PMID- 26544909
OWN - NLM
STAT- MEDLINE
DCOM- 20160707
LR  - 20240324
IS  - 1520-6882 (Electronic)
IS  - 0003-2700 (Print)
IS  - 0003-2700 (Linking)
VI  - 87
IP  - 23
DP  - 2015 Dec 1
TI  - Method for Sorting and Pairwise Selection of Nanobodies for the Development of 
      Highly Sensitive Sandwich Immunoassays.
PG  - 11907-14
LID - 10.1021/acs.analchem.5b03561 [doi]
AB  - 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.
FAU - Rossotti, Martin A
AU  - Rossotti MA
AD  - Catedra de Inmunologia, DEPBIO, Facultad de Quimica, Instituto de Higiene, UDELAR 
      , Montevideo, Uruguay.
FAU - Pirez, Macarena
AU  - Pirez M
AD  - Catedra de Inmunologia, DEPBIO, Facultad de Quimica, Instituto de Higiene, UDELAR 
      , Montevideo, Uruguay.
FAU - Gonzalez-Techera, Andres
AU  - Gonzalez-Techera A
AD  - Catedra de Inmunologia, DEPBIO, Facultad de Quimica, Instituto de Higiene, UDELAR 
      , Montevideo, Uruguay.
FAU - Cui, Yongliang
AU  - Cui Y
AD  - Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, 
      University of California , Davis, California, United States.
FAU - Bever, Candace S
AU  - Bever CS
AD  - Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, 
      University of California , Davis, California, United States.
FAU - Lee, Kin S S
AU  - Lee KS
AD  - Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, 
      University of California , Davis, California, United States.
FAU - Morisseau, Christophe
AU  - Morisseau C
AD  - Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, 
      University of California , Davis, California, United States.
FAU - Leizagoyen, Carmen
AU  - Leizagoyen C
AD  - Parque Lecocq, IMM, Montevideo, Uruguay.
FAU - Gee, Shirley
AU  - Gee S
AD  - Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, 
      University of California , Davis, California, United States.
FAU - Hammock, Bruce D
AU  - Hammock BD
AD  - Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, 
      University of California , Davis, California, United States.
FAU - Gonzalez-Sapienza, Gualberto
AU  - Gonzalez-Sapienza G
AD  - Catedra de Inmunologia, DEPBIO, Facultad de Quimica, Instituto de Higiene, UDELAR 
      , Montevideo, Uruguay.
LA  - eng
GR  - D43 TW005718/TW/FIC NIH HHS/United States
GR  - 1K99ES024806-01/ES/NIEHS NIH HHS/United States
GR  - K99 ES024806/ES/NIEHS NIH HHS/United States
GR  - P42 ES004699/ES/NIEHS NIH HHS/United States
GR  - P30 ES023513/ES/NIEHS NIH HHS/United States
GR  - P42 ES04699/ES/NIEHS NIH HHS/United States
GR  - TW05718/TW/FIC NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20151118
PL  - United States
TA  - Anal Chem
JT  - Analytical chemistry
JID - 0370536
RN  - 0 (Antigens)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Single-Domain Antibodies)
RN  - EC 3.3.2.- (Epoxide Hydrolases)
SB  - IM
MH  - Antigen-Antibody Reactions
MH  - Antigens/chemistry/metabolism
MH  - *Enzyme-Linked Immunosorbent Assay
MH  - Epoxide Hydrolases/chemistry/metabolism
MH  - Humans
MH  - Recombinant Proteins/chemistry/metabolism
MH  - Single-Domain Antibodies/*immunology/*isolation & purification
MH  - Solubility
PMC - PMC4666776
MID - NIHMS738809
EDAT- 2015/11/07 06:00
MHDA- 2016/07/09 06:00
PMCR- 2016/12/01
CRDT- 2015/11/07 06:00
PHST- 2015/11/07 06:00 [entrez]
PHST- 2015/11/07 06:00 [pubmed]
PHST- 2016/07/09 06:00 [medline]
PHST- 2016/12/01 00:00 [pmc-release]
AID - 10.1021/acs.analchem.5b03561 [doi]
PST - ppublish
SO  - Anal Chem. 2015 Dec 1;87(23):11907-14. doi: 10.1021/acs.analchem.5b03561. Epub 
      2015 Nov 18.