Use of a Monoclonal antibody (5H5) recognizing FH but not FHL-1 and FHRs to study FH in aHUS patients

Factor H (FH) is a negative regulator of Complement that protects host cells from complement attack. Deficiencies due to mutations in the CFH gene are frequently associated with a number of human diseases, such as membranoproliferative glomerulonephritis (MPGN), atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD). Genetic characterisation is available for all these diseases, but functional data at protein levels are often missing. The study of FH is complicated by the presence of a shorter splicing variant (FHL-1) and FH-related proteins (FHRs) that share a high degree of similarity rendering its purification and direct analysis challenging. The aim of the study was to develop quantitative and functional FH-specific assays, using a monoclonal antibody (mAb 5H5) produced in our laboratory. Specificity of 5H5 for FH was first verified by Western Blot (WB) on human serum samples and compared with other antibodies also recognising FHL-1 or FHRs. The antibody was then used to purify FH from serum by affinity chromatography. The 5H5-affinity purified FH was shown to be free of contaminating FHL-1 or FHRs by WB and its biological activity, verified in a cofactor assay, was intact after the purification procedure. A home-made ELISA for the correct quantification of FH, making use of 5H5 as a catcher together with our chicken polyclonal anti-FH antibody as a tracer was developed and used to screen a group of aHUS patients revealing a deficit in four of them. A micro-method for the affinity-purification of FH starting from low amounts of sample was also developed. This method was as efficient as the column approach and turned out to be a useful tool to study FH especially in small-volume samples such as those coming from aHUS-affected children. Furthermore, the ability of 5H5 to recognise with the same affinity both the intact and the cleaved form of FH was demonstrated by immunoprecipitation. This important feature was exploited to develop a structural integrity test that was used to screen aHUS patients. The methods developed based on 5H5 are an important tool to study patient’s FH at the protein level: the functional data can complement the genetic characterisation of patients, thus allowing for a so far unavailable detailed description of the FH structure-function relationship.