Detection of different soluble HLA-G isoforms in samples from patients with melanocytic lesions by in-house quantitative ELISA method

Human leukocyte antigen-G (HLA-G) is a non-classical MHC class I molecule with seven described isoforms, three soluble and four membrane-bound. HLA-G confers maternal-fetal tolerance and is associated with tumor immune escape, being related to a worse prognosis in different types of neoplasia. Different anti-HLA-G antibodies are commercially available; however, they cannot differentiate all isoforms, making it difficult to understand their role in different pathological conditions. Commercial kits for detecting soluble HLA-G generally use the antibody that only recognizes HLA-G isoforms 1 and 5. The objective of this study was to develop a quantitative sandwich immunoenzymatic assay (ELISA) using the anti-HLA-G antibody clone 4H84 capable of recognizing all HLA-G isoforms. Clone 4H84 was used as the capture antibody and the same clone was labeled with peroxidase as the secondary antibody. To prepare the standard curve, a serial dilution of the recombinant HLA-G (100 ng/mL to 3.25 ng/mL) at a ratio of 2 was used. Then, 14 samples from patients with melanocytic lesions (nevi or melanoma) were used to validate the in-house method in duplicate (CAAE: 64852022.1.0000.5243). After different conditions were evaluated, we found ideal concentrations for analysis and the standard curve presented an excellent coefficient of determination (R2) with a value of 0.998. In the initial validation stage, we found the mean HLA-G concentration for samples from patients with nevi (n=6) and melanoma (n=9) of 18.0 ± 7.6ng/mL and 15.8 ± 2.4ng/mL, respectively. With these preliminary results found, the in-house quantitative sandwich ELISA for the detection of different HLA-G isoforms showed promise for use in the evaluation of this molecule as a prognostic marker in melanocytic lesions. However, new samples will be evaluated and correlations between different stages of the disease will be performed.