A colorimetric–enzymatic microassay for the quantitation of antibody-dependent complement activation

Abstract

In this report we describe a reliable, sensitive, safe, and easy way to assess antibody-dependent complement-mediated hemolysis. The assay is based on the quantitation of hemoglobin (Hb) released from lysed erythrocytes indirectly, through the generation of fluorene blue, a compound formed from 2-7 diaminofluorene in an enzymatic reaction catalyzed by the Hb molecule. The fact that Hb is the most abundant protein within a mature RBC (∼10 11 molecules per cell) and possesses pseudoperoxidase activity, makes the fluorene blue-coupled assay more sensitive than the simple estimation of Hb adsorption at 410 nm (Soret adsorption maxima of Hb), and as sensitive and reliable as its radioactive equivalent based on the release of 51 Cr from previously loaded RBCs. Using this assay chimeric mouse–human anti-dansyl antibodies, comprising all the human IgG isotypes, were tested for their ability to mediate complement activation. The results obtained agreed with previously reported data, confirming that the fluorene blue-coupled assay is reliable. This assay also has significant advantages over the radioactive-based assay in that the reagents used are inexpensive, and the concerns of using radioactivity and the associated hazards are obviated. Because there is no need for loading the target cells with the analyte to be detected since RBCs are already loaded with Hb, the fluorene blue-coupled assay is simpler and eliminates many steps in comparison to the 51 Cr release based assay.