Abstract
Antibody–enzyme complexes (AECs) are ideal molecular recognition elements for immunosensing applications. One molecule possesses both a binding ability to specific targets and catalytic activity to gain signals, particularly oxidoreductases, which can be integrated into rapid and sensitive electrochemical measurements. The development of AECs using fragment antibodies rather than intact antibodies, such as immunoglobulin G (IgG), has attracted attention for overcoming the ethical and cost issues associated with the production of intact antibodies. Conventionally, chemical conjugation has been used to fabricate AECs; however, controlling stoichiometric conjugation using this method is difficult. To prepare homogeneous AECs, methods based on direct fusion and enzymatic conjugation have been developed, and more convenient methods using Catcher/Tag systems as coupling modules have been reported. In this review, we summarize the methods for fabricating AECs using fragment antibodies developed for sensing applications and discuss the advantages and disadvantages of each method.
Highlights
Antibodies have been widely used as sensing elements and for therapeutic purposes because of their high specificity and affinity for target capture [1]
Takazawa et al fabricated a complex of anti-hen egg-white lysozyme (HEL) single-chain Fv (scFv) and Alkaline phosphatase (ALP), using microbial transglutaminase (MTG) isolated from Streptomyces mobaraensis, to detect HEL, a food allergen [37]
To overcome the limitations of the conventional method, we developed a new method for fabricating antibody–enzyme complexes (AECs) using the Catcher/Tag systems, which spontaneously form irreversible covalent bonds by mixing protein domains (Catchers) with each counter peptide tag (Tags)
Summary
Antibodies have been widely used as sensing elements and for therapeutic purposes because of their high specificity and affinity for target capture [1]. Alkaline phosphatase (ALP) and horseradish peroxidase (HRP) are often used because of their high enzymatic activities; for example, the reported activities of ALP from calf intestine and HRP are 2500 U/mg and 263 U/mg, respectively [18], which are desirable for labeling [19,20] These enzymes have been used as models in immobilization studies [21,22]. The variable domain of heavy chain of heavy-chain antibodies (VHH) derived from camelids has been generally used (Figure 1b) These fragment antibodies can be produced using cost-effective bacterial expression systems without ethical issues. Their small molecular size may contribute to the detection of targets that cannot be accessed by intact antibodies because of steric hindrance.
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