Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols.

Robust and sensitive ELISPOT protocols are commonly applied concomitant with the development of new immunotherapeutics. Despite the knowledge that individual serum batches differ in their composition and may change properties over time, serum is still commonly used in immunologic assays. Commercially available serum batches are expensive, limited in quantity and need to be pretested for suitability in immunologic assays, which is a laborious process. The aim of this study was to test whether serum-free freezing media can lead to high cell viability and favorable performance across multiple ELISPOT assay protocols. Thirty-one laboratories from ten countries participated in a proficiency panel organized by the Cancer Immunotherapy Immunoguiding Program to test the influence of different freezing media on cell quality and immunologic function. Each center received peripheral blood mononuclear cells which were frozen in three different media. The participants were asked to quantify antigen-specific CD8+ T-cell responses against model antigens using their locally established IFN-gamma ELISPOT protocols. Self-made and commercially available serum-free freezing media led to higher cell viability and similar cell recovery after thawing and resting compared to freezing media supplemented with human serum. Furthermore, the test performance as determined by (1) background spot production, (2) replicate variation, (3) frequency of detected antigen-specific spots and (4) response detection rate was similar for serum and serum-free conditions. We conclude that defined and accessible serum-free freezing media should be recommended for freezing cells stored for subsequent ELISPOT analysis.

A novel, sensitive and specific real-time PCR for the detection of traces of allergenic Brazil nut (Bertholletia excelsa) in processed foods.

Food-allergic individuals have to strictly avoid the offending food because no causative immunotherapies are available. Thus, reliable labelling of allergenic ingredients or precautionary labelling of cross-contacts with potential allergens is of major importance. Verification of compliance with labelling requirements and identification of cross-contacts demand test methods that enable the specific and sensitive detection of the analyte. Brazil nut (Bertholletia excelsa) is such a food commodity with allergenic potential. We describe the development of a novel qualitative real-time polymerase chain reaction (PCR) specific for Brazil nut DNA and its comparison with a qualitative commercially available lateral flow device (LFD) that detects Brazil nut protein. Specificity was investigated with 58 foods, and no false-positive reactions were observed in real-time PCR. The sensitivity was investigated with spiked chocolate and incurred dough samples as well as cookies baked thereof. The simultaneous spiking of matrices with identical amounts of Brazil nut and peanut between 5 and 100,000 mg/kg allowed the verification of the spike quality with two peanut-specific enzyme-linked immunosorbent assay. The real-time PCR detected Brazil nut in all three matrices down to the lowest investigated spike level of 5 mg/kg. The real-time PCR results from the analysis of 15 retail samples were confirmed by LFD results and were in concordance with the labelling of products. The real-time PCR showed unparalleled specificity, and primary data indicated potentially quantitative features in spiked and retail samples. Because of entirely reproducible chemistry of this real-time PCR, this is the first generally available Brazil nut-specific detection method with an appropriate sensitivity to help avoid severe allergic reactions for Brazil nut-allergic individuals.