An Electrochemical Fiveplex Biochip Assay Based on Anti-Idiotypic Antibodies for Fast On-Site Detection of Bioterrorism Relevant Low Molecular Weight Toxins

Abstract

Modern threats of bioterrorism force the need for multiple detection of biothreat agents to determine the presence or absence of such agents in suspicious samples. Here, we present a rapid electrochemical fiveplex biochip screening assay for detection of the bioterrorism relevant low molecular weight toxins saxitoxin, microcystin-LR, T-2 toxin, roridin A and aflatoxin B1 relying on anti-idiotypic antibodies as epitope-mimicking reagents. The proposed method avoids the use of potentially harmful toxin-protein conjugates usually mandatory for competitive immunoassays. The biochip is processed and analyzed on the automated and portable detection platform pBDi within 13.4 min. The fiveplex biochip assay revealed toxin group specificity to multiple congeners. Limits of detection were 1.2 ng/mL, 1.5 ng/mL, 0.4 ng/mL, 0.5 ng/mL and 0.6 ng/mL for saxitoxin, microcystin-LR, T-2 toxin, roridin A or aflatoxin B1, respectively. The robustness of the fiveplex biochip for real samples was demonstrated by detecting saxitoxin, microcystin-LR, HT-2 toxin, roridin A and aflatoxin B1 in contaminated human blood serum without elaborate sample preparation. Recovery rates were between 52–115% covering a wide concentration range. Thus, the developed robust fiveplex biochip assay can be used on-site to quickly detect one or multiple low molecular weight toxins in a single run.

A new chapter for anti-idiotypes in low molecular weight compound immunoassays

“…The development of detection methods for LMW compounds has implications for a range of sectors. Steroid and thyroid hormones are examples of LMW compounds monitored within clinical diagnostics, while toxic compounds, such as saxitoxin, are considered as chemical weapons and are of interest to security sectors [1,2]. In the food safety and environmental sectors, LMW compounds include natural toxins such as marine, freshwater, plant, and mycotoxins, as well as anthropogenic compounds, including pesticides and veterinary drug residues.…”

“…Ab2s have been used as coating or detection reagents in a range of competitive assays, including ELISAs, surface plasmon resonance, microarrays, electrical biochips, and immunochromatographic assays (ICAs) for a variety of LMW compounds, again with a mixed effect on assay performance (Table 1). Ab2 use in multiplex platforms, suitable for in-field use, has been demonstrated on an ICA, namely lateral flow assay, for the detection of aflatoxins and zearalenone, and an electrochemical biochip for the detection of five toxins; saxitoxin, microcystin-LR, T-2, roridin A, and aflatoxin B1 [2,14]. Whether Ab2s were used as a coating or detection reagent in the electrochemical biochip was altered for the different toxins.…”

Multiplex Immunoassay Techniques for On-Site Detection of Security Sensitive Toxins

“…Recently, the detection capability of the system was broadened to security sensitive low molecular weight toxins [ 94 ]. Therefore, a five-plex assay for simultaneous detection of bioterrorism relevant low molecular weight toxins saxitoxin, microcystin-LR, T-2 toxin, roridin A and aflatoxin B1 was established on the pBDi platform.…”

“…LODs were 1.2 ng/mL, 1.5 ng/mL, 0.4 ng/mL, 0.5 ng/mL and 0.6 ng/mL for saxitoxin, microcystin-LR, T-2 toxin, roridin A or aflatoxin B1, respectively, applying a fully automated detection regime with a total assay time of 13.4 min. The robustness of the assay was demonstrated by analyzing human serum samples for the presence of the analyzed low molecular weight toxins without elaborate sample preparation steps [ 94 ].…”

“…Copyright (2006) American Chemical Society). ( C ) Assay scheme of multiplex competitive immunoassay for detection of security relevant low molecular weight toxins (Reprinted from [ 94 ]. MDPI (2019)).…”