A monoclonal antibody and its use in preparing a bovine early pregnancy diagnostic reagent

Abstract

The application relates to the field of bovine early pregnancy diagnosis technology, in particular to a monoclonal antibody and application thereof in preparation of a bovine early pregnancy diagnosis reagent. The monoclonal antibody provided by the application comprises a monoclonal antibody B6. The application takes EPF as a research target, applies hybridoma cell technology, and obtains the specific antibody of EPF through cell fusion screening and subcloning and AKTA purification. Based on the principle that EPF-HRP competes with the EPF in actual bovine serum samples for the solid-phase EPF antibody, an antigen competition ELISA detection method is established. When the cut-off value of bovine serum PI is set as 0.5415, the early pregnancy detection method shows the best sensitivity (82.61%) and specificity (81.48%).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of early pregnancy diagnosis of cattle, in particular to a monoclonal antibody and application thereof in preparation of a reagent for early pregnancy diagnosis of cattle. BACKGROUND

[0002] Simple and effective early pregnancy diagnosis can improve the reproduction of animals such as cattle and sheep, shorten the interval between births, and improve the economic benefits of breeding. It has been a major problem in intensive breeding production for a long time. At present, the main methods for early pregnancy diagnosis include observation, rectal detection, ultrasonic detection, progesterone detection, and detection of early pregnancy diagnosis markers (interferon tau-induced genes (ISGs), miRNA in exosomes). Observation is generally used as a supplementary method for early pregnancy diagnosis. Rectal detection and ultrasonic detection may lead to miscarriage if not properly detected. Progesterone detection technology has gradually matured, and many studies have reported on this. However, progesterone is a steroid hormone and a lipid-soluble substance, and there are large differences in progesterone levels among different individuals. 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, so it is difficult to determine the criteria for this method, and it has not been widely applied so far. ISGs detection is non-invasive, but changes in physiological conditions such as stress and inflammation can affect the expression of ISGs. These interference factors may reduce the specificity of ISGs as a pregnancy diagnosis indicator. As a non-invasive and repeatable biomarker, exosomal miRNA has the potential to diagnose pregnancy loss in dairy cows, but only a small number of studies have explored the application of exosomal miRNA in pregnancy diagnosis in dairy cows. More clinical trials are needed to verify the feasibility of exosomal miRNA as a pregnancy diagnosis marker. Compared with the above methods, immunological ELISA detection is widely used because of its scientificity, high sensitivity, simple operation, and ability to detect changes in the content of pregnancy-related proteins in the blood of cattle. Currently, commercial ELISA kits for early pregnancy detection in cattle are mainly produced by the United States of America and the United States of America Biotracking company, but the import cost is high, and they are rarely used in domestic breeding plants. Although there are some products on the market for early pregnancy detection, most of them are quantitative detection, and the detection results of different products differ greatly, which cannot clearly distinguish the problem of conception and cannot meet the actual needs of rapid early pregnancy diagnosis in the breeding industry. Therefore, it is urgent to develop a simple and fast early pregnancy detection method for cattle that can achieve batch detection and high efficiency.

[0003] Currently, the most concerned pregnancy detection technology is to detect the content of pregnancy-specific protein B (Pregnancy-Specific Protein B, PSP-B), pregnancy serum protein 60 (Pregnancy-Specific Protein 60, PSP60), pregnancy-associated glycoprotein (Pregnancy-associated glycoproteins, PAG) and early pregnancy factor (Early Pregnancy Factor, EPF) in the blood of pregnant cows by immunological method. The immunological detection method of this blood early pregnancy protein not only has high accuracy, is not disturbed by personnel factors, but also can avoid the abortion caused by direct contact with the embryo too early. In addition, batch detection can be realized, the efficiency is extremely high, and it is especially suitable for use in large-scale ranches. Among them, EPF is the earliest detected protein related to pregnancy. Mice are mated for 6h, and EPF can be detected from the maternal serum 24h after fertilization of sheep, cattle, pigs and humans. Studies have shown that EPF can be used to judge whether the pregnant body is aborted, detect whether embryo transplantation fails, monitor the fetal status of the pregnant mother, etc., and has become a hot spot for early pregnancy detection research and development. SmartYC et al. determined the EPF activity of the serum collected every month of pregnant women, judged that EPF can exist in most of the whole pregnancy period. If the pregnancy is terminated, the content of EPF in the blood rapidly decreases and decreases to a negative level within 5-7 days. Sungwoo Park et al. prepared a pig EPF monoclonal antibody, and detected the urine and blood of a pregnant 20-day Duroc sow, and the accuracy rate reached 70%; that is, the existence of EPF can be used as a pregnancy marker and an important indicator for early pregnancy diagnosis. Therefore, it is necessary to prepare a monoclonal antibody with excellent sensitivity and specificity for early pregnancy diagnosis of cattle. SUMMARY

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

[0005] In order to achieve the above purpose, the application provides the following technical scheme:

[0006] The application provides a monoclonal antibody, which comprises a 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 application provides a diagnostic reagent for cattle early pregnancy, which comprises the monoclonal antibody provided in the above technical scheme.

