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Aim Public speaking seems to be one of the most anxiety-provoking situations for individuals with autism spectrum disorder (ASD). However, there are few evidence-based interventions. We developed Autism-Focused Public Speech Training using Simple Virtual Audiences (APSV), which differs from a general virtual audience in terms of its simple facial expressions and emphasis on the importance of the eyes. The present study aimed to evaluate the feasibility of APSV as an educational method for individuals with ASD. Methods Fifteen male individuals with ASD were randomly assigned to two groups: one group received APSV (n = 8), and the other group (n = 7) received independent study (IS). From Days 2 to 6, participants in the APSV and IS groups were encouraged to read and answer questions often asked in actual public speaking events. Participants in the APSV study group performed this activity in front of the APSV system, while those in the IS group performed in an empty room. Before and after the intervention (Days 1 and 7), the participants in the two groups had a mock public speaking experience in front of 10 people for approximately 10 min. Results After the training sessions, the participants' self-confidence had improved and salivary cortisol levels were significantly decreased in the APSV group as compared to those in the IS group. APSV improved self-confidence and decreased public speaking stress in individuals with ASD. Conclusion APSV appears to be useful in improving self-confidence and decreasing public speaking stress in individuals with ASD.

Discrepancies in efficacy between single-dose and repeated administration of oxytocin for autism spectrum disorder have led researchers to hypothesize that time-course changes in efficacy are induced by repeated administrations of the peptide hormone. However, repeatable, objective, and quantitative measurement of autism spectrum disorder’s core symptoms are lacking, making it difficult to examine potential time-course changes in efficacy. We tested this hypothesis using repeatable, objective, and quantitative measurement of the core symptoms of autism spectrum disorder. We examined videos recorded during semi-structured social interaction administered as the primary outcome in single-site exploratory (n = 18, crossover within-subjects design) and multisite confirmatory (n = 106, parallel-group design), double-blind, placebo-controlled 6-week trials of repeated intranasal administrations of oxytocin (48 IU/day) in adult males with autism spectrum disorder. The main outcomes were statistical representative values of objectively quantified facial expression intensity in a repeatable part of the Autism Diagnostic Observation Schedule: the maximum probability (i.e. mode) and the natural logarithm of mode on the probability density function of neutral facial expression and the natural logarithm of mode on the probability density function of happy expression. Our recent study revealed that increases in these indices characterize autistic facial expression, compared with neurotypical individuals. The current results revealed that oxytocin consistently and significantly decreased the increased natural logarithm of mode on the probability density function of neutral facial expression compared with placebo in exploratory (effect-size, −0.57; 95% CI, −1.27 to 0.13; P = 0.023) and confirmatory trials (−0.41; −0.62 to −0.20; P < 0.001). A significant interaction between time-course (at baseline, 2, 4, 6, and 8 weeks) and the efficacy of oxytocin on the natural logarithm of mode on the probability density function of neutral facial expression was found in confirmatory trial (P < 0.001). Post hoc analyses revealed maximum efficacy at 2 weeks (P < 0.001, Cohen’s d = −0.78; 95% CI, −1.21 to −0.35) and deterioration of efficacy at 4 weeks (P = 0.042, Cohen’s d = −0.46; 95% CI, −0.90 to −0.01) and 6 weeks (P = 0.10, Cohen’s d = −0.35; 95% CI, −0.77 to 0.08), while efficacy was preserved at 2 weeks post-treatment (i.e. 8 weeks) (P < 0.001, Cohen’s d = −1.24; 95% CI, −1.71 to −0.78). Quantitative facial expression analyses successfully verified the positive effects of repeated oxytocin on autistic individuals’ facial expressions and demonstrated a time-course change in efficacy. The current findings support further development of an optimized regimen of oxytocin treatment.

