Preparation of the diagnostic kit for measuring of free Thyroxin (FT4) in human blood by RIA method

Introduction: The main hormone of thyroid gland is T4. Only 0.3 % of T4 is free and the rest has been binded mostly to Thyroxine Binding Globulin (TBG), then Throxine Binding Pre Albumin (TBPA)& Throxine Binding Albumin(TBA). While 99.7% of T4 is bound to protein, it is the free fraction (FT4) which is considered to be biologically active. The normal range of FT4 is about 10 - 24 pico mole per liter. FT4 levels in serum or plasma have been recognized as an important indicator of thyroid status.T4 secretion from the thyroid gland is regulated by a sensitive feedback system which involves the hypothalamus and the pituitary gland. Methods: Radio Immunoassay is one of the sensitive & specific methods for measuring the hormones. It is one example of saturation analysis, a particular type of immunoassay (an analytical technique which utilizes antibody). An analyte in a saturation analysis is called a ligand and the specific reagent in such assay is called a binder. T4 is labelled with I-125 by chloramines-T method .After purification by coloumn Chromatography (Sephadex G-25) and dilution, we adjust the activity of labelled T4 (or tracer) for a 50% binding with T4-Ab. The tracer competes with the unlabeled T4 in binding to the limit amount of T4-Ab which have already been coated in polystyrene tubes. After the incubation period, the Ag -Ab complex which has been formed in solid phase (polystyrene tubes) could easily be measured in Gamma counter. By plotting the standard curve, we can estimate the concentration of free T4 in unknown samples. Results & conclusion: After running the assay we obtained a good standard curve. All the controls were within the range and the sensitivity (Minimum detectable dose) of the kit was 3.5 pmole/L , also the amount of coefficient variation (%CV) was less than 10% . All these results show that the local kit could be accepted as a cost effective & reliable kit for measuring of the FT4 in human serum. Although the incubation period during the assay is a little longer than it's equivalents which imported from the abroad, but however if we have a better Ab with higher affinity this period could be potentially decreased