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.
Goldman
Ellen R
ER
Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, 4555 Overlook Ave SW, Washington, DC 20375, USA. ellen.goldman@nrl.navy.mil
Anderson
George P
GP
Bernstein
Rachael D
RD
Swain
Marla D
MD
D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000276:immunology; D002161:Camelids, New World; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D019151:Peptide Library; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012276:Ricin / Q000276:immunology; D012680:Sensitivity and Specificity
D018033:Antibodies, Bispecific; D000939:Epitopes; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D012276:Ricin; D019274:Botulinum Toxins, Type A