Anti-idiotypic VHH mediated environmentally friendly immunoassay for citrinin without mycotoxin

Abstract

Competitive enzyme-linked immunosorbent assay (ELISA) is the most widely used method for rapidly screening of mycotoxins in contaminated food products; however, it involves the use of toxin-protein conjugates as a competitive antigen and toxin standards as calibrators. In this work, a single domain heavy chain antibody (VHH)-based environmentally friendly ELISA (VHH-ELISA) was developed to measure citrinin concentration in Monascusfermented rice. With this novel indirect competitive VHH-ELISA, the use of a synthetic antigen and pure toxins for operation and environmental safety simultaneously avoided. The assay exhibited a limit of detection of 2.25 ng/mL, with a linear range of 5.0 −500.0 ng/mL. VHH-ELISA was then utilized for sample testing, and excellent agreement was observed between VHH-ELISA and high-performance liquid chromatography as a reference. This study provided a novel platform to develop environmentally friendly immunoassays for other toxic small molecules in the application of anti-idiotypic VHHs.

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

“…There is only one example of this, for citrinin. The affinity of the Ab2 nanobodies for Ab1 was assessed and the Ab2 with the lowest affinity was used as a coating antigen, while the Ab2 with the highest affinity was used as a standard replacement [32]. Collectively, this work demonstrates the need for Ab2s with a range of affinities to the Ab1 and the screening of only high-affinity binders should be avoided.…”

“…Indeed, Ab2 nanobodies produced for aflatoxin M1 [33] and aflatoxins [34], which used low Ab1 coating concentrations during biopanning (2.5-0.5 μg/ml and 10-1 μg/ml, respectively), had reduced assay sensitivity when used as coating reagents. Coating Ab2 nanobodies that resulted in a higher or equal assay performance for ochratoxin A [28], deoxynivalenol [30], citrinin [27,32], and fumonisin B1 [29] had Ab1 coating concentrations ranging from 100 to 10 μg/ml during biopanning.…”

Evaluation of methods for the detection of hazardous substances in food based on machine learning

“…2.2.2 Evaluation index system for the detection methods of biological hazards. For the evaluation of the detection methods of biological hazards, we also have to consider the cost of the detection method, the time spent, the amount of reagent used and their impact on the environment, 29 whether they are easy to obtain and whether the technical route is reasonable. Besides, a detection method can be used for biological hazards in different matrices, for example a colloidal gold immunochromatographic test strip (ICTS) can detect a variety of Salmonella.…”

Bispecific Single-Domain Antibodies as Highly Standardized Synthetic Calibrators for Immunodiagnosis

“…In this way, recombinant calibrators can be generated by combining a nanobody against the antigen of interest with a nanobody against the antigen-binding site of the secondary antibody. The production of this type of recombinant calibrators is facilitated by the fact that it is fairly easy to generate anti-idiotype nanobodies because the paratope of the monoclonal antibody used for immunization turns out to be the most immunogenic region for the immunized animal. , …”

“…The production of this type of recombinant calibrators is facilitated by the fact that it is fairly easy to generate anti-idiotype nanobodies because the paratope of the monoclonal antibody used for immunization turns out to be the most immunogenic region for the immunized animal. 15,16 As a proof of concept, in this study, we developed recombinant calibrators for an allergy test for the antibiotics aztreonam and amoxicillin as outlined in Figure 1. The chosen models represent typical examples in which, as it occurs for other allergies against antibiotics, the absence of reference sera makes it difficult to compare the results obtained in different laboratories.…”