Amplification of immunoassays using phage-displayed single domain antibodies

Single domain antibodies (sdAb) are recombinantly produced variable domains derived from the heavy-chain only antibodies found in camelids. Previously, we selected sdAb that were specific for both ricin and botulinum A (BoNT A) toxin complex from phage display libraries of sdAb and evaluated the solubly expressed protein. Here, phage-displayed sdAb were used as reporter reagents and compared to soluble, unfused sdAb. We found that using phage-displayed sdAb as reporter elements in immunoassay formats gave improved detection over using unfused, soluble sdAb reporters. In enzyme-linked immunosorbent assays (ELISAs), the lowest level of toxin detected for both ricin and BoNT A toxoid complex was decreased by one to two orders of magnitude using phage-displayed sdAb as reporter reagents. Use of the phage preserved the ability to discriminate ricin and RCA120 by at least a factor of 10 fold. In an effort to reduce the number of steps in the assays, we directly labeled phage displaying sdAb with a Cy-3 fluorescent dye. Signal was greatly decreased using the dye-labeled phage compared to biotinylated phage followed by streptavidin-phycoerythrin. In these assays the use of phage-displayed sdAb gives more sensitive detection than soluble sdAb alone, however directly dye labeling the phage failed to provide responses of a similar magnitude.