Development of Bioluminescent Enzyme Immunoassay for S-Equol Using Firefly Luciferase and Its Application to the Assessment of Equol-Producer Status

Abstract

Equol [7-hydroxy-3-(4Ј-hydroxyphenyl)-chroman] is a metabolite produced in vivo from the soy isoflavone daidzein by the action of enterobacteria. It is known to be estrogenic, so exposure to equol could have significant biological effects on humans. Epidemiological studies suggest that it might be beneficial in the prevention of many diseases, including breast cancer, 1) prostate cancer, 2,3) and osteoporosis.4) The production of equol in humans varies: only 30-50% of any population group can produce equol after ingestion of soy foods, and they are called equol producers.4) It is thought that certain bacteria in the intestinal microflora are greatly involved in equol bioconversion. Recently, bacteria that convert daidzein to equol were isolated from human feces (ex. Lactocossus garvieae).5-7) A clinical trial intended to identify soy health benefits is taking place, using S-equol. Ishiwata et al. reported that S-equol supplement improved moodrelated symptoms in premenopausal/postmenopausal equol non-producers. 8) Jackson et al. reported using pure synthetic S-equol for the clinical indications of vasomotor symptoms (VMS, hot flashes) in women and benign prostatic hyperplasia (BPH, enlarged prostate) in men, as well as osteoporosis. 9)The major methods used today for the determination of equol are high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). 10)These methods are useful for basic study that measures multiple molecules, such as equol, daidzein and genistein, and they show excellent specificity. However, these methods require sample extraction and have relatively low throughput, but the measurement of a large number of samples is necessary for epidemiological study to examine the evidence that the clinical efficacy of isoflavones in humans depends on the production of an enterobacterial metabolite, equol. As an alternative method, enzyme immunoassay is the most useful. Brouwers et al. reported a time-resolved fluoroimmunoassay (TR-FIA) method that uses rabbit polyclonal antibodies. 11)Talbot et al. reported successful development of monoclonal antibodies and applied them to a TR-FIA method.12) As a result, a large number of samples have become measurable.Equol, unlike the soy isoflavones daidzein and genistein, has a chiral center, and therefore it can occur as two distinct diastereoisomers, R-equol and S-equol. When equol is chemically synthesized, it is the (Ϯ) equol that is usually obtained. S-Equol has a high affinity for estrogen receptor b (K i ϭ 0.73Ϯ0.2 nmol/l), whereas R-equol is relatively inactive (K i ϭ15.4Ϯ1.3 nmol/l). The exclusive product of human enterobacterial synthesis from soy isoflavones is S-equol. 13)Therefore, a measurement system that can specifically measure S-equol is anticipated in clinical research. However, conventional methods have drawbacks with regard to their specificity for S-equol.The measurement of urine samples is known to be influenced by the matrix effect. The extraction of equol from urine reduces this influence, but it re...

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

“…Therefore, this fusion was suggested as a perspective reagent for the use in bioanalytical systems based on biotin-streptavidin interactions. Although the use of luciferase-streptavidin fusion proteins is economically feasible, most studies describe the preparation and use of noncovalent complexes of biotinylated luciferase and streptavidin (7,36,(42)(43)(44)(45)(46).…”

A Novel Streptavidin–luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA

“…The preliminary experiments pointed to the major limitation of using the firefly luciferase as a label in heterogeneous analysis, namely a high background signal resulting from the nonspecific adsorption of luciferase on a hydrophobic support surface. Therefore, it is necessary to use blocking agents (11)(12)(13)(14)(15)(16)(17). Various BSA-based mixtures are commonly used to block the centers of nonspecific protein binding (11,(14)(15)(16)(17).…”

“…Therefore, it is necessary to use blocking agents (11)(12)(13)(14)(15)(16)(17). Various BSA-based mixtures are commonly used to block the centers of nonspecific protein binding (11,(14)(15)(16)(17). We compared the traditional plate treatment with 1% BSA-casein mixture and the treatment of polystyrene surface with Pluronic F108, which is known to significantly reduce nonspecific adsorption of proteins (44), and the firefly luciferase in particular (45).…”

“…Although the use of luciferase-streptavidin fusion proteins is economically feasible, most studies describe the preparation and use of noncovalent complexes of biotinylated luciferase and streptavidin (11)(12)(13)(14)(15)(16)(17). Apparently, it is difficult to prepare luciferasestreptavidin fusions.…”

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

“…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).…”