[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 a microplate with the monoclonal antibody described in the above technical solution 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 a bovine serum solution and an EPF-HRP solution to the blocking plate obtained in step 2) and incubating the plate, followed by washing 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, measure the 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 a positive pregnant bovine serum; when the inhibition rate PI value is greater than 0.5415, it is a 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 was 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 5% skimmed milk powder solution at 37°C for 1 h.

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

[0021] The adding amount of the EPF-HRP solution in the hole of the closed plate is 50 μL / hole, the EPF-HRP solution is obtained after the srEPF protein is cut by SUMO enzyme, is labeled with HPR and is diluted 4000 times, and the amino acid sequence of the srEPF protein is shown as SEQ ID No. 3.

[0022] Preferably, the incubation condition of the step 3) includes that the time is 1 h and the temperature is 37℃.

[0023] Preferably, the adding amount of 3,3',5,5'-tetramethylbenzidine in the hole of the incubated plate in the step 4) is 100 μL;

[0024] The continued incubation condition includes that the temperature is 20-30℃ and the time is 15 min.

[0025] The reaction is terminated by using a 2 mol / L sulfuric acid solution.

[0026] The beneficial effects of the present application are as follows:

[0027] In the present application, EPF is taken as a research target, a specific antibody of EPF is obtained by using hybridoma cell technology, cell fusion screening and subcloning, AKTA purification and sequencing of the variable region sequence of the antibody, the antibody is used as a detection probe, and an antigen competition ELISA detection method is established based on the principle that EPF-HRP competes with EPF in actual bovine serum samples for solid-phase EPF antibodies. When the cutoff value of bovine serum PI is set as 0.5415, the early pregnancy detection method shows the best sensitivity (82.61%) and specificity (81.48%). The detection method established in the present application has great potential for the detection of bovine serum samples, lays a foundation for subsequent EPF basic research and early pregnancy detection based on EPF, and is crucial for reducing empty cycle, maintaining the economic sustainability of dairy farms and increasing the fertility efficiency of cattle herds. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0029] Figure 1 In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

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

[0031] Figure 3 Figure 2. EPF mAb purification and reactivity; a. B6 purification using AKTA protein purification system. b. EPF mAb subtype identification. c. EPF mAb affinity identification. d. Western-blot verification of srEPF recombinant protein reactivity with three EPF mAbs. e. Western-blot verification of bovine pregnancy serum natural EPF protein reactivity with three EPF mAbs;

[0032] Figure 4 Figure 3. Evaluation of EPF mAbs in antigen competition ELISA; a. ELISA verification of B6, F8, F6 specific binding with EPF-HRP conjugate. b. Chessboard method optimization of B6 coating concentration and EPF-HRP dilution. c. Optimization of ELISA coating conditions. d. Determination of OD 450nm values of bovine pregnancy serum samples at different dilutions;

[0033] Figure 5 Figure 4. Detection of serum samples based on B6 antigen competition ELISA; 27 positive and 23 negative bovine pregnancy serum samples were detected using the ELISA method. a. ROC curve analysis of antigen competition ELISA. The AUC of B6 based antigen competition ELISA was 0.833. b. Box plot comparison of B6 based antigen competition ELISA detection of bovine pregnancy serum positive and negative samples. The difference was significant. c. Box plot comparison of Bovine EPF ELISA KIT detection of bovine pregnancy serum positive and negative samples. There was no significant difference.

[0034] Figure 6For the prediction analysis results of the interaction between the EPF mAb and the 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 score result of the B6 and EPF complex predicted by AlphaFold, E is the visualization result of the B6 and EPF complex predicted by AlphaFold, and F is the visualization result of the B6 and EPF complex predicted by ClusPro. DETAILED DESCRIPTION

[0035] The present application provides a monoclonal antibody, including a 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 is specifically as follows:

[0037] SEQ ID No. 1:

[0038] MECSWILPFILSVTSGVYSLVQLQQSGAELARPGASVKLSCKASGYTFTNYWMQWVKQRPGQGLEWIGAIYPGDFDTRYTQKFKGKATLTADKSSNTAYMQLSSLASEDSAVYYCVRWGWGQGAYWGQGTTLTVSS;

[0039] SEQ ID No. 2:

[0040] MSVPTQVLGLLLLWLTGARCDIQMTQSSASQSASVGETVTITCRASENIY SYLAWFQQRQGKSPQLLIYNAETLAEGVPSRFSGSGSGTQFSLKINSLQPEDF GTYYCQHHYGSPWTFGGGTKLEIK.

[0041] The present application also provides a diagnostic reagent for early pregnancy of cattle, which includes the monoclonal antibody described in the above technical solution. The present application does not have special limitations on other components in the diagnostic reagent, and the person skilled in the art can set it according to the conventional monoclonal antibody.

