A-031 Development of novel ELISAs for total GDF-15 and H-specific GDF-15

Abstract

Abstract Background To develop sensitive and specific ELISAs for the quantitative measurement of total human GDF-15 and H-specific (H202D mutant) GDF-15 in human serum, and other biological fluids. Growth/differentiation Factor 15 (GDF-15) is a divergent member of the TGF-β superfamily of growth factors. It is encoded in humans by a gene in chromosome 19. The human GDF-15 gene encodes for a protein of 308 amino acid residues which consists of a signal sequence (residues 1-29), pro-domain (30-194), and mature growth factor domain (195-308). The molecular weight of a mature GDF-15 dimer is 25 kDa. Approximately 25% of humans have a missense polymorphism in the GDF-15 gene resulting in mutation of histidine 202 to aspartate (histidine 6 in the mature domain), close to the N-terminus of the mature growth factor. This variant is associated with phenotypes in prostate cancer, hyperemesis gravidarum, (severe morning sickness in pregnancy), and rheumatoid arthritis. Methods Highly specific and reproducible total GDF-15 (AL-1014-r) and H-specific GDF-15 (AL-1018-r) ELISAs have been developed using specific monoclonal antibodies to help estimate the total and mutant (DD) concentration in serum in the respective assays. The ELISAs use 10 uL sample volume and are calibrated to recombinant human GDF-15 from R & D Systems (Biotechne, USA). These ELISAs were validated for their specificity using HH, DD, HD recombinant preparations and their circulating levels in male, female, 1st, and 2nd-trimester serum samples. Results The Total GDF-15 ELISA assay detects total GDF-15 including histidine 202 to aspartate mutation (HH, HD & DD variant) in the mature domain in equimolar proportions. The H-specific GDF-15 assay is specific to histidine at 202aa in the GDF-15 sequence and does not detect histidine 202 to aspartate mutation (DD variant). Median Levels of total GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 847.9 pg/mL, 1182.4 pg/mL, 12759.7 pg/mL, and 12951.2 pg/mL. Median Levels of H-specific GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 723.2 pg/mL, 641.3 pg/mL, 8381.4 pg/mL and 4652.1 pg/mL. Method comparison between total GDF-15 and commercial GDF-15 assay using serum samples (HH, wild type) in the range of 400-2500 pg/mL yielded a slope of 0.98 (r=0.97). The HD and DD mutant samples in the total assay recovered at twice the concentrations of the commercial assay. The total and intact assays are highly reproducible with the total coefficient of variation less than 7%. Conclusions Use of total GDF-15 assay in combination with the GDF-15 H-specific assay (DD nondetectable) ELISA can accurately estimate the mutant (DD) concentration in serum. This will enhance the knowledge of the GDF-15 measurements in the literature as the commercial assays were compromised for its immunoreactivity to the HD DD genotypes which is highly prevalent.