Assessment of Humoral Alloimmunity in Mixed Lymphocyte Reaction

Humoral alloimmunity remains a significant and unresolved problem that constrains allograft survival. Thus, there is a need for the development of an easy, preferably non-radioactive, and inexpensive protocol for assessing the effect of various drug treatments on humoral alloimmunity. In order to satisfy this demand, we developed such a protocol in which de novo alloantibodies production is induced in one-way mixed lymphocyte reaction (MLR). The amount and capacity of the generated alloantibodies in the supernatant of each one-way MLR is assessed using an antibody-mediated cell dependent cytotoxicity (CDC) assay. The principle of the assay relies on the assessment of cellular survival of resting PBMCs isolated from the same donors, supplemented with the alloantibodies from the one-way MLR supernatant. The lesser is the cellular survival, the higher the production of alloantibodies in one-way MLR and consequently the more potent the humoral alloimmunity.