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

[0043] The present application provides a method for detecting early pregnancy of cattle for non-diagnostic purposes, which includes the following steps:

[0044] 1) coating a microplate with the monoclonal antibody described in the above technical solution 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 a bovine serum solution and an EPF-HRP solution to the blocking plate obtained in step 2) and incubating the plate, followed by washing 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, measure the 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 a positive pregnant bovine serum; when the inhibition rate PI value is greater than 0.5415, it is a 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 has no special limitation on 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 coated plate is washed, blocked, and washed to obtain a blocked plate. The plate is preferably washed and washed three times using PBST buffer. The plate is preferably blocked with a 5% by weight skim milk solution 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 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 srEPF protein cleaved by SUMO and labeled with HPR and then diluted 4000 times. The amino acid sequence of srEPF after removing the immunosuppressive peptide (FRDGDILGKYV) is as shown in SEQ ID No. 3, specifically as follows:

[0052] MAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVVAV GSGSKGKGGEIQPVSVKVGDKVLLPEYGGTKVVLDDKDYFL.

[0053] The present invention adds 3,3',5,5'-tetramethylbenzidine to the obtained incubation plate and continues to incubate. After terminating the reaction, the 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 a positive pregnant bovine serum; when the inhibition rate PI value is greater than 0.5415, it is a 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 wells 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 for 15 minutes. In the present invention, the reaction is preferably terminated using a 2 mol / L sulfuric acid solution.

[0055] In order to further illustrate the present invention, the present invention is described in detail below with reference to the 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 Co., Ltd.

[0061] 1.1.2 Materials and reagents

[0062] IPTG (isopropyl thiogalactoside) was purchased from Solabio Technology Co., Ltd. of China; Rainbow pre-stained protein marker was purchased from Beijing Joinergy Biotechnology Co., Ltd.; M5 HiPer ECL Western HRP Substrate was purchased from Beijing Joinergy Biotechnology Co., Ltd.; TMB color developing solution A and B were purchased from Beijing Meixinwande Biotechnology Co., Ltd.; goat anti-mouse IgG secondary antibody was purchased from Immunoway; skimmed milk powder was purchased from Shengong Bioengineering Co., Ltd.; 0.22 μm PVDF membrane and 0.5 mL ultrafiltration tube were purchased from Merck Millipore; 96-well ELISA plate was purchased from Guangzhou Jietian Biological Filtration Co., Ltd.; HRP conjugation kit was purchased from Beijing Meixinwande Biotechnology Co., Ltd.; mouse monoclonal antibody subclass identification ELISA kit was purchased from Luoyang Biaotong Experimental Material Center; Bovine EPF ELISA kit was purchased from Shanghai Lanpai Biological Technology Co., Ltd.; bovine serum was collected from Jiulong Cattle Farm in Changchun, Jilin Province and treated by routine 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 with the inhibitory region removed was selected as the target gene. The sequence was added with polyhistidine (6×His) at the N-terminus. Primer Primer 5.0 was used to design the upstream and downstream primers, and Nde I and Xho I enzyme digestion sites were added, respectively. The target gene was synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. The target gene was amplified by PCR, connected to the pMD18-T vector, transformed into E. coli DH5α competent cells, and plated and cultured. Single colonies were picked and the bacterial liquid was identified correctly. The recombinant plasmid was double-digested, and the target gene was connected to the pET-22b vector and transformed into E. coli BL21 (DE3) competent cells. The cells were inoculated in liquid LB medium containing ampicillin and cultured at 37°C to the logarithmic growth phase. The OD 450nmWhen the value is about 0.5, 1 mM IPTG is added for induction for 5 h, and the bacterial solution is collected, subjected to SDS-PAGE gel electrophoresis and stained. Then the protein gel is added into a 0.25 mol / L KCl solution for complete soaking, reacted at 4°C for 5 min, and the target band is cut and put into a dialysis bag, added with a regular electrophoresis solution for complete soaking of the target band, sealed, placed in an electrophoresis tank with an ice-water mixture, and subjected to 80 V voltage for 2 h to obtain the purified rEPF protein. The purified rEPF protein (SEQ ID No. 3) (only the EPF sequence with the inhibitory region removed is provided in this part) is identified by SDS-PAGE and Western blot.

[0066] Amplification of the target fragment:

[0067] Table 1 PCR system

[0068] Reagents Volume (μL) 10 x PCR buffer 5 2.5 mM dNTP 4 22b EPF-276-F 2 22b EPF-276-R 2 Synthetic plasmid template 1 EXTaq enzyme 0.5 ddH2O 35.5 Total 50

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

[0070] The purified target fragment is connected to a pMD18-T vector. The connection pMD18-T system is 10 μL: PCR gel recovery product 4 μL, pMD18-T vector 1 μL, Solution I 5 μL. The connection pMD18-T conditions are 16°C, overnight. The connection product is transformed into E. coli DH5α competent cells, plated, and single colony strains are picked and amplified in LB liquid medium. The correct single colony strain is verified by PCR, and the correct positive pMD18-T-DH5a is sent to Shanghai Shenguo for sequencing and further verification.

