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

ABUD, J.E.; SANTAMARIA, C.G.; LUQUE, E.H.; RODRIGUEZ, H.A.

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2017

0003-2697

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 fordetection 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) wererationally selected and the optimal assay conditions of sandwich ELISAs were established. TheELISA prototypes were evaluated with standards calibrated with WHO 2nd International ReferencePreparation for hTSH and in comparison with a commercial ELISA Kit. Although the limit ofdetection (LOD) was 0.1 µIU/ml in all cases, B-9-ELISA showed an analytical performance similarto commercial ELISA Kit. Therefore, we selected the B-9 ELISA to develop a hTSH-IPCR assayapplying an ?Universal-IPCR? format in standard PCR tubes without pretreatment. The signalamplification was achieved through the interaction between the biotinylated detection MAb andmono-biotinylated DNA probe pre-self-assembled with neutravidin. The hTSH-IPCR assay showeda significant increase in terms of the slope definition of sensitivity in low levels range. Our resultssupport the potential of IPCR technique for being applied in clinical diagnosis of thyroid states.