A kind of kit for detecting chromium ion based on direct competitive ELISA and its application

A technology of enzyme-linked immunoassay and detection kit, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., and can solve the problems of long detection time

Active Publication Date: 2019-07-19
HENAN INST OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research on immunological detection technology of heavy metal chromium ion pollution at home and abroad is very active, and the patent whose notification number is CN103439508B discloses a kind of indirect competitive enzyme-linked immunoassay kit for detecting chromium ion and its composition and detection method, and its detection sensitivity is 2ng / mL, and the detection time is longer

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  • A kind of kit for detecting chromium ion based on direct competitive ELISA and its application
  • A kind of kit for detecting chromium ion based on direct competitive ELISA and its application
  • A kind of kit for detecting chromium ion based on direct competitive ELISA and its application

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preparation example Construction

[0042] In the present invention, the preparation method of the detection plate coated with goat anti-mouse IgG secondary antibody preferably includes the following steps:

[0043] Ⅰ. Add 100~150μL / well of goat anti-mouse IgG coating solution with a coating concentration of 50~200μg / mL goat anti-mouse IgG to the detection well of the detection plate, and incubate for 1.5~3h at 35~40℃ for the first time, after incubation Remove the coating liquid, wash the plate for the first time with the washing liquid 2 to 5 times;

[0044] Ⅱ. Add 200-300 μL / well of pig negative serum at a mass concentration of 3 to 6% to the test plate obtained in step I, incubate for the second time at 35 to 40°C for 1.5 to 3 hours, and remove the coating solution after incubation , Wash the plate for the second time with washing solution 2 to 5 times, and dry the test plate naturally at 23-27°C to obtain a test plate coated with goat anti-mouse IgG secondary antibody.

[0045] In the present invention, the type ...

Embodiment 1

[0105] Synthesis of Cr-ITCBE-cBSA immunogen ( figure 1 )

[0106] (1) Weigh 10mg ITCBE dissolved in 1mL dimethylsulfoxide (DMSO) to form a metal chelating agent solution; Weigh 16.1mg chromium chloride (CrCl 3 .6H 2 O) Dissolve in 1 mL of HEPES buffer pH 8.0 (non-toxic to cells. It is a hydrogen ion buffer that can control a constant pH range for a long time) (10mmol / L) to form Cr 3+ Solution; the metal chelating agent solution and Cr 3+ The solution is mixed, and the pH value is adjusted to 7.0 with NaOH, and then placed on a shaker at room temperature for 12 hours to form a Cr-ITCBE chelate hapten.

[0107] (2) Weigh 66mg BSA and 11.6mg EDC and dissolve them in 5mL PBS buffer. Under stirring conditions, slowly add 7mg ethylenediamine (dissolved in 3mL PBS+DMF solution in advance), and react with shaking at 37°C for 2h. The reaction solution was dialyzed with PBS for 4 days, and the activated carrier protein BSA was freeze-dried and preserved, namely cBSA.

[0108] (3) Weigh 30 mg o...

Embodiment 2

[0110] Preparation of anti-chromium ion monoclonal antibody

[0111] The anti-chromium ion specific monoclonal antibody is prepared by immunizing Balb / C mice with Cr-ITCBE-cBSA immunogen, and is achieved by the following steps:

[0112] (1) Mouse immunization: 5 female Balb / C mice aged 6-8 weeks were immunized with Cr-ITCBE-cBSA, the dose was 60 μg / mouse, and the volume was 0.2 mL. The immunogen diluted with PBS and an equal volume of FCA were completely emulsified for the first immunization, and then boosted every 4 weeks, and replaced with FIA emulsification. After 5 times of immunization, the tail was cut off and blood was collected to separate the serum on the 7th day, and the mice with high titer and good blocking effect were screened by indirect ELISA and indirect competitive ELISA (ciELISA) as spare mice for fusion. The mice were superimmunized 3 days before fusion, and 50 μg immunogen was injected into the tail vein and the abdominal cavity, each with a volume of 100 μL.

...

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Abstract

The invention provides a kit for detecting chromium ions on the basis of a direct competitive enzyme linked immunosorbent assay. The kit comprises a detection plate coated with a goat-anti-mouse IgG secondary antibody, an enzyme-labeled Cr-ITCBE chelate hapten with mass concentration of 20 to 50mu g / mL, chromium ion resistant monoclonal antibody solution with mass concentration of 10 to 20mu g / mL, 1.0 to 3.0mol / L of stop buffer, sample diluent, substrate developing solution, cleaning solution, chromium ion standard substance solution, and EDTA conditioning fluid with mass concentration of 10 to 20mg / mL, wherein the detection plate is packaged in a vacuum sealed mode; coating concentration of the goat-anti-mouse IgG secondary antibody is 50 to 200mu g / mL; the chromium ion resistant monoclonal antibody solution is prepared from Cr-ITCBE-cBSA immunogen immunized Balb / C mice. The kit provided by the invention has the characteristics of high sensitivity and high specificity, and meanwhile, has the advantage of short detection time.

Description

Technical field [0001] The invention belongs to the technical field of environmental detection, and specifically relates to a chromium ion detection kit based on direct competition enzyme-linked immunoassay, and a preparation method and application thereof. Background technique [0002] In recent years, drinking water sources in many parts of our country have been polluted, and various heavy metal ion pollution incidents have occurred frequently, and people's physical and mental health have been seriously threatened. Among them, the three industrial wastes (waste water, waste gas, waste residue) have caused great damage to the environment. For example, industrial waste water from industries such as mining, battery, electroplating, and chemical industries contains a large amount of heavy metal ions. Among various pollution sources of heavy metal ions, chromium is a kind of toxic element that exists widely, which seriously threatens the health of livestock and humans. When chromiu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 范国英姜金庆贺永惠孙亚伟柳东阳杨雪峰胡建和葛亚明齐永华丁菡
Owner HENAN INST OF SCI & TECH
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