Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay

Hee Joo Kim, Mark R. McCoy, Zuzana Majkova, Julie E Dechant, Shirley J. Gee, Sofia Tabares-Da Rosa, Gualberto G. González-Sapienza, Bruce D. Hammock

Research output: Contribution to journalArticlepeer-review

67 Scopus citations


Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC 50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC 50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.

Original languageEnglish (US)
Pages (from-to)1165-1171
Number of pages7
JournalAnalytical Chemistry
Issue number2
StatePublished - Jan 17 2012

ASJC Scopus subject areas

  • Analytical Chemistry


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