CN108192875B - Hybridoma cell line and monoclonal antibody and detection kit of glycocholic acid based on hybridoma cell line - Google Patents

Abstract

The present invention relates to detection technique fields, in particular to hybridoma cell strain and glycocholic acid monoclonal antibody and detection kit based on hybridoma cell strain.The deposit number of the hybridoma cell strain is CGMCC No.15292.The technical effect that the present invention has are as follows: the glycocholic acid antibody titer generated by hybridoma cell strain of the present invention is higher, it can be used for enzyme linked immunosorbent assay analysis method (ELISA) detection glycocholic acid, have the characteristics that convenient sample operation, low in cost, rapid reaction, high sensitivity, high specificity.

Description

Hybridoma cell lines and monoclonal antibodies to glycocholic acid based on hybridoma cell lines test kit

technical field

The invention relates to the technical field of detection, in particular to a hybridoma cell line, a monoclonal antibody to glycocholic acid based on the hybridoma cell line, and a detection kit.

Background technique

Glycolic acid is one of the conjugated cholic acids formed by the combination of cholic acid and glycine. It is synthesized by hepatocytes, enters the intestinal tract with bile, and returns to the liver through the portal vein. When the hepatocytes are damaged, the liver cells have the ability to absorb glycocholic acid. decreased, resulting in an increase in blood levels. Glycolic acid is the largest amount of organic acid secreted into bile by the liver. It enters the intestinal lumen to help fat digestion and absorption. Most of it is reabsorbed in the ileum and colon. It enters the liver through the portal vein. Hepatocytes can efficiently absorb a large amount of glycocholic acid from the portal vein. acid, so that the amount of glycocholic acid in the blood is less than 1.9g/mL. The reabsorbed glycocholic acid enters the absorption cycle again, and through this mechanism, the body can make full use of this glycocholic acid. Once hepatocellular lesions, the concentration of glycocholic acid in the blood increases, among which acute hepatitis, chronic hepatitis slightly increase, cirrhosis, liver cancer patients significantly increase. When hepatocytes are damaged, the ability of hepatocytes to absorb serum cholyglycine (CG) decreases, resulting in an increase in the content of CG in blood; when bile stagnation is stagnant, the liver excretion of bile acid is hindered. The increased CG content in the reflux blood circulation also increases the CG content. Therefore, glycocholic acid is one of the sensitive indicators for evaluating the function of liver cells and the circulation of hepatobiliary substances.

In addition, in the middle and late pregnancy of pregnant women, due to the enlargement of the baby, it will oppress the liver and cause the liver to excrete cholic acid, resulting in high glycocholic acid. It can induce changes in the hepatobiliary system, make pregnant women susceptible to intrahepatic cholestasis of pregnancy (ICP), and have a serious impact on the fetus. The uterine birth rate is increased, and severe cases can lead to fetal death. Therefore, the determination of serum glycocholic acid in pregnant women is of great significance for early detection of ICP and understanding of the severity of ICP. The determination of glycocholic acid can be used as an indicator for screening and follow-up of ICP. , thereby reducing perinatal mortality.

At present, the quantitative determination of glycocholic acid in vitro mainly uses radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA) and so on. Radioimmunoassay requires professional radioimmunization facilities, which is difficult to carry out in ordinary laboratories, and the accuracy of radioimmunization method is low. Radioactive rays will also cause great harm to the health of operators, and are rarely used internationally. Chemiluminescence method has high sensitivity, but the determination speed is slow, the accuracy of test results and the stability of reagents are poor, and it requires expensive special chemiluminescence detection equipment, which is not conducive to the development of routine laboratories, and has obvious limitations in clinical application. Enzyme-linked immunosorbent assay (ELISA) has the characteristics of convenient sample operation, low cost, rapid reaction, high sensitivity and strong specificity, and can be better used for the detection of glycocholic acid. However, the currently available monoclonal antibodies to glycocholic acid have the problem of low titer.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a hybridoma cell line, a glycocholic acid monoclonal antibody and a detection kit based on the hybridoma cell line. The glycocholic acid monoclonal antibody has high titer, strong specificity and high sensitivity.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a hybridoma cell line, whose deposit number is CGMCC NO.15292.

