Immunoassay for detection of oligomeric proteins

Abstract

The mass concentration of specific proteins is often used as a biomarker and play an important part in diagnostics of inflammatory diseases. Monodisperse proteins are robustly measured in immunoassays, but it is considerably more complicated to measure polydisperse oligomeric proteins. The degree of protein oligomerization is critical for functional aspects. For such proteins, information on both the mass concentration as well as the degree of oligomerization is important. Here, a time-resolved immunofluorometric assay (TRIFMA) with sensitivity for protein structure to detect homo-oligomeric and polydisperse proteins is presented. An established TRIFMA for mannan-binding lectin (MBL) was modified by implementing an additional blocking step prior to coating with capture antibodies, leading to a decrease in coating density. Recombinant human MBL was sorted into small, intermediate, and large complexes, using gel permeation chromatography. Small MBL complexes were poorly detectable by TRIFMA with a sparse antibody coating, while larger complexes produced a strong response. From comparison of molecular dimensions, this difference can be related to the size of oligomers. In conclusion, it is possible to design oligomer-size-sensitive immunoassays by regulating the inter-molecular distance of capture antibodies on a scale comparable to the size of the oligomers.

Keywords: Biomarker; Immunoassay; Mannan-binding lectin; Oligomers; Protein structure.