[0071] The positive pMD18-T-DH5a with correct verification result is plasmid-extracted and subjected to enzyme digestion to recover the target fragment. The pET22b target gene and expression enzyme digestion system and conditions are as follows: positive EPF-pMD18-T / empty vector pET22b 25 ul; Nde I 1 ul; EcoR I 1 ul; 10×QuickCut Buffer 3 ul; 37°C, 60 min. The positive EPF-pMD18-T / empty vector pET22b is transformed into E. coli BL21 (DE3) competent cells, inoculated into liquid LB medium containing ampicillin, and cultured at 37°C 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] CATATGGCAGGACAGGCATTTAGAAAGTTTCTTCCCCTCTTTGACCGAGTATTAGTTGAAAGAAGTGCGGCCGAAACTGTAACCAAAGGAGGGATTATGCTTCCAGAAAAATCACAAGGAAAAGTATTGCAAGCAACGGTGGTAGCTGTTGGATCAGGCTCTAAAGGAAAGGGTGGAGAGATTCAACCAGTTAGTGTGAAAGTTGGAGATAAAGTTCTTCTCCCAGAATATGGAGGCACCAAAGTAGTTCTAGACGACAAGGATTATTTCTTATTTAGAGATGGTGACATTCTTGGGAAATATGTCGACCTCGAGCACCACCACCACCACCACTGA.

[0074] Upstream and downstream primer sequences: according to the primer with upstream Nde I and downstream Xho I enzyme cutting site, the fragment after removing the inhibition zone (276 bp) (connecting pET-22b), the primer sequence is 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 srEPF detection original

[0079] The research group has successfully constructed pET-28a-srEPF positive plasmid (Wang C, Wang R, Zhang H L, et al. Prokaryotic expression and preparation of polyclonal antibody of recombinant early pregnancy factor [J]. Hubei Agricultural Sciences, 2023, 62(05): 129-34.) and transferred into BL21(DE3) competent cells. The correct positive bacteria liquid was resuscitated, srEPF was prepared and purified according to the literature method, and the amino acid sequence is shown as SEQ ID No. 3.

[0080] 1.2.3 Preparation of EPF mAbs

[0081] The immunogen rEPF was used to immunize 6-8 week old female Balb / c mice subcutaneously on the back three times, with an interval of 14 days between immunizations and an immunization dose of 50 μg / mouse. The serum antibody titer was detected by indirect ELISA, and mice with higher titers were taken for booster immunization. After the immunization, the immunized mice were euthanized, and the spleen cell suspension was fused with the SP2 / 0 cells that had been revived 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 and 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 1 week later, 1x10 6 The mouse abdomen was swollen and the ascites was extracted after the abdomen was swollen. The ascites was centrifuged at 8000 rpm for 10 minutes and frozen.

[0082] 1.2.4EPF ascites antibody purification

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

[0084] 1.2.5 Identification of the Immunological Properties of EPF mAbs

[0085] Titer determination: 1 μg / mL recombinant protein was coated on an ELISA plate at 100 μL / well at 4°C overnight; the plate was blocked with 5% skim milk powder for 1 hour; EPF mAb was diluted at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, and 1:1024000, and incubated at 100 μL / well for 1 hour; HRP-labeled goat anti-mouse IgG antibody was added at 100 μL / well; after 1 hour, substrate was added for color development for 10-15 minutes; the reaction was terminated with stop solution, and the OD was measured. 450nm The absorbance value was set at 0.05, and the absorbance ratio of the monoclonal antibody to the negative control (P / N) was detected. If P / N>2.1, it was determined to be positive, and the positive result with the largest dilution ratio was used 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 series for indirect ELISA detection. The reaction curve was drawn and the affinity constant Ka was 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 calf serum were mixed with loading buffer, respectively, and subjected to 15% SDS-PAGE electrophoresis. The proteins were transferred from the gel to a 0.22μM PVDF membrane using wet transfer at 200mA for 1h30min. Blocking was performed with 5% skim milk at room temperature for 2h. Subsequently, EPF monoclonal antibody was diluted 1:1000 and incubated at 4°C for 12h. The membrane was washed three times with TBST (10min each). HRP-labeled goat anti-mouse IgG was diluted 1:5000 and incubated at 37°C for 1h. The membrane was washed four times with TBST and developed using M5 HiPer ECLWestern HRP Substrate (ultrasensitive ECL luminescent solution).