The present invention also provides a monoclonal antibody to glycocholic acid, which is produced by the hybridoma cell line of the present invention.

In the present invention, the preparation method of the glycocholic acid monoclonal antibody is as follows: immunizing mice with glycocholic acid as an immunogen, fusing the spleen cells and myeloma cells of the immunized mice, using limiting dilution method and indirect ELISA screening technology, A positive monoclonal hybridoma cell line was obtained, ascites was induced, and after purification, the monoclonal antibody to glycocholic acid was obtained.

Preferably, the myeloma cells are SP2/0 myeloma cells.

Preferably, the mice are Balb/C mice.

The invention also provides the application of the glycocholic acid monoclonal antibody in the preparation of the glycocholic acid detection kit.

The present invention also provides a glycocholic acid detection kit, comprising the glycocholic acid monoclonal antibody provided by the present invention.

Preferably, the glycocholic acid detection kit further includes a coating antigen.

Preferably, the coating antigen is a conjugate of ovalbumin and glycocholic acid.

Preferably, the glycocholic acid detection kit further includes a glycocholic acid standard solution, a coating solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody, a substrate chromogenic solution and a stop solution.

Preferably, the washing solution is 0.05% PBST.

Preferably, the enzyme-labeled secondary antibody is a goat anti-mouse enzyme-labeled antibody.

Preferably, the glycocholic acid detection kit further includes a solid phase carrier.

In the embodiments provided by the present invention, the solid-phase carrier is an ELISA plate.

The present invention also provides a kind of indirect ELISA detection method of glycocholic acid, comprising the following steps:

The ovalbumin and glycocholic acid conjugates are coated on the solid-phase carrier and sealed to obtain the solid-phase carrier coated with the coated antigen;

Incubate the glycocholic acid standard, the monoclonal antibody of glycocholic acid and the solid-phase carrier coated with the coated antigen together, add the enzyme-labeled secondary antibody to develop color, and obtain the standard curve;

The sample to be tested, the monoclonal antibody to glycocholic acid and the solid phase carrier coated with the coated antigen are incubated together, an enzyme-labeled secondary antibody is added to develop color, and the concentration of glycocholic acid in the sample to be tested is obtained according to the standard curve.

The present invention provides a hybridoma cell line, a monoclonal antibody to glycocholic acid based on the hybridoma cell line, and a detection kit. The deposit number of this hybridoma cell line is CGMCC NO.15292. The technical effect of the invention is as follows: the monoclonal antibody of glycocholic acid produced by the hybridoma cell line with the deposit number of CGMCC NO.15292 has a higher titer, and can be used for qualitative or quantitative detection by enzyme-linked immunosorbent assay (ELISA). The content of glycocholic acid has the characteristics of convenient sample operation, low cost, rapid reaction, high sensitivity and strong specificity.

Biological Preservation Instructions

Classification name: Glycolic acid hybridoma cell line, which was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Management Committee on January 25, 2018. The address of the collection center is No. 1, No. 1 Beichen West Road, Chaoyang District, Beijing, China. No., the deposit number is CGMCC NO.15292.

Description of drawings

Fig. 1 is the SDS-PAEG electrophoresis pattern of the purified glycocholic acid monoclonal antibody of the present invention, wherein 1 is the electrophoresis pattern of 1 mg/ml of ascites fluid before purification, 2 is the electrophoretic pattern of the purified and diluted to 1 mg/ml, and after purification The IgY is composed of two chains, which are the heavy chain at about 55KDa and the light chain at about 25KDa respectively. In 2, the band around 25KDa of the light chain can be seen a little fuzzy, and the analysis is that the concentration is too low;

Fig. 2 is a graph showing the results of measuring the optical density value of monoclonal antibody glycocholate ascites indirect ELISA enzyme-labeled hole of the present invention;

Figure 3 is the competition ELISA curve of the monoclonal antibody glycocholic acid ascites and the standard glycocholic acid of the present invention, the ascites is diluted 2000 times, wherein K represents ×1000.

