Development of a quantitative immuno-polymerase chain reaction assay to detect and quantify low levels of human thyroid stimulating hormone

Abstract

In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and a highly sensitive immuno-polymerase chain reaction (IPCR) assay specific for detection of human thyroid stimulating hormone (hTSH). Several anti-hTSH monoclonal antibodies (MAbs) were generated using hybridoma technology. Two pairs of MAbs (B-4 and B-9) were rationally selected and the optimal assay conditions of sandwich ELISAs were established. The ELISA prototypes were evaluated with standards calibrated with WHO 2nd International Reference Preparation for hTSH and in comparison with a commercial ELISA Kit. Although the limit of detection (LOD) was 0.1 μIU/ml in all cases, B-9-ELISA showed an analytical performance similar to commercial ELISA Kit. Therefore, we selected the B-9 ELISA to develop a hTSH-IPCR assay applying an “Universal-IPCR” format in standard PCR tubes without pretreatment. The signal amplification was achieved through the interaction between the biotinylated detection MAb and mono-biotinylated DNA probe pre-self-assembled with neutravidin. The hTSH-IPCR assay showed a significant increase in terms of the slope definition of sensitivity in low levels range. Our results support the potential of IPCR technique for being applied in clinical diagnosis of thyroid states.