Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins
Location: Foodborne Contaminants Research
Title: Evaluation and comparison of three enzyme-linked immunosorbent assay formats for the detection of ricin in milk and serum
Submitted to: Biocatalysis and Agricultural Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 29, 2011
Publication Date: January 9, 2012
Citation: He, X., Mcmahon, S.A., Rasooly, R. 2012. Evaluation and comparison of three enzyme-linked immunosorbent assay formats for the detection of ricin in milk and serum. Biocatalysis and Agricultural Biotechnology. 1(2):105-109. doi:10.1016/j.bcab.2011.08.016.
Interpretive Summary: Simple, rapid, reliable, and high-throughput methods are essential for food defense, to detect foodborne contaminants and to facilitate remediation and recovery from potential toxin-related incidents. Ricin is a highly toxic protein found in castor beans and the industrial by-products of castor oil production, and has been used for intentional poisoning. The enzyme-linked immunosorbent assays (ELISAs) have been used broadly for the detection of proteins and are well-known to be simple, rapid, sensitive and robust. We evaluated and compared three formats of ELISA for quantification of ricin in milk and serum utilizing the same pair of antibodies and found that the ELISA using a biotinylated primary detection antibody and streptavidin-HRP system performed the best among all assays tested, it detected as little as 25 pg/mL of ricin in PBS, 50 pg/mL in non-fat milk, mouse serum, and 100 pg/mL in whole milk. This assay will be valuable to food safety research and clinical diagnosis.
When applied to the detection of a specific protein toxin in food or biological fluids in the incidence of a potential contamination, it is crucial that the assay be both sensitive and specific. In order to identify an immunoassay which is sensitive, simple, and accurate for the detection of ricin in milk and serum, three formats of enzyme-linked immunosorbent assay (ELISA) were compared utilizing the same pair of antibodies. The ELISA using a biotinylated primary detection antibody and streptavidin-linked horseradish peroxidase (HRP) system was shown to be the most sensitive assay with limits of detection (LOD) 25 pg/mL in phosphate buffered saline (PBS), 50 pg/mL in non-fat milk, mouse serum, and 100 pg/mL in whole milk. The second to the best was the ELISA using a streptavidinylated primary detection antibody and biotin-HRP system, the LOD for ricin was 100 pg/mL in PBS and milk, and 1 ng/mL in serum. The ELISA using a non-tagged primary detection antibody and HRP-labeled secondary antibody performed the poorest among of all and the LOD was 1 ng/mL in all matrices tested. Estimation of the precision of these immunoassays using the Coefficient of Variability (CV) showed that the most sensitive ELISA format also had the lowest inter- (4.28%) and intra-assay CV (2.15%) although the inter- and intra-assay CV for the other two ELISAs were less than 10% and 6%, respectively, well below the acceptable level. To conclude, the ELISA using a biotinylated primary detection antibody and streptavidin- HRP system is the best assay for detection of ricin in PBS, milk and serum among three ELISA formats tested and the application of this assay will be valuable to food safety research and clinical diagnosis.