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Figure 2. Correlation between the urinary equol concentrations measured employing the HPLC method and the ELISA method using different combinations of (R,S)-equol and S-equol for the standard and enzyme-labeled antigen.

Table 2. Cross-reactivity of the equol ELISA compared between Methods 2 and 5.

collected. The standard curve showed a favorable line within a range of 36.3 - 8910 ng/ml (Figure 3). On precision profile analysis the concentration at which the coefficient of variation (CV) was lower than 10% was 330 ng/ml (data not shown), based on which the measurement range was set at 330 - 8910 ng/ml. When 1 volume of equol at a known concentration was added to 9 volumes of urine, the recovery rate of equol was 71.1- 121.9% (Table 3). The CVs of the intraand inter-assay reproducibility of human urinary equol (537 - 4008 ng/ml) measurement was 6.2% - 8.1% (n = 8) and 3.5% - 4.0% (n = 12), respectively (Table 4).

The time-course change in urinary equol concentration after isoflavone ingestion was measured as a clinical application model (Figure 4). The urinary equol concentration reached its first peak of 790 ng/ml at 12.5 hours after isoflavone ingestion and then declined to below the detection limit at 14 hours. The concentration increased again, to 475 ng/ml at 16 hours, then reached its second peak of 729 ng/ml at 30.5 hours, and declined below the detection limit at 37 hours. The appearance of two peaks in the urinary equol level may have been due to enterohepatic circulation [19].

4. Conclusion

In this study we developed S-equol diastereomer specific ELISA (Method 5) by selecting S-equol for both the standard and the antigen labeled enzyme, which was improved from another novel invention of the simple ELISA (Method 1). Since the simple ELISA (Method 1) can measure equol without the deconjugation process, measuring equol in urine sample became more convenient, and its application for other methods such as immunochromatography is expected.

Since the specific ELISA (Method 5) not only shows a low cross reaction to R-equol and daidzein but also handles a large number of samples within a short period of time, the laboratory possessing only general equipments are able to carry out the studies on equol without special apparatus used for HPLC and LC/MS/MS. The utilizetion of this method in broad fields including epidemiological and biokinetic studies is expected.

We prepared antiserum with low stereospecificity, instead of S-equol-specific antibody by immuni-zation with

Figure 3. Standard curve of S-equol measured by the S-equol specific ELISA (n = 16, mean ± 2.6 SD).

Table 3. Recovery test using urine samples in the S-equol specific ELISA.

Table 4. Reproducibility in the S-equol Specific ELISA.

Figure 4. Time-course changes in the urinary equol concentration. The urinary equol concentration was measured before and after the ingestion intake of a soybean bar containing 19 mg of isoflavone, regarding the time of ingestion as 0 hour.

(R,S)-equol, and developed an S-equol-specific immunoassay system by a combination of S-equol HRP conjugate and this low stereospecific antiserum. This system is expected to be applied for a simple stereospecific immunoassay in which specific antibody preparation is difficult.

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