Poster Board III-457 Surface Plasmon Resonance (SPR) Analysis Detects Clinically Significant Low Avidity HPA-1a Antibodies Not Identified by Conventional Serology.

Abstract 3520

Poster Board III-457

Neonatal alloimmune thrombocytopenia (NATP) is caused by passively transmitted maternal antibodies specific for fetal platelet antigens inherited from the father. The diagnosis is made by demonstrating a maternal antibody specific for a paternal platelet alloantigen inherited by the affected infant. It is not unusual for mothers of suspected NATP cases to be antibody-negative using standard serologic methods. Assays routinely used to detect alloantibodies (flow cytometry, glycoprotein-specific solid phase assays) require repeated washing of the target cell or antigen to remove unbound antibody, followed by detection of bound antibody with a labeled secondary probe. Low avidity antibodies could dissociate from their targets during these washing steps and might, therefore, not be detected; however, the extent to which such antibodies cause human disease is controversial. Surface plasmon resonance (SPR) directly measures the accretion of ligand on an immobilized receptor in real time and could therefore lend itself to detection of low avidity, clinically significant human antibodies. In fact, Socher et al recently described use of SPR to detect maternal antibodies specific for the HPA-1a (PlA1) platelet antigen in two NATP cases not diagnosed by standard serology (Transf 49:943, 2009).

We have also studied the utility of SPR for detection of clinically important human antibodies. HPA-1a/a-positive and -negative GPIIb/IIIa was isolated from platelets and was immobilized in the lanes of a CM5 Biacore chip. IgG used for testing was doubly purified from serum to remove vitronectin and other proteins that might react with the target integrin through RGD binding sites, thereby masking antibody-specific signals. The range for binding of IgG from normal subjects to these targets was defined with IgG from 30 healthy donors.

In preliminary studies, we found that SPR analysis is about 5 times as sensitive as standard serology for detection of known HPA-1a-specific antibodies. We then studied maternal sera from 45 cases of suspected NATP in which the mother was HPA-1a-negative but no maternal antibody was detected. Seven of these sera were found to contain IgG that recognizes HPA-1a-positive, but not HPA-1a-negative GPIIb/IIIa. A comparison of the reaction kinetics of these antibodies with those of “conventional” HPA-1a-specific antibodies showed that the former have a significantly higher off rate, as would be expected of low avidity antibodies. We conclude that low avidity antibodies specific for HPA-1a are capable of causing NATP and may not be rare and that SPR may provide a unique tool for their detection. The extent to which SPR can be useful in studying other antibody-mediated human disorders deserves exploration.