Development of Enzyme-Linked Immunosorbent Assay for the Heavy Metal Lead
|School||Tianjin University of Science and Technology|
|Course||Nutrition and Food Hygiene|
|Keywords||Lead Polyclonal antibody ELISA|
In this paper, a reliable enzyme-linked immunosorbent assay method was developed for detecting of heavy metal lead. Lead connected with keyhole limpet hemocyanin using ITCBE, which was used as immunogen. A polyclonal antibody with high affinity and specificity was obtained after immunization of rabbits. Lead connected with Bovine serum albumin using ITCBE, which was used as coating antigen. We established a high sensitivith indirect competition ELISA detection method, the influence of the experimental factors (including antigen incubation concentration, ionic strength, pH, etc.) were studied, the standard curve obtained the highest sensitivity and stability when antigen incubation concentration was0.1μg/well, ionic strength was1mmol/L PBS, pH7.5. The concentration of lead causing15%inhibition is0.32μg/L,50%inhibition is3.48μg/L. The cross-reactivity against other metal ionic is all low.Tap water, Haihe water, dahurica, carrots, mantis shrimp were chosen for detection. tap water was spiked with Pb (II) at three different levels (0.2μg、2μg、6.5μg). Top water was diluted five-fold with1mmol/L PBS (pH7.5) and directly applied to the IC-ELISA analysis. River water samples was spiked with Pb (II) at three different levels (0.32μg、3.2μg、10.4μg). The river water was initially passed through0.22μm filter film and was diluted eight-fold with1mmol/L PBS (pH7.5). Dahurica, carrots and mantis shrimp were ground using traditional Chinese medicine pulper, and1g of ground samples put into the high-pressure digestion cans. These samples spiked with Pb (II) at three different levels (0.024μg、0.240μg、0.78μg).10mL of nitric acid and2mL of perchloric acid were added into high-pressure digestion cans overnight, and the obtained solution was heated to boiling using heating plate at160℃-180℃until the solution became to clear and was solved completely. The pH was adjusted to7.4with trihydroxymethyl aminomethane (Tris), and the solution was passed through0.22μm filter film. The resulting solution was diluted with double distilled water to60mL of volume, and finally was used to analyze by IC-ELISA.Adding three different concentrations of lead, recovery rate was between78.5-121%. Limit of detection in tap water was1.6μg/L, in Haihe water was2.56μg/L, in other samples was19.2μ/kg. Inter-and Intra-variation were less than16%, which meant the method established in this experiment had high accuracy and precision to meet the detection.