Multiplexed analytical approaches to beta-lactam allergy in vitro testing standardization.

The suspicion of beta-lactam allergy directly contributes to the prescription of antibiotics that diverge from the guidelines, increasing antimicrobial resistance, one of the biggest threats to global health. In vitro quantification of specific IgE is very useful for monitoring allergy, as it confirms or rules out immediate beta-lactam drug allergy and helps find safe alternative antibiotic stewardship. However, reliable in vitro quantification of specific IgE to beta-lactam antibiotics by immunoassay is challenging because of the difficulty of having selective immunoreagents, mainly beta-lactam antigens, and its low concentration levels in serum. Thus, reliable and sensitive in vitro tests for multiplex detection of allergy to different beta-lactam antibiotics is currently essential for clinical diagnosis. Nevertheless, the lack of standardization of quantitative in vitro methods makes the comparison and interpretation of the results difficult. Here, as proof of concept, we report an improved multiplex microimmunoassay for beta-lactam allergy in vitro testing standardization. The results revealed that homologous calibration allows reliable quantification of specific IgE in human serum at very low concentrations (144 ng L). Moreover, the reproducibility of the results increases 2-fold using an internal standard, achieving accurate quantitative information: 93% and 106% recovery for penicillin and amoxicillin, respectively. We simultaneously evaluated the reliability of the improved multiplexed in vitro method in a cohort of 40 human serum samples and achieved excellent agreement (0.99) with a currently used in vitro test.