Quantification of α-tubulin isotypes by sandwich ELISA with signal amplification through biotinyl-tyramide or immuno-PCR

Microtubules formed by αβ-tubulin dimers represent cellular structures that are indispensable for the maintenance of cell morphology and for cell motility generation. Microtubules in intact cells are in highly regulated equilibrium with cellular pools of soluble tubulin dimers. Sensitive, reproducible and rapid assays are necessary to monitor tubulin changes in cytosolic pools after treatment with anti-mitotic drugs, during the cell cycle or activation and differentiation events. Here we describe new assays for α-tubulin quantification. The assays are based on sandwich ELISA, and the signal is amplified with biotinyl-tyramide or immuno-PCR. Matching monoclonal antibody pair recognizes phylogenetically highly conserved epitopes localized outside the C-terminal isotype-defining region. This makes it possible to detect α-tubulin isotypes in different cell types of various species. Biotinyl-tyramide amplification and immuno-PCR amplification enable detection of tubulin at concentrations 2.5ng/ml and 0.086ng/ml, respectively. Immuno-PCR detection shows enhanced sensitivity and wider dynamic range when compared to ELISA with biotinyl-tyramide detection. Our results on taxol-treated and activated bone marrow-derived mast cells demonstrate, that the assays allow sensitive quantification of tubulin in complex biological fluids.

3,3′,5,5′-tetramethylbenzidine.; BMMC; BSA; BSS; Biotinyl-tyramide; C(q); ELISA; Immuno-PCR; Mast cells; PBS; PCR; TBS; TBS containing 0.05% Tween 20; TBST; TMB; Tris buffer solution; bone marrow-derived mast cell; bovine serum albumin; buffered saline solution; enzyme-linked immunosorbent assay; phosphate buffer solution; polymerase chain reaction; quantification cycle; α-Tubulin isotypes.