Practical application of bioluminescence enzyme immunoassay using enhancer for firefly luciferin–luciferase bioluminescence

Abstract

Firefly luciferin-luciferase bioluminescence is known for its high quantum yield (41.0 ± 7.4%). Given this high quantum yield, application of this bioluminescence is expected to be useful in the field of clinical diagnostics. The kinetic profile of this bioluminescence exhibits an instant rise (<1 s) and a rapid decay in light emission (decreased to 42% after 5 s). In this study, we applied four enhancers including coenzyme A, inosine5'-triphosphate sodium salt, sodium tripolyphosphate and potassium pyrophosphate to prolong light emission. When these enhancers were used, luminescence was only decreased to 89, 83, 87 and 82% after 5 s, respectively. These materials modified the kinetic profile of bioluminescence so that the luminescence is more suitable for clinical application. It becomes more suitable because they enable highly sensitive integration and simplification of a device by separating luminescence measurements from dispensing of reagents. Using these enhancers, we then developed a bioluminescent enzyme immunoassay (BLEIA) for hepatitis B virus surface antigen (HBsAg) that employed firefly luciferase as a labeling enzyme. We compared the results obtained from the HBsAg BLEIA method with the conventional chemiluminescent enzyme immunoassay method, and found a satisfactory correlation (r=0.984, n=118).

Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

“…An enzyme-labeled anti-HBs rabbit polyclonal antibody (I.L. Japan, Tokyo, Japan) and an anti-HBs MAb, HBs335, were prepared using a previously described method (36,37). In brief, through the reaction with maleimide-activated streptavidin (MP Biomedicals, Solon, OH), the Fab= fragments conjugated with thermostable biotinylated luciferase (bL248; Kikkoman, Chiba, Japan) were purified using Superdex 200 HR 10/30 (GE Healthcare Japan, Tokyo, Japan) and then by affinity absorption again to remove nonspecifically bound materials, using a gel coated with normal human serum (37).…”

“…Antibodies with streptavidin and thermostable biotinylated luciferase bind through an avidinbiotin interaction, allowing for the conjugation of luciferase on an antibody without decreasing the reactivity of luciferase. Some measurement systems using luciferase as a labeling enzyme have already been reported (36)(37)(38). In a previous study, we developed a measurement system for HBsAg with a high sensitivity of 10 mIU/ml on the basis of a bioluminescent enzyme immunoassay (BLEIA) using firefly luciferase with a high quantum yield (36,37).…”

“…Some measurement systems using luciferase as a labeling enzyme have already been reported (36)(37)(38). In a previous study, we developed a measurement system for HBsAg with a high sensitivity of 10 mIU/ml on the basis of a bioluminescent enzyme immunoassay (BLEIA) using firefly luciferase with a high quantum yield (36,37). However, this preliminary assay system failed to detect a G145R mutant due to the low reactivity of the immobilized antibodies with the mutant.…”

The Effect of Surface Charge Saturation on Heat‐induced Aggregation of Firefly Luciferase

“…The emission of visible light through by the two‐step oxidation of firefly luciferin, in the presence of ATP, Mg 2+ , and molecular oxygen has prepared the susceptibility of accomplishment of ATP‐related assays , analytical assays and in vivo molecular imaging . Meanwhile, luciferase can act as a molecular probe for protein‐protein interactions and different analytes , and as well as a label for immunoassays .…”

Approaches to Engineer Stability of Beetle Luciferases

“…This makes them a widely used tool in a variety of in vitro and in vivo applications: in ATP-related assays from direct ATP measurements to estimation of bacterial contamination and pyrosequencing [4, 5], in in vivo molecular imaging and as a genetic reporter in molecular biology [6–8]. This enzyme was also shown to be a promising tool for molecular sensing of protein-protein interactions and different analytes [9–11], in analytical assays based on real time monitoring of polynucleotide amplification [12] and a label for immunoassays [13]. …”