Detailed ways

The invention discloses a hybridoma cell line, a glycocholic acid monoclonal antibody and a detection kit based on the hybridoma cell line, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

Indirect ELISA detection method operating procedures:

Take a 96-well microtiter plate, add 100 μL of the coating antigen ovalbumin and glycocholic acid conjugate (OVA-CG) at a concentration of 5 μg/mL diluted with coating buffer to each well, and coat at 4°C After overnight, the plate was washed 3 times with washing solution 0.05% PBST and patted dry;

Press 250 μL of the prepared blocking solution per well, incubate at a constant temperature of 37 °C for 1 h; wash the plate 3 times and pat dry;

The antiserum was diluted with PBS at a specific concentration, and 2 wells of negative control and blank control were set at the same time, each well was 100 μL, and incubated at 37°C for 1 h; the solution in the wells was dried, washed three times with PBST washing solution, and patted dry;

Add an enzyme-labeled secondary antibody (goat anti-mouse enzyme-labeled antibody, diluted 1:5000), incubate at 37°C for 1 h; shake off the solution in the well, wash 3 times with PBST washing solution, and pat dry;

Add 100 μL of substrate chromogenic solution to each well and incubate at 37°C for 15 min in the dark for color development. Finally, 50 μL of stop solution was added to stop the reaction. The OD value (450nm) was measured with a microplate reader.

The method for detecting glycocholic acid and its derivatives and content according to the present invention is preferably an indirect competitive ELISA method, and the glycocholic acid immunization antigen in the sample can react with the coating antigen (OVA-CG) to a limited amount of antibodies. Competitive binding, according to the value of its absorbance, the content of glycocholic acid in the sample can be calculated from the standard curve.

The hybridoma cell line provided by the present invention, the monoclonal antibody based on the hybridoma cell line, and the reagents or instruments used in the detection kit can be purchased from the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1: Monoclonal Antibody Preparation

(1) Animal immunity

The immunogen was pure glycocholic acid, which was directly dissolved in PBS to prepare a concentration of 1 mg/mL. Take 0.5mL of the above solution and emulsify it with an equal volume of Freund's complete adjuvant, and inject it into Balb/C mice (5 6-week-old Balb/C female mice, 18-22g.), 0.5mL/mouse, take subcutaneously Multipoint injection and intraperitoneal injection. The initial immunization was emulsified with immunizing antigen and Freund's complete adjuvant, and then emulsified with incomplete Freund's incomplete adjuvant, and the time interval of each immunization was 15 days. On the 15th and 29th, booster immunization was carried out, and the immunization dose was the same as the initial immunization. Three days before fusion, a final shock immunization was performed using unadjuvanted antigen. After the third immunization, blood was collected from the tail vein of the mice, and the serum was isolated, and the antibody titer of the serum was detected by an antigen-coated indirect ELISA method. The mice that prepared monoclonal antibodies were selected from Balb/C mice with the highest antibody titers after the last immunization.

(2) Preparation of hybridoma cells

Recovery and culture of SP2/0 cells: SP2/0 cells were recovered two weeks before fusion. The myeloma cell (SP2/0) cryopreservation tube was taken out from the refrigerator at -80°C, rapidly thawed in a 40°C water bath for 2 minutes, centrifuged at 1000 rmp for 5 minutes, and room temperature. Discard the supernatant, gently blow off the cells with RPMI1640 medium (containing 10% fetal bovine serum + 1% double antibody), and then suck the cells into the cell culture flask, supplement with an appropriate amount of RPMI1640 medium (containing 10% fetal calf serum). bovine serum + 1% double antibody), put it into a 37°C 5% CO ₂ cell incubator for culture. Change the medium according to the observed growth state of the cells. To ensure that the SP2/0 cells used are sensitive to HAT selection medium, 1% 8-azaguanine (100*) can be added to the medium. One day before fusion, one flask of cells was passaged to two flasks, and the culture was continued, so that the cells were in the logarithmic growth phase and had the best viability. When confluent, pour off the complete medium, blow off with an appropriate amount of RPMI1640, count, the total number of cells is about 1-1.2× ¹⁰⁷ , and transfer to a 50mL centrifuge tube; 1000rpm, 5min, centrifuge and wash 2-3 times, resuspend back up.

