P026

• Published in: Human immunology Vol. 75; p. 66
• Main Authors: Roberts, Tiffany K, Tinckam, Kathryn, Tumer, Gizem, Gebel, Howard M, Bray, Robert A
• Format: Journal Article
• Language: English
• Published: 01-10-2014
• Subjects: Allergy and Immunology
• ISSN: 0198-8859
• DOI: 10.1016/j.humimm.2014.08.088

Aim Microparticle-based, solid phase assays are the preferred method to detect and identify HLA antibodies employing both Luminex-based and standard flow cytometric approaches. However, discrepant results between platforms underscore the challenges associated with data interpretation. Therefore, the goal of this study was to develop a more sensitive and robust SAB assay. Methods A biotin-streptavidin system for HLA antibody detection (BIO-SAB) was compared to the standard SAB (STD) assay. A total of ten informative sera (five for Class I and five for Class II) were identified as being positive by FlowPRA[TM] and negative by STD-SAB. An additional ten sera (five for Class I and five for Class II) that had been previously identified to be EDTA-sensitive were also tested using both assays. Results Our findings revealed that the BIO-SAB method increased assay sensitivity approximately 4-fold over the conventional assay for Class I and Class II antibodies in all sera tested. Therefore, the BIO-SAB provided a better correlation with flow cytometry-based assays. Importantly, our modified assay was unaffected by complement-mediated inhibition (“prozone effect”). The BIO-SAB yielded results similar to EDTA-treated sera run by STD-SAB, thereby eliminating the need for pre-test serum manipulations. Conclusions Our modified SAB assay using a biotin/streptavidin detection system overcomes complement-mediated inhibition while improving detection of low level HLA antibodies, especially those recognizing public epitopes. Laboratories currently using SABs as the sole methodology for antibody detection would benefit from implementation of the BIO-SAB as it reduces the risk of missing a potentially clinically relevant antibody. For laboratories currently using both a SAB as well as a phenotype-based bead product, implementation of the BIO-SAB method would reduce the rate of discrepant results between methods as well as decrease the need for pre-testing manipulations. Implementation of this modified method to detect HLA antibodies will increase assay sensitivity and reduce the frequency of false negative results.