[0091] 1.2.6 Performance Analysis of Early Pregnancy Diagnosis Based on EPF and mAb Antigen Competitive ELISA

[0092] In order to evaluate the potential use of EPF mAbs as diagnostic reagents for early pregnancy detection in cattle, an antigen competition ELISA based on EPFmAb was designed. The EPF after SUMO digestion and purification of srEPF was labeled with HRP (Beijing Meikewande Biotechnology Co., Ltd.). After verification of successful labeling, antigen competition ELISA was performed by using the free EPF antigen in the serum to compete with EPF-HRP for solid-phase EPF mAb. Eight positive and five negative serum samples from pregnant cattle were used, and antigen competition ELISA was performed after serial dilution with PBS at a ratio of 1:10-1:80. The OD values ​​were analyzed. 450nmValue. After the conditions were optimized, the specific detection was as follows: B6 was fixed in a transparent 96-well microplate (Guangzhou Jiete Biofiltration Co., Ltd.) at 0.1 μg per well in CBS (0.05 mol / L, pH 9.6) at 4°C for 12 hours. 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 a wavelength of 450 nm was measured and analyzed by a spectral analyzer (UV-ELISA).

[0093] 1.2.7 Repeatability Evaluation of Competition ELISA

[0094] Five positive and five negative serum samples from pregnant bovines were tested using ELISA plates coated from the same batch. Each sample was tested three times, and the mean of the three replicates was calculated. The results were statistically analyzed, and the within-plate relative standard deviation (RSD) was calculated as RSD (%) = (standard deviation / mean) × 100%. The reproducibility of this method was evaluated by calculating the inter-plate RSD using ELISA plates coated from three different batches. The samples were tested using the same method, and the inter-plate RSD was calculated.

[0095] 1.2.8 Practical Evaluation 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 practicality of this method.

[0097] 2. Results and Analysis

[0098] 2.1 Results of immunogen rEPF-induced expression

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

[0100] 2.2 Detection of the induced expression results of the original srEPF

[0101] After activation of the positive pET-28a-srEPF bacterial solution, IPTG is used for induction expression, and SDS-PAGE analysis is performed after ultrasonic crushing, and the result is shown in Figure 2 In the middle a, the srEPF protein supernatant is expressed, which is consistent with the expectation; after crushing and affinity chromatography purification of the supernatant, the result is shown in Figure 2 In the middle b, imidazole can be eluted at a concentration of 80-300 mmol / L, and the purity is high, the eluate is collected, srEPF is concentrated by 10 kDa ultrafiltration tube, and imidazole is replaced with PBS. After purification, srEPF is shown in Figure 2 In the middle c, there is a clear band at 26 kDa, which is consistent with the expected result.

[0102] 2.3 Preparation and property identification of EPF mAb

[0103] Three strains of specific monoclonal antibody positive hybridoma cell strains capable of stably secreting EPF protein are screened in the application, and are named 8E6BB6, 8E6BF8 and 11E3GF6. Part of the positive cell strains are frozen for standby, and part of them are expanded and cultured, and EPF mAb is prepared by mouse in vivo induced ascites method. The collected mouse ascites is mixed with an equal volume of binding buffer, and purified by AKTA protein purification system, and the result is shown in Figure 3 In the middle a, the ascites passes through the affinity chromatography column, the antibody is captured, the flow passes through the impurity peak, and the single antibody peak after elution is the target peak. The purified EPF mAb B6, F8 and F6 are taken to measure the titer, and the results are shown in Tables 2 and 3, and the titers of the three antibodies all reach 10 -6 The above.

[0104] Table 2 EPF mAb titer determination

[0105]

[0106]

[0107] The prepared three strains of EPF mAbs were subjected to subclass identification using a mouse mAb subclass kit, and the results are shown in Table 1. Figure 3 Table 1: Subclass identification of three strains of EPF mAbs 450nm The OD values of the three strains of EPF mAbs were the highest when the subclass was IgG1, and thus it was determined that the three strains of mAbs were of the IgG1 subclass.

[0108] The EPF mAb affinity curve is shown in Table 2. Figure 3 Table 2: EPF mAb affinity curve -10 For example, when the coating antigen concentration was 0.5 μg / mL and 1.0 μg / mL, the [Ab]t and [Ab']t were 1.35 x 10-8 mol / L and 9.22 x 10-8 mol / L, respectively, which were calculated according to the formula, and the Ka was 1.4 x 10-7 L / mol, while the affinity constants of F8 and F6 were 1.2 x 10-7 L / mol and 8.7 x 10-7 L / mol, respectively, which were measured simultaneously, indicating that the EPF mAbs prepared in the present application have good affinity. -11 9 9 8

[0109] To detect the reaction specificity of the EPF mAbs with the natural EPF protein, SDS polyacrylamide gel electrophoresis was performed using the srEPF recombinant protein and pregnant cow serum as antigens, and Western-blot detection was performed using the obtained EPF mAbs after membrane transfer, and the results are shown in Table 3. Figure 3 Table 3: Western-blot detection of EPF mAbs Figure 3 Table 3: Western-blot detection of EPF mAbs