Background Autism spectrum disorder (ASD) is characterized by the core symptoms of impaired social interactions. Increasing evidence suggests that ASD has a strong genetic link with mutations in chromodomain helicase DNA binding protein 8 (CHD8), a gene encoding a chromatin remodeler. It has previously been shown that Chd8 haplodeficient male mice manifest ASD-like behavioral characteristics such as anxiety and altered social behavior. Along with that, oxytocin (OT) is one of the main neuropeptides involved in social behavior. Administration of OT has shown improvement of social behavior in genetic animal models of ASD. The present study was undertaken to further explore behavioral abnormalities of Chd8 haplodeficient mice of both sexes, their link with OT, and possible effects of OT administration. First, we performed a battery of behavioral tests on wild-type and Chd8+/∆SL female and male mice. Next, we measured plasma OT levels and finally studied the effects of intraperitoneal OT injection on observed behavioral deficits. Results We showed general anxiety phenotype in Chd8+/∆SL mice regardless of sex, the depressive phenotype in Chd8+/∆SL female mice only and bidirectional social deficit in female and male mice. We observed decreased level of OT in Chd+/∆SL mice, possibly driven by males. Mice injected by OT demonstrated recovery of social behavior, while reduced anxiety was observed only in male mice. Conclusions Here, we demonstrated that abnormal social behaviors were observed in both male and female Chd8+/∆SL mice. The ability of peripheral OT administration to affect such behaviors along with altered plasma OT levels indicated a possible link between Chd8 + /∆SL and OT in the pathogenesis of ASD as well as the possible usefulness of OT as a therapeutic tool for ASD patients with CHD8 mutations.

Oxytocin (OT) is a neuropeptide hormone. Single and repetitive administration of OT increases social interaction and maternal behaviour in humans and mammals. Recently, it was found that the receptor for advanced glycation end-products (RAGE) is an OT-binding protein and plays a critical role in the uptake of OT to the brain after peripheral OT administration. Here, we address some unanswered questions on RAGE-dependent OT transport. First, we found that, after intranasal OT administration, the OT concentration increased in the extracellular space of the medial prefrontal cortex (mPFC) of wild-type male mice, as measured by push-pull microperfusion. No increase of OT in the mPFC was observed in RAGE knockout male mice. Second, in a reconstituted in vitro blood-brain barrier system, inclusion of the soluble form of RAGE (endogenous secretory RAGE [esRAGE]), an alternative splicing variant, in the luminal (blood) side had no effect on the transport of OT to the abluminal (brain) chamber. Third, OT concentrations in the cerebrospinal fluid after i.p. OT injection were slightly higher in male mice overexpressing esRAGE (esRAGE transgenic) compared to those in wild-type male mice, although this did not reach statistical significance. Although more extensive confirmation is necessary because of the small number of experiments in the present study, the reported data support the hypothesis that RAGE may be involved in the transport of OT to the mPFC from the circulation. These results suggest that the soluble form of RAGE in the plasma does not function as a decoy in vitro.

Autism spectrum disorder (ASD) occurs in 1 in 160 children worldwide. Individuals with ASD tend to be unique in the way that they comprehend themselves and others, as well as in the way that they interact and socialize, which can lead to challenges with social adaptation. There is currently no medication to improve the social deficit of children with ASD, and consequently, behavioral and complementary/alternative intervention plays an important role. In the present pilot study, we focused on the neuroendocrinological response to participatory art activities, which are known to have a positive effect on emotion, self-expression, sociability, and physical wellbeing. We collected saliva from 12 children with ASD and eight typically developed (TD) children before and after a visual art-based participatory art workshop to measure the levels of oxytocin, a neuropeptide involved in a wide range of social behaviors. We demonstrated that the rate of increase in salivary oxytocin following art activities in ASD children was significantly higher than that in TD children. In contrast, the change rate of salivary cortisol after participatory art activities was similar between the two groups. These results suggest that the beneficial effects of participatory art activities may be partially mediated by oxytocin release, and may have therapeutic potential for disorders involving social dysfunction.

Although small-scale studies have described the effects of oxytocin on social deficits in autism spectrum disorder (ASD), no large-scale study has been conducted. In this randomized, parallel-group, multicenter, placebo-controlled, double-blind trial in Japan, 106 ASD individuals (18–48 y.o.) were enrolled between Jan 2015 and March 2016. Participants were randomly assigned to a 6-week intranasal oxytocin (48IU/day, n = 53) or placebo (n = 53) group. One-hundred-three participants were analyzed. Since oxytocin reduced the primary endpoint, Autism Diagnostic Observation Schedule (ADOS) reciprocity, (from 8.5 to 7.7; P

