An evaluation of the potential application of proteomic methods (using mass spectrometry) for the characterisation of human leukocyte antigen (HLA) antibodies

Abstract

The detection and definition of HLA antibodies is critical in organ transplantation to prevent failure of the transplanted organ. The current gold-standard test for this purpose is the single antigen bead (SAB) assay. This assay produces a list of HLA antibodies for each patient which is utilised by the clinical team in the pre-transplant risk assessment and for post-transplant monitoring of the organ.

However, the SAB assay has limitations: the assay is only semi-quantitative, denaturation of proteins within the assay can lead to false positive results and the detection of HLA antibodies is limited by the number of beads in the kit. Mass spectrometry (MS) technology is currently used in a wide range of fields and has advanced rapidly over recent years, leading to the ability to examine larger proteins. There are two types of approach to proteomics using mass spectrometry: bottom-up whereby the protein is digested into peptides using an enzyme before analysis, and top-down analysis which is mass spectrometer analysis without the initial enzymatic digestion step, thereby achieving a ‘birds-eye view’ of the protein.

We plan to use samples from volunteer renal patients to evaluate both MS approaches for the detection and definition of HLA antibodies. This technology has potential for highly specific and sensitive HLA antibody analysis, overcoming the limitations with the current testing and could herald a potential first step in the next generation of HLA antibody detection.