Immobilization of T4 antibodies on magnetic particles

An effecient method to separate bound from free antigen is an essential component of a radioimmunoassay system. In the present study solid phase separation method has been developed with the aim to avoid centrifugation and thus enabling more extensive use of radioimmunoassay kits in hospitals where centrifuges ar not available. Antibodies were immobilised on to magnetised particles as solid support by means of chemical binding using 1,1-carbonil-diimidazole (CDI). The antibodies to be immobilized should be in the form of purified T4-IgG. Purification was performed using protein-A-sepharose and a fraction containing 0.610 ug/ml of T4 was obtained. The tire of the immobilized antibodies was determined. Assay design was optimized by determining the reagent volumes and lenght of incubation time suitable for the desired working range. An assay desgn using 50 ul of T4 standard solution or sample, 50 ul of 125I-labelled T4 and 50 ul of magnetised T4-IgG suspension was found to yield rasonably senrive result in the working range of interest i.e between 25 - 1500 nmol/l. Further evaluation showed that the in-house kit performs comparably with those produced by DPC and Amersham. Stability testing was carried out to determine shelf life. Experimental results showed that up to about 5 mounths the kits are fairly stable. In general it can be concluded from the data obtained, tahr satisfactory results are obtained using a solid phase separation method employing magnetic particle. (authors). 6 refs.; 5 tabs.; 3 figs