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ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting methotrexate and application thereof

A methotrexate and reagent kit technology, applied in the field of immunoassay, can solve the problems of high detection cost, complex processing, cumbersome operation steps, etc., and achieve the effects of high sensitivity and accuracy, simplified operation steps, and good anti-interference performance

Inactive Publication Date: 2017-09-29
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods are difficult to detect, and now commonly used high-performance liquid chromatography mass spectrometry detection
However, the sample pretreatment of chromatography-mass spectrometry is complicated, and the operation steps are relatively cumbersome, and the cost of chromatography-mass spectrometer is high, and the detection cost is high, so it is not suitable for large sample detection and grass-roots development.

Method used

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  • ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting methotrexate and application thereof
  • ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting methotrexate and application thereof
  • ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting methotrexate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 of the present invention detects the ELISA kit for methotrexate, including the following reagents: 96-well microwell plate coated with methotrexate, methotrexate monoclonal antibody, antibody diluent, HRP-labeled goat anti-mouse II Antibody, Methotrexate Standard, Washing Solution Concentrate, Sample Diluent, TMB Chromogenic Solution and Stop Solution.

[0038] 1. The reagents are prepared as follows:

[0039] a. Coating CB buffer: pH9.6 carbonate buffer:

[0040]

[0041] b.25×PBS:

[0042]

[0043] When used, dilute 25×PBS 25 times to obtain 1×PBS.

[0044] c.10×TBST:

[0045]

[0046] When used, dilute 10×TBST 10 times to obtain 1×TBST.

[0047] d. Blocking solution:

[0048]

[0049] e. Dipping solution:

[0050]

[0051] f. Antibody diluent:

[0052]

[0053] g. Primary antibody diluent and enzyme-labeled secondary antibody diluent: antibody diluent

[0054]

[0055] h. Calibrator diluent and sample diluent: 1×PBS+0.0025% bromop...

Embodiment 2

[0095] Aspects 3-5 are similar to Embodiment 1, the difference is:

[0096] 1. Reagent preparation

[0097] a. Coating buffer: same as Example 1

[0098] b. 25×PBS: Same as Example 1, when used, dilute 25×PBS 25 times to obtain 1×PBS

[0099] c.10×TBST: Same as Example 1, when in use, 10×TBST is diluted 10 times to obtain 1×TBST

[0100] d. Blocking solution:

[0101]

[0102] e. Antibody diluent:

[0103]

[0104] f. Primary antibody diluent and enzyme-labeled secondary antibody diluent: antibody diluent

[0105] g. Calibrator diluent and sample diluent: 1×PBS++0.0025% bromophenol red indicator

[0106] 2. Preparation of MTX-OVA-coated microwell plates: a. Coating: Dilute the coated original methotrexate-OVA to a suitable concentration with CB buffer solution, add it to the microwells, 100ul per well, Seal with plate sealing film, coat overnight at 4°C or incubate at 37°C in an incubator for 2 hours; b. Seal: after overnight coating or coating at 37°C, shake off t...

Embodiment 3

[0108] Aspects 3-5 are similar to Embodiment 1, the difference is:

[0109] 1. Reagent preparation

[0110] a. Coating buffer: same as Example 1

[0111] b. 25×PBS: Same as Example 1, when used, dilute 25×PBS 25 times to obtain 1×PBS

[0112] c.10×TBST: Same as Example 1, when in use, 10×TBST is diluted 10 times to obtain 1×TBST

[0113] d. Blocking solution:

[0114]

[0115] e. Antibody diluent: 1×PBS+2%BSA+10% bovine serum+0.0025% bromophenol red indicator

[0116] f. Primary antibody diluent and enzyme-labeled secondary antibody diluent: antibody diluent

[0117] g. Calibrator diluent and sample diluent: 1×PBS++0.0025% bromophenol red indicator

[0118] 2. Preparation of MTX-OVA-coated microwell plates: a. Coating: Dilute the coated original methotrexate-OVA to a suitable concentration with CB buffer solution, add it to the microwells, 100ul per well, Seal with plate sealing film, coat overnight at 4°C or incubate at 37°C in an incubator for 2 hours; b. Seal: after ...

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Abstract

The invention provides an ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting methotrexate and application thereof. The kit is prepared from the following reagents: a methotrexate coated 96-pore microporous plate, a methotrexate monoclonal antibody, an antibody diluting solution, an HRP (Hypothalamic Regulatory Peptide)-labeled goat anti-mouse secondary antibody, a methotrexate standard product, a washing liquid concentrated solution, a sample diluting solution, a TMB (Tetramethylbenzidine) color-developing solution and a stopping solution. The stable, rapid and simple methotrexate detection kit is obtained through improving the combination of all reagents and improvement on parameters of the reagents; when the ELISA kit is applied, the standard deviation value of the kit is relatively small. The ELISA kit can be used for detecting the plasma drug concentration of patients who utilize the methotrexate with a large dosage and detecting the plasma drug concentration of patients who utilize the methotrexate with a small dosage.

Description

technical field [0001] The invention belongs to the field of immune detection, and in particular relates to an ELISA kit for detecting methotrexate and its application. Background technique [0002] Methotrexate (MTX) is a drug used to treat cancer and autoimmune diseases. It is designed as an antifolate to inhibit the metabolism of folic acid. In cancer therapy, methotrexate competitively inhibits dihydrofolate reductase (DHFR), which converts dihydrofolate to active tetrahydrofolate, by blocking folate binding. Inhibition of DHFR results in inhibition of purine and pyrimidine base synthesis, effectively limiting DNA and RNA synthesis and cancer cell growth. In autoimmune diseases, especially in the treatment of rheumatoid arthritis, methotrexate appears to affect several pathways leading to inhibition of T cell activation. These effects include inhibition of T cell expression of intercellular adhesion molecules, inhibition of methyltransferase activity and increased CD9...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/531
CPCG01N33/531G01N33/543
Inventor 方勇飞牟方祥吴红陈思闫彩霞陈欣
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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