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

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).

Adenosine abolishes MTX-induced suppression of osteoclastogenesis and inflammatory bone destruction in adjuvant-induced arthritis.

Methotrexate (MTX) is widely utilized for the treatment of patients with rheumatoid arthritis (RA); however, recent observation of the MTX-resistant patients proposed some difficulty in MTX-dependent therapeutic approach for RA. To access cellular events related to MTX resistance in RA in respect to inflammatory bone destruction, we investigated on an involvement of the potent inflammatory mediator adenosine in the regulation of osteoclastogenesis and inflammatory bone destruction. In rats with adjuvant-induced arthritis (AA rats), MTX efficiently suppressed bone destruction when it was administrated within 3 days after adjuvant injection, while it could not suppress inflammatory bone destruction if MTX was injected at the time of onset of inflammation (at day 10 after adjuvant injection). Time-course change in the level of plasma adenosine of AA rats was estimated by use of high-performance liquid chromatography and elucidated that adenosine level was markedly elevated till 10 days after adjuvant injection. In vitro bone marrow culture system for evaluating osteoclastogenesis, MTX markedly suppressed osteoclastogenesis in a stromal cell-dependent manner. This MTX-induced suppression of osteoclastogenesis was abrogated by the addition of adenosine. MTX suppressed the expression of mRNA for the receptor activator NF-κB ligand (RANKL), but it did not suppress the expression of osteoprotegerin (OPG). The addition of MTX and adenosine together markedly suppressed the level of OPG expression. Abolishment of MTX action by adenosine was significantly blocked by MRS1754, a highly selective antagonist for the A(2b) adenosine receptor (A(2b)AR), but not by caffeine, an antagonist for A1, A(2a), A3 AR (A1AR, A(2a)AR, and A3AR), which suggests that adenosine acts through A(2b)AR. Immunohistochemical studies showed abundant expression of A(2b)AR in cells localized in the bone-bone marrow boundary of the distal tibia in AA rats but not in control rats. When adenosine was injected in the ankle joints of MTX-treated AA rats, the suppressive effects of MTX on bone destruction was abolished. The current data therefore suggest that upregulation of adenosine production abolished the suppressive effect of MTX on osteoclastic bone destruction. Involvement of the adenosine-A(2b)AR system may explain MTX resistance in RA.

Development of bioluminescent enzyme immunoassay for s-equol using firefly luciferase and its application to the assessment of equol-producer status.

In this study, we developed a specific bioluminescent enzyme immunoassay (BLEIA) for S-equol, employing firefly luciferase as a labeling enzyme, as an alternative to HPLC methods. Satisfactory correlation (r=0.992) was shown when this S-equol BLEIA was compared with HPLC. The cross-reactivity with R-equol as its diastereoisomer is <5%, and that with daidzein, which is the substrate of equol, is 0.02%. Frequencies of Japanese equol producers determined using two distinct approaches were compared: a threshold value for urinary S-equol concentration of 232 ng/ml gave frequencies of 32% of men and 19% of women. These values correspond to the results for log(10)-transformed urinary S-equol to daidzein ratio threshold of -1.75, namely, 34% of men and 19% of women. When the changes in concentration of urinary equol and daidzein were measured after ingestion of isoflavone, the maximum concentration (C(max)) of urinary equol appeared after 9.6 h of isoflavone consumption; this C(max) was 2 h later than that for daidzein. The S-equol BLEIA documented in this study is expected to be an important tool for the assessment of equol producer status and demonstration of the bioavailability of isoflavone.

Development of ultra-high sensitivity bioluminescent enzyme immunoassay for hepatitis B virus surface antigen using firefly luciferase.

Hepatitis B virus (HBV) infection continues to be a global public health concern. Efficient diagnosis of HBV surface antigen (HBsAg) is useful for identification of infection, treatment and prevention of transfusion-transmitted viral infections. Seronegative window reduction afforded by a highly sensitive measurement methodology is necessary as a small quantity of virus with infection risk exists for the period characterized by undetectable HBsAg following HBV infection. In this study, a bioluminescent enzyme immunoassay (BLEIA) for HBsAg was developed employing firefly luciferase as a labeling enzyme and a two-step sandwich immunoassay method. The cut-off value (10 mIU/mL) was 50-fold more sensitive relative to conventional chemiluminescent enzyme immunoassay based on luminol luminescence involving peroxidase as the labeling enzyme and the identical antibodies. Preliminary clinical data for this BLEIA revealed that the HBV seroconversion panel derived sequentially from HBV-infected human blood was detected 11 days following window closure from the first bleed, whereas detection occurred 14-25 days following window closure with the three conventional commercial kits.