The line blot assay: Problems with titrating first and second antibodies for Western blot and immunohistochemistry assays?

Abstract

We describe a technique designed to assess the optimal dilution of primary and secondary antibodies, to be used in Western blot, dot blot, the multi‐antigen print immunoassay (MAPIA) and immunohistochemistry assays. The method that we call “line blot” is not an alternative but a practical, complementary tool for the above techniques that assures definitive results are obtained from single assays, so there is no need to repeat the assay. As with most immunoenzymatic assays, the line blot assay is very sensitive, allowing the detection of absolute amounts of antigen as low as 2.5 ng in the 0.5 cm‐long segment line (see Results), depending on the strength of the secondary, enzyme‐labelled antibody.

• Dot blot
• ELISA
• immunohistochemistry
• MAPIA
• Western blot

Acknowledgements

This report was funded by the Consejo Nacional de Ciencia y Tecnología (Project CONACYT 3844‐1 M) and Coordinación General de Postgrado e Investigación del IPN (Project CGPI 20050004). O.R.E. holds a fellowship from COFAA (IPN), EDI (IPN) and SNI.