Solid phase assays for HLA antibody detection in clinical transplantation

The complement dependent microlymphocytotoxicity assay has been used for over 40 years for detecting HLA antibodies in transplant patients. This method has been replaced recently by more sensitive solid phase assays such as ELISA and bead based technology including the Luminex method. The introduction of these techniques into clinical practice has revealed previously undetected sensitisation in some patients and allowed the accurate assignment of antibody specificities directed at HLA-DQ and HLA-DP which was not previously possible. However it is emerging that despite the advantage of sensitivity some HLA antibodies defined by these assays are not associated with hyperacute or acute rejection. The role in allograft rejection of antibody titre and non-complement fixing antibodies, which are also detected by these methods, are areas currently under consideration.

Introduction

The complement dependent cytotoxicity assay (CDC) developed over 40 years ago [1] has been the cornerstone technique for the detection of HLA antibodies since its introduction. Although this assay has served us well its limitations have been well documented. Firstly because it measures any antibody capable of binding to a cell surface and fixing complement it cannot discriminate between HLA and non-HLA antibodies. The assignment of HLA specificity is made by comparing the reactivity of the test serum with the HLA typing of the target cells. Since cells display co-dominant expression (i.e. two expressed products from each locus) and some alleles at neighbouring loci display linkage disequilibrium (LD) antibody specificity is assigned based on a probability calculation which is prone to error. Secondly the presence of autoantibodies in the test serum can be a confounding factor and CDC does not readily distinguish between IgG and IgM antibodies. Thirdly the relative low sensitivity of the CDC assay and its failure to detect low level antibody has come under question with the introduction of solid phase assays.

Solid phase antibody detection assays comprise three types. First introduced was an enzyme-linked immunosorbent assay (ELISA) modified for the detection of HLA antibodies [2, 3, 4]. The second major development was the introduction of dye-impregnated beads bound with HLA molecules extracted from cell lines. Two fluorescent dyes are mixed in differing proportions that gives each type of bead a unique fluorescent signal. Test serum containing HLA antibody binds to the relevant bead(s) that is then detected using a fluorescent-labelled anti-human IgG antibody. Two methods for the detection of bound antibody are used. In the first instance the beads are analysed in a flow cytometer and a channel shift indicates the binding of HLA antibody [5]. The second method [6] involves the use of a Luminex (Luminex Corporation, TX, USA) fluorocytometer which has two lasers. One laser excites the dye in the bead and the other excites the phycoerythrin bound to the second antibody. The combination of the two signals indicates firstly the binding of HLA antibody, and secondly the bead carrying a specific HLA molecule to which the antibody is bound.

There are three levels of testing for both HLA class 1 and class 2 with the beads at each level varying in the number of specific HLA molecules bound. The third level is the single antigen bead (SAG) in which just one HLA molecule produced by recombinant technology is bound to a specific bead type. SAG testing enables sera with complex mixtures of antibodies to be accurately dissected.

Solid phase and, in particular, the bead assays are able to detect pre-sensitisation not previously possible with CDC [7, 8, 9, 10, 11, 12, 13•, 14•, 15, 16, 17•]. This sensitivity has raised important issues concerning the clinical relevance of antibodies detected by these techniques. Since the bead assays are the most common assays now in use the emphasis of the review will be on that technique paying particular attention to the Luminex platform.