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Immunoassay method based on thioflavin T

An immunoassay and sulfur technology, applied in the field of immunoassay in multi-read mode, can solve the problems of lack of specificity, time-consuming, difficult to identify, etc., and achieve the effect of multi-selectivity and wide linear range.

Active Publication Date: 2018-11-23
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Because the clinical symptoms of Mycoplasma pneumoniae infection are atypical and lack of specificity, it is difficult to distinguish it from other bacterial and viral infections, and it is easy to be misdiagnosed
The traditional culture method takes more than 2 weeks to detect Mycoplasma pneumoniae, which takes a long time and has a low detection rate, which cannot guide clinical medication in time

Method used

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  • Immunoassay method based on thioflavin T
  • Immunoassay method based on thioflavin T
  • Immunoassay method based on thioflavin T

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] This example is used to illustrate the implementation steps of the thioflavin T-based immunoassay method provided by the present invention.

[0064] The specific operation is as follows:

[0065] (1) Coating: Coat Mycoplasma pneumoniae bacterial antigen (100 μl / well, 1 mg / mL) on a 96-well ELISA plate with serum albumin solution of corresponding dilution concentration, seal the plate with film, and leave overnight at 4°C.

[0066] (2) Plate washing: Add 100 μl of Tris-HCl (0.05 μmol / L) washing solution per well, wash the plate 3 times with an interval of 1 min each time, turn over the microplate and place it on absorbent paper and filter out the water.

[0067] (3) Blocking: add blocking solution, 200 μl per well, and block for 1 hour at room temperature.

[0068] (4) Wash the plate and repeat step 2.

[0069] (5) Add 0.1 mg of alkaline phosphatase ALP-labeled mouse anti-human MP-IgM standard substance and 1 ul of serum to be tested, and incubate at room temperature fo...

Embodiment 2

[0076] This example is used to illustrate the implementation steps of the thioflavin T-based immunoassay method provided by the present invention.

[0077] The specific operation is as follows:

[0078] (1) Coating: Coat Mycoplasma pneumoniae bacterial antigen (100 μl / well, 0.1 mg / mL) on a 96-well ELISA plate with serum albumin solution of corresponding dilution concentration, seal the plate, and place it at 37°C for 2 Hour.

[0079] (2) Plate washing: Add 100 μl of Tris-HCl (0.05 μmol / L) washing solution per well, wash the plate 3 times with an interval of 3 minutes each time, turn over the microplate and place it on absorbent paper and filter out the water.

[0080] (3) Blocking: add serum albumin solution, 200 μl per well, and block for 2 hours at room temperature.

[0081] (4) Wash the plate and repeat step 2.

[0082] (5) Add 0.1 mg of mouse anti-human MP-IgM labeled with alkaline phosphatase ALP and 1 ul of serum to be tested, 100 μl / well, and incubate at room tempera...

Embodiment 3

[0089] This example is used to illustrate the implementation steps of the thioflavin T-based immunoassay method provided by the present invention.

[0090] The specific operation is as follows:

[0091] (1) Coating: Coat Mycoplasma pneumoniae bacterial antigen (100 μl / well, 10 mg / mL) on a 96-well ELISA plate with serum albumin solution of corresponding dilution concentration, seal the plate with film, and leave overnight at 4°C.

[0092] (2) Plate washing: Add 100 μl of Tris-HCl (0.05 μmol / L) washing solution per well, wash the plate 3 times with an interval of 2 min each time, turn over the microplate and place it on absorbent paper and filter out the water.

[0093] (3) Blocking: add serum albumin solution, 200 μl per well, and block for 5 hours at room temperature.

[0094] (4) Wash the plate and repeat step 2.

[0095] (5) Add 0.1 mg of mouse anti-human MP-IgM labeled with alkaline phosphatase ALP and 1 ul of serum to be tested, 100 μl / well, and incubate at room temperat...

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Abstract

The invention provides an immunoassay method based on thioflavin T and application thereof. The method comprises the following steps of adding enzyme-responsive polypeptides on the basis of a conventional alkaline phosphatase reaction system ELISA to be used as a reaction substrate so that polypeptides form beta-folding structure under the enzyme catalysis; then, introducing fluorescent dye of thioflavin T; performing detection by using signals generated when the thioflavin T is combined with beta-folding polypeptides. Five kinds of read-out modes are provided for detecting mycoplasma pneumoniae antibodies; more selectivity is shown; an excellent method for detecting mycoplasma pneumoniae IgM antibodies is provided.

Description

technical field [0001] The invention relates to the field of immunoassay, in particular to an immunoassay method based on thioflavin T with multiple readout modes and its application. Background technique [0002] Multiple readout modes show more obvious advantages for disease diagnosis than single readout modes. Multiple modes of readout not only allow for more flexible reporting of disease under various resource-limited conditions, but also improve the reliability of detection by verifying the consistency of different readout results. Qualitative and quantitative dual analysis showed more advantages. Qualitative analysis assists in the rapid judgment of the disease. Quantitative analysis provides accurate and detailed data reporting on disease progression. Therefore, developing an immunoassay with multiple readouts is an attractive research direction in the field of disease diagnosis. Peptide self-assembly combined with fluorescent dyes offers us the possibility to dev...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56933
Inventor 蒋兴宇刘野王云云
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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