Abstract
Conventional HTSH radioimmunoassay kits requires long incubation time (four days for first incubation and one day for second incubation at low temperature) . This long incubation time is thought to reduce the clinical applicability of the HTSH radioimmunoassay kits. Recently rapid processing procedure of the kits is suggested by the manufacturer (two days method, Daiichi Radioisotopes Co., Ltd.) . The authors also tried to develop rapid processing procedure of the HTSH radioimmunoassay kits and performed basic study of the procedure.
Bound percent of the zero samples decreased exponentially according to the increase in first incubation volume (0.2-0.7 ml) . This decrease in bound percent was larger in low standard HTSH concentration than in high standard HTSH concentration.
Bound percent of the standard HTSH tubes differed little when first incubation temperature was changed from 20 to 30°C for 24 hours incubation, and it was considered that one could choose any temperature between 20 and 30°C in the first incubation for 24 hours. When sample HTSH concentration was expected to be low, especially under 5μU/ml, higher incubation temperature was thought to be desirable.
In the second incubation, bound percent of zero samples and samples of high, middle and low HTSH concentration differed little between incubation period of three hours and seven hours at 25°C. Bound percent of zero samples differed little between incubation temperature of 4 and 10°C for 24 hours.
Addition of HTSH free serum (carrier serum) to the standard HTSH tubes was necessary to correct the effect of nonspecific binding of HTSH-125I to the protein. This was especially important in assay of low (under 10μU/ml) HTSH samples.
From these results, rapid processing procedure of HTSH radioimmunoassay kits should be as follows: (1) First incubation volume should be 0.4 ml. (2) First incubation temperature could be chosen any points between 20 and 30°C for incubation period of 24±4 hours. (3) Second incubation temperature and period after addition of second antibody could be chosen from each one of the followings; (a) at 25°C for 5±2 hours (two days method when combined with first incubation at 25°C for 20 hours), (b) at 4°C for 24 hours (three days method) . (4) Centrifuge at 3000 rpm for 30 minutes immediately after addition of 0.5 ml of phosphate buffer.
Both of the above two rapid processing procedures revealed satisfactory reproducibility and recovery rates.
