Preparation Of Monoclonal Antibodies And Research Of Related Immunoassay Methods For Cd²⁺

In recent years,China's economy has been rapid promoted,and people's living standards have been improved significantly.However,in this process,humans have paid a very heavy price.In the worldwide,especially in China,many kinds of heavy metals such as mercury,cadmium,lead,chromium have already been causing serious pollution of the natural environment,which is the dependence of survival and development of mankinds since many years ago.At the same time,it also has brought the great threat to the health of the masses.Cadmium is a heavy metal,which has toxicity and high environmental risk,which can continued to pollute the environment.It can enter the body through the food chain and bioaccumulation,and its biological half-life is 10 to 30 years.Cadmium easy to accumulate in the kidney,liver,bone of the human body,and high concentration of cadmium in the human body have the great harmful to organ function.Traditional methods of heavy metal ions exists deficiencies,such as complex and time-consuming operation,testing costly,inconvenient for on-site testing.Therefore,establish sensitive,rapid,simple,low cost and can be used on-site testing methods to detect heavy metal ions in the environment is very meaningful.Compared with traditional methods of heavy metal ions,heavy metal immunological detection have the advantages,such as high sensitivity,selectivity,speed,and easy operation,low cost,easy on-site testing.Heavy mental immunological detection methods include competitive ELSIA detection method,KinExA immunoassay method,fluorescence polarization immunoassay method and colloidal gold immune chromatography assay?CGICA?method.This paper established the indirect competitive enzyme-linked immunosorbent assay?ELISA?method and colloidal gold immunochromatography assay method for Cd²⁺,and the main contents are as follows:Chapter 1:This chapter gives an overview of the definition,distribution,pollution and hazards,and pollution prevention and control of heavy metals;describes heavy metal immunoassay method and ELISA method;introduces principle and research progress of colloidal gold immunochromatography rapid detection technology.On this basis,describes the purpose and main content of this paper.Chapter 2:To prepare monoclonal antibody?mAb?against cadmium ion and develop an indirect competitive Enzyme-Linked Immunosorbent Assay?ELISA?method to rapidly detect the cadmium ion in water samples.Cadmium ion was respectively coupled to carrier protein?bovine serum albumin,BSA;ovalbumin,OVA?via a phenyl isothiocyanate-EDTA?ITCBE?to form the immunogen?Cd-ITCBE-BSA?and coating antigen?Cd-ITCBE-OVA?.BALB/c mice were immunized with Cd-ITCBE-BSA for the preparation of monoclonal antibody?mAb?against cadmium ion by hybridoma technique.By optimizing experimental conditions,the mAb-based indirect competitive ELISA for rapidly detecting cadmium ion in water samples was developed when the concentration of Cd-EDTA among 1-1000 ng/mL.The 50%inhibitory concentration?IC50?was 13.67 ng/mL and the detection limit was 0.48 ng/mL.In addition to the cross-reactivity rate of the antibody with mercury ion was 79.29%,no cross-reactivity was found with Zn²⁺,Cr³⁺,Pb²⁺,Cu²⁺,Ni²⁺,Al³⁺,Co²⁺,Fe³⁺,Mg²⁺,Ca²⁺and Ba²⁺.Acceptable recovery rates of 87.78%?103.3%and relative standard deviation of 1.60%?11.41%were obtained in ultra-pure water,tap water and lake water samples.The results show that we have developed mAb-based ELISA for sensitive,rapid detection of cadmium ion in water samples.Chapter 3:To prepare a colloidal gold immunochromatography strip for to rapidly detect the cadmium ion in water samples.Competitive assay was adopted.Colloidal gold binding cushion was coated with colloidal gold-Cd²⁺monoclonal antibody complex,and coating antigen Cd-ITCBE-OVA and goat anti-mouse IgG were streaked onto nitrocellulose membrane as the test line?T line?and control line?C line?,the test result was observed 3?5 min later and judged according to the color of the bands.The prepared strip was verified for sensitivity,specificity and accuracy,and used for determination of water samples.By optimizing experimental conditions,when the dilution of goat anti-mouse IgG was 1:200,and the concentration of coating antigen was 0.25 mg/mL,the minimum detection limit of the strip for cadmium ion was 100 ng/mL.Except that with Hg-EDTA,no cross-reactivity was found with Zn²⁺,Cr³⁺,Pb²⁺,Cu²⁺,Ni²⁺,Al³⁺,Co²⁺,Fe³⁺,Mg²⁺,Ca²⁺ and Ba²⁺.The determination result of water samples which containing cadmium ion standard solution by the prepared strip was consistent with that by ELISA.The results show that the developed colloidal gold immunochromatography strips is quick,simple,stable and reliable,which may be used as an effective means for on-site testing of cadmium ion in water samples.