Methodological aspects of Universal immuno-PCR on standard tubes

• Published in: Analytical biochemistry 2019-04, Vol.570, p.56-61
• Main Authors: Abud, J.E., Santamaría, C.G., Oggero, M., Rodriguez, H.A.
• Format: Article
• Language: English
• Subjects: Biotinylation; Diagnostic; DNA Probes - chemistry; DNA Probes - metabolism; Human TSH; Humans; Immune-PCR; Immunoassay; Immunoassays; Limit of Detection; Monoclonal antibodies; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Reproducibility of Results; Thyrotropin - analysis; Thyrotropin - genetics
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2019.02.007

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. •Quantification of hTSH is highly useful in diagnostic of thyroid diseases.•We improved the analytical sensitivity of an IPCR assay to measure hTSH (hTSH-IPCR).•This hTSH-IPCR assay might be very useful to measure very low levels of GST is necessary.