This work aimed to develop a new indirect ELISA for detecting the soybean allergen Gly m TI in processed and complex food matrices and to investigate the influence of antibody batch variability on immunoassay perfor-m...This work aimed to develop a new indirect ELISA for detecting the soybean allergen Gly m TI in processed and complex food matrices and to investigate the influence of antibody batch variability on immunoassay perfor-mance.During method development,inconsistencies in Gly m TI detection prompted a comparative evaluation of several batches of anti-Gly m TI IgG using both the newly developed ELISA and an electrochemical immuno-sensor.The ELISA was optimised in terms of protein extraction,antibody concentration,and linear dynamic range,and successfully applied to model foods simulating biscuits,sausages,and cooked hams,achieving a limit of detection(LOD)of 1,000 mg/kg irrespective of matrix or processing.Given this limited sensitivity,antibody batch variability was further assessed across both ELISA and immunosensor.Antibodies obtained approximately at the same time showed comparable performance,whereas those acquired five years earlier differed substan-tially.In particular,antibodies of older batches(Ab1/Ab3)outperformed the most recent ones(Ab2/Ab4),exhibiting wider dynamic ranges,lower LOD(0.1 mg/kg),and requiring lower antibody concentrations.These results highlight the significant variability of commercial antibodies and its critical impact on immunoassay sensitivity and reproducibility in food allergen detection.展开更多
Centella asiatica is a plant well recognised by its use in traditional medicines owing to its vast therapeutic applications,being also used in plant food supplements(PFS).The increased popularity of this species has p...Centella asiatica is a plant well recognised by its use in traditional medicines owing to its vast therapeutic applications,being also used in plant food supplements(PFS).The increased popularity of this species has prompted the need of tools to authenticate the botanical origin of derived products.This work proposes the first real-time PCR method to detect and quantify C.asiatica in herbal products and PFS.The method targeted a nuclear marker with a TaqMan probe,addressing the influence of plant matrix.C.asiatica-specific assay detected 10 pg/reaction of DNA.The influence of six different plant species(artichoke,bacopa,horsetail,green tea,ginger and ginkgo)was evaluated in C.asiatica detection and quantification.Based on the low matrix effect and relevance,green tea(Camelia sinensis)was selected for reference mixtures to propose a calibration model within 0.5–25%(w/w)of C.asiatica in C.sinensis.The method was successfully validated using blind mixtures and further applied to analyse 15 commercial samples,suggesting that 36%of the infusion samples and 100%of PFS were adulterated/mislabelled due to species substitution.Herein,a novel approach is proposed as a quantitative powerful tool to authenticate C.asiatica herbal products,ensuring the compliance of labelling.展开更多
基金funded by national funds(FCT/MECI,Fundaçao para a Ciencia e Tecnologia and Ministerio da Educaçao,Ciencia e Inovaçao)through project HYPOALLERGEN(PTDC/BAA-AGR/4005/2021 DOI 10.54499/PTDC/BAA-AGR/4005/2021)ALLEVIATE(2023.12193.PEX DOI 10.54499/2023.12193.PEX)+3 种基金the strategic funding of UID/50006/2025(DOI:10.54499/UID/50006/2025-LAQV)UID/PRR/00081/2025(DOI:10.54499/UID/PRR/00081/2025-CIQUP)and LA/P/0056/2020(DOI:10.54499/LA/P/0056/2020-IMS)and European Regional Development Fund(COMPETE/FEDER)through project IMMUNOGATE(operation no.14906,COMPETE2030-FEDER-00848700)CD thanks FCT for funding PhD fellowship(2024.04195.BD)funding through the Individual Call to Scientific Employment Stimulus(2023.07644.CEECIND/CP2842/CT0011 DOI 10.54499/2023.07644.CEECIND/CP2842/CT0011,2021.03670.CEECIND/CP1662/CT0011 DOI 10.54499/2021.03670.CEECIND/CP1662/CT0011,and 2021.03583.CEECIND/CP1662/CT0012 DOI 10.54499/2021.03583.CEECIND/CP1662/CT0012,respectively).
文摘This work aimed to develop a new indirect ELISA for detecting the soybean allergen Gly m TI in processed and complex food matrices and to investigate the influence of antibody batch variability on immunoassay perfor-mance.During method development,inconsistencies in Gly m TI detection prompted a comparative evaluation of several batches of anti-Gly m TI IgG using both the newly developed ELISA and an electrochemical immuno-sensor.The ELISA was optimised in terms of protein extraction,antibody concentration,and linear dynamic range,and successfully applied to model foods simulating biscuits,sausages,and cooked hams,achieving a limit of detection(LOD)of 1,000 mg/kg irrespective of matrix or processing.Given this limited sensitivity,antibody batch variability was further assessed across both ELISA and immunosensor.Antibodies obtained approximately at the same time showed comparable performance,whereas those acquired five years earlier differed substan-tially.In particular,antibodies of older batches(Ab1/Ab3)outperformed the most recent ones(Ab2/Ab4),exhibiting wider dynamic ranges,lower LOD(0.1 mg/kg),and requiring lower antibody concentrations.These results highlight the significant variability of commercial antibodies and its critical impact on immunoassay sensitivity and reproducibility in food allergen detection.
文摘Centella asiatica is a plant well recognised by its use in traditional medicines owing to its vast therapeutic applications,being also used in plant food supplements(PFS).The increased popularity of this species has prompted the need of tools to authenticate the botanical origin of derived products.This work proposes the first real-time PCR method to detect and quantify C.asiatica in herbal products and PFS.The method targeted a nuclear marker with a TaqMan probe,addressing the influence of plant matrix.C.asiatica-specific assay detected 10 pg/reaction of DNA.The influence of six different plant species(artichoke,bacopa,horsetail,green tea,ginger and ginkgo)was evaluated in C.asiatica detection and quantification.Based on the low matrix effect and relevance,green tea(Camelia sinensis)was selected for reference mixtures to propose a calibration model within 0.5–25%(w/w)of C.asiatica in C.sinensis.The method was successfully validated using blind mixtures and further applied to analyse 15 commercial samples,suggesting that 36%of the infusion samples and 100%of PFS were adulterated/mislabelled due to species substitution.Herein,a novel approach is proposed as a quantitative powerful tool to authenticate C.asiatica herbal products,ensuring the compliance of labelling.
