Coating method for indirect antigen-coated elisa plate of ELISA kit

A technology for coating microtiter plates and kits, which can be used in measurement devices, instruments, scientific instruments, etc., and can solve the problems of high price, unstable sources, and short shelf life of ELISA kits, and achieve a wide range of applications and improve stability. performance, improve shelf life

Active Publication Date: 2016-03-09
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
View PDF4 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the quality of domestic ELISA kits is unstable, mainly due to the poor stability of direct or indirect antigen-coated microtiter plates, resulting in a short shelf life of ELISA kits; and the antigen stabilizers on the market are not only expensive, but also unstable sources , seriously affected the development of ELISA diagnostic kits in my country

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Coating method for indirect antigen-coated elisa plate of ELISA kit
  • Coating method for indirect antigen-coated elisa plate of ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0019] (3) The preparation and identification of nonstructural protein 3B monoclonal antibody refer to the following documents:

[0020] Li Yongliang, Tian Meina, Lu Zengjun, et al. Preparation and Identification of Monoclonal Antibody to Nonstructural Protein 3B of Foot-and-Mouth Disease Virus[J]. Journal of Jiangsu Agricultural Science, 2009,25(2):296-300.

Embodiment 1

[0021] Embodiment 1: the coating method of foot-and-mouth disease virus 3A monoclonal antibody coating capture 3ABC microtiter plate

[0022] (1) Coating: Dilute 3A monoclonal antibody with coating buffer (0.05mol / L pH9.6 carbonate buffer) to a working concentration of 1:10000, add 100 μL to each well, and overnight at 4°C. Wash 5 times with PBS.

[0023] (2) Blocking: 100 μL of blocking solution (PBS solution containing 1% fish gelatin) was added to each well, blocked at 37° C. for 1 hour, and washed 3 times with PBST.

[0024] (3) Capture 3ABC protein: dilute 3ABC protein with 0.01mol / LPBS buffer to a working concentration of 1 μg / mL, add 100 μL per well to the microtiter plate, react at 37°C for 1 hour, and wash 3 times with PBS.

[0025] (4) Solid-phase antigen stabilizer: prepare 0.01mol / L phosphate buffer containing different concentrations of sucrose (50g / L and 100g / L) and different concentrations of bovine serum albumin (10, 30, 50 and 100g / L) solution (pH7.5), to de...

Embodiment 2

[0032] Embodiment 2: the coating method of foot-and-mouth disease virus 2C polyclonal antibody coating captures 2C3AB microtiter plate

[0033] The difference from Example 1 is only that the polyantibody to the foot-and-mouth disease virus 2C epitope region is used to coat the microtiter plate (1:20000) to capture the non-structural protein 2C3AB (1 μg / mL), and other reaction conditions are the same as in the reagent example 1.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a coating method for an indirect antigen-coated elisa plate of an ELISA kit. The method comprises the following steps that 1, coating is performed; 2, closing is performed, wherein a 0.01-mol / L LPBS buffer solution of 10-g / Lfish gelatin is adopted for closing; 3, a target antigen is captured; 4, a solid-phase antigen stabilizer of a phosphate buffer solution which contains 5% of sucrose and 1%-5% of bovine serum albumin and has the concentration of 0.01 mol / L and the pH of 7.2-7.6 is added, extracting is performed, and then the indirect antigen-coated elisa plate is obtained. According to the method, the retention period of the elisa plate obtained through coating can reach one year or above, the stability of the indirect antigen-coated ELISA kit is significantly improved, and the retention period of the indirect antigen-coated ELISA kit is significantly prolonged.

Description

technical field [0001] The invention relates to a coating method of an enzyme plate, in particular to a coating method of an ELISA kit antigen indirectly coating an enzyme plate. Background technique [0002] Antigen indirect coating refers to first coating the microtiter plate with an antigen-specific antibody (monoclonal antibody or polyclonal antibody), and then capturing the target antigen on the microtiter plate through the specific binding of the antigen and antibody for ELISA assembly Reagent test kit. The advantages of indirect coating of antigens to prepare microtiter plates are as follows: First, it helps to promote the formation of the natural conformation of the target antigen protein expressed by genetic engineering methods, and further purifies the coated antigen through the specific affinity reaction of antigen and antibody. The process makes the protein structure closer to the natural conformation, thereby improving the sensitivity and specificity of the dia...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityApplications(China)
IPC IPC(8): G01N33/543
CPCG01N33/54393
Inventor卢曾军付元芳曹轶梅孙普白兴文李平花包慧芳李冬陈应理刘在新
OwnerLANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI