Date of Award
Doctor of Philosophy
Department of Biological Sciences
Dr. John O'Mullane
A flow-through enzyme-linked immunosorbent assay was developed based on affinity chromatography using the determination of ferritin in serum as a model system. In this method, samples and standards are introduced to separate columns containing immobilised anti-ferritin antibody, and antigen bound by solid-phase antibody is subsequently detected using an anti-ferritin-alkaline phosphatase conjugate. To detect immobilised label, p-nitrophenyl phosphate is added and product is developed in the column at room temperature. Following elution of product from the column, the absorbance is measured and the columns are regenerated using a low pH elution. The final developed system requires approximately 1.5 h for the simultaneous assay of standards and up to forty samples. The lower limit of detection using a 200 μl assay volume is 1.82 x 10-15 mol or 9.11 x 10-12 mol/l (4.1 μg/l). However, there is potential to increase the assay sensitivity further through the use of amplification systems for alkaline phosphatase label. In addition, the present assay gives accurate results, good precision, and is easy to perform. The immunoaffinity columns have been shown to be stable for at least ten assays and presumably could be used for an even greater number. By using different immobilised and labelled antibodies, this method could easily he adapted for use with other analytes.
Spillane, Declan Gerard, "Development and Validation of Flow-Injection (Continuous-Flow) ELISA Techniques." (1998). Theses [online].
Available at: https://sword.cit.ie/allthe/357