The receptor for advanced glycation end-products (RAGE) binds oxytocin (OT) and transports it from the blood to the brain. As RAGE's OT-binding capacity was lost in RAGE knockout (KO) mice, we predicted that circulating concentrations of unbound (free) OT should be elevated compared to wild-type (WT) mice. However, this hypothesis has not yet been investigated. Unfortunately, the evaluation of the dynamics of circulating free and bound plasma OT is unclear in immunoassays, in part because of interference from plasma proteins. A radioimmunoassay (RIA) is considered the gold standard method for overcoming this issue, but is more challenging to implement; thus, commercially available enzyme-linked immunosorbent assays (ELISAs) are more commonly used. Here, we developed a pre-treatment method to remove the interference-causing components from plasma before performing ELISA. The acetonitrile protein precipitation (PPT) approach was reliable, with fewer steps needed to measure free OT concentrations than by solid-phase extraction of plasma samples. PPT-extracted plasma samples yielded higher concentrations of OT in RAGE KO mice than in WT mice using ELISA. After peripheral OT injection, free OT plasma levels spiked immediately then rapidly declined in WT mice, but remained high in KO mice. These results suggest that plasma samples with PPT pre-treatment appear to be superior and that circulating soluble RAGE can most likely serve as a buffer for plasma OT, which indicates a novel physiological function of RAGE.

The nonapeptide hormone oxytocin (OT) has pivotal brain roles in social recognition and interaction and is thus a promising therapeutic drug for social deficits. Because of its peptide structure, however, OT is rapidly eliminated from the bloodstream, which decreases its potential therapeutic effects in the brain. We found that newly synthesized OT analogues in which the Pro7 of OT was replaced with N-(p-fluorobenzyl)glycine (2) or N-(3-hydroxypropyl)glycine (5) exhibited highly potent binding affinities for OT receptors and Ca2+ mobilization effects by selectively activating OT receptors over vasopressin receptors in HEK cells, where 2 was identified as a superagonist (EMax = 131%) for OT receptors. Furthermore, the two OT analogues had a remarkably long-acting effect, up to 16–24 h, on recovery from impaired social behaviors in two strains of CD38 knockout mice that exhibit autism spectrum disorder-like social behavioral deficits, whereas the effect of OT itself rapidly diminished.

Job interviews are significant barriers for individuals with autism spectrum disorder because these individuals lack good nonverbal communication skills. We developed a job interview training program using an android robot. The job interview training program using an android robot consists the following three stages: (1) tele-operating an android robot and conversing with others through the android robot, (2) a face-to-face mock job interview with the android robot, and (3) feedback based on the mock job interview and nonverbal communication exercises using the android robot. The participants were randomly assigned to the following two groups: one group received a combined intervention with “interview guidance by teachers and job interview training program using an android robot” ( n = 13), and the other group received an intervention with interview guidance by teachers alone ( n = 16). Before and after the intervention, the participants in both groups underwent a mock job interview with a human interviewer, who provided outcome measurements of nonverbal communication, self-confidence, and salivary cortisol. After the training sessions, the participants who received the combined interview guidance by teachers and the job interview training program using an android robot intervention displayed improved nonverbal communication skills and self-confidence and had significantly lower levels of salivary cortisol than the participants who only received interview guidance by teachers. The job interview training program using an android robot improved various measures of job interview skills in individuals with autism spectrum disorder.

金沢大学医薬保健研究域医学系, Oxytocin sets the stage for childbirth by initiating uterine contractions, lactation and maternal bonding behaviours. Mice lacking secreted oxcytocin (Oxt -/-, Cd38 -/-) or its receptor (Oxtr -/-) fail to nurture. Normal maternal behaviour is restored by peripheral oxcytocin replacement in Oxt -/- and Cd38 -/-, but not Oxtr -/- mice, implying that circulating oxcytocin crosses the blood-brain barrier. Exogenous oxcytocin also has behavioural effects in humans. However, circulating polypeptides are typically excluded from the brain. We show that oxcytocin is transported into the brain by receptor for advanced glycation end-products (RAGE) on brain capillary endothelial cells. The increases in oxcytocin in the brain which follow exogenous administration are lost in Ager -/- male mice lacking RAGE, and behaviours characteristic to abnormalities in oxcytocin signalling are recapitulated in Ager -/- mice, including deficits in maternal bonding and hyperactivity. Our findings show that RAGE-mediated transport is critical to the behavioural actions of oxcytocin associated with parenting and social bonding., 30820471

