Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle

An enzyme-labeled antigen and avidin technology, applied in the fields of immunology and biology, can solve the problems of losing the ability of antibody binding, uncertain structure and function, affecting antibody binding, etc., and achieves retention of reactivity, good reactivity, detection Sensitive effect

Active Publication Date: 2014-03-12
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the number of chemical groups available for labeling in the antigen molecule is small or if they are in the internal structure of the molecule, labeling will be inefficient; if these groups are on or near the epitope, the labeling may change The spatial structure of the epitope and the steric hindrance will affect the binding to the antibody and affect the detection effect
The structures of various antigens are diverse, and many of their main functional regions are still uncertain, so it is not sure whether the labeling process will affect their structure and function, thereby reducing or even losing their ability to bind to antibodies

Method used

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  • Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle
  • Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle
  • Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] This example uses the method of the present invention to prepare the enzyme-labeled antigen for detecting porcine circovirus type 2 antibodies, establishes a double-antigen sandwich ELISA method, and compares it with the double-antigen sandwich ELISA established by the enzyme-labeled antigen prepared by the traditional chemical coupling method, The reliability and advantages of this method are illustrated by the detection effect.

[0023] 1) Preparation of enzyme-labeled antigen for detection of circovirus 2 antibody by the method of the present invention

[0024] a. Selection of avidin binding peptide and fusion with antigen molecule Cap⊿41 protein

[0025] The two SBPs whose sequences were selected as DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP and YNCHPMNNLCKE were fused with the antigen molecule of porcine type 2 circovirus——Cap⊿41 protein (without signal peptide) to obtain two SBP-Cap⊿41 fusion antigens, respectively ACap ⊿41 and BCap ⊿41. The specific process is as fo...

Embodiment 2

[0036] This example is to use the method of the present invention to prepare an enzyme-labeled antigen for detecting the gB protein antibody of pseudorabies virus (the effect of the enzyme-labeled antigen prepared by traditional chemical coupling method is poor, and it cannot be used for the establishment of ELISA method), and establish a double-labeled antigen for detecting the antibody. Antigen sandwich ELISA method, and compared with the commercial kit for detecting the antibody to determine the effect of the enzyme-labeled antigen prepared by this method.

[0037] 1) Using the method of the present invention to prepare an enzyme-labeled antigen for detecting pseudorabies virus gB protein antibody

[0038] a. Selection of avidin-binding peptides and fusion with antigen molecules

[0039] The fusion expression of SBP whose sequence is SNWSHPQFEK and the antigen molecule of pseudorabies virus—gB4 protein (820-917 amino acids of gB) was performed to obtain SBP-gB4 fusion antig...

Embodiment 3

[0050] This example is to use the method of the present invention to prepare an enzyme-labeled antigen for detecting the gE protein antibody of pseudorabies virus (the effect of the enzyme-labeled antigen prepared by the traditional chemical coupling method is poor, and cannot be used for the establishment of the ELISA method), and to establish a double-labeled antigen for the detection of the antibody. Antigen sandwich ELISA method, and compared with the commercial kit for detecting the antibody to determine the establishment effect of the enzyme-labeled antigen prepared by this method.

[0051] 1) Using the method of the present invention to prepare an enzyme-labeled antigen for detecting pseudorabies virus gE protein antibody

[0052] a. Selection of avidin binding peptide and fusion with antigen molecule gE protein

[0053] The SBP whose sequence is selected as DVEAWLGAR is fused with the antigen molecule of pseudorabies virus—gEa protein (amino acids 22-336 of gE protein)...

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Abstract

The invention discloses a method for preparing an ELISA enzyme-labelled antigen through utilizing an avidin binding peptide and avidin combining principle. The enzyme-labelled antigen is formed by recombining, antigenically fusing and mixing an enzyme-labelled streptavidin (one type of avidins) with a streptavidin binding peptide (SBP). The method comprises steps of: utilizing the principle that the enzyme-labelled streptavidin and biotin can be specifically bound with each other, and adopting a genetic engineering method to structure the streptavidin binding peptide (SBP) with a simulated biotin structure with antigenic protein molecules so as to form a fusing protein; and binding the SBP in the fusing protein with the enzyme-labelled streptavidin through affinity, and thus forming a novel enzyme-labelled antigen with antigenic activity and enzymatic activity. The method does not need special chemical groups as a coupling target position and has wide applicability; and moreover, the antigenic molecules are not directly labeled, the influence on an antigenic structure is reduced, and the steric hindrance of a labeled object to antigen-antibody reaction is decreased, so that the detection sensitivity is improved.

Description

technical field [0001] The invention belongs to the field of immunology and biotechnology, and specifically relates to a method for constructing and preparing an ELISA enzyme-labeled antigen using the principle of combining an avidin-binding peptide with avidin. The enzyme-labeled antigen prepared by this method can be used for detection Antibody enzyme-linked immunosorbent assay method was established. Background technique [0002] Enzyme-labeled antigens are used in the detection of antibodies in enzyme-linked immunosorbent assay (Enzyme Linked Immunosorbent Assay, ELISA). The traditional enzyme-labeled antigen preparation method is based on the chemical coupling method, that is, the chemical reaction between the antigen molecule and the cross-linking agent forms a covalent bond and connects. Different cross-linking agents react to different chemical groups (mainly primary amines, thiols, carboxyls, etc.) on the antigen molecule. Therefore, the labeling efficiency and th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535
Inventor 余兴龙葛猛李润成
Owner HUNAN AGRICULTURAL UNIV
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