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Kit for detecting chromium ions on basis of direct competitive enzyme linked immunosorbent assay and application thereof

An enzyme-linked immunosorbent assay and detection kit technology, which can be used in measurement devices, instruments, scientific instruments, etc., and can solve problems such as long detection time.

Active Publication Date: 2017-05-10
HENAN INST OF SCI & TECH
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  • Summary
  • 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

Method used

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  • Kit for detecting chromium ions on basis of direct competitive enzyme linked immunosorbent assay and application thereof
  • Kit for detecting chromium ions on basis of direct competitive enzyme linked immunosorbent assay and application thereof
  • Kit for detecting chromium ions on basis of direct competitive enzyme linked immunosorbent assay and application thereof

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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 comprises the following steps:

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

[0044] Ⅱ. Add 200-300 μL / well of pig negative serum with a mass concentration of 3-6% to the detection plate obtained in the above step 1, and incubate for the second time at 35-40°C for 1.5-3 hours, and remove the coating solution after incubation , washing the plate for the second time with washing solution for 2-5 times, and drying the detection plate naturally at 23-27° C. to obtain a detection plate coated with goat anti-mouse IgG secon...

Embodiment 1

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

[0106] (1) Weigh 10mg ITCBE and dissolve it in 1mL dimethyl sulfoxide (DMSO) to form a metal chelating agent solution; weigh 16.1mg chromium chloride (CrCl 3 .6H 2 O) Dissolve in 1mL HEPES buffer (10mmol / L) of pH 8.0 (has no toxic effect on cells. It is a hydrogen ion buffer and can control a constant pH range for a long time) (10mmol / L) 3+ solution; the metal chelating agent solution and Cr 3+ The solutions were mixed, and the pH value was adjusted to 7.0 with NaOH, and then reacted on a shaker at room temperature for 12 hours to form the Cr-ITCBE chelate hapten.

[0107] (2) Weigh 66 mg of BSA and 11.6 mg of EDC and dissolve in 5 mL of PBS buffer, slowly add 7 mg of ethylenediamine (dissolved in 3 mL of PBS+DMF solution in advance) under stirring conditions, and shake at 37°C for 2 hours. The reaction solution was dialyzed against PBS for 4 days, and the activated carrier protein BSA was lyophilized and stored as...

Embodiment 2

[0110] Preparation of anti-chromium ion monoclonal antibody

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

[0112] (1) Mice immunization: 5 6-8 week old female Balb / C mice were immunized with Cr-ITCBE-cBSA, the dose was 60 μg / mouse, and the volume was 0.2 mL. The immunogen diluted with PBS was completely emulsified with an equal volume of FCA for the first immunization, and then boosted every 4 weeks, and emulsified with FIA. 7 days after immunization for 5 times, the blood was collected by docking the tail to separate the serum, 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. 3 days before the fusion, the hyperimmunized mice were injected with 50 μg of immunogen in the tail vein and intraperitoneally, each with a volume of 100 μL.

[0113] (...

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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 in particular relates to a kit for detecting chromium ions based on a direct competitive enzyme-linked immunosorbent assay and a preparation method and application thereof. Background technique [0002] In recent years, drinking water sources in many parts of my country have been polluted, and pollution incidents of various heavy metal ions have occurred frequently, and people's physical and mental health have been seriously threatened. Among them, the three industrial wastes (waste water, waste gas, and waste residue) have caused great damage to the environment, such as industrial waste water from industries such as mining, battery, electroplating, and chemical industry, which contain a large amount of heavy metal ions. Among all kinds of heavy metal ion pollution sources, chromium is a kind of widespread toxic element, which seriously threatens the health of domestic animals...

Claims

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Application Information

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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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