Background: Oxytocin (OT), a neuropeptide, has positive effects on social and emotional processes during group activities. Because cooking is an integrated process in the cognitive, physical, and socio-emotional areas, cooking in a group is reported to improve emotion and cognition. However, evidence for efficacy in group cooking has not been well established at the biological level. Methods: To address this shortcoming, we first measured salivary levels of OT and cortisol (CORT), a biomarker of psychological stress, before and after group cooking for approximately 1 h by people who know each other in healthy married or unmarried men and women. We then compared the initial OT and CORT concentrations with those during individual non-cooking activities in isolation. Results: Baseline OT concentrations before group and non-group sessions did not significantly differ and OT levels increased after both types of activity in men and women. In men, however, the percentage changes of OT levels in the first over the second saliva samples were significantly small during cooking compared with those in individual activities. In women, however, such a difference was not observed. In contrast, the mean salivary CORT concentrations after group cooking were significantly decreased from the baseline level in both sexes, though such decreases were not significant after individual activity sessions. The sex-specific differences were marital-status independent. Conclusion: These results indicate that OT and CORT concentrations after two activity sessions by a familiar group changed in opposite directions in a sex-specific manner. This suggests that, because cooking is experience-based, we need to consider the sex-specific features of group cooking if we apply it for intervention.

金沢大学子どものこころの発達研究センター, Many emotionally-disturbed children who have been maltreated and are legally separated from their parents or primary caregivers live in group homes and receive compulsory education. Such institutions provide various special intervention programs. Taiko-ensou, a Japanese style of group drumming, is one such program because playing drums in a group may improve children’s emotional well-being. However, evidence for its efficacy has not been well established at the biological level. In this study, we measured salivary levels of oxytocin (OT), a neuropeptide associated with social memory and communication, in three conditions (recital, practice, and free sessions) in four classes of school-aged children. Following the sessions, OT concentrations showed changes in various degrees and directions (no change, increases, or decreases). The mean OT concentration changes after each session increased, ranging from 112% to 165%. Plasma OT concentrations were equally sensitive to drum playing in school-aged boys and girls. However, the difference between practice and free play sessions was only significant among elementary school boys aged 8–12 years. The results suggest that younger boys are most responsive to this type of educational music intervention.

Oxytocin (OT) is a critical molecule for social recognition that mediates social and emotional behaviors. OT is released during stress and acts as an anxiolytic factor. To know the precise molecular mechanisms underlying OT release into the brain during stress is important. It has been reported that intracellular concentrations of free calcium in the hypothalamic neurons are elevated by simultaneous stimulation of cyclic ADP-ribose (cADPR) and heat. We have reported in vitro and in vivo data that supports the idea that release of OT in the brain of male mice is regulated by cADPR and fever in relation to stress conditions. 1) Significantly higher levels of OT release were observed in hypothalamus cultures isolated from subordinate mice in group-housed males compared to dominant males after cage-switch stress; 2) OT concentrations in micro-perfusates at the paraventricular nucleus upon perfusion stimulation with cADPR were enhanced in subordinate mice compared to dominant mice; 3) The OT concentration in the cerebrospinal fluid (CSF) was higher in endotoxin-shock mice with fever compared to controls with no body temperature increase; and 4) In mice exposed to new environmental stress, the CSF OT level transiently increased 5 min after exposure, while the rectal temperature increased from 36.6 °C to 37.8 °C from 5 to 15 min after exposure. In this review, we examine whether or not cADPR and hyperthermia co-regulate hypothalamic OT secretion during social stress through the elevation of intracellular free Ca2+ concentrations involved in CD38-dependent Ca2+ mobilization and TRPM2-dependent Ca2+ influx. Finally, we propose that the interaction between CD38 and TRPM2 seems to be a new mechanism for stress-induced release of OT, which may result in anxiolytic effects for temporal recovery from social impairments in children with autism spectrum disorder during hyperthermia.

