| Radioimmunoassay (RIA) has played an important role in such diverse fields as clinic diagnosis, medicine, cytology, forensics, environmental sciences, and amongst others. The ubiquitous use of RIA is mainly due to the high specificity and sensitivity of this technique. Even though, various kinds of non-isotopic immunoassays (NRIAs) have been rapidly developed in the past two decades, with an attempt to circumvent the well-known methodological and environmental problems of RIA. As one of the most important NRIAs, TRFIA has found its widest and most successful applications, due to its high sensitivity, wide dynamic range and precise detection; moreover, TRFIA can be applied to multi-analyte immunoassay (MALA) more conveniently.Different from ordinary immunoassay concept, MAIA means that two or more analytes are analyzed simultaneously in a single assay procedure with only once sample addition. Presently, the commonly used modes for performing MAIA mainly include multiple-probe approach, single-probe spatial resolution approach and dual-probe spatial resolution approach. All these techniques require a discrimination of the specific signal that is specific to different analytes.The objectives of immunoassay, as is the same for other analytical methods, would be accurate, rapid, simple and inexpensive; MAIA provides a way to achieve this goal.This thesis consists of three parts: the preface, the development of dual-label TRFIA for simultaneous detection of serum TSH/TT4 and the synthesis and evaluation of several fluorescent ?-diketonate-europium chelates.The preface is a comprehensive review on the principles and methodological features of different immunoassay techniques, especially on TRFIA and MAIA. As improving analytical sensitivity represents one of the major methodological challenges for developing serum TSH/TT4 TRFIA, the strategies for improving analytical sensitivity of immunoassay was described briefly.In the second part, we investigated the features of different assay formats for designing competitive serum TT4 TRFIA, and studied different strategies for improving the analytical sensitivity of serum TSH TR-IFMA. Based onthe use of double detection-antibody and biotin-streptavidin system, a highly sensitive serum TSH TR-IFMA was developed. Taken the advantages of a novel 'co-coating' protocol, a highly active surface anti-T4 and anti-TSH antibodies was prepared, and serum TSH/TT4 TRFIA was developed by employing Eu3+ and Sm3+ as labels.In the third part of the thesis, several ?-diketonate-europium chelates were synthesized based on the work originally described by Yuan JL, et al. The chelates synthesized in our work have improved fluorescence features compared to the originally ones. The relation of the fluorescence properties and the structures of these chelates were analyzed. The potential use of the chelates as labels in TRFIA was evaluated and the general problems associated with ligand label used in TRFIA were discussed briefly.The following results have been obtained from above experiments:1 In order to ensure an accurate measurement of serum TT4 in high concentration range, the serum TT4 TRFIA based on surface-antigen requires abundant surface T4-derivatives to be immobilized on the micro-well surface.2 Serum TT4 TRFIA based on surface S-Ab (Second antibody) requires a high capacity of the surface S-Ab for capturing the primary anti-T4 antibody; otherwise, the serum TT4 TRFIA will suffer from matrix interference, causing significant measurement bias. Surface streptavidin (SA) with an improved binding capacity for the biotinylated anti-T4 antibody can be prepared via the biotin-streptavidin interaction. With the use of streptavidin-biotin separation technique, the matrix interference was removed.3 For multiple-labeling, SA-poly(l) conjugate was synthesized via the maleimide-thiol method. Labeling SA-poly(l) with DTTA-Eu3+ in a one-step procedure could not give stable SA-poly(l)-Eu3+, while labeling with DTPAA as bi-functional coupling agent proved to be...
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