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Quantitative enzyme-linked immunoabsorbent assay method based on moving reaction boundary electrophoresis titration

A technology of mobile reaction and detection method, which is applied in the direction of chemical reaction of materials and material analysis by observing the influence on chemical indicators, so as to reduce costs and simplify ELISA detection.

Active Publication Date: 2016-12-21
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, MRB-ET technology has not been applied to enzyme-linked immunosorbent assay analysis, so it is necessary to develop a new technology to quantitatively detect the concentration of enzyme catalytic products by MRB-ET and then quantitatively detect target antibodies, antigens, or haptens

Method used

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  • Quantitative enzyme-linked immunoabsorbent assay method based on moving reaction boundary electrophoresis titration
  • Quantitative enzyme-linked immunoabsorbent assay method based on moving reaction boundary electrophoresis titration
  • Quantitative enzyme-linked immunoabsorbent assay method based on moving reaction boundary electrophoresis titration

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Such as Figure 1~3 As shown, this embodiment performs ELISA-MRB-ET detection on standard samples of SEB type enterotoxins with different concentrations and known SEB-type enterotoxins under the same conditions, and establishes the functional relationship between MRB moving distance and substrate concentration, along the electrophoretic channel Mark the concentration scale; in the sample detection, use the same conditions for ELISA-MRB-ET detection, and the concentration scale corresponding to the stop position of the formed MRB is the substrate concentration in the sample; specifically, the following steps are included:

[0026] Step 1. Fill the gel in the electrophoresis channel and perform primary antibody coating and blocking.

[0027] The electrophoresis channel is a straight electrophoresis channel, and its length, width and height are 20mm, 1.0mm and 0.5mm respectively.

[0028] The two ends of the electrophoresis channel are respectively provided with a cathode...

Embodiment 2

[0053] The standard sample in this embodiment is glycosylated hemoglobin, and the HbA1C in human whole blood is determined by the double-antibody sandwich method.

[0054] The primary antibody of this example is 10 mg / L, 200 μL of haptoglobin, and the cathode chamber is coated overnight at 4° C., and washed 3 times with phosphate buffer.

[0055] Blocking in this embodiment refers to: using 200 μL of 3% BSA to block the blank sites on the plate, incubating at 37° C. for 1 h, washing with phosphate buffer solution 3 times, and removing excess liquid.

[0056] The difference compared with Example 1 is that the serial dilution concentrations of the standard samples in this example are 5 μg / L, 2.5 μg / L, 1.5 μg / L, 0.625 μg / L, 0.3125 μg / L and 0 μg / L respectively .

[0057] The glycosylated hemoglobin can reflect the patient's blood sugar control in the past 8 to 12 weeks, and is a new standard for diabetes diagnosis and a "gold standard" for treatment monitoring.

Embodiment 3

[0059] The standard sample of this embodiment is residual sulfur phosphorus on lettuce.

[0060] Said parathion is a broad-spectrum insecticide, and my country requires that the maximum residue of agricultural products should be less than 0.1 mg / kg, and must not be detected on vegetables and fruits.

[0061] The primary antibody in this example is a goat anti-rabbit antibody, which was diluted by 2000 times, coated the cathode chamber at 200 μL and 4° C. overnight, and washed 3 times with phosphate buffer.

[0062] Compared with Example 1, the difference is that the dilution concentrations of the sulfur and phosphorus standard samples in this example are 500ng / mL, 100ng / mL, 25ng / mL, 12.5ng / mL, 6.5ng / mL and 0ng / mL respectively.

[0063] The experimental conditions of the ELISA in this example are as follows: 10 μL of standard samples, 45 mL of enzyme-labeled substances and 45 mL of antibodies were incubated in the cathode chamber at 37° C. for 30 min, and washed three times wit...

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Abstract

The invention discloses a quantitative enzyme-linked immunoabsorbent assay method based on moving reaction boundary electrophoresis titration. ELISA-MARB-ET is carried out on known standard samples with different antigen concentrations respectively under a same condition, so that a mathematical model of the moving distance and antigen concentration of a moving reaction boundary is established, and an antigen concentration ruler is labeled along an electrophoresis channel; ELISA-MRB-ET is carried out on samples to be detected under the same condition in a test stage, so that a measured concentration scale corresponding to a stopping position of the moving reaction boundary is the antigen concentration in the sample. The method is reasonable in design, and quantitative detection can be realized without expensive instruments, so that the cost is reduced, and high-throughput detection can be realized.

Description

technical field [0001] The present invention relates to a technology in the field of ELISA detection, in particular to a quantitative ELISA detection method based on moving reaction boundary-electrophoresis titration (MRB-ET). Background technique [0002] In 1971, Engvall and Perlmann introduced an enzyme-linked immunosorbent assay (enzyme-linkedimmune sorbentassay, ELISA) method for the quantitative detection of IgG, which developed the enzyme-labeled antibody technology previously used for antigen localization into the detection of trace substances in liquid specimens Methods (E. Engvall, P. Perlmann, 1971, Immunochemistry, 8, 871‐874.). The basic principle of this method is: bind the primary antibody to the surface of a solid phase carrier; link the enzyme and the secondary antibody to form an enzyme-labeled antibody; reaction, and finally the amount of enzyme combined on the solid phase carrier is proportional to the amount of the antigen to be detected in the sample; ...

Claims

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Application Information

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IPC IPC(8): G01N21/79
CPCG01N21/79
Inventor 曹成喜孔凡志钟冉曹馨语张强李国庆肖华樊柳荫
Owner SHANGHAI JIAO TONG UNIV
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