Development of a high sensitivity immuno-pcr assay to detect and quantify low levels of thyroid stimulating hormone (tsh) in human serum samples.

HORACIO A. RODRÍGUEZ; CLARISA G. SANTAMARÍA; ENRIQUE H. LUQUE; JULIÁN E. ABUD ; MA. ALEJANDRA CARDOZO

Congreso; Reunión Conjunta de Biociencias.; 2017

Thyroid gland function is regulated by the TSH, a glycoproteinhormone secreted by the pituitary gland. Measurement of TSH inhuman fluids is utilized as a ?frst line? thyroid test, in order to assistclinical decision-making. Most of the current TSH methods used inclinical laboratories are two-site ?sandwich? heterogeneous immunoassays. Some differences in terms of analytical performance andmeasured TSH concentrations still exist among the commerciallyavailable methods. The between-methods variability is the largest atlow TSH concentrations (below 0.4 µIU/ml), due to limit of detectionand lower analytical sensitivity of some immunoassay methods. Wedeveloped a highly sensitive immuno-PCR (IPCR) assay specifc fordetection of TSH in human serum samples. First, several anti-TSHmonoclonal antibodies (MAbs) were generated using hybridomatechnology. Pairs of MAbs were rationally selected and the performance characteristic (specifcity, sensitivity, precision and accuracy)were established by sandwich ELISA. Next, we develop a TSH-IPCR assay applying a ?Universal-IPCR? format in standard PCR tubes. The signal amplifcation was achieved through the interactionbetween the biotinylated detection MAb and mono-biotinylated DNAprobe pre-self-assembled with neutravidin. The IPCR prototypewas evaluated with standards calibrated with WHO 2nd International Reference Preparation for TSH, with human serum samples andin comparison with a commercial ELISA Kit. The TSH-IPCR assayshowed a signifcant increase in terms of the slope defnition of sensitivity in low levels range, providing better quantitative resolution fora given amount of measurement error or, conversely, higher sensitivities can tolerate larger measurement errors for a given amountof quantitative resolution. The sensitivity (m = 1.25), LOD (0.01 ng/ml), and other aspects of our results support the potential of IPCRtechnique for being applied in clinical diagnosis of thyroid states.