Development of xMAP Assay for Detection of Six Protein Toxins

Abstract

xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2-to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3−200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.

Ricin detection: Tracking active toxin

“…The use of microspheres generated an increase in collective surface area, providing improvements in sensitivity and assay time, while a magnetoelastic sensor surface also helped to reduce total assay time. The use of microarrays, which include panels of antibodies for simultaneous detection of a variety of antigenic targets of interest, allowed multiplexed detection of ricin in parallel with other harmful toxic agents, such as cholera toxin, staphylococcal enterotoxins A and B, Bacillus globigii, botulinum toxin A, Yersinia pestis, and heat labile toxin of Escherichia coli, and provided dramatic improvements in assay utility and flexibility (Delehanty and Ligler, 2002;Garber et al, 2010;Simonova et al, 2012;Wadkins et al, 1998;Weingart et al, 2012). In other works, the toxin capture antibodies used as receptors were substituted by DNA/RNA aptamers (Haes et al, 2006;Kirby et al, 2004;Lamont et al, 2011), single domain antibodies (Anderson et al, 2013;Shia and Bailey, 2013;Stine et al, 2005), and sugar-conjugated materials (Huebner et al, 2013;Liu et al, 2011).…”

Impact of Aerosol Dust on xMAP Multiplex Detection of Different Class Pathogens

“…On the other hand, the sensitivity of viruses detection is comparable to that of toxins detection if it is measured in mass values rather than virions. According to several papers ( Garber et al, 2010 ; Simonova et al, 2012 ) describing toxin detection by xMAP technology, the sensitivity is typically around few nanograms/ml. This is still not enough for viruses because 1 ng/ml of the virus roughly corresponds to 10 6 vir/ml.…”

“…Milk used as a matrix led to a significant sensitivity decrease. LOD decreased 2- to 5-fold for most toxins and 30-fold for LT ( Simonova et al, 2012 ). This research demonstrates the importance of investigating the matrix effect during multiplex immune assay development.…”

Single Microbead‐Anchored Fluorescent Immunoassay (SMFIA): A Facile and Versatile Platform Allowing Simultaneous Detection of Multiple Antigens

“…[25][26][27][28][29][30] Particularly,t he Luminex xMAP technology,w hich allows simultaneous detection of up to 100 analytes based on the flow cytometric analysiso fd ifferent set of patented color-coded microbeads, has been widely used in biomedical studies. [31][32][33] However,f or conventional beadsbased fluorescenta ssays including the Luminex xMAP-based methods, thousands of or even millions of beads are generally used in each reaction. In such cases, if the target-generated fluorescencer eporters are less than or only comparable to the number of theu sed beads, the diluted fluorescencem olecules at each bead cannot be detectable.…”