Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

Details

• Personal Author:
  Bever CS ; Cui Y ; Gee S ; González-Sapienza G ; Gonzalez-Techera A ; Hammock BD ; Lee KSS ; Leizagoyen C ; Morisseau C ; Pirez M ; Rossotti MA
• Description:
  Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays. [Description provided by NIOSH]
• Subjects:
  Antibodies Chemistry, Analytic Enzymes Occupational Health Safety
• Keywords:
  Analytical Chemistry ELISA Enzyme-linked Immunosorbent Assay Immunoassays Protein Chemistry Proteins
• 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:
  87
• Issue:
  23
• NIOSHTIC Number:
  nn:20062186
• Citation:
  Anal Chem 2015 Dec; 87(23):11907-11914
• Contact Point Address:
  Gualberto González-Sapienza, Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR, Montevideo, Uruguay
• Email:
  ggonzal@fq.edu.uy
• Federal Fiscal Year:
  2016
• 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-512:4680c719329f68f5217199beddcd4bf57b5978e8a3466e3e27ca3155ffe6dc9ddedf8fcdb7c3df1fc226ca7758a76f6a2332d2179c2884769ccb34582423c28b
• Download URL:
  https://stacks.cdc.gov/view/cdc/225483/cdc_225483_DS1.pdf
• File Type:
  [PDF - 1.52 MB ]