Preparation of feeder cells: macrophages from mouse peritoneal cavity were taken as feeder cells. One day before cell fusion, 8-10 week old Balb/C mice were taken and killed by de-neck, immersed in 75% alcohol for 5 min, fixed on a sterile foam board on an ultra-clean workbench, and cut the abdominal skin with sterile scissors , expose the peritoneum, carefully cut the peritoneum, use a sterile dropper to draw a small amount of RPMI1640 culture medium and pipet the mouse peritoneal cavity, and the peritoneal cells fully enter the culture medium, and repeatedly pipette into a 50mL sterile centrifuge tube until the color of the pipette is the same as that of RPMI1640 Consistent color. Centrifuge the suctioned cell fluid at 1000 rpm for 5 min. Discard the supernatant, resuspend with HAT medium, count the cells, adjust to 1 x 10 ⁵ cells/mL, add to a sterile 96-well plate, 100 μL per well, place cells in 37%, 5% CO ₂ Cultivate in an incubator.

Preparation of immune spleen cells: Three days after the mice with the highest titer were immunized by pulse, the eyeballs were removed to take blood, and the serum was collected as a positive control for antibody detection. Hold the head of the mouse, pull the tail to kill, immerse it in 75% alcohol for 5 minutes, fix the mouse to the left on a sterile foam board on a sterile operating table, and cut the abdominal skin of the mouse with sterile scissors. Tear the skin up and down along the incision, and you can see where the spleen is located; change another sterile scissors and forceps, cut the peritoneum to fully expose the mouse spleen, and fix the skin with sterile nails; change the scissors and forceps, forceps Gently clamp the spleen, carefully separate the spleen with scissors, remove the connective tissue around the spleen as much as possible, wash the spleen cells in a sterile petri dish with 8-10mL RPMI1640 medium, and put the spleen into another In a sterile petri dish, poke a few times with a 1mL sterile syringe needle, and use a 2mL sterile syringe to draw 8-10mL of RPMI1640 medium to wash the mouse spleen, so that the spleen cells can be fully washed out from the spleen to make a spleen cell suspension , transfer the liquid to another 50mL centrifuge tube. Count, approximately 1 x 10 ⁸ /mouse. To the ultra-clean workbench for spare.

Preparation of myeloma cells: Consistent with the recovery and culture process conditions of SP2/0 cells, the myeloma cells were placed on an ultra-clean workbench for use.

Cell fusion: Mix 1×10 ⁸ splenocytes and 1×10 ⁷ SP2/0 cells in a 50 mL centrifuge tube, add incomplete medium RPMI1640 medium to 20 mL, mix well, centrifuge, 1000 rpm, 5min. Take the centrifuged spleen cells and SP2/0 cells, remove the supernatant (be careful to keep this position) and gently stick the culture medium on the tube wall with a filter paper strip (to ensure that PEG1450 is not diluted). Tap lightly to mix well. Put the centrifuge tube into a 40°C water bath (use a small beaker to make a water bath) (preheat PEG1450), add 1 mL of 50% PEG1450 preheated to 40°C for 45 seconds (mix gently while adding, about 3 seconds a drop ), mix gently for 30 seconds, and stand still for 1 min. Slowly add a pipette of pre-warmed serum-free culture medium to the cells to terminate the fusion, and the action should be as gentle as possible, while stirring (now the cells have changed shape and shrunken under the action of PEG1450, if the solution is added too quickly or too much movement) Thickness will cause cells to rupture and die.). The specific method of adding liquid is to add slowly first and then fast. Use a sterile dropper to slowly add 1 mL of preheated RPMI1640 medium dropwise in the first minute, add 2 mL in the second minute, and quickly drop in the third minute. 8 mL was added dropwise, and finally RPMI1640 medium was supplemented to 20 mL. The solution was placed in a 37°C water bath for 10 min, and then centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Add 100 mL of HAT medium containing 20% fetal bovine serum, gently pipette the pelleted cells to suspend and mix them, add to the 96-well plate with feeder cells, 100 μL per well, place the cells at 37°C, Culture in 5% CO ₂ cell incubator.

Example 2: Screening of hybridoma cells and their subcloning

Change 100 μL of HAT medium half solution every three days after fusion, do not pipette. After 10 days, the entire HAT medium was replaced with the HT medium, and the HT medium was replaced every 3 days. After culturing for about 20 days, it was changed to general complete medium.