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

[0111] B6, F8 and F6 were coated at appropriate concentrations, and different dilution multiples of EPF-HRP were added to verify whether the coupling was successful by ELISA. The results are shown in Table 4. Figure 4 Table 4: Coupling of EPF-HRP to B6, F8 and F6 450nm ​​​​The absorbance value decreased with the increase of the dilution degree of EPF-HRP. EPF was successfully coupled with 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. The checkerboard method was used. The coating concentrations of EPF mAb B6 were 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL and 0.125 μg / mL, and the dilution degrees of EPF-HRP were 500 times, 1000 times, 2000 times, 4000 times and 8000 times, respectively. The results are shown in Figure 4 Fig. 2a. 450nm When the absorbance value was about 1, the detection was more sensitive. Therefore, the OD value was selected to be about 1. Considering the principle of saving antibodies, the coating concentration of EPF mAb B6 was finally determined to be 1 μg / mL, and the corresponding dilution degree of EPF-HRP was 4000 times. According to the optimized coating amount of EPF mAb and the optimal dilution degree of EPF-HRP, four coating conditions were set: 4°C for 12 h, 37°C for 1 h, 37°C for 2 h and 37°C for 4 h. The negative control group was set to perform the antigen competition ELISA experiment. The coating conditions were taken as the horizontal coordinates, and the OD 450 values and the inhibition rates PI = (blank OD 450nm - sample OD 450nm ) / blank OD 450nm were taken as the vertical coordinates, respectively. The results are shown in Figure 4 Fig. 2c. When the coating was performed at 4°C for 12 h, the PI value was the highest. Therefore, 4°C for 12 h was selected as the coating condition. Eight positive serum samples of pregnant cows and five negative serum samples were diluted with PBS at a ratio of 1:10 to 1:80 in a continuous volume ratio for detection. The absorbance of the positive and negative serum samples at OD 450nm was analyzed at each dilution degree. The detection results are shown in Figure 4 Fig. 2d. When the dilution ratio was 1:10, 1:40 or 1:80, there was no significant difference in the OD 450nm values of the positive and negative serum samples. When the dilution ratio was 1:20, the OD 450nm values of the positive and negative serum samples were significantly overlapped, indicating that the difference was significant at this dilution degree, and the positive and negative serum samples could be distinguished.

[0112] 2.5 Evaluation of the repeatability and reproducibility of the competition ELISA

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

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

[0115]

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

[0117] The established antigen competition ELISA and a similar commercial kit (Bovine EPF ELISA KIT) were used to test a total of 50 serum samples in parallel, 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 receiver operating characteristic (AUC) curve for the DC-ELISA was 0.833, with a standard error (SE) of 0.06 and a 95% confidence interval (CI) of 0.714-0.951. ROC curve analysis showed that when the PI cutoff value of the competitive ELISA was 0.5415 (PI < 0.5415, indicating positive pregnant bovine serum), the specificity and sensitivity were optimal, at 82.61% and 81.48%, respectively, both exceeding 70%, indicating that the test performed well. Figure 5 As shown in b, the difference between positive and negative pregnant bovine serum detected by DC-ELISA is significant. 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 Structure of Monoclonal Antibody Variable Regions

[0119] Given that the B6 antibody has good reaction characteristics with EPF, the present invention sequenced the variable regions of the B6 antibody and used bioinformatics software to perform structure prediction and interaction analysis to preserve cell and gene resources for subsequent antibody applications.

[0120] Cell culture and RNA extraction

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

[0122] Antibody variable region sequence composition:

[0123] Sequencing analysis revealed that 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 modeling and analyze the structure and interaction of B6 monoclonal antibody and EPF protein. AlphaFold is a protein structure prediction method based on deep learning, which can integrate local and global features according to protein sequence information to generate high-precision three-dimensional structure prediction results. PyRosetta is a molecular modeling tool based on the Rosetta algorithm, which 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 (blue area in middle D), indicating 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 is above the medium level (ipTM>0.5 is considered reliable), the spatial arrangement of the interface residues is reasonable, but the local conformation such as the side chain orientation still has room for optimization; pTM = 0.72: the overall folding has a high similarity to the native 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 and molecular docking); in the EPE heat map ( Figure 6 In the right side of D), 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) shows that the two proteins form a reasonable three-dimensional conformation, with high confidence in key interface residues and a compact and stable overall fold. The heavy chain of the B6 monoclonal antibody is marked in green, the light chain in yellow, and the EPF protein in purple.

[0132] PyRosetta was further used to perform overall structure prediction and energy evaluation on B6 mAb (heavy chain and light chain sequences), and the structure of B6 mAb predicted by PyRosetta was obtained (see Figure 6 C in the middle: 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 center): B6 heavy chain in green, light chain in yellow, and EPF protein in blue.) Combined with data analysis results, the complex structure energy is approximately -373.3, indicating that this complex is the most stable and exhibits stable binding. This suggests that the binding between the B6 monoclonal antibody and the EPF protein is highly reliable and will serve as a focus for subsequent research.

