On solid-phase antibody assays

Detection of anti-human leukocyte antigen (HLA) antibodies has traditionally involved the use of peripheral blood leukocytes or cell lines that constitutively express HLA on the cell membrane (upper flow diagram). Anti-HLA antibodies are detected by the serum's ability to lyse the cells in the presence of complement (complement-dependent cytotoxicity [CDC]) or by fluorescence-based techniques using flow cytometry. Recently (lower flow diagram), purified HLA molecules bound onto a solid matrix (e.g., microbeads) are used as the substrate and the antibodies are detected by either enzyme-linked immunosorbent assays (ELISA) or again by flow cytometry. A recent consensus conference organized by the International Society for Heart and Lung Transplantation⁹ reported that 78% of centers performing thoracic organ transplantation used Luminex assays (solid-phase HLA microbead assays) for the detection of circulating antibodies.

This schematic illustrates the basic steps involved in flow cytometric antibody detection. (1) Incubation of recipient serum with microbeads coated with purified human leukocyte antigen (HLA) molecules. If the serum contains anti-HLA antibodies specific to the HLA on the beads, the antibodies will remain bound to the HLA-bead complex. (2) Incubation of the microbeads with fluorescent-tagged anti-human globulin. (3) Detection of the fluorescent tagged anti-human globulin using a flow cytometer.

Simplified example, using only 2 class I human leukocyte antigen (LCA) loci of a positive and negative virtual crossmatch (VXM). The sensitized imaginary recipient has had antibodies detected against A1, A11, and B7 by Luminex single-antigen beads. The first donor offered to the recipient has been typed as A1, A25; B55 and B57. Because the class I antibody (anti-A1) detected by the SPA corresponds to the donor typing as A1, the VXM is considered positive. The second donor does not express a class I allele that corresponds to the antibodies detected in this recipient. Thus, the VXM is negative in the second example.