Method for detecting small molecule protein and peptides by using sandwich immunization sensing method

A sandwich immunoassay and small molecule technology, which is applied in the field of sandwich immunoassays based on sensing membranes, can solve the problems of long detection period, cumbersome operation, and difficulty, and achieves low reagent cost, high sensitivity, and no background interference. Effect

Inactive Publication Date: 2004-03-10
JILIN UNIV
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Problems solved by technology

[0003] The existing technology is to add liposomes that can be connected to the second antibody through the affinity of avidin-biotin to amplify the optical reaction signal to improve the detection sensitivity, so the operation is cumbersome, time-consuming, and the detection period is long. In the case of using labeled particles, the connection between liposomes and antibodies is also more difficult than colloidal gold particle-labeled antibodies

Method used

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  • Method for detecting small molecule protein and peptides by using sandwich immunization sensing method
  • Method for detecting small molecule protein and peptides by using sandwich immunization sensing method
  • Method for detecting small molecule protein and peptides by using sandwich immunization sensing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Sandwich immunosensing method detection of rabbit skeletal muscle troponin C (TnC)

[0021] (1) Formation of immunologically active assemblies

[0022] 1) Activation of the gold film: first add 6.7mmol / L dimercaptoacetic acid or mercaptopropionic acid prepared with ethanol to the sample cell; add 0.87mol / L N-hydroxysuccinimide (NHS) and 0.52 mol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution was reacted for 15 min, and the flow cell was washed 3 times with phosphate buffered saline (PBS).

[0023] 2) After each step of the reaction is completed (the resonance wavelength reaches a stable value), wash the flow cell with phosphate buffer (PBS, 10mmol / L, pH7.4, containing 8.5g / L NaCl) for 3 times to remove non-specific adsorption, Then inject the next reaction reagent. Except for the specific anti-ThC monoclonal antibody reaction for 30 minutes, each step can be completed in 20 minutes. Add 0.2g / L avidin prepared with PBS; add...

Embodiment 2

[0034] Example 2: Specificity of rabbit skeletal muscle troponin C (TnC) sandwich immunosensing assay

[0035] In order to illustrate the specificity of the sandwich immunoassay results, BSA was used instead of TnC as the sample to be tested for immunoassay, and the whole process of Example 1 was repeated. In Example 2, the two strains of anti-TnC monoclonal antibodies and BSA used are unpaired antigen antibodies or non-related antibodies, which do not have selective recognition, so they cannot specifically bind, nor can they form a three-layer sandwich immune complex , that is, the corresponding optical signal cannot be generated, and the resonance wavelength does not change, indicating that the sample to be tested does not contain TnC, and non-related proteins in the sample will not cause non-specific reactions. The sandwich immunoassay is highly specific.

Embodiment 3

[0036] Example 3: Sandwich immunosensing detection of basic fibroblast growth factor (bFGF)

[0037] Same as the steps in Example 1, the difference is that the first antibody and the second antibody are anti-bFGF monoclonal antibodies, and the antigen to be tested can only be bFGF. Similarly, the specimen containing bFGF is used as the sample to be tested, and the content of bFGF in the sample can be measured through detection. image 3 As the detection result of Example 3, the combination of the second anti-bFGF monoclonal antibody increases the resonance wavelength by 2.9nm, it can be judged that the sample contains bFGF, and the concentration of bFGF in the sample can be obtained by comparing with the standard reference.

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Abstract

The invention belongs to immunity detection area, especially related to method for quantitative measuring proteins and peptides with molecular weight under 20kDa. Gold-coated slide glass or triangular prism is as detection substrate. The method includes following procedures: forming an assembling body, forming duality immune complex of first antibody - antigen, forming sandwich ternary immune complex of first antibody - antigen - second antibody as well as resonant wavelength testing. The antigens to be detected are insulin bFGF, T3 or T4 etc. the first antibody and second antibody are first and second monoclone antibodies possessing specificities and complementarity for anti each antigen to be detected. The invention monitors dynamic reaction procedure of biomoleculars, and tests change of concentration of object to be detected with high sensitivity. The combination of second anti body generates stronger optical signal, creating biologic amplified activate.

Description

technical field [0001] The invention belongs to the field of immunodetection, and relates to a method for quantitatively detecting proteins or peptides with a molecular weight below 20kDa, in particular to a sandwich immunoassay method established on a sensing membrane. Background technique [0002] The prior art close to the present invention is a sensitive method for the detection of trace interferon by the double monoclonal antibody sandwich method with liposome as the middle layer published in 1998. See the article by Wink T, Anal Chem., 1;70(5):827-32. Liposome-mediated enhancement of the sensitivity in immunoassays of proteins and peptides in surface plasmon resonance spectrometry. The analytical method is carried out using surface plasmon resonance (SPR) technology on a thin polystyrene layer about 20nm thick and coated with a gold film. After coating the capture antibody (primary antibody), adding the tested sample, adding biotinylated detection antibody (secondary ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/68
Inventor 魏景艳房学迅李善玉罗贵民
Owner JILIN UNIV
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