Generation of recombinant alpaca VHH antibody fragments for the detection of the mycotoxin ochratoxin A

Abstract

To develop sensor technologies based on genetically engineered recognition elements, recombinant antibodies characterised by high stability are a prerequisite. Here we describe the first successful isolation of recombinant alpaca single-domain antibody fragments with high affinity to the mycotoxin ochratoxin A (OTA). Variable domains (VHH) of heavy-chain antibodies binding to OTA were cloned from an immunised alpaca. Selected VHH clones clustered in four major groups, based on protein sequence similarity. Six representative VHH antibody fragments were produced in Escherichia coli and characterised for their sensitivity and specificity by indirect competitive enzyme-linked immunosorbent assays. OTA concentrations causing 50% inhibition (IC50) ranged from 12 ng/ml for antibody fragment OCH-62 to 442 ng/ml for antibody fragment OCH-40. The IC50 of OCH-62 for ochratoxin B, an OTA analogue, was 476 ng/ml. No significant cross-reactivity was observed with common food constituents with structural similarities with parts of OTA, such as L-phenylalanine and coumarin. The performance of OCH-62 for detection of OTA in food matrices was comparable to the performance in buffer (white wine: IC50=17 ng/ml; red wine: IC50=21 ng/ml; instant coffee: IC50=17 ng/ml). In a membrane-based flow-through immunoassay, used for fast visual screening of samples, OCH-62 showed a cut-off level of 10 ng/ml OTA. Thereby, OCH-62 ranked among the best recombinant antibody fragments described for mycotoxins and is an excellent candidate for the design of food contamination sensors based on nanotechnology.