A detection reagent for frozen semen of guizhou black pig

Abstract

The application provides a detection reagent for frozen semen of Huai pig, and first acquires the raw semen, and then carries out the following steps: ammonium sulfate fractionation precipitation, DEAE cellulose filter gel column chromatography, CM Sepharose FastFlow gel chromatography and Sephadex G-100 chromatography to separate and purify the NAGase of the semen of the Huai pig, and monoclonal antibodies JN21 and JN37 are prepared by taking the NAGase as an antigen, and the monoclonal antibodies can specifically recognize the NAGase of the Huai pig. The prepared double-antibody sandwich ELISA kit can rapidly and quantitatively detect the NAGase content in the semen of the Huai pig, and provides a new thought for Huai pig breeding and purification and rejuvenation. The kit is convenient to operate, low in use cost, good in repeatability and suitable for being widely applied.

Description

TECHNICAL FIELD

[0001] The present application relates to a monoclonal antibody of NAGase derived from hog and a detection method thereof, in particular to a detection method of NAGase in hog semen. BACKGROUND

[0002] Hog is a local pig breed widely distributed in the southwest mountainous area of Fujian Province, mainly produced in Shanghang, Zhangping and Pinghe, etc. According to the "Shanghang County Annals·Product Annals", the hog is a local breed, with moist fur and thin skin, sweet and delicious meat, and has been raised in Shanghang County for more than 1000 years. The body is short, the chest is wide and deep, the back is wide and concave, the abdomen is round and large, the whole is oblique, the hips are not full, mostly lying, the thighs are thick, and the tail root is thick. It has the characteristics of roughage tolerance, early maturity, strong adaptability, good stress resistance, high intermuscular fat content and delicious and tender meat, which is deeply loved by local consumers. How to maintain and develop the germplasm resources of local pig breeds has become the top priority of conservation and development.

[0003] β-N-acetylglucosaminidase (β-N-acetylglucosaminidase, NAGase for short, EC 3.2.1.52) is an enzyme belonging to the chitin catabolic cascade. As one of the key enzymes for hydrolyzing glycosidic bonds, NAGase is widely distributed in nature and participates in many biological processes, and can hydrolyze the β-N-acetylglucosaminidase bond in polysaccharides, protein polysaccharides and glycoproteins. Further studies have found that NAGase also widely exists in the reproductive organs of vertebrates and male semen, with high enzyme activity, mainly hydrolyzing the β-1, 4-glycosidic bond of N-acetyl-β-D-glucosamine, not only participating in the decomposition and metabolism of sugar substances, but also being an oligosaccharide chain modification enzyme in glycoprotein and glycolipid biosynthesis, and also related to the degradation of glycoprotein, and also plays an important role in the process of sperm-egg combination. There is a strong correlation between the content and sperm motility, the combination of sperm and egg in the process of fertilization, and the pregnancy rate of female animals.

[0004] Applicant in the previous study with local characteristics of pig breeds Huai pig semen as the material, separation and purification of N-acetyl-beta-D-glucosaminidase closely related to the reproductive physiology of Huai pig, and further study of the physical and chemical properties, kinetic properties, explore the functional groups of enzyme active center, catalytic mechanism and other enzymatic characterization. The activity analysis of NAGase in the prior art is mainly based on p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNp-beta-D-GlcNAc) as the substrate, under the condition of partial acidity, the ability of enzyme catalyzing the hydrolysis of the substrate. This method can effectively, simply and quickly detect the presence and activity of NAGase, but this method is not applicable to the purification and preparation of the enzyme, electrophoretic activity staining of the enzyme in different tissues of the organism and different growth and physiological periods. How to directly and quantitatively detect the NAGase content in the Huai pig semen, especially in the frozen semen, and establish the relationship between the NAGase content and the sperm activity and the sperm-egg binding force still need to be solved. SUMMARY

[0005] In order to solve the above problems, the application provides a double antibody sandwich ELISA kit for NAGase content in Huai pig semen, which is simple to operate, good in stability and repeatability, and has good reliability.

[0006] Firstly, the application provides a method for separating and purifying NAGase in Huai pig semen, which comprises: obtaining of the raw semen, ammonium sulfate fractionation precipitation, DEAE cellulose filter gel column chromatography, CM Sepharose FastFlow gel chromatography, and Sephadex G-100 chromatography.

