Anticuerpoa Alpaca
Enviado por beredufo • 20 de Junio de 2013 • 3.909 Palabras (16 Páginas) • 294 Visitas
Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain
Antibodies for Development of Sensitive Immunoassay
Hee-Joo Kim,
†
Mark R. McCoy,
Sofia Tabares-da Rosa,
†
‡
§
†
Zuzana Majkova,
Gualberto G. Gonza
́
lez-Sapienza,
†
Julie E. Dechant,
§
‡
Shirley J. Gee,
and Bruce D. Hammock*
Department of Entomology and UCD Cancer Center, University of California, Davis, California 95616, United States
Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, California 95616,
United States
§
Ca
́
tedra de Inmunología, Facultad de Química, Instituto de Higiene, UDELAR, Av. A. Navarro 3051, Piso 2, Montevideo 11600,
Uruguay
*
S
Supporting Information
ABSTRACT: 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
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 4hydroxylated
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.
S
ince the first radioimmunoassay was reported,
countless
immunoassays have been developed and proven to be
invaluable analytical methods for in vitro diagnostics and
environmental monitoring for wide array of substances such as
viruses, bacteria, disease biomarkers, food toxins, and environmental
pollutants including endocrine disruptors and pesticides
and their metabolites.
2−6
For an immunoassay to be applied to
a real sample, it should have high sensitivity and robustness in
the matrix in which it is detected. These properties are largely
dependent on the availability of antibodies with high affinity
and specificity to their target analyte along with a high stability
in the matrix. Monoclonal antibodies (MAbs) mostly derived
from murine hybridoma cell lines, along with polyclonal
antibodies (PAbs) from sera of rabbits, goats, sheep, and other
species, are traditional reagents used in immunoanalytical
techniques.
Conventional antibodies (IgG subclass) have an average
molecular weight of 150 kDa, and they are composed of two
identical heavy and light chains connected by disulfide bonds.
Each antibody contains two antigen binding pockets. Although
PAbs can be easily obtained at a low production cost, they are
finite, which requires subsequent antibody characterization and
1
50
†
,†
assay optimization because of animal to animal variation in
immune response. This can be a limiting factor for the use of
PAbs in development of an assay for large scale production or
commercialization. MAbs obtained from hybridoma cell lines
can overcome the reproducibility issues of PAbs. An established
hybridoma
...