Detection efficacy of soluble HLA-A, B antigens using 1D-IEF.
Soluble HLA class I alloantigens (sHLA class I) can be typed according to their isoelectric points (IEP) after immunoprecipitation by w6/32 monoclonal antibody (mAb) coupled to immunomagnetic beads and focusing. In order to prove the large scale efficacy of this methodology, EDTA-plasma samples from 344 probands HLA-A, B typed by serology were analysed by one-dimensional isoelectric focusing and HLA class I specific Westernblot (1D-IEF). In addition, detergent solubilized HLA class I membrane molecules from approximately one half of the probands were studied too. Soluble HLA-A24,B7,B18,B62 antigens were identified in nearly all experiments, whereas A28, B13, and B51 could be detected in about 50%. A third group of HLA antigens (A26, B8, B44) could be visualized rarely. The difficulties of detection might be due to the different affinity of mAb w6/32 to certain sHLA class I gene products or to variable amounts of sHLA class I in the plasma specimens. Some modifications of the antigen capture technique have already led to a slightly better degree of antigen recognition in 25 probands tested. Thus, HLA-A, B typing using sHLA molecules and 1D-IEF in the assay format presented does not yet seem to be a definitive alternative for HLA class I serology or biochemistry of membrane-bound HLA class I molecules but it should be a promising technique if no cells are available or donor-derived sHLA allotypes are to be monitored after HLA mismatched organ transplantation.
Kubens
B S
BS
Institute of Immunology, University Hospital Essen, Medical School, Germany.
Pässler
M
M
Grosse-Wilde
H
H
D000911:Antibodies, Monoclonal; D005069:Evaluation Studies as Topic; D015234:HLA-A Antigens / Q000302:isolation & purification*; D015235:HLA-B Antigens / Q000302:isolation & purification*; D006650:Histocompatibility Testing; D006801:Humans; D018189:Immunomagnetic Separation; D007525:Isoelectric Focusing / Q000379:methods*; D015203:Reproducibility of Results; D012995:Solubility; D014181:Transplantation Immunology
D000911:Antibodies, Monoclonal; D015234:HLA-A Antigens; D015235:HLA-B Antigens