Validation Procedure for Multiplex Antibiotic Immunoassays Using Flow-Based Chemiluminescence Microarrays.

Small molecules like antibiotics or other pharmaceuticals can be detected and quantified, among others, with indirect competitive immunoassays. With regard to multiplex quantification, these tests can be performed as chemiluminescence microarray immunoassays, in which, in principle, the analyte in the sample and the same substance immobilized on the chip surface compete for a limited number of specific antibody binding sites. The amount of the specific primary antibody that has been bound to the surface is visualized by means of a chemiluminescence reaction.Validated quantitative confirmatory methods for the detection of contaminants, for example drug residues, in food samples usually comprise chromatographic analysis and spectrometric detection, e.g., HPLC-MS, GC-MS, or GC with electron capture detection. Here, we describe a validation procedure (according to the Commission Decision of the European Communities 2002/657/EC) for multiplex immunoassays performed as flow-through chemiluminescence microarrays, using the example of a small molecule microarray for the simultaneous detection of 13 antibiotics in milk. By this means, we suggest to accept multianalyte immunoassays as confirmatory methods as well, to benefit from the advantages of a fast automated method that does not need any pretreatment of the sample. The presented microarray chip is regenerable, so an internal calibration is implemented. Therefore, the analytical results are highly precise, combined with low costs (the aim for commercialization is less than 1 € per analyte per sample, this is significantly less than HPLC-MS).

New route for fast detection of antibodies against zoonotic pathogens in sera of slaughtered pigs by means of flow-through chemiluminescence immunochips.

The research on fast screening methods for antibodies against zoonotic pathogens in slaughter animals is important for food safety in farming and meat-processing industries. As a proof-of-concept study, antibodies against the emerging zoonotic pathogen hepatitis E virus (HEV) and enteropathogenic Yersinia spp. were analyzed in parallel using immobilized recombinant antigens (rAgs) of HEV genotypes 1 and 3 and Yersinia outer protein D (YopD) on a flow-through chemiluminescence immunochip. These rAgs are usually part of commercially available line immunoassays (LIAs) used for human diagnostics. In this study, sera from slaughtered pigs were tested on the microarray analysis platform MCR 3 to detect anti-HEV and anti-Yersinia IgG. The new method was characterized regarding signal reproducibility and specificity. The analytical performance was compared with in-house enzyme-linked immunosorbent assay (ELISA) and a LIA based on recomLine HEV (Mikrogen) or the ELISA test kit pigtype Yersinia Ab (Qiagen), respectively. The immunochip revealed the highest analytical sensitivity and was processed in 9 min automatically on the MCR 3. A comparative screening of swine serum samples from Bavarian slaughterhouses regarding anti-HEV and anti-Yersinia IgG seroprevalence was conducted. By using the LIA, 78% of the sera were tested positive for HEV antibodies. The immunochip and the ELISA identified anti-HEV IgG in 96% and 93% of the tested samples using the O2C-gt1 and O2C-gt3 rAg, respectively. The screening for anti-Yersinia IgG resulted in 86% positive findings using the immunochip and 57% and 48% for the ELISA methods, respectively, indicating a higher detection capability of the new method. Serum samples of slaughtered pigs could be analyzed faster and in an automated way on the microarray analysis platform MCR 3 which shows the great potential of the new immunochip assay format for multiplexed serum screening purposes.