Hypothalamic oxytocin (OT) is released into the brain by cyclic ADP-ribose (cADPR) with or without depolarizing stimulation. Previously, we showed that the intracellular free calcium concentration ([Ca2+]i) that seems to trigger OT release can be elevated by β-NAD+, cADPR, and ADP in mouse oxytocinergic neurons. As these β-NAD+ metabolites activate warm-sensitive TRPM2 cation channels, when the incubation temperature is increased, the [Ca2+]i in hypothalamic neurons is elevated. However, it has not been determined whether OT release is facilitated by heat in vitro or hyperthermia in vivo in combination with cADPR. Furthermore, it has not been examined whether CD38 and TRPM2 exert their functions on OT release during stress or stress-induced hyperthermia in relation to the anxiolytic roles and social behaviors of OT under stress conditions. Here, we report that OT release from the isolated hypothalami of male mice in culture was enhanced by extracellular application of cADPR or increasing the incubation temperature from 35°C to 38.5°C, and simultaneous stimulation showed a greater effect. This release was inhibited by a cADPR-dependent ryanodine receptor inhibitor and a nonspecific TRPM2 inhibitor. The facilitated release by heat and cADPR was suppressed in the hypothalamus isolated from CD38 knockout mice and CD38-or TRPM2-knockdown mice. In the course of these experiments, we noted that OT release differed markedly between individual mice under stress with group housing. That is, when male mice received cage-switch stress and eliminated due to their social subclass, significantly higher levels of OT release were found in subordinates compared with ordinates. In mice exposed to anxiety stress in an open field, the cerebrospinal fluid (CSF) OT level increased transiently at 5 min after exposure, and the rectal temperature also increased from 36.6°C to 37.8°C. OT levels in the CSF of mice with lipopolysaccharide-induced fever (+0.8°C) were higher than those of control mice. The TRPM2 mRNA levels and immunoreactivities increased in the subordinate group with cage-switch stress. These results showed that cADPR/CD38 and heat/TRPM2 are co-regulators of OT secretion and suggested that CD38 and TRPM2 are potential therapeutic targets for OT release in psychiatric diseases caused by social stress. © 2016 Zhong, Amina, Liang, Akther, Yuhi, Nishimura, Tsuji, Tsuji, Liu, Hashii, Furuhara, Yokoyama, Yamamoto, Okamoto, Zhao, Lee, Tominaga, Lopatina and Higashida.

A sensitive immunosensor for the detection of pregnancy marker, human chorionic gonadotropin hormone (hCG), was developed using the direct electrical detection of Au nanoparticles. We utilized disposable screen-printed carbon strips (SPCSs) for the development of our immunosensor, which provided cost-effective tests with the required antigen sample volume as small as 2 μL. After the recognition reaction between the surface-immobilized primary antibody and hCG, the captured antigen was sandwiched with a secondary antibody that was labeled with Au nanoparticles. Au nanoparticles were exposed to a preoxidation process at 1.2 V for 40 s, which was subsequently followed with a reduction scan on the same surface using differential pulse voltammetry (DPV). We could observe Au nanoparticle-labeled antigen-antibody complexes immobilized on the surface of SPCS using scanning electron microscopy (SEM). Additionally, the number of Au nanoparticles on the immunosensor was determined using SEM images, and showed a linear relationship with the current intensity obtained from the DPV measurements with a detection limit of 36 pg/mL hCG (612 fM, 3.6×10−4 IU/mL). Our immunosensor system, a combination of the screen-printing technology with Au nanoparticles provides a promising biosensor for various applications in life sciences.

The neurotoxic property of organophosphate pesticides due to the inhibition of acetylcholinesterase (AChE) was utilized to develop disposable detection chips for easy and fast detection of the pesticides. An enzymatic assay involving acetylcholinesterase (AChE) was developed using a stable substrate specific to the enzymatic reaction product, thiocholine. The generated thiocholine reacted with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) to produce 5′-mercapto-2′-nitrobenzoic acid, which was measured at 410 nm. Optimum pH, buffer types and concentrations, substrate concentrations and optimum conditions of the color reaction were investigated. The substrate specificity, test interferences were evaluated. Our simple detection system does not require any expensive instruments. Diazinon-oxon (DZN-oxon) could be detected by a visual color change from white to yellow on the surface of the chip. The total amount of time required for the detection of residual DZN-oxon in apple and orange juices was about an hour. This method provides excellent specificity, reproducibility, a wide measurement range and minimal interference from endogenous substances in juice matrices. Since the reagents are stable after preparation, our method would be useful for routine pesticide screening by non-professional end-users.

