Double-antigen sandwich antibody detection method

A double-antigen sandwich and antibody detection technology, which is applied in the field of molecular biology, can solve the problems of antigen-antibody failure to become a sandwich state, strong positive missed detection, and high background, so as to reduce the interference of endogenous organisms, reduce research and development costs, and improve The effect of sensitivity

Active Publication Date: 2019-03-08
GUANGDONG FAPON BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the one-step method will have a hook effect (HOOK effect), that is, when the antibody content in the serum is high, the amount of coated antigen and labeled antigen will be less than that of the antibody in the serum, resulting in the failure of the antigen and antibody to become a sandwich state, resulting in missed detection of strong positive
The two-step method has many reaction steps and a long reaction time. Since the coated antigen and the labeled antigen react with the antibody in the serum in two steps, the coated antigen may occupy the antibody site and cannot form a sandwich. The sensitivity of low values ​​is lower than that of the one-step method. , and the background is higher than that of the one-step method

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Experimental program
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Effect test

Embodiment 1

[0058] 1). Compare the detection sensitivity of the one-step double-antigen sandwich method, the two-step double-antigen sandwich method and the double-antigen sandwich comprehensive method (contents in the examples).

[0059] In order to compare the advantages and disadvantages of the three reaction modes, 20 negative samples and 20 positive samples were selected for detection using the two reaction modes at the same time. The test results are as follows:

[0060] It can be seen from the table below that the detection sensitivity of the one-step two-step combination method is equivalent to that of the one-step method, and higher than the two-step method, so the low-value positive test value is higher, and the two-step method has a risk of missing detection for weakly positive samples; strong positive samples The measured value of the one-step method is low, and the measured value of the two-step method and the comprehensive method is higher, which is caused by the hook effect,...

Embodiment 2

[0093] This embodiment relates to a detection method for Treponema pallidum.

[0094] 1. Preparation of the kit.

[0095] Treponema pallidum antibody chemiluminescent immunoassay assay kit of the present invention: comprises the following steps:

[0096] 1) Preparation of Treponema pallidum-specific recombinant chimeric antigen:

[0097] The Treponema pallidum-specific recombinant chimeric antigen is connected by two or more antigens in TpN15, TpN17, TpN47 and TmpA through molecular biological methods, more preferably two or three of TpN15, TpN17 and TpN47 , the above-mentioned chimeric antigen is transformed into Escherichia coli for expression, and finally the specific chimeric protein antigen of Treponema pallidum is obtained by protein purification technology.

[0098] 2) Magnetic bead system:

[0099] Beads are Fe 2 o 3 or Fe 3 o 4 A complex of magnetic nanoparticles and organic polymer materials, with a particle size of 0.1-5 μm, and -COOH functional groups on its...

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Abstract

The invention relates to a double-antigen sandwich antibody detection method. According to the method, detection is finished in a manner of forming a solid-phase support-a first antigen Ag1-a to-be-detected antibody-a second antigen Ag2, wherein the Ag2 is coupled with a marker for displaying signal strength; the method comprises the following steps: 1), enabling the Ag1 and the Ag2 to be in contact with a to-be-detected object under the condition that antigen/antibody reaction can occur with enough Ag1, Ag2 and the to-be-detected object, so as to form an immune compound, wherein the content of the Ag1 is greater than that of the Ag2 based on mol number; 2) washing the unbound to-be-detected antibody; 3) adding the Ag2 to bind the Ag2 with residual antigen binding sites in the immune compound; 4) detecting the markers to indicate the existence and/or content of the to-be-detected antibody. Compared with the prior art, the method provided by the invention ensures the low-value sensitivity of detection, also can solve a hook effect and reduce the miss rate.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for detecting antibodies in a double-antigen sandwich. Background technique [0002] Antibody detection is one of the main methods for general screening of pathogens at present. Serological examination is usually used. Immuno-serological examination has the advantages of convenience, speed and accuracy, and has been widely used in the field of medical testing and diagnosis. [0003] From the perspective of immunological principles, there are generally two methods for antibody detection: indirect method and sandwich method. The principle of the indirect method is that the antibody in the serum recognizes and binds to the antigen coated on the stationary phase. After removal of impurities, the secondary antibody that can recognize the antibody binds to the serum antibody bound to the antigen on the stationary phase to form a stationary phase-antigen- Antibody-an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/532
CPCG01N33/532G01N33/54326G01N33/54346G01N33/6854G01N2333/47G01N2446/90
Inventor 叶小琴代双夏良雨赵存洋陈艳华潘少丽
Owner GUANGDONG FAPON BIOTECH CO LTD
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