[0007] Further, the application provides a monoclonal antibody for NAGase in the above-mentioned Huai pig semen, which is obtained by immunizing BALB / c mice with purified Huai pig NAGase through a specific immunization program, and is fused and screened through hybridoma technology. The monoclonal antibody is secreted by JN21 and JN37 hybridoma cells, and can specifically recognize Huai pig NAGase.

[0008] The amino acid sequence of the light chain variable region of JN21 is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2.

[0009] The amino acid sequence of the light chain variable region of JN37 is shown in SEQ ID NO. 3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4.

[0010] Further, the application provides a double antibody sandwich ELISA kit, which comprises:

[0011] (1) an enzyme-labeled plate coated with a capture antibody;

[0012] (2) Enzyme-labeled detection antibody.

[0013] Wherein, the capture antibody is obtained by secretion of hybridoma cell strain JN21, and the detection antibody is obtained by secretion of hybridoma cell strain JN37.

[0014] Further, the application provides an ELISA method for rapidly and quantitatively detecting the NAGase content in the semen of the Huai pig by using the kit, wherein the concentration of the coating antibody, the coating liquid, the blocking liquid, the standard diluent, the enzyme-labeled antibody diluent and the enzyme-labeled antibody dilution concentration are optimized. The formula for establishing a standard curve is Y=0.00526X+0.1303, R 2 =0.98, and the linear range of detection is 5-400 ng / mL.

[0015] Advantages

[0016] The separation and purification method for the NAGase in the semen of the Huai pig provided by the application is simple and fast, and a protein with high purity can be obtained without additional processing. The monoclonal antibodies JN21 and JN37 prepared by using the antigen can specifically recognize the NAGase in the Huai pig. The double-antibody sandwich ELISA kit prepared by using the same can be used for rapidly and quantitatively detecting the NAGase content in the semen of the Huai pig, further clarifies the reproductive physiological function of the NAGase in the pig, provides a theoretical basis for the research on other related enzymes in the fertilization process of mammals, provides a theoretical basis and technical reference for the research on other related enzymes in the fertilization process of mammals, and is also beneficial to finding an effective way for improving the quality of the semen of the Huai pig and increasing the fertilization rate, and provides a new idea for the preservation and purification of the Huai pig. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Separation and purification of NAGase in the semen of the Huai pig, wherein A is a DEAE cellulose chromatography protein concentration and enzyme activity map; B is a CM Sepharose FastFlow gel chromatography protein concentration and enzyme activity map; C is a Sephadex G-100 chromatography protein concentration and enzyme activity map; and D is a PAGE method test result of the purified NAGase.

[0018] Figure 2 WB detection result of the monoclonal antibody.

[0019] Figure 3 Standard curve of the quantitative ELISA detection method for the NAGase in the Huai pig. DETAILED DESCRIPTION

[0020] The following examples further illustrate the present application, but should not be construed as limiting the application. Modifications or variations of the method, steps or conditions of the application can be made by those skilled in the art without departing from the spirit and scope of the application. If not specified, the technical means used in the examples are the conventional means known to those skilled in the art.

[0021] Some of the reagents involved include: TGL-40B table low speed centrifuge; KFLOW pure water machine; ZD-9556 horizontal shaker; 96-well 8x12 detachable enzyme plate; Multiska Mks enzyme reader, Thermo Labsystems company; adjustable pipette, Thermo Labsystems company; tetramethyl benzidine (TMB); H2SO4, all reagents are analytical reagents.

[0022] Example 1 Isolation and purification of NAGase from sophy sperm

[0023] Take several fresh sperm of sophy (from Fujian Shanghang County Lvqi Sophy Breeding Farm) and mix them, then store them in a -80℃ refrigerator. Freeze-thaw the sperm repeatedly and perform ultrasonic disruption of sperm cells. Filter the gelatinous material with a gauze to obtain 500 ml of raw sperm plasma. Add 4℃ pre-cooled 0.01 mol / L pH 7.4 Tris-HCl buffer (containing 0.2 mol / L NaCl) at a ratio of 1:1 (V:V), extract at 4℃ for 12 h, and centrifuge at 4℃ at 10000xg for 20 min to obtain supernatant.

