Regeneration of Recombinant Antigen Microarrays for the Automated Monitoring of Antibodies against Zoonotic Pathogens in Swine Sera

Abstract

The ability to regenerate immobilized proteins like recombinant antigens (rAgs) on surfaces is an unsolved problem for flow-based immunoassays on microarray analysis systems. The regeneration on microarray chip surfaces is achieved by changing the protein structures and desorption of antibodies. Afterwards, reactivation of immobilized protein antigens is necessary for reconstitution processes. Any backfolding should be managed in a way that antibodies are able to detect the protein antigens in the next measurement cycle. The regeneration of rAg microarrays was examined for the first time on the MCR3 flow-based chemiluminescence (CL) microarray analysis platform. The aim was to reuse rAg microarray chips in order to reduce the screening effort and costs. An antibody capturing format was used to detect antibodies against zoonotic pathogens in sera of slaughtered pigs. Different denaturation and reactivation buffers were tested. Acidic glycine-SDS buffer (pH 2.5) and 8 M guanidinium hydrochloride showed the best results in respect of denaturation efficiencies. The highest CL signals after regeneration were achieved with a carbonate buffer containing 10 mM DTT and 0.1% BSA for reactivation. Antibodies against Yersinia spp. and hepatitis E virus (HEV) were detected in swine sera on one immunochip over 4 days and 25 measurement cycles. Each cycle took 10 min for detection and regeneration. By using the rAg microarray chip, a fast and automated screening of antibodies against pathogens in sera of slaughtered pigs would be possible for zoonosis monitoring.

Keywords

• chemiluminescence microarrays
• indirect microarray immunoassays
• regeneration
• automated analysis platforms
• food safety
• zoonotic pathogens
• recombinant antigen
• meat processing
• serology