Quantitative dot-blot assay for low titer anti-lipopolysaccharide antibodies in human plasma

Abstract

Low titer antibodies in plasma are very hard to detect by enzyme-linked immunosorbent assay (ELISA) mainly because of high nonspecific binding of various plasma proteins to the plastic substratum. In this report we present a sensitive and quantitative dot-blot assay which overcomes the high nonspecific binding problem and enables the detection of very low antibody titers in plasma. Natural low titer antibodies to Gram negative bacteria's lipopolysaccharide in plasma of healthy donors could not be detected by ELISA. However, by using nitrocellulose membrane as the carrier for the antigen and enhanced chemiluminescence as the detection method, we could detect and quantify low titers of anti-lipopolysaccharide antibodies even in undiluted plasma with no background interference. The dot-blot assay is linear, in semilogarithmic plot, over a broad range of plasma dilutions. This assay will enable the early detection of antigen specific antibodies in immune processes such as in infectious diseases and vaccination.