[0024] Take solid ammonium sulfate, grind it into powder repeatedly, and then add it gently to the continuously stirred supernatant until the saturation of ammonium sulfate in the supernatant reaches 35%. Place it in a 4℃ refrigerator for 4 h, then use a high-speed centrifuge to centrifuge at 4℃ at 10000g for 25 min, discard the precipitate and keep the supernatant. Again, gently add ammonium sulfate powder to the continuously stirred supernatant until the saturation of ammonium sulfate in the supernatant reaches 75%. Place it in a 4℃ refrigerator for 4 h, then use a high-speed centrifuge to centrifuge at 4℃ at 10000g for 25 min, discard the supernatant and keep the precipitate. Dissolve the precipitate with 4℃ pre-cooled 0.01 mol / L pH 7.4 Tris-HCl buffer, and then place it in a pre-soaked dialysis bag. Seal it and suspend it in 1.5 L of 4℃ pre-cooled 0.01 mol / L pH 7.4 Tris-HCl buffer, maintain 4℃ continuous stirring dialysis, and replace the dialysate every 2 h until there is no SO4 2- is detected, centrifuge at 4℃ at 12000xg for 30 min, and collect the supernatant to obtain a crude enzyme preparation.

[0025] The crude enzyme preparation was added to a DEAE-cellulose filter gel column equilibrated with 0.01 mol / L pH 7.4 Tris-HCl buffer. The column was eluted with 0.01 mol / L Tris-HCl (pH 5.7) buffer in an amount of 2.5 times the column volume to remove the impurities. Gradient elution was then performed using 0.01 mol / L Tris-HCl (pH 5.7) containing NaCl at a concentration of 0-2 mol / L, with a flow rate of 0.5 mL / min, and an automatic collector was used to collect one tube of eluate every 6 min. The protein concentration and enzyme activity of the eluate were plotted (see Fig. A), and the activity peaks were combined. Figure 1 A), and the activity peaks were combined.

[0026] The collected activity peaks were again dialyzed thoroughly and added to a CM Sepharose Fast Flow gel column equilibrated with 0.01 mol / L pH 7.4 Tris-HCl buffer. Gradient elution was then performed using 0.01 mol / L Tris-HCl (pH 5.7) containing NaCl at a concentration of 0-2 mol / L, with a flow rate of 0.5 mL / min, and an automatic collector was used to collect one tube of eluate every 6 min. The protein concentration and enzyme activity of the eluate were plotted (see Fig. B), and the activity peaks were combined. Figure 1 B), and the activity peaks were combined.

[0027] The eluate of the enzyme activity peaks was collected and loaded into a 13000 dp concentrating tube, and the eluate was centrifuged at 5000 g for 20 min at 4°C using a high-speed centrifuge to obtain an enzyme concentrate. After thorough dialysis, the concentrate was added to a Sephadex G-100 column equilibrated with 0.01 mol / L pH 7.4 Tris-HCl buffer, and gradient elution was performed using 0.01 mol / L Tris-HCl (pH 5.7) containing NaCl at a concentration of 0-2 mol / L, with a flow rate of 0.5 mL / min, and an automatic collector was used to collect one tube of eluate every 6 min. The protein concentration and enzyme activity of the eluate were plotted (see Fig. C), and the activity peaks were combined. The enzyme solution of the activity peaks was dialyzed thoroughly against buffer, centrifuged at 12000 x g for 30 min at 4°C, and the supernatant was collected and concentrated to obtain a pure enzyme preparation of the NAGase from the semen of the Chinese toad. Figure 1 C), and the activity peaks were combined. The enzyme solution of the activity peaks was dialyzed thoroughly against buffer, centrifuged at 12000 x g for 30 min at 4°C, and the supernatant was collected and concentrated to obtain a pure enzyme preparation of the NAGase from the semen of the Chinese toad.

[0028] The obtained product was detected for purity and property, wherein the enzyme activity of NAGase was determined as 2 mL of enzyme activity determination system: 1 mL of 0.1 mol / L PBS (pH 5.7), 80 μL of 5 mmol / L p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG), 20 μL of pure enzyme preparation of Jinhua pig semen NAGase (about 0.22 ng), and distilled water was added to 2 mL. The substrate pNP-NAG, distilled water and 0.1 mol / L PBS (pH 5.7) were preheated in a 37°C water bath for five minutes, then 20 μL of pure enzyme preparation of Jinhua pig NAGase was added, and the reaction was carried out at 37°C for ten minutes. 2 mL of 0.5 M NaOH solution was added to terminate the reaction, and the optical density value (OD405) at a wavelength of 405 nm was detected by a DU-800 nucleic acid protein analyzer.

