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Hybridoma cell strain and glycocholic acid monoclonal antibody and detection kit based on hybridoma cell strain

A hybridoma cell line and monoclonal antibody technology, applied in the field of detection, can solve the problem of low titer, achieve high sensitivity, fast response, and convenient sample operation

Active Publication Date: 2018-06-22
广东南粤药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current commercially available monoclonal antibodies to glycolic acid have the problem of low titer

Method used

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  • Hybridoma cell strain and glycocholic acid monoclonal antibody and detection kit based on hybridoma cell strain
  • Hybridoma cell strain and glycocholic acid monoclonal antibody and detection kit based on hybridoma cell strain
  • Hybridoma cell strain and glycocholic acid monoclonal antibody and detection kit based on hybridoma cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of Monoclonal Antibody

[0042] (1) Animal immunity

[0043] The immunogen is pure glycocholic acid, which is directly dissolved in PBS to make 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, inject it into Balb / C mice (5 6-week-old Balb / C female mice, 18-22g.), 0.5mL / mouse, take subcutaneous Multiple injections and intraperitoneal injections. The immune antigen was emulsified with complete Freund's adjuvant for the first immunization, and then emulsified with incomplete Freund's incomplete adjuvant, with an interval of 15 days between each immunization. Booster immunization was carried out on the 15th and 29th day, and the immunization dose was the same as the initial immunization. Three days before fusion, a final pulse immunization was performed with antigen without adjuvant. After the third immunization, blood was collected from the tail vein of the mice, and ...

Embodiment 2

[0050] Example 2: Screening of hybridoma cells and their subcloning

[0051] After fusion, change 100 μL of the HAT medium half solution every three days, and do not pipette. After 10 days, replace all the HAT medium with HT medium, and change the whole HT medium every 3 days. After culturing for about 20 days, replace it with a general complete medium.

[0052] Observe the cell growth every day. On the 10th day, the cells cover 1 / 2 of the culture well. Take the supernatant for indirect ELISA assay to detect whether antibodies are produced.

[0053] Prepare hybridoma cell suspension: Gently blow off the hybridoma cells in the positive wells detected by ELISA, pipette the hybridoma cells into a 10mL centrifuge tube, count, calculate, and dilute the cell concentration with complete medium to 200 cells / mL, take out 2 mL and add it to 2 mL of complete medium containing feeder cells, and the concentration of hybridoma cells becomes 100 cells / mL;

[0054] Limiting dilution: 100 μ...

Embodiment 3

[0056] Example 3: Ascites induction and antibody purification

[0057] Induction of ascites: 5 Balb / C mice were taken, and each mouse was intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant about ten days before hybridoma cell inoculation. At the same time, culture the monoclonal antibody-secreting cell line in a cell culture flask to the logarithmic phase, centrifuge at 1000rpm for 5min, take the precipitate and resuspend it with 1640 basal medium, and adjust the number of cells to 1×10 6 cells / mL, inject 0.5mL hybridoma cells intraperitoneally into the above mouse, and wait for the mouse abdomen to be obviously enlarged and move slowly, about 10-13 days later. Disinfect the abdominal skin with alcohol cotton balls, extract the ascites with a 5mL syringe, centrifuge the ascites at 4°C and 1000rpm for 10min, take the supernatant, aliquot it and store it in a -20°C refrigerator for later use.

[0058] Purification of Glycocholic Acid Antibodies: Antibodies f...

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Abstract

The invention relates to the technical field of detection, in particular to a hybridoma cell strain and a glycocholic acid monoclonal antibody and detection kit based on the hybridoma cell strain. Thepreservation number of the hybridoma cell strain is CGMCC No.15292. The hybridoma cell strain has the advantages that the titer of the glycocholic acid monoclonal antibody generated from the hybridoma cell strain is high, the hybridoma cell strain can be used for detecting glycocholic acid through enzyme linked immunosorbent assay (ELISA), sample operation is convenient, the cost is low, reactions are quick, and the sensitivity and specificity are high.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a hybridoma cell line, a glycocholic acid monoclonal antibody and a detection kit based on the hybridoma cell line. Background technique [0002] Glycocholic acid is one of the conjugated bile acids formed by the combination of bile acid and glycine. It is synthesized by liver cells, enters the intestinal tract with bile, and returns to the liver through the portal vein. When liver cells are damaged, the ability of liver cells to absorb glycocholic acid decreased, leading to an increase in blood levels. Glycocholic acid is the most abundant organic acid secreted by the liver into the bile. It enters the intestinal lumen to help fat digestion and absorption. It is reabsorbed in the ileum and colon, and enters the liver through the portal vein. The liver cells can efficiently absorb a large amount of glycocholate from the portal vein. acid, so that the amount of glycocholic acid...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
CPCC07K16/44C07K2317/35G01N33/577
Inventor 赵肃清陈莉莉陈莹珊沈定贺攀
Owner 广东南粤药业有限公司
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