Evaluation of a 1 step TRAb Assay for the Detection of High-affinity Components to hTSHR: Evidences Indicating Superiority of the Assay in the Lower TRAb Range

Abstract

Abstract. The aim of this study was to develop an assay to selectively detect high-affinity components among TRAb. Using an rhTSHR-coated tube system, a 1 step TRAb assay method was developed that included 1) co-incubation with 125 I-bTSH, 2) a 50 ml serum sample, 3) an increased incubation volume (450 ml), and 4) a 1 hour incubation time. Sixtyone TRAb positive Graves' sera were studied. When the regular TRAb assay (Reg) results were quantitatively compared to the 1 step assay (1 step) results, certain dispersions and overestimations using the latter were seen. Further, some 1 step positive results were observed in the low Reg range. Overestimations were considered mostly due to the differences between TRAb standard and patients' serum TRAb in the binding competition against co-incubated 125 I-bTSH, which was shown from a modified assay mimicking the 1 step conditions. Therefore, the 1 step results were decided to be expressed by % inhibition against 125 I-bTSH. As for data dispersions, TRAb absorptions during the regular 1 st incubation were studied. Individually, the absorption rates varied from 11 to 69%, and higher absorptions were observed in lower Reg range, especially in those negative by the 1 step. Observed 1 step positive results in the low Reg range were of interest, and 1 step/Reg ratios were calculated. The ratios with 1 step negative samples were significantly lower than those of 1 step positive samples. In conclusion, the 1 step assay was proved to detect a particular and biologically active TRAb, especially in those with low TRAb. The clinical significance of the 1 step results should be of future interest.

Third generation radioimmunoassay (RIA) for TSH receptor autoantibodies (TRAb) – one step less, similar results?

“…As a fact, the three GD a patients with discrepant TRAb values showed low TRAb levels by the two-step TRAb RIA (< 2 IU/L). A similar phenomenon was reported by comparison of a 2 nd generation TRAb assay using 125 I-labelled bovine TSH for the inhibition of patient TRAb to solid-phase immobilized human TSHR in a simultaneous reaction environment with its corresponding consecutive assay 9 21 They concluded that the one-step TRAb assay detected particularly biologically active TRAb in patients with low TRAb levels. In contrast, the consecutive TRAb assay presumably also identified low affinity TRAb that lack thyroid stimulatory activity and did not reflect clinical symptoms.…”