Fabrication of Fragment Antibody–Enzyme Complex as a Sensing Element for Immunosensing

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.

The Dawn of a New Era: Targeting the “Undruggables” with Antibody-Based Therapeutics

“…Genetic mutation of human enzymes to react with a specific substrate that cannot be activated by wild-type enzymes is one of the solutions . In addition to their broad therapeutic applications, AECs are also useful for target detection . Among various known AECs, some of them have specific target-binding abilities and possess excellent catalytic activities capable of release and amplification of detectable signals.…”

“…For example, AECs conjugated to oxidoreductases are capable of rapidly liberating electrochemical luminescence. Sode et al have successfully utilized an anti - EGFR V H H conjugated to glucose dehydrogenase (GDH) to detect EGFR in a dose-dependent manner from tumor cells. , …”

“…344 In addition to their broad therapeutic applications, AECs are also useful for target detection. 345 Among various known AECs, some of them have specific target-binding abilities and possess excellent catalytic activities capable of release and amplification of detectable signals. For example, AECs conjugated to oxidoreductases are capable of rapidly liberating electrochemical luminescence.…”

“…Sode et al have successfully utilized an anti-EGFR V H H conjugated to glucose dehydrogenase (GDH) to detect EGFR in a dose-dependent manner from tumor cells. 345,346 3.2.3. Radiolabeled Antibodies.…”

Rapid and sensitive immunoassay for alpha-fetoprotein in serum by fabricating primary antibody–enzyme complexes using protein self-assembly

“…In recent years, primary antibody–enzyme complexes (PAECs), which are able to recognize analytes and catalyze substrates to generate detection signals, have drawn widespread attention for being considered as ideal immunoassay probes. 10 The PAECs can avoid the use of enzyme-labeled secondary antibodies in immunoassay, thereby simplifying the immunoassay procedure and improving the consistency of the analysis results. Yu et al developed a series of nanobodies that specifically recognize the biomarker (Glypican-3, GPC3) of hepatocellular carcinoma through phage display and prepared two PAECs (GPN2-Nluc and GPN2-Ap) using the best-performing nanobody through fusion protein expression.…”