Production and Use of Thyroxine antisera in Radioimmunoassay Technique

Abstract

This study describes production of antisera from sheep and its use in determination of thyroxine hormone (T4) level in serum using Radioimmunoassay (RIA) technique. Two local sheep (Ovis aris) were subjected to immunization against human T4 immunogen, sera obtained from both sheep after each injection were subjected to evaluation through titration in a purified and non-purified form. The produced antibodies were used to assemble a kit for determination of total human serum thyroxine. Different separation techniques were tried, (second antibody polyethylene glycol (PEG) assisted precipitation, polystyrene beads and magnetisable particles solid phases. For the PEG assisted precipitation, local antiserum and that produced by the North East Thamus Region Immunoassay (NETRIA donkey anti-sheep serum (DASS) as second antibodies) were tried. The final dilutions of the anti-T4 antibody used were 1/4000 in a liquid phase using second antibody PEG assisted separation, 1/3000 using magnetisable particles and a dilution of 1/10,000 using polystyrene beads as solid phase for separation. Optimization of T4 assay conditions including incubation temperature and reaction time were done. Tests for T4 assay validation (linearity, recovery and reproducibility) were carried out. For linearity and recovery tests, the regression coefficient ranges were found to be from (0.8 to 0.9) and (0.88 to 0.98), respectively. The assay was found to be reproducible where the coefficients of variation within and between assays were less than 10%. The locally developed assay was found comparable with NETRIA assay as a reference method with a correlation coefficient of 0.88, 0.93 and 0.87 for PEG assisted separation, magnetisable particles and polystyrene bead techniques, respectively. The clinical validation tests showed a reliable sensitivity, specificity and efficiency with values of 97%, 94% and 96%, respectively. When the T4 concentrations measured using the locally produced kit, the overall profile of the thyroid-pituitary axis for thyroid function tests was found to be acceptable.