An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10−19 mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

To minimize patient suffering, the smallest possible volume of blood should be collected for diagnosis and disease monitoring. When estimating insulin secretion capacity and resistance to insulin in diabetes mellitus (DM), increasing insulin assay immunosensitivity would reduce the blood sample volume required for testing. Here we present an ultrasensitive ELISA coupled with thio-NAD cycling to measure immunoreactive insulin in blood serum. Only 5 μL of serum was required for testing, with a limit of detection (LOD) for the assay of 10(-16) moles/assay. Additional recovery tests confirmed this method can detect insulin in sera. Comparisons between a commercially available immunoreactive insulin kit and our ultrasensitive ELISA using the same commercially available reference demonstrated good data correlation, providing further evidence of assay accuracy. Together, these results demonstrate our ultrasensitive ELISA could be a powerful tool in the diagnosis and treatment of not only DM but also many other diseases in the future.

Although participatory workplace improvement programs are known to provide favorable effects on high stress occupations like nursing, no studies have confirmed its effect using biomarkers. The aim of this study was to determine whether a participatory workplace improvement program would decrease stress-related symptoms as evaluated by biomarkers and self-reported stress among hospital nurses. Three actions to alleviate job stress, which were determined through focus group interviews and voting, were undertaken for two months. A total of 31 female Japanese nurses underwent measurement of inflammatory markers, autonomic nervous activity (ANA), and perceived job stress (PJS) at three-time points; before the program (T1), within a week after the completion of the program (T2), and three months after the program (T3). A series of inflammatory markers (Interferon-γ, Interleukin (IL)-6, and IL-12/23p40) decreased significantly at T2, and IL-12/23p40 and IL-15 significantly decreased at T3 compared to T1, while ANA and PJS remained unchanged. Our participatory program exerted beneficial effects in reducing inflammatory responses, but not for ANA and PJS. Further investigations with a better study design, i.e., a randomized controlled trial, and a larger sample size are warranted to determine what exerted beneficial effects on inflammatory markers and why other outcomes remained unchanged.

Many female nurses leave their jobs because of major life events. However, the mental health status and related factors among nurses who assume the roles of wives and/or mothers have been insufficiently examined. Therefore we examined the mental health levels and related factors among such nurse. We conducted a questionnaire survey on 763 female nurses working at general hospitals with over 200 beds in Fukuoka Prefecture. Of 402 responses, 108 were divided into two groups: nurses who had left because of marriage, childbirth, or childrearing (leaving group), and those who had not (non-leaving group). The following were assessed: work satisfaction level, the Brief Job Stress Questionnaire, The General Health Questionnaire (GHQ) 28, and the Sense of Coherence (SOC) scale. Results showed that nurses who had assumed the roles of wives and/or mothers had lower mental health status than general women, and nurses who retained their jobs had higher mental health status and sense of comprehensibility on the SOC scale than those who left. Multiple regression analyses using the total GHQ score as an objective variable showed that only the sense of comprehensibility on the SOC scale correlated with mental health status in the non-leaving group. For the leaving group, having support, high work and life satisfaction levels, and several work stressors were correlated. These findings strongly suggest that to maintain and improve the mental health of nurses who assume the role of wives and/or mothers, greater support, higher satisfaction, reduced stressors, and maintenance and improvement of the sense of comprehensibility are required.