[0029] The specific activity of the enzyme was defined as the number of enzyme activity units per milligram of enzyme protein. It refers to the amount of enzyme required to catalyze the hydrolysis of 1 μmol of product per minute per liter of solution under specific conditions, i.e., the ratio of enzyme unit activity to unit enzyme protein content. The unit enzyme protein content was determined according to the Lowry method.

[0030] The purity of the enzyme was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) polyacrylamide PAGE (see Figure 1 D). The results showed that the NAGase pure enzyme preparation obtained by ammonium sulfate fractionation and multi-stage chromatography of Jinhua pig semen had only one band near 70 KDa. The final pure enzyme preparation had a specific activity of 2982.52 U / mg.

[0031] Example 2 Screening and identification of Jinhua pig NAGase monoclonal antibody

[0032] Screening antibodies were prepared using conventional immunization methods in the field, summarized as follows: The purified *Sophora japonica* NAGase preparation (diluted with physiological saline to a 1 mg / mL solution) was used as the immunogen to immunize 6-week-old BALB / c mice five times, with each immunization spaced 2-3 weeks apart. The first immunization used *Sophora japonica* NAGase preparation plus an equal volume of Freund's complete adjuvant, at a dose of 100 μg / mouse, administered via multiple subcutaneous injections at multiple points on the neck and back. Subsequent immunizations used Freund's incomplete adjuvant. 12-15 days after the fourth immunization, a small amount of blood was collected from the tail, and serum titer was determined by indirect ELISA. If the serum titer was satisfactory, a booster immunization was performed three days before fusion, using *Sophora japonica* NAGase preparation injected intraperitoneally at a dose of 100 μg / mouse. One week after the final immunization, blood was collected from the tail, and antiserum titer was determined using an indirect non-competitive enzyme-linked immunosorbent assay (ELISA). Two wells (JN21 and JN37) with high ELISA values ​​and strong fluorescence intensity were selected for hybridoma fusion. Positive cells were screened using indirect ELISA, and subcloning was performed on the positive wells using limiting dilution. Hybridoma cell culture supernatant and monoclonal antibody ascites fluid were serially diluted 1:500 starting from 1:500. The titers of the monoclonal antibody supernatant and ascites fluid were determined by indirect ELISA. The titer results for the monoclonal antibody supernatant and ascites fluid are shown in Tables 1 and 2. Ascites fluid was purified using the caprylic acid-saturated ammonium sulfate method. After dialysis, the monoclonal antibody was obtained. Its concentration was determined using micro-UV method, aliquoted, and stored at -20℃.

[0033] Table 1. ELISA titers of monoclonal hybridoma cell culture supernatant

[0034]

[0035] Table 2 Ascites titer

[0036] Dilution fold 1:16000 1:32000 1:64000 1:128000 1:256000 JN21 2.141 1.942 1.785 1.207 0.916 JN37 2.315 2.049 1.903 1.460 1.226 Dilution fold 1:512000 1:1024000 1:2048000 1:4096000 1:8192000 JN21 0.622 0.524 0.394 0.184 0.076 JN37 0.903 0.716 0.428 0.192 0.083

[0037] The positive and negative cutoff values ​​for indirect ELISA (OD) 450 A value greater than 0.1 is considered positive. Therefore, the titer of JN21 and JN37 in the supernatant can reach 1:128000, and the titer in the ascites can reach 1:4096000.

[0038] Western blotting was performed using ascites fluid from JN21 and JN37 as the primary antibody and HRP-goat anti-mouse IgG as the secondary antibody. The results were as follows: Figure 2 The results showed that the above-mentioned monoclonal antibodies could specifically recognize Sophora japonica NAGase. The purified monoclonal antibodies were sent to BGI Genomics (Beijing) for sequencing, and the results were as follows:

[0039] The heavy chain variable region sequence of JN21 antibody is shown in SEQ ID NO:1, and its light chain variable region sequence is shown in SEQ ID NO:2.

