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Methodological aspects of Universal immuno-PCR on standard tubes
J E Abud1, C G Santamaría1, M Oggero2
1Instituto de Salud y Ambiente Del Litoral ISAL, CONICET-UNL, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional Del Litoral, Ciudad Universitaria, Ruta Nacional N°168, km 472, CPA S3000ZAA, Santa Fe, Argentina.
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.
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