HLA class I epitopes: recognition of binding sites by mAbs or eluted alloantibody confirmed with single recombinant antigens.

Development of beads coated with single recombinant HLA antigens has permitted the confirmation and further definition of HLA class I epitopes. In this study, monoclonal antibodies (mAbs) or alloantibodies eluted from recombinant cell lines were tested for reactivity with Luminex beads individually coated with 79 recombinant HLA class I single antigen (rHLA SA). Published amino acid sequences were used to map epitopes common to sets of antigens reactive with each antibody. While several epitopes have already been demonstrated, this study confirmed them by adsorption of allosera with transfectants or SA beads having a single HLA antigen and specific binding of the eluted antibody on SA beads. The allosera and mAbs used in this study recognized a total of at least 58 HLA class I epitopes, as demonstrated by their different adsorption/reactivity patterns. Of these, 25 epitopes were characterized by a single unique common amino acid, 30 shared 2 signature amino acids in close proximity, and 3 epitopes involved 3 specific amino acids in a non-linear sequence. Since these epitopes may be targets for antibody-mediated allograft rejection, epitope analysis should complement HLA and CREG assignment for defining complex antibodies and identifying suitable donors for highly sensitized transplant patients.

Clinical cytometry and progress in HLA antibody detection.

For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes.

HLA class II DQ epitopes.

1. DQ epitopes were defined by studying the distinct reaction profiles of 17 allosera and 35 DQ-specific mouse monoclonal antibodies to HLA class II single antigen beads coated with purified recombinant DQ molecules. 2. DQ molecules are heterodimers with a polymorphic alpha chain and beta chain. Sixteen of the defined epitopes correlated to various DQB specificities and one defined epitope correlated to DQA1*0201. 3. Among the 16 DQB epitopes, 7 sites were recognized by both alloantibodies and DQ monoclonal antibodies, 5 sites were identified by DQ monoclonal antibodies and 4 sites by alloantibodies. The only unequivocal DQA1*0201 epitope found was identified by an alloantibody from a patient with a failed kidney graft. 4. Of the 17 DQ epitopes, 10 sites have a very restricted possible choice involving one to three amino acid(s) to form the unique sites; while the other 7 sites (6 DQB sites and 1 DQA site) have multiple potential sites. 5. The amino acid residues for all of 16 DQB epitope sites are unique and not shared with DQA, DR or DP antigens; while the DQA1*0201 epitope site is not shared with DQB, DR or DP antigens. 6. Except epitope #2008, all the other 16 epitopes consist of unique amino acids that appear to be exposed on the surface of DQ molecules as viewed from the 3-D model. 7. The existence of antibodies to multiple epitopes on the same HLA molecule invites molecular epitope analysis of serum reaction profiles. A single antigen cannot be considered to be a single epitope.