Seven male children with autism spectrum disorder (ASD), aged 8-12 years, attending special education classrooms for ASD and disabled children, were assigned to receive touch therapy. Their mothers were instructed to provide gentle touch in the massage style of the International Liddle Kidz Association. The mothers gave massages to their child for 20 min every day over a period of 3 months, followed by no massage for 4 months. To assess the biological effects of such touch therapy, saliva was collected before and 20 min after a single session of massage for 20 min from the children and mothers every 3 weeks during the massage period and every 4 weeks during the non-massage period, when they visited a community meeting room. Salivary oxytocin levels were measured using an enzyme immunoassay kit. During the period of massage therapy, the children and mothers exhibited higher oxytocin concentrations compared to those during the non-massage period. The changes in oxytocin levels before and after a single massage session were not significantly changed in children and mothers. The results suggested that the ASD children (massage receivers) and their mothers (massage givers) show touch therapy-dependent changes in salivary oxytocin concentrations. © 2015 Tsuji, Yuhi, Furuhara, Ohta, Shimizu and Higashida.

In prostate cancer screening, prostate-specific antigen (PSA) has been utilized as a valuable biomarker. There are routinely used procedures based on enzyme-linked immunosorbent assay (ELISA) for PSA detection. The procedures based on ELISA, however, are time consuming, complicated, and costly. We have developed a rapid, very simple, cost effective and sensitive immunochromatographic assay using gold nanoparticles and evaluated its applications for first screening of prostate cancer in serum samples. The sensitive immunochromatographic assay requires only 40 μL of the serum sample. The assay used is rapid and simple, that it totally takes approx 15 min to complete. The method for sensitive immunochromatographic assay has the other advantage of decreasing the antibody concentration that is used for the test line. In this study, we show the advantage to decrease the antibody concentration and the evaluation of our sensitive immunochromatographic assay for the semiquantitative detection of PSA in serum. The results obtained from 163 serum samples using sensitive immunochromatographic assay are compared with the results obtained using the chemiluminescent enzyme immunoassay (CLEIA) and normal immunochromatographic assay. The results obtained in the sensitive immunochromatographic assay correlated well with the values obtained in CLEIA. We concluded that our sensitive immunochromatographic assay is applicable to the first screening test for the diagnosis of prostate cancer. Our developed sensitive immunochromatographic assay is a promising candidate for diagnosis or research use, which may become commercially available in the near future.

The immunochromatographic assay is a well-known and convenient diagnostic system. In this report, the development of a novel enhancement assay for the test strips is described. Additionally, this highly sensitive immunochromatographic assay was applied to detect human chorionic gonadotropin hormone (HCG) as the model case. The primary antibody-conjugated gold nanoparticles were used as the enhancer of the standard method. The primary antibodies were immobilized within a defined detection zone (test line) on the diagnostic nitrocellulose membrane. The secondary antibodies were conjugated with colloidal gold nanoparticles. In combination with an effective sample pretreatment, the gold-conjugated antibodies and the primary antibodies formed a sandwich complex with the target protein. Within the test line, the sandwich complex was immobilized, and furthermore, concentrated by the enhancer resulting in a localized surface plasmon resonance (LSPR) phenomenon and a distinct red color on the test line. The intensity of color of the red test line (signal intensity), which correlated directly with the concentration of the target protein in the standard or spiked samples, was assessed visually and by computer image analysis using a three-determination analysis. Under optimum conditions, the limit of detection (LOD) for HCG assay was 1 pg/mL. When using human serum, 10 pg/mL of HCG could be detected. We have also spiked total prostate-specific antigen (TPSA) in female serum. The LOD for TPSA was determined as 0.2 ng/mL. With this method, the quantitative determination of the target protein could be completed in less than 15 min. Our novel immunochromatographic strips using the enhancing method based on LSPR of gold nanoparticles are useful as a rapid and simple screening method for the detection of important analytes for medical applications, environmental monitoring, food control, and biosecurity.

A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip®. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect low concentrations of target antigens in urine samples. In addition, the washing procedure could also be performed easily to reduce the background signal levels. For analytical evaluations, the color intensity was captured by a flatbed scanner, and processed by a software. We have achieved the detection of human chorionic gonadotropin (hCG) and prostate-specific antigen (PSA). The detection limit of hCG was 0.5 ng/ml (0.05 ng/tip), which is comparable to that of other conventional immunochromatographic systems. Moreover, the detection of PSA was greatly improved over the existing systems with the application of different sample volumes, such as 1 ng/ml (0.2 ng/tip) in a 200 μl sample volume, and 1 ng/ml (0.3 ng/tip) in 300 μl sample volume. Our bioanalysis system is a promising candidate for application to point-of-care tests with its simple handling and high sensitivity.

