Labelling of antibodies and detection by laser ablation inductively coupled plasma mass spectrometry PART III. Optimization of antibodylabelling for application in a Western blot procedure

Abstract

The labelling of three different polyclonal antibodies (chicken anti-bovine serum albumin, rabbit anti-chicken lysozyme and rabbit anti-bovine casein) with iodine and lanthanides has been investigated and the reaction conditions were optimized for application in a Western blot assay. For this purpose target protein standards (bovine serum albumin, chicken lysozyme and bovine β-casein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto NC membranes. After the immuno-reaction of the labelled antibodies with the antigen the element label is detected by laser ablation ICP-MS directly on the Western blot membrane. In this study it turned out that only the labelling of antibodies using p-SCN-Bn-DOTA (2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) lanthanide chelates could be applied successfully in the Western blot approach whereas iodine labelling of the antibody showed unspecific binding and a too low sensitivity. For validation a conventional method, chemiluminescence detection, was applied and similar limits of detection at ng levels of antigen were achieved by both methods. In comparison to chemiluminescence detection, the Western blotting procedure in combination with LA-ICP-MS detection is less time consuming and the elemental signatures on the blots show long term stability. Last but not least LA-ICP-MS offers multiplexing capabilities and therefore simultaneous detection of three differentially labelled antibodies in one single Western blot assay is demonstrated.