[0133] AlphaFold predicts that B6 and EPF complex ( Figure 6 Middle E) and ClusPro predicted B6 and EPF complex ( ​ Compared to (center F), the complex exhibits a relatively loose conformation in some regions, possibly due to limitations in the ClusPro sampling algorithm or insufficient processing of binding site flexibility. However, the overall folding and energy distribution indicate that the complex maintains a reasonable three-dimensional structure and good stability, as expected.

[0134] In summary, the interaction predictions for B6 mAb and EPF protein using multiple computational modeling and analysis strategies (AlphaFold, PyRosetta, and ClusPro) demonstrate good binding characteristics, with various scoring and energy analyses supporting stable binding. These predictions provide a reliable theoretical basis for antibody affinity maturation and the optimization of corresponding detection methods.

[0135] 3. Discussion

[0136] Early and super early pregnancy diagnosis of cattle is an important link of preventing miscarriage, reducing empty pregnancy, increasing production performance and improving reproductive rate. The ideal early pregnancy diagnosis technique of cattle should have the characteristics of showing diagnosis effect within one estrus cycle after mating, diagnosis accuracy rate of 85% or more for pregnancy and non-pregnancy, safety to mother and fetus, simple and convenient method, economic and practical, etc. The traditional diagnosis methods of ultrasonic detection and rectal detection in the past will cause invasive injury to cattle, and improper operation will also cause embryo death and cause great economic loss. In recent years, with the development of analysis technology, it is found that animal embryos can produce progesterone, testosterone, EPF, pregnancy-specific protein, pregnancy-related protein and pregnancy serum protein. EPF is a pregnancy-related factor that appears in the serum of the mother's body after fertilization, which can appear in a few 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 be used as a marker for embryo status in the later stage of pregnancy. The research of EPF has great significance for dairy farming industry.

[0137] EPF is an active protein with immunosuppression in the serum of the mother's body after fertilization. In 1998, Clara et al. characterized and identified EPF, and found that EPF is an immunomodulatory active peptide with Fc receptor. Studies have shown that EPF affects the function of lymphocytes by inhibiting the synthesis of lymphocyte DNA, thereby down-regulating the immune function mediated by T cells, so that the fetus is not eliminated as a foreign body in the mother's body. Therefore, in order to prevent the interference of immunosuppression in the preparation of EPF monoclonal antibody and enhance its immunogenicity, the protein of EPF gene without inhibitory region was expressed by using the conventional molecular cloning technology of the research group, and the EPF monoclonal antibody was prepared by hybridoma technology. The titer analysis showed that the expressed recombinant antigen had good immunogenicity and could stimulate the body to produce good immune response, which provided a good experimental basis for the preparation of hybridoma and the acquisition of antibody.

[0138] It is reported that the basic activity of EPF is a polypeptide containing 101 amino acids, but due to the complexity of serum itself, different laboratories use different methods to isolate and purify EPF in different species at different gestational ages, and the molecular weight obtained is also different. Wilson et al. purified two polypeptides with EPF activity from the serum of pregnant ewes at 3-8 weeks of gestation, with molecular weights of 20 kDa and 67 kDa, respectively. Metha et al. isolated and purified EPF active polypeptide from the serum of pregnant women at 5-12 weeks of gestation. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weight of the purified EPF was about 21 kDa. However, analysis by gel permeation chromatography (HPLC) showed that its molecular weight was 28 kDa. Xiangya et al. isolated and purified EPF polypeptide with high activity from pregnant cow blood, with a molecular weight of 44 kDa. In this study, we prepared EPF ascites mouse monoclonal antibodies with high titer and high affinity, which were identified by Western-blot. At 25 kDa and 45 kDa, three antibodies can specifically bind to pregnant cow serum. The molecular weight of EPF is different, and it is speculated that it may carry some carriers itself, or the protein itself forms a dimer and a trimer, and the molecular form is complex, but the specific way that leads to the difference in molecular weight has not been reported in detail, and further in-depth study is needed.

[0139] AlphaFold uses a deep learning strategy to predict the three-dimensional structure of proteins based on sequence information. It has high prediction accuracy for both monomeric proteins and multi-protein complexes. PyRosetta is a molecular modeling tool based on the Rosetta algorithm, which can optimize protein structure, docking, and energy evaluation. The core idea is to gradually optimize the structure to make the system tend to a more stable low-energy state. In general, 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 different conformations generated by docking and evaluates the stability and reliability of the conformations based on energy scores. Using the above three modeling and docking strategies to analyze the structure and interaction of B6 mAb and EPF protein, we found that monomeric protein prediction and complex docking AlphaFold both showed high confidence and accuracy; PyRosetta further predicted and energy-optimized B6 mAb to obtain a more stable antibody structure;

