A high-affinity anti-heavy metal cadmium monoclonal antibody and its application

A monoclonal antibody and anti-heavy metal technology, applied in the fields of application, chemical instruments and methods, botanical equipment and methods, etc., can solve the problems of inability to achieve rapid detection, achieve high titer and affinity, good sensitivity, and low cost effects

Active Publication Date: 2017-12-29
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ELISA method also has its own defects, that is, it still takes at least 3 hours to complete the detection of a batch of samples, which cannot meet the requirements of rapid detection; and the selection of antigen or antibody is a very important factor, which determines the specificity and sensitivity of detection.

Method used

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  • A high-affinity anti-heavy metal cadmium monoclonal antibody and its application
  • A high-affinity anti-heavy metal cadmium monoclonal antibody and its application
  • A high-affinity anti-heavy metal cadmium monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Synthesis of heavy metal cadmium complete antigen

[0040] Cd-iEDTA-BSA (immunogen) and Cd-iEDTA-OVA (detection source) were synthesized by the isothiocyanate method, and the synthesis schematic is attached figure 1 Shown, concrete preparation method is as follows:

[0041] (1) Dissolve 1 mg of iEDTA in 200 μL DMSO, then add 200 μL 6.4 mg / mL Cd dropwise 2+ Standard solution, to ensure that Cd 2+ The molar ratio of the standard solution to iEDTA is 1.1-1.2:1, placed in a shaker at 24°C and 125rpm for about 5 hours, until the solution turns slightly yellow.

[0042] (2) Dissolve 5mg BSA (or 3.4mg OVA) in 2mL Tris-HCl buffer (pH7.4), stir and add the reacted Cd-iEDTA dropwise, and add triethylamine dropwise to adjust the pH to 9.0, Then add 10 μL of PEG600, shake at 24° C., 125 rpm in a shaker for about 36 hours, until the solution turns slightly yellow. After the reaction, the unchelated Cd was removed by ultrafiltration 5 times through Centricon-30 ultrafi...

Embodiment 2

[0043] Example 2 Screening of hybridoma cell line 1H9 and preparation of monoclonal antibody

[0044] The preparation method of anti-heavy metal cadmium mouse-derived monoclonal antibody is as follows:

[0045] 1. Animal immunity

[0046] (1) Dilute the immunogen Cd-iEDTA-BSA to 200 μg / mL with physiological saline, then add the same volume of Freund’s complete adjuvant, and emulsify the complete antigen by the double push method of the syringe;

[0047] (2) Number 5 BALB / c mice (6-8 weeks old), and the immunization dose of each mouse is 100 μg, 200 μL, and multi-point immunization is carried out through the subcutaneous neck and abdomen;

[0048] (3) After 3 weeks of immunization, mix 0.7 mL of 200 μg / mL complete antigen and 0.7 mL of Freund's incomplete adjuvant, and immunize with the above-mentioned immunization method, and immunize once in the next 2 weeks;

[0049] (4) After the mice were immunized for three times, blood was collected by docking the tail on the 10th day ...

Embodiment 3

[0100] Example 3 Direct competition ELISA detection

[0101] Based on the monoclonal antibody 1H9, a direct competitive ELISA detection method was established, the operation process and detection principle are attached Figure 6 shown.

[0102] The specific operation is as follows:

[0103] (1) Dilute the prepared cadmium monoclonal antibody to the working concentration of 100 μL per well, incubate at 37°C for 3 hours, and then wash with PBST buffer 3 times.

[0104] (2) Then use 5% skimmed milk powder and 2% polyethylene glycol 20000 as the mixed blocking solution to carry out constant temperature blocking at 37°C for 1 hour, and wash 3 times with PBST buffer.

[0105] (3) Add 50 μL triple-distilled water as the control well and 20 ng / mL Cd 2+ The standard product was used as the competition well, and then 50 μL of enzyme standard substance Cd-iEDTA-HRP diluted in working concentration was added, and reacted in a 37°C incubator for 10 minutes.

[0106] (4) After washing w...

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Abstract

The invention discloses a hybridoma cell line 1H9 secreting a high-affinity anti-heavy metal cadmium monoclonal antibody and the monoclonal antibody 1H9 secreted therefrom. The hybridoma cell line 1H9 was deposited in the China Center for Type Culture Collection on March 4, 2015, with the preservation number CCTCC C201521. The ascitic fluid Elisa titer of the monoclonal antibody 1H9 reaches 1:1×106, and can specifically recognize the heavy metal cadmium. The direct competition Elisa detection method (one-step competition method) based on the monoclonal antibody 1H9 is specific and sensitive. , Accurate, fast, safe and convenient, stable reagents and low cost, etc. The results can be obtained in 30 minutes, and quantitative detection can be performed, with a sensitivity of up to 2.1ng / mL. It is especially suitable for the rapid detection of samples, and can be widely used in the analysis and testing of environmental water samples containing heavy metal cadmium and rice samples and the rapid screening of actual samples.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a high-affinity anti-heavy metal cadmium monoclonal antibody and its application. Background technique [0002] In nature, cadmium mostly exists in the form of cadmium salt, which can form inorganic compounds with sulfur, chlorine, oxygen, etc. The oxidation state of cadmium is +1 or +2, and it is easily oxidized in the air, and a brown oxide layer will appear when heated; under high temperature conditions, cadmium can react violently with halogen to produce cadmium halide; cadmium can also directly react with sulfur reaction to produce toxic and carcinogenic yellow or orange cadmium sulfide. In addition, various cadmium compounds such as cadmium oxide, cadmium bromide, cadmium selenide, cadmium iodide, cadmium chloride, cadmium cyanide, and cadmium nitrate are toxic. Since most cadmium compounds are soluble in water, they can be absorbed and accumulated by the bod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44C12N15/13G01N33/577G01N33/543
Inventor 向军俭刘艳梅钟辉黄建芳
Owner JINAN UNIVERSITY
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