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 can produce MAb indefinitely. However mAb

technology is expensive, and sometimes frozen hybridoma cell

lines are hard to recover and generation of high quality MAbs

to small molecules is difficult. Because of the size, the

requirement for two chains, and sophisticated post-translational

modification, functional conventional antibodies are difficult to

express recombinantly. Many antibody-derived proteins that are

derivatives of an intact antibody molecule have been developed,

including monovalent fragments such as Fab, scFv, and

engineered variants including diabodies, triabodies, minibodies,

and single-domain antibodies.

7−9

However, these constructs

often have lower affinity and are less stable than the intact

Received: November 14, 2011

Accepted: December 12, 2011

Published: December 12, 2011

© 2011 American Chemical Society 1165 dx.doi.org/10.1021/ac2030255 | Anal. Chem. 2012, 84, 1165−1171

Article

pubs.acs.org/ac

Analytical Chemistry Article

antibody. Production can be problematic because the limited

solubility and aggregation of the expressed antibody fragments

can reduce yields of expressed proteins.

10,11

In addition, the

diversity of phage antibody libraries from hybridoma cell lines

should be very large to increase the possibility of selecting a

desirable antibody fragment due to the high level of

nonfunctional association of VHs and VLs.

12

In 1993, Hamers-Casterman discovered a new subclass of

antibodies in members of the Camelidae family (i.e., camels,

llamas, and alpacas) that have an antigen binding pocket

comprised solely of a variable region of the heavy chain and

completely devoid of light chains.

13,14

Recombinant expression

of these heavy chain variable domains yields single domain

antibodies (VHHs). The single domain nature of VHHs

provides many advantages over the other recombinant antibody

fragments; in particular, ease of expression in various expression

systems, high thermal stability, excellent solubility, resistance to

proteolysis, and ease of genetic manipulation.

15−17

Antigenbinding

patterns of VHHs are very unique compared to

conventional antibodies. Since the binding area of a conventional

antibody is comprised of a heavy chain and a light chain,

they tend to bind to proteins through a large surface area and

they encompass small molecules in a deep cleft formed at an

interface of a heavy and a light chain. Because heavy chain

antibodies need to compensate for the missing light chain, their

complementary determining region 3 (CDR3) is normally

longer, which allows the formation of a “finger-like” shape and

subsequent binding in a groove of the protein target.

This

allows for heavy chain antibodies to be used as enzyme

inhibitors.

19

When binding to small molecules, their binding

occurs through a lateral recognition involving the coordinated

contribution of the three hyper variable loops and the

framework regions (FRs).

20−22

It has also been observed that

the binding of a VHH against caffeine is accompanied by a

hapten-induced dimerization of the VHHs.

23

To date, most reports about biotechnological applications of

VHHs are related to protein or other macromolecular

antigens,

23,24

while few reports refer to small molecules.

Some VHH-based assays have been developed for the detection

of small haptens but, with the exception of two VHHs against

azo-dye Reactive Red

20

and ochratoxin A,

26

the μM range

sensitivities of the assays are somewhat unsatisfactory for

practical application to real samples.

27−32

We recently

demonstrated that the generation of VHHs to triclocarban

(TCC) is not favored in the llama immune response but that

high-affinity anti-TCC VHHs can be isolated by competitive

selection from phage libraries.

33

The VHH-based ELISA

showed an IC

value of 3.5 ng/mL. To further test this

hypothesis and evaluate the potential of VHHs for the

development of practical applications, we present in this work

the isolation of alpaca VHHs to 3-phenoxybenzoic acid (3PBA)

(MW = 214) and their use in the immunodetection of

this compound in human urine samples. 3-PBA is a human

urinary metabolite of pyrethroid insecticides and can be used as

a biomarker to monitor human exposure to these pesticides.

We have previously developed a PAb-based enzyme-linked

immuosorbent assay (ELISA) for 3-PBA with IC

50

values of

120 ng/mL for a homologous ELISA and 1.6 ng/mL for a

heterologous ELISA.

34

The same antigen was used in this study

to immunize an alpaca, and the VHHs were isolated

...