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

[0141] ELISA is a sensitive, specific, simple method for detecting bacteria, proteins and viruses, which is widely used in biological and medical fields due to its accuracy, repeatability, stable reagent and cheap equipment. There are some research reports on the method of detecting EPF in pregnant serum based on ELISA technology. Chen Feihu et al. used EPF-McAb to detect the serum of clinically diagnosed pregnant women and non-pregnant women, and used ELISA and Ea-RIT to detect EPF antigen at the same time. The detection rate of ELISA was 92.98%, which was significantly higher than that of Ea-RIT. Yi Chuzhu et al. prepared anti-EPF monoclonal antibody to establish a double monoclonal antibody sandwich ELISA method to detect the activity of EPF, with a sensitivity of 90.2%. Based on this, this study first used HRP labeling kit to couple the detection of EPF with activated HRP to prepare competitive antigen. Ascites monoclonal antibody B6 was used as solid-phase antibody to coat in the microwell plate, and the free target antigen in the detection was used to compete with the HRP-coupled antigen for the solid-phase EPF monoclonal antibody. After optimizing the reaction conditions, the positive and negative serum of pregnant cattle was detected. The detection method established in this study has the advantages of good differentiation between positive and negative, and the detection rate of competitive ELISA in positive samples is 81.48%(22 / 27), which has good detection performance and provides reference for early pregnancy detection in cattle farm. The EPF content in positive and negative pregnant cattle serum detected by parallel determination of commercial quantitative detection kit has no significant difference. It is analyzed that EPF-A exists in the form of precursor in the serum near the ovulation period in mice, humans, sheep and pigs, and its production does not depend on fertilization or pregnancy. However, in pregnant mice, the existence time of EPF-A is longer than that in non-pregnant mice. Therefore, combined with this detection method, we speculate that the non-pregnant cattle serum also contains EPF, and its content may be slightly lower than that of pregnant cattle. If EPF is used to distinguish pregnant and non-pregnant, more specific antibodies and more accurate detection of EPF content are needed. In the follow-up study, our research group will determine the epitope 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 breeding farm, monitor the normal development of fetus in real time, improve the reproductive efficiency of animals, and increase the economic benefit of animal husbandry.

[0142] 4. Conclusion

[0143] Three monoclonal antibodies of EPF were prepared by using hybridoma cell technology, cell screening and subcloning, AKTA purification, after the identification of the characteristics of the monoclonal antibodies of EPF by ELISA, Western blotting and other technologies, the ELISA method based on antigen competition was developed for the detection of EPF in pregnant bovine serum, when the PI cutoff value was 0.5415, the detection specificity and sensitivity reached as high as 82.61% and 81.48%. At present, China's dairy farming industry is developing towards scale, intensification and standardization, and the dairy farming industry has become a breakthrough in the development of animal husbandry. Dairy farming is a high input and high output industry, in the daily feeding management, improving the milk yield and reproduction rate of dairy cows is the key to improve economic efficiency. With the improvement of people's understanding, more and more people realize the importance of early pregnancy diagnosis, and early pregnancy diagnosis technology will develop rapidly. The monoclonal antibodies of EPF produced in the application can provide a detection strategy for the development of a bovine pregnancy diagnosis kit using blood samples, and provide a reference for monitoring fetal development and early prediction of pregnancy outcome in dairy cows.

[0144] Although the above embodiment makes a detailed description of the present application, it is only a part of the embodiment of the present application, not all the embodiments, and people can obtain other embodiments under the premise of no creativity according to the embodiment, and these embodiments all belong to the protection scope of the present application.

Claims

1. Use of monoclonal antibody B6 in the preparation of a bovine early pregnancy diagnostic reagent, wherein 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 method for detecting early pregnancy in cattle for non-diagnostic purposes, characterized in that: The following steps are involved: 1) Coating the monoclonal antibody B6 in a microplate to obtain a coated plate; 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) washing, blocking, and washing the coated plate obtained in step 1) to obtain a blocked plate; 3) adding a bovine serum solution and an EPF-HRP solution to the blocking plate obtained in step 2) and incubating the plate, followed by washing 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, measure the 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.

3. The method according to claim 2, characterized in that The microplate in step 1) is a transparent 96-well microplate; The coating amount of the monoclonal antibody B6 in the wells of the microplate was 0.1 μg / well.

4. The method according to claim 2, characterized in that The coating conditions of the monoclonal antibody B6 in step 1) include: coating at 4° C. for 12 hours.

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

6. The method according to claim 2, characterized in that In step 3), the amount of bovine serum solution added to the wells of the blocking 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 it 4000 times. The amino acid sequence of the srEPF protein is shown in SEQ ID No.

3.

7. The method according to claim 2, characterized in that The incubation conditions in step 3) include: a time of 1 hour and a temperature of 37°C.

8. The method according to claim 2, 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: temperature of 20-30°C and time of 15 minutes; The reaction was terminated with 2 mol / L sulfuric acid solution.

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