[0040] QVQLQASAGAELTKCKASPAASVCMSGYTFT SACMH WVKQRAGQLEWIG INPGTGAYEYNQSFD KATLAADAKYMQ ALSSLTSESTADSAVCAS RHYGADADY WGCGTAVTVS (SEQ ID NO: 1)

[0041] DIVMKHMQTSHASTSVCDRVITC KAGAAVQDTA WYQQSPPAKGQLLIY WASTRHT GVPDRFTSGSGGTDHTLTISN VQSEDLDYFAC QYAPFST FGSGKLSIK (SEQ ID NO: 2)

[0042] The antibody heavy chain variable region sequence of JN37 is shown as SEQ ID NO: 3, and the light chain variable region sequence is shown as SEQ ID NO: 4:

[0043] DVQLVESGAGALVQKLSPGGSTFRCAASGAFSSF MGHAWAVR WVQTPEKGLEA YIASGGAYADSSTAI PVKG RFTI SSAEDTRDNPKNTLFLQMTSLRAMYYCAR GWADAGAWAFAF WGQGTQVVSA (SEQ ID NO: 3);

[0044] DIVMTQSHVGKFMSTSDRVSITC KASQDAVTSAA WYQQKQSPKLPGLIY WARHATS GVPDRFTGSGTDYTLTISGS SVQLYYSEDLTC QQAHPLRST FGAKLGTELN (SEQ ID NO: 4).

[0045] Example 3 Establishment of quantitative ELISA detection method for sophora NAGase

[0046] (1) Coat 96-well enzyme-labeled plates with JN21 at a series of dilutions (3 μg / mL, 1.5 μg / mL, 0.75 μg / mL, 0.375 μg / mL) in coating buffer (0.05 M carbonate buffer, pH 9.6), 100 μL / well, at 4°C overnight. The next day, take out the enzyme-labeled plate and return to room temperature, inject 200 μL PBST solution into each well, shake on the shaker for 3 min, shake off the washing solution, and dry on the blotting paper, continue to wash 2 times. The following washing method is the same.

[0047] (2) After washing thoroughly, the enzyme-labeled plate was blocked with blocking buffer (CBS containing 0.2% gelatin) at 200 μL / well, and incubated in a 37°C incubator for 2 h, then taken out and dried for use.

[0048] (3) The reaction plate was washed with PBST for 3 times, 200 μL / well, 3 min each time, and then the reaction plate was spun dry.

[0049] (4) The E. coli β-alkaline phosphatase solution was diluted with PBS to a series of concentrations of 6.25, 12.5, 25, 50, 100, 200, 400 ng / mL, and a PBS blank control was set. 100 μL of sample (mixed with lysis solution Triton X-100 in equal volume) was added to each well, and incubated at 37°C for 1 h.

[0050] (5) The plate was washed, and the step (3) was repeated.

[0051] (6) Enzyme-labeled monoclonal antibody JN37 was added, diluted by 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 100 μL / well, and reacted at 37°C for 45 min, and then washed as above.

[0052] (7) Freshly prepared TMB substrate was added for color development, 100 μL / well, and reacted at 37°C for 15 min.

[0053] (8) After color development, 50 μL of 2 mol / mL H2SO4 was added to each well to stop the reaction;

[0054] (9) The OD was detected by an enzyme-labeled instrument 450 .

[0055] After optimization, it was confirmed that the coating dilution of JN21 was 3 μg / mL, the dilution of enzyme-labeled monoclonal antibody was 4 μg / mL, the formula of the standard curve was Y=0.00526X+0.1303, R 2 =0.98, and the linear range of detection was 5-400 ng / mL.

[0056] Example 4 Consistency detection of sophy NAGase content and sperm activity

[0057] The semen stored in a 17°C constant temperature box was taken out and restored to room temperature, and 7 portions of sperm with decreasing sophy NAGase content (L1-L7, in order of decreasing content) were screened according to the method of Example 2 for subsequent experiments.

[0058] The sperm quality is the key factor to complete the fertilization, and is the most basic factor to judge and evaluate the boar's fertilization ability. The effective screening of the excellent boar semen can improve the breeding capacity of the farm, and is also beneficial to the breed cultivation. There are many related evaluation standards for the pig semen quality. Among them, the sperm motility and acrosome integrity are two indexes with significant representativeness and importance. The sperm has sufficient motility, which is the premise to carry out the fertilization process, such as penetrating the zona pellucida, and the acrosome integrity is the important basis for the sperm to complete the fertilization process.

[0059] Sperm motility determination: using the flat plate pressing method, a drop of semen (10 μl) was taken on a preheated (38℃) glass slide, covered with a cover glass, and observed under a 400x phase contrast microscope to estimate the motility (ten grade scoring method).

