Analytical characterization of IgG–cTpp and IgG–Mn-cTpp conjugates

Abstract

Herein, the conjugation of carboxylated tetraphenylporphyrin or its derivative containing manganese cation and model protein — immunoglobulin G is presented. The obtained IgG–cTpp and IgG–Mn-cTpp conjugates were subsequently used for model immunoassays construction. The IgG–cTpp formation was confirmed using size-exclusion chromatography. Thanks to the unique properties of applied labels the assay analysis was carried out with both spectrophotometric and spectrofluorimetric detection. The assays were performed creating semi-quantitative detection system using 96-well plates. The incubation time, ensuring full saturation of the surface with secondary antibodies was also optimized. Moreover, in the case of IgG–Mn-cTpp conjugates we present the possibility of both direct and indirect determination of the label, the latter based on the catalytic activity of Mn-cTpp, which allows for amplification of the measured signal. We proved that both cTpp and Mn-cTpp may be successfully used for protein labeling and serve as universal tracers for various formats of affinity assays and sensors.