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Preparation of heavy metal cadmium polyclonal antibody and method for measuring enzyme linked immunity absorption

An enzyme-linked immunosorbent (ELISA), polyclonal antibody technology, applied in anti-animal/human immunoglobulin, biological testing, material testing products, etc., can solve the problems of long cycle, high preparation cost, high risk, etc., and achieve easy remedial and return to work, significant practical effects

Inactive Publication Date: 2008-08-13
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with polyclonal antibodies, the preparation of monoclonal antibodies is more costly, longer, and riskier
Although more clonal antibodies are slightly higher in specificity, as far as the established detection method is concerned, polyclonal antibodies can also meet the detection requirements, and the detection limit and sensitivity of the method are close to those of monoclonal antibody-based detection methods

Method used

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  • Preparation of heavy metal cadmium polyclonal antibody and method for measuring enzyme linked immunity absorption

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Experimental program
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Embodiment 1

[0026] Antibody preparation

[0027] Step 1: Synthesis of complete antigen

[0028] Weigh 10 mg ITCBE and dissolve in 10 mL pH 9.5 sodium phosphate buffer solution to form a 2.275 mM metal chelator solution. Weigh 40mg BSA protein and dissolve in pH 7.4 PBS (137mM NaCl, 3mM KCl, 8mM NaCl 2 HPO 4 , 1 mM KH 2 PO 4 ) buffer solution to form a 40mg / mL protein solution. Cadmium (99.999%) was dissolved in hot nitric acid and diluted to 4.7mM for use.

[0029]Take 2.5mL ITCBE and react with 1.5mL 4.7mM cadmium working solution to obtain Cd-ITCBE complex. The complex was added dropwise to 1 mL of 40 mg / mL BSA protein solution. The reaction solution was reacted at room temperature under slow stirring for 24 hours, then put into EDTA pretreated dialysis bags, dialyzed three times with PBS buffer, twice with pure water, and dialyzed at 4°C for three days to remove unreacted Cd, ITCBE and Cd-ITCBE complex. Accurately measure the dialysate, which is the immunogen Cd-ITCBE-BSA, and...

Embodiment 2

[0039] ELISA method

[0040] Step 1: Determination of Potency

[0041] Use the square matrix titration method to determine the optimal working concentration of the antigen and antibody: the coated antigen (Cd-ITCBE-OVA) is serially diluted to 1500 times, 2500 times, 5000 times, 10000 times the AB, CD, EF of the coated enzyme plate, GH line; use 1% OVA as blocking solution; antiserum was diluted to 20000 times, 40000 times, 80000 times, 160000 times, 320000 times and 640000 times respectively, and added to 1 2, 3 4, 5 6, 7 8 of the microtiter plate, 9 10, 11 12 columns; add goat anti-rabbit-horseradish peroxidase enzyme-labeled secondary antibody (1:10000, diluted with 1% OVA); develop color with TMB, stop with 2M sulfuric acid, measure OD450 value. The antigen-antibody concentration with an OD450 value of 0.8-1.2 was used as the optimal working concentration. Finally, it was determined that the optimal coating original Cd-ITCBE-OVA and antibody concentrations were 1.05 μg / mL...

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Abstract

The invention discloses a preparation of polyclonal antibody of heavy metal cadmium, and a measurement method for enzyme linked immunosorbent. The preparation of polyclonal antibody of heavy metal cadmium includes steps of : (A) synthesis of complete antigen, which is critical for preparation of the polyclonal antibody; (B) preparation of the antibody. The measurement method for enzyme linked immunosorbent includes: (1) determining optimal reaction concentration of coating source Cd-ITCBE-OVA and antibody by matrix titration, and coating 96-well enzyme label plate by coating source Cd-ITCBE-OVA with optimal concentration; (2) direct competitive enzyme-linked immunosorbent assay; (3) examining impact of various reaction condition on CI-ELISA, and optimizing reaction condition; (4) establishing CI-ELISA, drawing standard inhibition curve under optimized reaction condition. Detection limit of the invention is 0.04 mu g / L, linear range is 10-1-103mu g / L. The invention is specific to cadmium, suitable for measurement of trace cadmium in ambient water. The invention also provides technical support in fast in-situ detection for sudden accident of heavy metal pollution, in order to carry out remedy and recovery for pollution accident in time.

Description

technical field [0001] The invention relates to the preparation of heavy metal cadmium polyclonal antibody and the antibody-based enzyme-linked immunosorbent assay method (ELISA). Background technique [0002] The pollution caused by heavy metals is called heavy metal pollution. At present, the most serious heavy metals to the environment are lead, cadmium, mercury, chromium and arsenic, referred to as "five poisons". After heavy metals enter the human body, they are difficult to excrete and accumulate gradually. When they exceed the physiological load of the human body, they will cause changes in physiological functions, lead to acute and chronic diseases, or cause long-term harm. In recent years, with the rapid development of my country's economy, more and more environmental accidents have occurred frequently. The main type of pollutants is heavy metal pollutants, especially food poisoning, drinking water poisoning, and sudden pollution accidents of water bodies. Therefor...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/53
Inventor 孙成何欢杨绍贵刘贤进刘振宇张川陈成
Owner NANJING UNIV
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