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Method for improving sensitivity of competitive immunoassay

An immunoassay and sensitivity technology, applied in the field of biomedical detection, can solve the problems of being unsuitable for high-sensitivity detection and low sensitivity, and achieve the effects of low cost, high sensitivity and improved sensitivity

Active Publication Date: 2012-10-10
SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the deficiencies in the prior art above, the purpose of the present invention is to provide a method for improving the sensitivity of competitive immunoassay, aiming at solving the problem that the existing immunoassay method has low sensitivity and is not suitable for high-sensitivity detection

Method used

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  • Method for improving sensitivity of competitive immunoassay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Whether the existing pre-reaction method can improve the sensitivity (taking the detection of ciprofloxacin by ELISA as an example)

[0038] Coat the ELISA plate with ciprofloxacin-BSA conjugate, 1 μg / mL, 100 μL per well, and block overnight at 4 °C. Block with 3% skim milk powder the next day. Prepare standard 0ppb, 1ppb, 3ppb, 9ppb, 27ppb, 81ppb of ciprofloxacin with washing solution (PBST).

[0039] The experiments were divided into two groups:

[0040] Group 1: Pre-incubate 60 μL standard and 60 μL diluted ciprofloxacin antibody at 37°C for 20 minutes; then take 100 μL and add it to the ELISA plate coated with ciprofloxacin conjugate, react at 37°C for 30 minutes; wash Add enzyme-labeled secondary antibody, react at 37°C for 30 minutes, wash the plate; add 100 μL of chromogenic reagent, react at 37°C for 15 minutes; finally add 50 μL of stop solution, and measure OD450 with a microplate reader.

[0041] Group 2: Add 50 μL of standard substance and 50 μL...

Embodiment 2

[0044]Example 2 Effects of different re-reaction times (taking the detection of ciprofloxacin by ELISA as an example)

[0045] Coat the ELISA plate with ciprofloxacin-BSA conjugate, 1 μg / mL, 100 μL per well, and block overnight at 4 °C. Block with 3% skim milk powder the next day. Prepare ciprofloxacin standard 0ppb, 1ppb, 5ppb, 10ppb with washing solution.

[0046] The experiment was divided into 5 groups, each group took 100 μL standard and 100 μL ciprofloxacin antibody, pre-reacted in a microwell plate at 37°C for 20 minutes, and then added to the ciprofloxacin enzyme plate:

[0047] Group 1, Group 2, Group 3, and Group 4 took 120 μL of the pre-reaction product into the ciprofloxacin ELISA plate, and then reacted for 30 min, 20 min, 10 min, and 5 min respectively; washed the plate; added enzyme-labeled secondary antibody, and reacted for 30 min at 37°C , wash the plate; add chromogenic reagent 100 μL, react at 37°C for 15 min; finally add 50 μL stop solution, measure OD45...

Embodiment 3

[0051] Example 3 Effects of different pre-reaction times (taking the detection of ciprofloxacin by ELISA as an example)

[0052] Coat the ELISA plate with ciprofloxacin-BSA conjugate, 1 μg / mL, 100 μL per well, and block overnight at 4 °C. Block with 3% skim milk powder the next day. Prepare ciprofloxacin standard 0ppb, 1ppb, 5ppb, 10ppb with washing solution.

[0053] The experiment was divided into 4 groups, each group took 100 μL of standard substance and 100 μL of ciprofloxacin antibody, pre-incubated in a microwell plate at 37°C for 5min, 10min, 15min, and 20min respectively, and then took 20μL of the mixture of antibody and standard substance into the ring In the Profloxacin ELISA plate, react for 1 min, pour off the liquid in the well, pat dry on absorbent paper, then add 20 μL of the mixed solution to react for 1 min, repeat 6 times in total; wash the plate; add enzyme-labeled secondary antibody, and react for 30 min at 37°C , wash the plate; add chromogenic reagent 1...

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Abstract

The invention discloses a method for improving the sensitivity of competitive immunoassay. The method is mainly characterized in that a micromolecular substance to be detected is fully reacted with an antibody / receptor firstly and then reacted with a second molecule which participates in competition and is located on a solid phase in a short time, thus the sensitivity of the competitive immunoassay can be greatly improved. The method disclosed by the invention has the advantages of simplicity in operation, low cost, no need of expensive equipment and professional technicians, high sensitivity, and applicability to a plurality of immunoassay methods.

Description

technical field [0001] The invention relates to the field of biomedical detection, in particular to a method for improving the sensitivity of competitive immunoassay. Background technique [0002] Immunological detection methods are a series of experimental methods designed by applying immunological theory to detect antigens, antibodies, immune cells and their secreted cytokines. The antigen is specifically bound by the complementarity of the surface antigenic determinant and the hypervariable region of the antibody molecule in the spatial configuration. The same antigenic molecule may have many different antigenic determinants, if two different antigenic molecules have one or more identical antigenic determinants, cross-reaction may occur when reacting with antibodies. [0003] In addition to complementarity in spatial configuration, antigen-antibody binding mainly uses non-covalent methods on the surface of molecules such as hydrogen bonds, electrostatic attraction, van d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/53G01N33/54306G01N33/54393
Inventor 朱海李细清李金峰
Owner SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
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