The cell growth was observed every day, and about 1/2 of the culture well was covered by the cells on the 10th day, and the supernatant was taken for indirect ELISA assay to detect whether the antibody was produced.

Preparation of hybridoma cell suspension: Gently blow off the hybridoma cells in the positive wells detected by ELISA, pipette the hybridoma cells into a 10 mL centrifuge tube, count, calculate, and dilute the cell concentration with complete medium When it reaches 200 cells/mL, 2 mL is taken out and added to 2 mL of complete medium containing feeder cells, and the concentration of hybridoma cells becomes 100 cells/mL;

Limiting dilution: Add 100 μL of the above hybridoma cell suspension per well to the first and second rows of a 96-well cell culture plate, and the concentration of hybridoma cells in these two rows is 10 per well. Add an equal volume of feeder cell suspension to the remaining cell suspension, and add it to the third and fourth rows after mixing. The concentration of hybridoma cells in these two rows is 5 per well. Repeat the above steps, the fifth The concentration of lines 6 and 6 is 2.5 per well, and the concentration of lines 7 and 8 is 1.25 per well. After culturing for 3 days, observe the cell growth under an inverted microscope, and carefully aspirate 100 μL of the culture medium on the culture plate, and then add 100 μL of complete medium. The supernatant in the wells was detected by indirect ELISA, and the cell culture wells with high titer were selected for re-cloning, and the monoclonal wells were selected as much as possible. Until two consecutive cloned monoclonal wells are all positive.

From the antibody-positive wells, select cell wells with high antibody titer, strong specificity, and good cell condition, and continue recloning to obtain monoclonal antibody cell lines. in the refrigerator. And it was deposited in China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center, CGMCC), and the deposit number is CGMCC NO.15292.

Example 3: Ascites Induction and Antibody Purification

Induction of ascites: 5 Balb/C mice were taken, and 0.5 mL of incomplete Freund's adjuvant was intraperitoneally injected into each mouse about ten days before inoculation with hybridoma cells. At the same time, the monoclonal antibody-secreting cell line was placed in a cell culture flask and cultured to log phase, centrifuged at 1000 rpm for 5 min, the pellet was resuspended in 1640 basal medium, and the number of cells was adjusted to 1×10 ⁶ cells/mL. The mice were intraperitoneally injected with 0.5 mL of hybridoma cells, and the abdomen of the mice was obviously swollen and the action was slow, about 10-13 days later. Disinfect the abdominal skin with alcohol cotton balls, extract ascites with a 5mL syringe, centrifuge the ascites at 4°C and 1000 rpm for 10 min, take the supernatant, aliquot and store in a -20°C refrigerator for later use.

Purification of glycocholic acid antibody: purification of mouse ascites antibody by ammonium octanoate sulfate method. Take out the mouse ascites from the refrigerator, thaw at room temperature, and filter with double-layer filter paper; slowly add 0.06 mol/L NaAC (pH 4.5) solution with 2 times the serum volume; slowly add n-octanoic acid dropwise to precipitate impurities (denatured precipitation) During the process, you will find that a white substance is slowly produced in the serum. The content of IgG in the supernatant can reach more than 90% after the addition of n-octanoic acid for complete precipitation. The amount of n-octanoic acid added to different substances is also different: 40 μL of n-octanoic acid was added to 1 mL of mouse serum, and 33 μL of n-octanoic acid was added to 1 mL of mouse ascites. After the dropwise addition was completed, after 1-2 hours at 4°C, centrifugation at 8000-10000rpm for 15min at 4°C was performed, the precipitate was discarded, and the supernatant was taken. Add 1M 10×PBS to the pellet, the volume is 1/10 of the supernatant. The pH was quickly adjusted back to neutral with 1M NaOH to reduce the damage to the antibody; 1 volume of saturated ammonium sulfate (pH=7.5) was added at 4°C, and the volume was the previous total volume. The concentration of ammonium sulfate after the addition was 50%. The process of adding should be slow, and the local salt concentration should not be too high while stirring and ice bathing. To a certain extent the solution became cloudy and IgG began to precipitate. After adding, stir on a magnetic stirrer for 6h or stand at 4°C overnight; centrifuge at 8000-10000rpm for 15min at 4°C, discard the supernatant, and dissolve the precipitate in PBS (0.01M, pH=7.4) with the same volume as the original ascites. Or in normal saline, add 1/2 volume of saturated ammonium sulfate solution to make the concentration of ammonium sulfate 33%, place at 4°C for 3h; centrifuge at 8000-10000rpm at 4°C for 15min, discard the supernatant, and precipitate with PBS (0.01M, pH=7.4) and resuspended to the original volume; then placed in 0.01M PBS for dialysis, dialyzed at 4°C for 2 days, during which the water was changed more than 4 times. The dialyzed solution was centrifuged at 8000 rpm for 30 min to remove the insoluble sediment, and then aliquoted into small tubes of 1 mL and stored frozen at -80 °C for later use. Figure 1 shows the SDS-PAEG electrophoresis pattern of the purified monoclonal antibody.

