPF in pregnant serum based on ELISA technology. Chen Feihu et al. used EPF-McAb to detect the serum of clinically confirmed pregnant women and non-pregnant serum, and used ELISA and Ea-RIT to simultaneously detect EPF antigen. The detection rate of ELISA was 92.98%, which was significantly higher than that of Ea-RIT. Yi Chuanzhu et al. established a double monoclonal antibody sandwich ELISA method by preparing anti-EPF monoclonal antibodies to detect the activity of EPF, with a sensitivity of 90.2%. Based on this, this study first used an HRP labeling kit to couple the original EPF with activated HRP to prepare a competitive antigen. Ascites monoclonal antibody B6 was coated in a microplate as a solid-phase antibody, and a direct competitive ELISA was initially established by using the free target antigen in the test object to compete with the HRP-coupled antigen for the solid-phase EPF monoclonal antibody. At the same time, the positive and negative serum of pregnant cattle was detected after optimizing the reaction conditions. Compared with the commercial quantitative detection kit, the detection method established in this study has the advantage of being able to distinguish positive and negative sex well, and the detection rate of competitive ELISA in positive samples is 81.48% (22 / 27), which has good detection performance and provides a reference for early pregnancy detection in cattle on pastures. The commercial quantitative detection kit was measured in parallel, and there was no significant difference in the EPF content in the serum of positive and negative pregnant cattle. The analysis may be due to the presence of EPF-A in the non-pregnant period. In studies on mice, humans, sheep and pigs, it has been found that EPF-A exists in the serum near the ovulation period in the form of precursors, and its production does not depend on fertilization or pregnancy. However, in pregnant mice, EPF-A exists longer than that in non-pregnant mice. Therefore, combined with this detection method, we speculate that EPF is also contained in the serum of non-pregnant cattle, and its content may be slightly lower than that of pregnant cattle. If EPF is used to distinguish between pregnant and non-pregnant cattle, more specific antibodies may be needed to more accurately detect the EPF content. In subsequent research, this research group will determine the epitopes recognized by each antibody, pair the antibodies, and establish a double-antibody sandwich ELISA detection method, in order to better realize the rapid detection of early pregnancy in cattle, reduce the cost of pregnancy diagnosis in large-scale farms, monitor the normal development of the fetus in real time, improve the reproductive efficiency of animals, and increase the economic benefits of the breeding industry.

[0142] 4. Conclusion

[0143] The present invention uses prokaryotically expressed rEPF immunized mice, utilizes hybridoma cell technology, and prepares three EPF monoclonal antibodies through cell screening and subcloning and AKTA purification. After the characteristics of the EPF monoclonal antibody are identified by ELISA, Western blotting and other technologies, an ELISA method based on antigen competition is developed for EPF detection in pregnant bovine serum. When the PI cutoff value is 0.5415, the detection specificity and sensitivity are as high as 82.61% and 81.48%. At present, my country's dairy farming industry is developing in the direction of scale, intensiveness and standardization, and dairy farming has become a breakthrough in the development of animal husbandry. Dairy farming is a high-input and high-output industry. In daily feeding and management, improving the milk production and reproduction rate of dairy cows is the key to improving economic benefits. With the improvement of people's level of understanding, more and more people are aware of the importance of carrying out early pregnancy diagnosis, and early pregnancy diagnosis technology will surely be greatly developed. The EPF monoclonal antibody produced in the present invention can provide a detection strategy for developing a bovine pregnancy diagnostic kit using blood samples, and provide a reference for monitoring fetal development and early prediction of pregnancy outcomes in dairy cows.

[0144] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody, characterized in that Includes monoclonal antibody B6; The amino acid sequence of the heavy chain variable region of the monoclonal antibody B6 is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. A diagnostic reagent for early pregnancy in cattle, characterized in that: Comprising the monoclonal antibody according to claim 1.

3. Use of the monoclonal antibody according to claim 1 in the preparation of a diagnostic reagent for early pregnancy in cattle.

4. A method for detecting early pregnancy in cattle for non-diagnostic purposes, characterized in that: The following steps are involved: 1) coating the monoclonal antibody according to claim 1 in a microplate to obtain a coated plate; 2) washing, blocking and washing the coated plate obtained in step 1) to obtain a blocked plate; 3) adding bovine serum solution and EPF-HRP solution to the closed plate obtained in step 2) and incubating the plate, and washing the plate to obtain an incubation plate; 4) Add 3,3′,5,5′-tetramethylbenzidine to the incubation plate obtained in step 3) and continue incubation. After terminating the reaction, detect OD 450 The inhibition rate PI value was calculated according to the formula: inhibition rate PI value = (blank control OD 450 -Bovine serum solution OD 450 ) / blank control OD 450 , when the inhibition rate PI value is less than 0.5415, it is positive pregnant bovine serum; When the inhibition rate PI value is greater than 0.5415, it is negative pregnant bovine serum; When the inhibition ratio PI value was equal to 0.5415, the final result was confirmed by repeated determination.

5. The method according to claim 4, characterized in that In step 1), the microplate is a transparent 96-well microplate; The coating amount of the monoclonal antibody in the wells of the microplate is 0.1 μg / well.

6. The method according to claim 4, characterized in that The coating conditions of the monoclonal antibody in step 1) include: coating at 4° C. for 12 hours.

7. The method according to claim 4, characterized in that In step 2), the plate was washed with PBST buffer for 3 times; The cells were blocked with a 5% skimmed milk powder solution at 37°C for 1 h.

8. The method according to claim 4, characterized in that In step 3), the amount of bovine serum solution added to the wells of the sealing plate is 50 μL / well, and the bovine serum solution is obtained by diluting the bovine serum 20 times; The amount of EPF-HRP solution added to the wells of the closed plate is 50 μL / well. The EPF-HRP solution is obtained by srEPF protein after SUMO digestion and HPR labeling and then diluting 4000 times. The amino acid sequence of the srEPF protein is shown in SEQ ID No.

3.

9. The method according to claim 4, characterized in that The incubation conditions of step 3) include: time of 1 hour and temperature of 37°C.

10. The method according to claim 4, characterized in that In step 4), the amount of 3,3′,5,5′-tetramethylbenzidine added to the wells of the incubation plate is 100 μL; The conditions for continued incubation include: a temperature of 20 to 30° C. and a time of 15 min; The reaction was terminated using 2 mol / L sulfuric acid solution.

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