Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins

Abstract

Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg mL(-1) (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg mL(-1) (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg mL(-1) (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation <20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little cross-reactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry

“…3E). 59,[74][75][76][77][78] The mean inter-assay precision for the four ELISAs ranged from 8.2 to 24.4% (Table 3), again similar to other BoNT-specific ELISAs. 3B-E, left panel).…”

“…The different antibody combinations resulted in four highly specific sandwich ELISAs, each recognising one of the four related BoNTseither BoNT/C, D, CD, or DCwith virtually no cross-reactivity among each other (Fig. 59,74,[76][77][78] The high specificity and sensitivity of the four ELISAs developed in this work represent a significant technical improvement over other immunoassays described for BoNT/C, CD, D, or DC that are non-discriminatory on the mosaic variants. Fig.…”

Characterization of Ricin and R. communis Agglutinin Reference Materials

“…Various ELISAs have been developed by different groups [ 70 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 ]. Only a few of them are able to quantify ricin with detection limits down to a few pg/mL with limits of detection of 10 fg/mL [ 84 ], 2 pg/mL [ 70 ], 8 pg/mL [ 85 ], and 40 pg/mL [ 86 ]. Several ricin-specific ELISAs developed in different laboratories have been evaluated in a common ricin proficiency test panel organized in the framework of the EQuATox project, and results are presented in different manuscripts in this special issue of Toxins [ 42 , 87 ].…”

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

“…Usually, they have restricted sensitivity compared to conventional ELISAs and are more prone to matrix interferences [ 31 ]. A number of interesting new developments have been published in recent years which aim at miniaturizing BoNT detection on the protein or functional level, among them microarray or biosensor applications, centrifugal microfluidic disk platforms and portable devices [ 51 , 52 , 53 , 54 , 55 ].…”