Methodological aspects of Universal immuno-PCR on standard tubes.

ABSTRACT

One of the most used formats in inmuno-polymerase chain reaction (IPCR) is known as "Universal" IPCR (signal-generating complexes is based on conjugates of biotinylated DNA, biotinylated IgG and avidin). In the present study, we evaluated the utility of using mono- and bi-biotinylated DNA probes, pre-self-assembled DNA-neutravidin complex, blocking step and glutaraldehyde pretreatment of standard PCR tubes to improve the analytical performance of the hTSH-IPCR assay. The use of pre-self-assembled mono-biotinylated DNA-neutravidin complex enhances both the sensitivity and the reproducibility of the hTSH-IPCR assay, even without blocking step hTSH-IPCR assay showed an improved limit of detection (LOD 0.01 µIU/ml), calibration sensitivity (SEN 2.4) and analytic sensitivity (γ 9 µIU/ml-1) in comparison with both a self-made ELISA and a commercial one.