Example 4: Identification of Monoclonal Antibodies - Indirect ELISA Method

Operation procedure of indirect ELISA detection method: Take a 96-well ELISA plate, and add the coating antigen ovalbumin and glycocholic acid conjugate (OVA-CG) diluted with coating buffer to each well at a concentration of 5ug/ mL 100 μL, after coating overnight at 4°C, wash the plate 3 times with washing solution 0.05% PBST, and pat dry; press 250 μL of the prepared blocking solution per well, incubate at 37°C for 1 h; wash the plate 3 times, pat dry; use PBS The antiserum was diluted at a specific concentration, and 2 wells of negative control and blank control were set at the same time, each well was 100 μL, and incubated at 37°C for 1 h; the solution in the well was dried, washed three times with PBST washing solution, and patted dry; added enzyme-labeled secondary antibody (goat anti-mouse enzyme-conjugated antibody, diluted 1:5000), incubated at 37°C for 1 h; dried the solution in the wells, washed 3 times with PBST washing solution, and patted dry; added 100 μL of substrate chromogenic solution to each well, incubated at 37°C Protect from light for 15min. Finally, 50 μL of stop solution was added to stop the reaction. The OD value (450nm) was measured with a microplate reader, and the results are shown in Figures 2-3 and Table 1. At the same time, the monoclonal antibody of glycocholic acid reported in other literatures was used as a control.

Table 1 embodiment 3 glycocholic acid monoclonal antibody ascites indirect ELISA enzyme-labeled hole optical density value measurement results

The relevant literature reports are as follows:

①"Glycolic acid immunogen, anti-glycolic acid specific antibody and detection reagent" application number: CN201410047903.5, the titer of the specific antibody is 1:30000

② "A Glycolic Acid Immunoassay Reagent and Its Preparation and Detection Method" Application No.: CN20141047767.X, the titer of the specific antibody is 1:30000

③ "An immunodetection reagent for glycocholic acid based on anti-glycolic acid specific antibody and its preparation method" Application No.: 201510717368.4, the titers of the specific antibody are 1:20000 and 1:30000

It can be seen that the titer of the glycocholic acid monoclonal antibody prepared by the present invention can reach 1:250,000, which is significantly higher than that of the glycocholic acid monoclonal antibody and the glycocholic acid derivative-specific antibody reported in other documents.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (7)

1. A hybridoma cell line, characterized in that the deposit number is CGMCC No.15292. 2 . A monoclonal antibody to glycocholic acid, characterized in that it is produced by the hybridoma cell line of claim 1 . 3. The application of glycocholic acid monoclonal antibody as claimed in claim 2 in the preparation of glycocholic acid detection kit. 4. A glycocholic acid detection kit, characterized in that, comprising the glycocholic acid monoclonal antibody as claimed in claim 2. 5 . The glycocholic acid detection kit according to claim 4 , further comprising a coating antigen. 6 . 6 . The glycocholic acid detection kit according to claim 5 , wherein the coating antigen is a conjugate of ovalbumin and glycocholic acid. 7 . 7. glycocholic acid detection kit according to claim 4 or 5, is characterized in that, also comprises glycocholic acid standard solution, coating liquid, washing solution, blocking solution, enzyme-labeled secondary antibody, substrate color developing solution and stop solution.