Anticuerpoa Alpaca

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

...