Homogeneous Electrogenerated Chemiluminescence Immunoassay Using A Luminophor-labeled Hapten

This thesis includes a review and a research section. The review includes the principle and characteristics of electrogenerated Chemiluminescence immunoassay (ECLIA), the apparatus of electrogenerated Chemiluminescence (ECL), and the labeling methods, separating technologies, immobilizing technologies and coupling technologies in ECLIA. The research comprises of two subunits. In the research section, two ECLIA methods have been developed, which are based on the signal changes derived from steric hindrance and the separation by a film electrode, respectively. Digoxin and luminol are used as a model for the ECLIA systems. Digoxin and anti-digoxin antibody are determined in sub-trace level based on the changes of the ECL signal.Electrogenerated Chemiluminescence analysis has been developed as a highly sensitive analytical method. In an ECL process, the reactive species are generated from stable precursors at the surface of an electrode. The immunoassay is an important analytical methodology that has been widely applied due to high specificity. ECLIA combines the advantages of ECL and immunoassay, so it has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for label are extremely low; the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most the chemiluminescent systems; the labels small molecules, can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immuno-reactivity, solubility, or ability to hybridize; and measurement is simple and rapid, requiring only a few seconds. ECLIA is promising and has been widely employed in protein, clinical, pharmaceutical, and biochemical fields. The review summarized the principle and characteristics of ECLIA, described the apparatus of ECL in detail and the labeling methods, the separating technologies, immobilizing technologies and coupling technologies in ECLIA.The research section includes two sections. A novel homogeneous ECLIA foranti-digoxin antibody and digoxin hapten was developed using a luminol-labeled digoxin hapten in the first section. ECL emission was electrochemically generated from the luminol-labeled digoxin and decreased markedly in the presence of anti-digoxin antibody due to steric hindrance. The anti-digoxin antibody concentration was determined in the range from 1/5000 to 1/500 dilution. Digoxin hapten was determined throughout its therapeutic range with a detection limit of 0.1 ng/mL and in a 3 orders of magnitude range by competitive immunoreaction between luminol-labeled digoxin and digoxin with fixed quantity of anti-digoxin antibody. The proposed method has been applied to determine anti-digoxin antibody and digoxin in human control serum with satisfactory results.A novel homogeneous ECLIA for anti-digoxin antibody and digoxin hapten was developed on a thin film electrode in the second section. ECL emission was electrochemically generated from the luminol-labed digoxin and decreased markedly in the presence of anti-digoxin antibody because the complex of digoxin-anti-digoxin antibody could not pass through the film to the surface of electrode. The anti-digoxin antibody concentration was determined in the range from 1/5000 to 1/500 dilution. Digoxin hapten was determined throughout its therapeutic range with a detection limit of 0.1 ng/mL and in a 3 orders of magnitude range by competitive immunoreaction between luminol-labeled digoxin and digoxin with fixed quantity of anti-digoxin antibody. The proposed method has been applied to determine anti-digoxin antibody and digoxin in human control serum with satisfactory results.