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

[EN] 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-1). 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.M.J.J acknowledges financial support from the FPIprogram from Universitat Politecnica de Valencia. This research was supported by the H2020 program (project COBIOPHAD, grant agreement No. 688448) , being an initiative of the Photonics Public Private Partnership; Agencia Estatal de Investigacian (PID2019-110713RB-I0 0, FEDER) ; program UPVLa FE 2019 (P105 VALBIOAL).Juárez-Rodríguez, MJ.; Ibáñez-Echevarría, E.; Hernández-Fernández De Rojas, D.; Maquieira, A.; Morais, S. (2021). Multiplexed analytical approaches to beta-lactam allergy in vitro testing standardization. Analytica Chimica Acta. 1173:1-8. https://doi.org/10.1016/j.aca.2021.33865618117

Synthesis of skeletally diverse ß-lactam haptens for the in vitro diagnosis of IgE-mediated drug allergy

[EN] We present the first synthesis of beta-lactam-derived haptens, leveraging the principles of diversity-oriented synthesis to discover compounds for drug allergy in vitro testing. We designed, synthesised, and performed in vitro immunological evaluation on 18 structurally diverse haptens derived from beta-lactam antibiotics. The antigens obtained with the synthesised haptens allow for the detection of specific anti-beta-lactam immunoglobulins G and E. Excellent diagnostic sensitivity (83%) and specificity (100%) were achieved when the panel of antigens was tested against a cohort of 31 human serum samples using a multiplexed compact disc-based in vitro testing tool. We posit that adopting this strategy could aid beta-lactam delabeling initiatives.This work was supported by the H2020 program (project COBIOPHAD, grant agreement No. 688448), Agencia Estatal de Investigacion (PID2019-110713RB-I00, FEDER awarded to S.M.); Generalitat Valenciana (PROMETEO/2020/094 awarded S.M.); and the National Institute of General Medical Sciences (GM- 1R35GM127045 awarded to S.L.S.). E.P.M. was supported by an FPU fellowship from the Ministerio de Educacio¿n, Cultura y Deporte (FPU15/01738 and EST18/00360). B.K.H. was supported by a fellowship from the National Science Foundation (DGE1144152 and DGE1745303).Peña-Mendizábal, E.; Hua, BK.; Ibáñez-Echevarría, E.; Hernández-Fernández De Rojas, D.; Maquieira Catala, A.; Schreiber, SL.; Morais, S. (2022). Synthesis of skeletally diverse ß-lactam haptens for the in vitro diagnosis of IgE-mediated drug allergy. Chemical Communications. 58(40):5964-5967. https://doi.org/10.1039/D2CC01677J59645967584

An ultra-sensitive homologous chemiluminescence immunoassay to tackle penicillin allergy

[EN] Penicillin is one of the most widely used antibiotics to treat bacterial infections in clinical practice. The antibiotic undergoes degradation under physiological conditions to produce reactive compounds that in vivo bind self- proteins. These conjugates might elicit an immune response and trigger allergic reactions challenging to di- agnose due to the complex immunogenicity. Penicillin allergy delabeling initiatives are now part of antibiotic stewardship programs and include the use of invasive and risky in vivo tests. Instead, the in vitro quantification of specific IgE is highly useful to confirm immediate allergy to penicillins. However, discrepant results associated with the low sensitivity and accuracy of penicillin allergy in vitro tests have limited their routine diagnostic use for delabeling purposes. We aimed to develop a homologous chemiluminescence-based immunochemical method for the reliable determination of specific IgE to penicillin G, using unprecedented synthetic human-like Analytica Chimica Acta 1214 (2022) 339940 2standards. The synthetic standard targets the major antigenic determinant of penicillin G and the paratope of Omalizumab, acting as human-like specific IgE. It is a potent calibrator, highly stable, easy, and inexpensive to produce, overcoming the limitations of the pooled human serum preparations. The developed method achieved a good agreement and strong positive relationship, reaching a detection limit below 0.1 IU mLP.Q-C acknowledges financial support from Generalitat Valenciana through the research staff-training program (GVA ACIF/2018/173). P.C. awards grants Ramon y Cajal and PID2019-110630 GB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe¿. S.M awards grants PID2019-110713RB-I00 funded by MCIN/ AEI/10.13039/501100011033 and by ERDF A way of making Europe. PROMETEO/2020/094 funded by Generalitat Valenciana, and program UPV-La FE 2019 (P105 VALBIOAL), FMV-ANII 148245, FMV-ANIII 156321 funded by Agencia Nacional de Investigacion and CSIC 2007- 348 UdelaR, Uruguay, are also acknowledged.Quintero-Campos, P.; Segovia-De Los Santos, P.; Ibáñez-Echevarría, E.; Hernández-Fernández De Rojas, D.; Casino, P.; Lassabe, G.; Gonzalez-Sapienza, G.... (2022). An ultra-sensitive homologous chemiluminescence immunoassay to tackle penicillin allergy. Analytica Chimica Acta. 1214:1-8. https://doi.org/10.1016/j.aca.2022.33994018121