Production of Antisera and Development of Radioimmunoassay for Serum T3, T4 and Ferritin

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Date
2015-06-22
Authors
Omer Mohamed Abdalla El Hag., El Hag
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Publisher
uofk
Abstract
Twelve local rabbits and 16 New Zealand rabbits were subjected to immunization against T3 and T4 immunogens. Two local sheep (Ovis aris) were immunized against human liver ferritin. T3 and T4 immunogens were prepared by conjugation of the haptens to carrier proteins (bovine serum albumin and horse serum protein), water soluble carbodiimide as coupling agent. The local and New Zealand rabbits were immunized against these conjugates emulsified in Freund's complete adjuvant in the first and second injections, and emulsified in Freund's incomplete adjuvant in the following injections. The blood samples obtained from rabbits after each injection were tested for antibodies as well as for the effect of immunization on rabbit biochemical and haematological parameters. The blood samples obtained from sheep were tested for anti- ferritin antibodies using crude antiserum. This antiserum was purified using ammonium sulphate. A part of it was adsorbed physically onto polystyrene beads while the part was linked chemically to magnitisable particles in order to develop two IRMAs. The purified antiferritin antibody was diluted 200,000 folds before being coated to polystyrene beads, and different dilutions were tried with coupling to magnetic solid phase. Optimization and validation procedures for the two IRMAs ferritin were performed. The results showed poor response of rabbits to immunization against T3 and T4 immunogenic conjugates, where the bound of tracer with the antibody ranged from 0.0 - 22% for local rabbits using charcoal separation technique and 0.0-2.9% using second antibody precipitation technique. The antisera from New Zealand rabbits ranged from 0.0-18.1% using second antibody precipitation technique. Serum T3, T4 and TSH of the immunized rabbits were measured and were not significantly different from the controls (P = 0.2211, 0.098 and 0.35, respectively). The body temperatures, ESR, serum albumin and basophils significantly increased after immunization (P < 0.05), where the total proteins, globulin, TWBCs, lymphocytes, monocytes and eosinophils did not significantly change after immunization. Optimization and validation steps for the two IRMAs serum ferritn showed that the polystyrene beads IRMA system was better than the magnitisable particles system. It was found that the minimum detectable dose in the bead system was 0.6 ng/ml compared to 6.6 ng/ml in the magnitisable particles one. In the beads system, the mean recovery of ferritin was 98.5%, while the linearity tests showed a correlation coefficient of 0.996. The comparison between the coated beads IRMA with an international commercial product (NETRIA) serum ferritin showed a correlation coefficient of 0.982.
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Production of Antisera and Development of Radioimmunoassay for Serum T3, T4 and Ferritin
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