P084 : THE HLA PROTEIN CHIP – A HIGH PERFORMANCE MICROARRAY PLATFORM FOR ANTI-HLA ANTIBODY DETECTION UTILIZING SOLUBLE HLA

Abstract

Aim Successful pairing of a transplant recipient with a donor organ depends on accurate prediction of risk factors for immunologic rejection. As such, there is much interest in accurate clinical measurement of HLA Ab specificities in recipient sera. However, it has become apparent that current methodologies are still surrounded by shortcomings. Here we offer a new solution that is based on recent advancements made in the application of microarray technology in clinical diagnostics, to increase the precision and sensitivity of HLA Ab serology. The aim of this study was to demonstrate the coupling of recombinant soluble HLA proteins to a novel microarray technology as a new, simplified, inexpensive and accurate approach to transplant serology. Methods In order to validate and optimize sHLA microarray performance, multiple 12 × 12 microarrays were fabricated by microfluidic spotting of highly purified soluble HLA class I and II molecules under a range of conditions chosen to optimize protein stabilization on the microarray surface. Of particular interest were the evaluation of maximal signal and background intensities, signal uniformity between different HLA alleles and the structural integrity of the HLA proteins subsequent to microfluidic attachment to the microarray surface. Results HLA protein printed in this array system yielded >65,000 MFI with essentially no background signal. Signal uniformity among alleles tested was consistently below a 10% spread. As assessed by structure-specific antibodies HC-10 and W6/32, it was determined that the native dimer configuration predominated subsequent to microarray printing. Accuracy and reproducibility required for a low cost HLA antibody-screening array was validated with over 200 samples, resulting in a high degree of concordance with known monoclonal Ab patterns and established clinical reference sera. Conclusion We have successfully demonstrated that the combination of microarray and soluble HLA technology has resulted in a simple, accurate, and low cost method for high-performance multiplexed HLA Ab detection, which with additional development in progress will be ready to accommodate very high resolution HLA Ab screening, at time of transplantation and as a routine follow-up test to monitor the earliest stages of graft rejection.