Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complex

Ellen R Goldman, George P Anderson, Jerry Conway, Laura J Sherwood, Melissa Fech, BaoHan Vo, Jinny L Liu, Andrew Hayhurst
Analytical Chemistry 2008 November 15, 80 (22): 8583-91
Immunoglobulins from animals of the Camelidae family boast unique forms that do not incorporate light chains. Antigen binding in these unconventional heavy-chain homodimers is mediated through a single variable domain. When expressed recombinantly these variable domains are termed single domain antibodies (sdAb) and are among the smallest naturally IgG-derived antigen binding units. SdAb possess good solubility, thermostability, and can refold after heat and chemical denaturation making them promising alternative recognition elements. We have constructed a library of phage-displayed sdAb from a llama immunized with a cocktail of botulinum neurotoxin (BoNT) complex toxoids and panned the library for binders for BoNT A complex toxoid. Six unique binders were isolated and found to specifically bind BoNT A complex in toxoid and untoxoided forms and when used in optimal combinations in buffer and milk could detect 100 pg/mL untoxoided complex. All sdAb retained their ability to specifically bind target after heating to 85 degrees C for 1 h, in contrast to conventional polyclonal sera. All of the sdAb were highly specific for subtype A1 rather than A2 and demonstrated binding to the 33 kDa hemagglutinin, potentially to a somewhat overlapping linear epitope. The unique properties of these sdAb may provide advantages for many diagnostic applications where long-term storage and in-line monitoring require very rugged yet highly specific recognition elements.

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