[0060] Acrosome integrity examination: after Giemsa staining, 200 sperm were randomly observed under a phase contrast microscope at 1000x, and the acrosome abnormality rate was calculated.

[0061] Giemsa staining preparation: ①Preparation: take 10 μl of semen on a glass slide, evenly spread the semen sample on the glass slide with a straight-edged glass slide at an angle of 35°, and naturally air dry to prepare a semen smear. ②Fixation: immediately take 1-2 ml of 10% formalin fixing solution and drop it on the naturally air-dried smear, fix for 15 min, gently rinse with water to wash off the fixing solution, and naturally air dry. ③Staining: drop Giemsa staining solution on the smear, stain for 1.5 h, then gently rinse with water, and air dry for examination.

[0062] The final results (Table 3) show that within a certain range, the content of NAGase in the semen can be used as an index to judge the motility and acrosome integrity of sperm cells, which is consistent with the previous research results. The NAGase enzyme activity promotes the motility and acrosome integrity of sperm cells, thereby improving the forwardness of sperm cells and the success rate of fertilization.

[0063] Table 3 NAGase content of 10 sperm samples and sperm motility consistency results

[0064] Detection index Arrangement order (from high to low) NAGase content L1 > L2 > L3 > L4 > L5 > L6 > L7 Sperm motility L1 > L3 > L2 > L4 > L6 > L5 > L7 Acrosome integrity L2 > L1 > L3 > L4 > L5 > L7 > L6

[0065] The above description of the embodiments is to facilitate the understanding and use of the present application by those skilled in the art. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present application is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A monoclonal antibody targeting β-N-acetylglucosidase in the semen of Sophora japonica, characterized in that, The amino acid sequence of the heavy chain variable region of monoclonal antibody JN21 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. The amino acid sequence of the heavy chain variable region of monoclonal antibody JN37 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

2. A double-antibody sandwich ELISA kit for detecting β-N-acetylglucosidase in the semen of Sophora japonica pigs, characterized in that... The antibody sandwich ELISA kit includes: (1) ELISA plate coated with capture antibody; (2) Enzyme-labeled antibody detection; The capture antibody is the monoclonal antibody JN21 as described in claim 1, and the detection antibody is the monoclonal antibody JN37 as described in claim 1.

3. An ELISA method for rapid quantitative detection of β-N-acetylglucosidase content in Sophora japonica semen using the double-antibody sandwich ELISA kit of claim 2 for non-disease diagnosis and treatment purposes.

4. The method of claim 3, comprising the following steps: (1) Dilute JN21 with coating buffer to 3 μg / mL and coat 96-well microplates, 100 μL / well, and incubate overnight at 4°C; the next day, remove the microplates and bring them to room temperature, inject 200 μL of PBST solution into each well, shake on a shaker for 3 min, shake off the washing solution vigorously, pat dry on absorbent paper, and wash twice more. The coating buffer was 0.05 M carbonate buffer with a pH of 9.6; (2) After thorough washing, block the microplate with blocking buffer, 200 μL / well, incubate at 37°C for 2 hours, then remove and dry for later use. The blocking buffer is CBS containing 0.2% gelatin. (3) Wash the reaction plate three times with PBST, 3 min each time, 200 μL / well, and then spin dry the reaction plate; (4) Dilute the Escherichia coli β-alkaline phosphatase stock solution with PBS to a series of concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 ng / mL, and set up a PBS blank control; add 100 μL of sample to each well and incubate at 37°C for 1 h. (5) Wash the plate, same as step (3); (6) Add enzyme-labeled monoclonal antibody JN37, serially diluted to 4 μg / mL, 100 μL / well, react at 37℃ for 45 min, and wash as above; (7) Add freshly prepared tetramethylbenzidine substrate for color development, 100 μL / well, react at 37°C for 15 min; (8) After color development, add 50 μL of 2mol / mL H2SO4 to each well to terminate the reaction; (9) Detect OD using an enzyme-linked immunosorbent assay (ELISA) reader 450 ; The formula for establishing the standard curve is: Y = 0.00526X + 0.1303, R0 2 =0.98, and the linear range of detection is 5–400 ng / mL.

5. A method for identifying the content of β-N-acetylglucosidase in the semen of a sow using the kit of claim 2 for purposes other than disease diagnosis and treatment.

6. The application of the monoclonal antibody of claim 1 or the kit of claim 2 in the breeding and preservation of Huai pigs.

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