Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain Antibodies for Development of Sensitive Immunoassay

Details

• Personal Author:
  Dechant JE ; Gee SJ ; González-Sapienza GG ; Hammock BD ; Kim H-J ; Majkova Z ; McCoy MR ; Tabares-da Rosa S
• Description:
  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 IC50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC50 = 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. This article has a correction: Anal Chem 2012 Aug; 84(15):6919 https://doi.org/10.1021/ac301927h. Incorrect primer sequences for the library construction were included in the Supporting Information of the original manuscript (page 3, lines 10-14). [Description provided by NIOSH]
• Subjects:
  Animals Antibodies Antigens Endocrine System Immune System Insecticides Occupational Health Safety
• Keywords:
  Animal Studies Endocrine Disruptors Immunoassays Immunochemistry Pesticides And Agricultural Chemicals
• ISSN:
  0003-2700
• Document Type:
  Text
• Funding:
  Cooperative Agreement
• Genre:
  Journal Article
• Place as Subject:
  California ; OSHA Region 9
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  84
• Issue:
  2
• NIOSHTIC Number:
  nn:20056709
• Citation:
  Anal Chem 2012 Jan; 84(2):1165-1171
• Contact Point Address:
  Bruce D. Hammock, Department of Entomology and UCD Cancer Center, University of California, Davis, California 95616, United States
• Email:
  bdhammock@ucdavis.edu
• Federal Fiscal Year:
  2012
• NORA Priority Area:
  Agriculture, Forestry and Fishing
• Performing Organization:
  University of California - Davis
• Peer Reviewed:
  True
• Start Date:
  20010930
• Source Full Name:
  Analytical Chemistry
• End Date:
  20270929
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:13d9b1d2ec6a248e10984bb295aef532b30fffe325a3e431ab62b1cab493f83140453907b32fd2fa8a73538bc29d42cf68ded76dd240446f9e5d9551f51dba4d
• Download URL:
  https://stacks.cdc.gov/view/cdc/214261/cdc_214261_DS1.pdf
• File Type:
  [PDF - 372.35 KB ]