Recombinant antibodies and their use in biosensors

Abstract

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

Fig. 1

Configuration of IgG and scFv recombinant antibodies, and locations of the antibody heavy and light-chain variable, constant, and idiotype regions, the antibody paratope framework (FW), and complementary determining (CDR) regions

Fig. 2

The antibody, and different antibody fragments [1]

Fig. 3

(a) Direct immunoassay: an antigen immobilized on a solid support is detected and quantified by use of an antigen-specific antibody conjugated with a reporter molecule; b) Sandwich immunoassay: an antibody specific for an antigen captures the antigen from a sample. The captured antigen is detected and quantified by use of an antibody labeled with a reporter molecule (e.g. dye, enzyme, isotope, etc.); c) Competition immunoassay: a capture antibody specifically captures an antigen from a sample. The antigen in the sample competes with a purified labeled antigen for binding to the capture antibody. As the concentration of unlabeled antigen in a sample increases, assay signal decreases

Fig. 4

Coupling of scFvs to the Au QCM sensor surface

Fig. 5

A10B scFv sensors with different linker sequences

Fig. 6

Signal vs. time curves of different A10B scFv QCM sensors for detection of 132 nmol L⁻¹ rabbit IgG

Fig. 7

Schematic diagram depicting the interactions and aggregations of scFv-conjugated gold nanoparticles with the antigen, IgG

Fig. 8

(a) scFv-based Fc sensor; (b) scFv sensor for detection of cancer cell surface receptor