Electrochemical Biochip Assays Based on Anti-idiotypic Antibodies for Rapid and Automated On-Site Detection of Low Molecular Weight Toxins

Phycotoxins and mycotoxins, such as paralytic shellfish poisoning toxins, type A trichothecenes, and aflatoxins are among the most toxic low molecular weight toxins associated with human poisoning incidents through the consumption of naturally contaminated food. Therefore, there is an utmost need for rapid and sensitive on-site detection systems. Herein, an electrochemical biochip for fast detection of saxitoxin, T-2 toxin as well as aflatoxin M1 and their corresponding congeners, respectively, using a portable and fully automated detection platform (pBDi, portable BioDetector integrated) was developed. Toxin analysis is facilitated upon the biochip via an indirect competitive immunoassay using toxin-specific antibodies combined with anti-idiotypic antibodies. The developed biochips enable detection in the low ng/mL-range within 17 min. Moreover, the assays cover a wide linear working range of 2–3 orders of magnitude above the limit of detection with an inter-chip coefficient of variation lower than 15%. The broad specificity of the employed antibodies which react with a large number of congeners within the respective toxin group allows efficient screening of contaminated samples for the presence of these low molecular weight toxins. With respect to the analysis of human urine samples, we focused here on the detection of saxitoxin, HT-2 toxin, and aflatoxin M1, all known as biomarkers of acute toxin exposure. Overall, it was proved that the developed biochip assays can be used to rapidly and reliably identify severe intoxications caused by these low molecular weight toxins

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibioticassociated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.Peer Reviewe

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

Biological toxins are a heterogeneous group of high molecular as well as low molecular weight toxins produced by living organisms. Due to their physical and logistical properties, biological toxins are very attractive to terrorists for use in acts of bioterrorism. Therefore, among the group of biological toxins, several are categorized as security relevant, e.g., botulinum neurotoxins, staphylococcal enterotoxins, abrin, ricin or saxitoxin. Additionally, several security sensitive toxins also play a major role in natural food poisoning outbreaks. For a prompt response to a potential bioterrorist attack using biological toxins, first responders need reliable, easy-to-use and highly sensitive methodologies for on-site detection of the causative agent. Therefore, the aim of this review is to present on-site immunoassay platforms for multiplex detection of biological toxins. Furthermore, we introduce several commercially available detection technologies specialized for mobile or on-site identification of security sensitive toxins

Nuclear CYLD expression in correlation to tumor grading in human HCC tissues.

<p>(<b>A</b>) Representative staining of positive and negative nuclear CYLD expression in HCC specimens. 10- and 40-fold magnification (^nucCYLD⁺; upper left and ^nucCYLD⁻, upper right panel). Representative staining of corresponding Ki67 expression in HCC specimens. 10-fold magnification (lower panels). (<b>B</b>) Nuclear expression of CYLD (IHS score 0–12) in HCC (G1 = 13, G2 = 54, G4 = 25 G4 = 3) and normal liver tissues (N = 7) correlated to grading (Spearman correlation coefficient: −0.423, P<0.001). (C) Nuclear expression of CYLD related to the ratio of Ki67 positive nuclei in HCC (^nucCYLD⁺; N = 47; ^nucCYLD⁻; N = 41) and normal liver tissues (N = 7) (Spearman correlation coefficient: −0.271, <i>P</i> = 0.006).</p

Survival of HCC patients after liver surgery according to subcellular CYLD expression.

<p>Kaplan-Meier analysis (N = 95) for overall survival (OS) of patients receiving liver resection, for the following subgroups: (^nucCYLD⁺/^cyt°CYLD⁻, ^nucCYLD⁺/^cyt°CYLD⁺, ^nucCYLD⁻/^cyt°CYLD⁺ and ^nucCYLD⁻/^cyt°CYLD⁻ (<i>P</i> = 0.06). Positive nuclear (^nucCYLD⁺) and cytoplasmic (^cyt°CYLD⁺) CYLD staining was defined as an immunohistochemical score (IHS) ≥3 for ^nucCYLD⁺ and ≥6 for ^cyt°CYLD⁺ (IHS ranging from 0 to 12, obtained by multiplication of the intensity and the quantity score).</p

Baseline characteristics of HCC patients corresponding to CYLD expression in the cytoplasma (^cyt°CYLD^+/−) or nucleus (^nucCYLD^+/−).

<p>Bilirubin levels >1 mg/dl were depicted as “increased”. AFP serum levels: normal range <8 IU/ml).</p><p>Baseline characteristics of HCC patients corresponding to CYLD expression in the cytoplasma (^cyt°CYLD^+/−) or nucleus (^nucCYLD^+/−).</p

Correlation of CYLD expression with outcome after liver resection <i>vs.</i> liver transplantation.

<p>(<b>A</b>) Kaplan-Meier analysis for overall survival (OS) of patients receiving liver resection with positive (^nucCYLD⁺, N = 33) and negative nuclear CYLD expression (^nucCYLD⁻; N = 29; <i>P</i> = 0.1). (<b>B</b>) OS of patients receiving liver transplantation with positive (^nucCYLD⁺; N = 16) or negative nuclear CYLD expression (^nucCYLD⁻; N = 11; <i>P</i> = 0.04).</p