P098 Characterization and quantitation of soluble HLA molecules in human plasma

Abstract

Aim The identification of prognostic parameters for the clinical course of disease states is of considerable scientific and therapeutic relevance. Serum levels of soluble HLA (sHLA) class I molecules have been advocated at various occasions possessing such predictive potential. However, lack of quantitation of sHLA levels, unavailability of appropriate assay standards and small population sizes investigated have dominantly contributed to the inability to establish the physiological importance of the presence of sHLA in body fluids and to make accurate disease associations. In this work, we have redesigned the assay approach for sHLA class I detection and introduced optimized assay conditions including a sHLA standard of highest purity and structural integrity. Methods Applying a standardized detection methodology, serum samples were screened from one hundred normal volunteer blood donors with known HLA-phenotypes. In addition, plasmapheresis samples from various patients were utilized for large-scale sHLA purification using a W6/32-affinity column. Eluted probes were further analyzed applying multiple assay strategies. Results Our findings showed an average serum level of sHLA class I in normal individuals of 494 ng/ml [190–1402 ng/ml]. No significant differences in sHLA serum levels were observed between different blood types or between male and female donors. Closer investigation of the sHLA content of several plasmapheresis samples indicated that more than 75% of sHLA is complexed with other proteins and not available in free form. HLA specificity testing using mAb matched the determined allotype of the patients confirming the self-origin of the purified sHLA. Conclusions The design and development of assay methods is crucial to meet analytical performance requirements for intact sHLA quantitation. Extraction of sHLA from human blood is highly feasible and offers a valid approach for the elucidation of the relevance of sHLA-mediated immunoregulation. Analysis of HLA-complexed antibody species or just the simple presence of elevated sHLA amounts can provide a source of information to establish a foundation for unique diagnostic approaches to gain insights into immunopathological processes for transplantation and for diseases such as cancer, autoimmunity, allergy, and viral diseases.