A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin-modified micropipette tip, namely as ZipTip®. The sandwich-type immunoassay was applied to our bioananalysis system. The first antibodies that were aspirated by the micropipette were immobilized on the immunochromatographic resin at the edge of micropipette tip. The blocking solution was also aspirated in the same fashion. The measurement operation was performed by aspirating the sample solution, and then the gold colloidal nanoparticles (Au naps) conjugated secondary antibody solution. Since this bioanalysis system utilizes a micropipette, it is possible to increase the sample volume, which would enable the detection of antigen at low concentrations. In addition, the washing procedure can also be performed easily to reduce the background level. After the antigen-antibody reaction, the color intensity of Au naps could be observed by the naked eye. For analytical evaluation, the color intensity was captured by a scanner, and processed by analysis software. We have achieved the detection of human chorionic gonadotropin hormone (hCG) and total prostate-specific antigen (TPSA), which are well-known as fundamental indicators of pregnancy and cancer. The limit of detection (LOD) for hCG was 8 ng/ml (0.8 ng/tip), which is comparable to that of other conventional systems based on immunochromatography. Moreover, the LOD for TPSA was improved over the existing systems with the application of different sample volumes, such as 5 ng/ml (1 ng/tip) in 200 μl sample volume, and 2 ng/ml (0.6 ng/tip) in 300 μl sample volume. Since our bioanalysis system requires a small amount of immobilized antigen, it would be greatly useful in basic research for screening the antigen–antibody reactions. Besides, our bioanalysis system can be applied to on-field screening, since its operation is simple, and the visual results can be obtained rapidly.

The immunochromatographic assay that is in widespread use for pregnancy diagnosis is a method for easy visual judging of the antigen–antibody reaction using gold nanoparticle. The rapid observation...

The concentration of salivary secretory immunoglobulin A (sIgA) is a well-known stress marker for humans. The concentration of salivary sIgA in dogs has also been reported as a useful stress marker. In addition, salivary sIgA in dogs has been used to determine the adaptive ability of dogs for further training. There are conventional procedures based on enzyme-linked immunosorbent assay (ELISA) for measuring salivary sIgA in dogs. However, ELISA requires long assay time, complicated operations and is costly. In the present study, we developed an immunochromatographic assay for measuring salivary sIgA in dogs using a dilution buffer containing a non-ionic surfactant. We determined 2500-fold dilution as the optimum condition for dog saliva using a phosphate buffer (50 mM, pH 7.2) containing non-ionic surfactant (3 wt% Tween 20). The results obtained from the saliva samples of three dogs using immunochromatographic assay were compared with those obtained from ELISA. It was found that the immunochromatographic assay is applicable to judge the change in salivary sIgA in each dog. The immunochromatographic assay for salivary sIgA in dogs is a promising tool, which should soon become commercially available for predicting a dog's psychological condition and estimating adaptive ability for training as guide or police dogs.

The concentration of salivary secretory immunoglobulin A (sIgA) is a well-known stress marker for humans. The concentration of salivary sIgA in dogs has also been reported as a useful stress marker. In addition, salivary sIgA in dogs has been used to determine the adaptive ability of dogs for further training. There are conventional procedures based on enzyme-linked immunosorbent assay (ELISA) for measuring salivary sIgA in dogs. However, ELISA requires long assay time, complicated operations and is costly. In the present study, we developed an immunochromatographic assay for measuring salivary sIgA in dogs using a dilution buffer containing a non-ionic surfactant. We determined 2500-fold dilution as the optimum condition for dog saliva using a phosphate buffer (50 mM, pH 7.2) containing non-ionic surfactant (3 wt% Tween 20). The results obtained from the saliva samples of three dogs using immunochromatographic assay were compared with those obtained from ELISA. It was found that the immunochromatographic assay is applicable to judge the change in salivary sIgA in each dog. The immunochromatographic assay for salivary sIgA in dogs is a promising tool, which should soon become commercially available for predicting a dog's psychological condition and estimating adaptive ability for training as guide or police dogs.

not Available.