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PMSG (pregnant mare serum gonadotropin) double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) kit

A gonadotropin, pregnant horse serum technology, applied in anti-hormone immunoglobulin, biological testing, anti-animal/human immunoglobulin, etc., can solve the problems of narrow detection range, expensive import, high price, etc. Simple and easy to implement, good stability and repeatability, and low cost of use

Active Publication Date: 2013-01-16
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high import price, a 96-well detection plate needs to cost 1800-2000 yuan. At the same time, because the detection range of the kit produced by DRG is 0-80IU, the highest content of some domestic horse breeds reaches 140IU, which is beyond its detection range. It is urgent to establish a fast, accurate, time-saving, easy-to-operate method for detecting PMSG content and develop a kit to solve the problems of expensive imports and narrow detection range

Method used

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  • PMSG (pregnant mare serum gonadotropin) double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) kit
  • PMSG (pregnant mare serum gonadotropin) double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) kit
  • PMSG (pregnant mare serum gonadotropin) double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation and Identification of Monoclonal Antibody to Serum Gonadotropin in Pregnant Horses

[0031] 1 material

[0032] 1.1 Experimental animals BALB / c mice were purchased from Beijing Weitong Lihua Experimental Animal Company; mouse myeloma cell line SP2 / 0 was purchased from the Institute of Zoology, Chinese Academy of Sciences.

[0033]1.2 Main instruments and reagents Microplate reader MK3 was purchased from Thermo Electric Company; microplate plate was purchased from Nunc Company; cell culture plate was purchased from Coster Company. Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) were provided by Beijing Qinbang Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody was purchased from Beijing Zhongshan Jinqiao Company; Pure PMSG, HAT and HT selection medium were purchased from Sigma Company; fetal bovine serum was purchased from Hyclone Company; other conventional chemical reagents wer...

Embodiment 2

[0090] The establishment of embodiment 2 pregnant horse serum gonadotropin ELISA detection method

[0091] 1 material

[0092] 1.1 PMSG monoclonal antibody

[0093] The hybridoma cell lines D-3 and A-4 were collected on September 5, 2012 at the General Microbiology Center of the China Committee for the Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences Institute, referred to as CGMCC, zip code 100101), classified as hybridoma cells, and the deposit numbers are CGMCC No.6450 and CGMCC No.6449.

[0094] 1.2 Consumables

[0095] PMSG standard products were purchased from Sigma Company of the United States; enzyme plate was purchased from Costar Company of the United States; substrate A and B solutions were purchased from Beijing Wanger Biotechnology Co., Ltd.; octanoic acid was purchased from Beijing Jinlong Chemical Reagent Co., Ltd.; saturated ammonium sulfate, Diol, sodi...

Embodiment 3

[0209] The composition of embodiment 3 test kits

[0210] The composition of the kit in this example is as follows:

[0211] ELISA plate: coated with monoclonal antibody secreted by D-3 strain

[0212] Enzyme-labeled antibody: horseradish peroxidase-labeled D-2 monoclonal antibody (D-2 is a purified monoclonal antibody secreted by A-4 hybridoma cells)

[0213] Substrate solution: Substrate chromogenic solution: The substrate chromogenic solution is composed of A liquid and B liquid, and the substrate chromogenic solution

[0214] Liquid A is carbamide peroxide; liquid B, the substrate chromogenic liquid, is tetramethylbenzidine.

[0215] Stop solution: (2mol / L H 2 SO 4 ) respectively take 177.8mL of distilled water and 22.2mL of concentrated sulfuric acid and mix them.

[0216] Add 0.5mL Tween-20 to the washing liquid 1000mL 10mmol / L PBS with pH 7.4;

[0217] Negative control: distilled water;

[0218] Positive control: PMSG standard.

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Abstract

The invention provides PMSG (pregnant mare serum gonadotropin) monoclonal antibody hybridoma strains D-3 and A-4 which respectively have the collection numbers of No. 6450 and No. 6449 in the CGMCC (China General Microbiological Culture Collection Center). The invention also provides a PMSG double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) kit which comprises a PMSG monoclonal antibody coated enzyme-labeled plate and an enzyme-labeled PMSG monoclonal antibody; a capture antibody and a detection antibody correspond to different antigenic determinants of the PMSG protein. The detection kit has the characteristics of high sensitivity, strong specificity, high detection determination speed, accurate quantification and good reproducibility, is easy to operate and can be used for the standard correction of the finished preparation. Compared with the existing commercialized PMSG detection kit, the detection kit provided by the invention has low cost and high detection sensitivity, has the lowest PMSG detection limit of 30-200mIU / ml and the detection range of 30-200 mIU and is suitable for popularization and application.

Description

technical field [0001] The invention relates to an ELISA detection kit, in particular to a double-antibody sandwich ELISA kit for detecting serum gonadotropin in pregnant horses. Background technique [0002] Pregnant Mare Serum Gonadotrophin PMSG is also called equine chorionic gonadotropin (eCG). PMSG is a glycoprotein hormone purified from the blood of a mare at 40-120 days pregnant. It is an economical and practical gonadotropin. It is often used in production to replace the more expensive follicle-stimulating hormone (FSH) and is widely used to induce estrus in animals. , superovulation or increase the ovulation rate (such as increasing the double lamb rate); it can also receive a certain effect on ovarian hypoplasia or male spermatogenic decline. Because the content of PMSG in the serum of pregnant horses is affected by many factors, different gestation periods, different breeds, different ages and parity of the same breed have a greater impact on it. Therefore, befo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/26G01N33/76G01N33/577
Inventor 韩国才苏晓倩邓亮
Owner CHINA AGRI UNIV
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