Common Salivary Protein 1 in Saliva of Diabetes Patients (II).

BACKGROUND: Human common salivary protein 1 (CSP1) is one of a variety of molecules in saliva but its function remains to be determined. The gold standard method for diagnosis of diabetes mellitus (DM) is to check levels of glucose or HbA1C in plasma or serum. The purpose of this study was to examine whether Salivary CSP1 concentration would be useful alternative for DM diagnosis. METHODS: The qualities of monoclonal antibodies (mAbs) to recombinant human CSP1 (rhCSP1) were tested by western blotting (WB) and immunohistochemistry. A sandwich ELISA was fabricated with the qualified capture and detector mAbs for measurement of CSP1 level in saliva. CSP1 levels of healthy adults and DM patients were measured by the sandwich ELISA and their results were statistically analyzed by Student's t-test. The receiver operating characteristic (ROC) curve was constructed and the area under the curve (AUC) was calculated. RESULTS: The tested mAbs recognized a 27-kDa CSP1 of saliva in WB and stained only a salivary gland in immunohistochemistry. Pearson's correlation coefficient with standard curve between OD450nm value vs. CSP level showed good linearity (r2 = 0.995). The median values (25th to 75th percentiles) of saliva CSP1 in 10 healthy adults and 18 DM patients using the sandwich ELISA were 3.92 µg/mL (3.15 - 4.02) and 4.35 µg/mL (3.94 - 5.11), respectively. Statistically, there was a significant difference of CSP1 level in two groups (p = 0.026). The sensitivity value of CSP1 was 64.71 while the specificity value was 88.89 with 0.784 of AUC (p = 0.003). These results suggested that the fabricated sandwich ELISA was a good diagnostic test tool for discriminating DM patients from healthy individuals. CONCLUSIONS: The present data showed a significant increase of CSP1 levels for DM patients compared with control group, indicating that CSP1 level in saliva could be used as a potential biomarker of detection or screening of DM patients. However, further studies are necessary to provide scientific and clinical validation.

Effects of virtual reality-based planar motion exercises on upper extremity function, range of motion, and health-related quality of life: a multicenter, single-blinded, randomized, controlled pilot study.

BACKGROUND: Virtual reality (VR)-based rehabilitation is considered a beneficial therapeutic option for stroke rehabilitation. This pilot study assessed the clinical feasibility of a newly developed VR-based planar motion exercise apparatus (Rapael Smart Board™ [SB]; Neofect Inc., Yong-in, Korea) for the upper extremities as an intervention and assessment tool. METHODS: This single-blinded, randomized, controlled trial included 26 stroke survivors. Patients were randomized to the intervention group (SB group) or control (CON) group. During one session, patients in the SB group completed 30 min of intervention using the SB and an additional 30 min of standard occupational therapy; however, those in the CON group completed the same amount of conventional occupational therapy. The primary outcome was the change in the Fugl-Meyer assessment (FMA) score, and the secondary outcomes were changes in the Wolf motor function test (WMFT) score, active range of motion (AROM) of the proximal upper extremities, modified Barthel index (MBI), and Stroke Impact Scale (SIS) score. A within-group analysis was performed using the Wilcoxon signed-rank test, and a between-group analysis was performed using a repeated measures analysis of covariance. Additionally, correlations between SB assessment data and clinical scale scores were analyzed by repeated measures correlation. Assessments were performed three times (baseline, immediately after intervention, and 1 month after intervention). RESULTS: All functional outcome measures (FMA, WMFT, and MBI) showed significant improvements (p < 0.05) in the SB and CON groups. AROM showed greater improvements in the SB group, especially regarding shoulder abduction and internal rotation. There was a significant effect of time × group interactions for the SIS overall score (p = 0.038). Some parameters of the SB assessment, such as the explored area ratio, mean reaching distance, and smoothness, were significantly associated with clinical upper limb functional measurements with moderate correlation coefficients. CONCLUSIONS: The SB was available for improving upper limb function and health-related quality of life and useful for assessing upper limb ability in stroke survivors. TRIAL REGISTRATION: The study was registered with the clinical research information service (CRIS) ( KCT0003783 , registered 15 April 2019; retrospectively registered).

Common Salivary Protein 1 in Serum of Diabetes Patients.

BACKGROUND: Recently, the human common salivary protein 1 (CSP1) was identified as an ortholog of the Demilune cell and parotid protein of mouse. However, its function remains to be determined. Here, we show that the serum CSP1 concentration of diabetes mellitus (DM) patients is much higher than that of healthy controls. METHODS: Recombinant human CSP1 was expressed as a Glutathione-S-transferase (GST)-tagged protein, and the purified fusion protein was used as an immunogen to generate monoclonal antibody (mAb) to CSP1. The produced mAb was tested as a probe in Western blotting of human saliva and in immunohistochemistry of various human tissues. The serum CSP1 levels of 31 DM patients and 38 normal adults were quantified by a house-fabricated CSP1 sandwich enzyme-linked immunosorbent assay (ELISA) system. RESULTS: Immunoblot analysis by mAb-hCSP1#4 showed that CSP1 in human saliva exists in a 27 kDa glycosylated form. Among the various human tissues tested, the salivary gland was the only tissue stained with mAb-hCSP1#4 by immunohistochemistry. Quantification of serum CSP1 concentration by CSP1 ELISA showed that the median values (25th-75th percentile) of DM patients and healthy adults were 22.2 (15.8-28.2) and 3.2 (0-11.4), respectively. Student's t-test results indicated that there was a statistically significant difference between the two groups (P < 0.01). CONCLUSION: The significant difference between the CSP1 levels of the two groups indicated that CSP1 would be a potential biomarker for detection or screening of DM patients.

Fluorescence immunoassay of human D-dimer in whole blood.

BACKGROUND: D-dimer is a widely used biomarker for the initial clinical assessment of suspected deep vein thrombosis and pulmonary embolism. Here, we presented a new fluorescence (FL) D-dimer assay system, which was developed with a platform of point-of-care test (POCT) for clinical applications. METHODS: Whole blood was mixed with FL-labeled anti-D-dimer detector antibody, and then loaded onto a disposable cartridge. After 12 min of incubation, the FL intensity was acquired by scanning of test cartridge and converted as level of D-dimer in a laser FL scanner. The analytical performance of FL immunoassay was evaluated by linearity, recovery, and precision tests. The comparability of the developed assay was examined with automated reference methods. RESULTS: The FL assay system showed a good linearity, and the analytical mean recovery of control was 103% in a dynamic working range. The imprecision of intra- and inter-as-say of coefficient of variations from assay system was less than 8%. The developed FL assay system showed strong correlation with two automated reference assays, Vidas D-dimer (r = 0.973) and Stalia D-dimer (r = 0.971). CONCLUSION: The new FL immunoassay for D-dimer is a user-friendly, precise, and reproducible platform of POCT in whole blood.

Point-of-care fluorescence immunoassay for cardiac panel biomarkers.

BACKGROUND: Myoglobin, creatine kinase-MB isoenzyme (CK-MB), and cardiac troponin I (cTnI) are cardiac biomarkers that are widely used to assist in the early and late diagnoses of acute myocardial infarction (AMI). Here, we present a clinically applicable fluorescence (FL) immunoassay for cardiac biomarkers. METHODS: Whole blood was mixed with FL-labeled detector Ab (antibody) and then loaded onto a capture Ab-immobilized strip in a test cartridge. The FL intensities at test and control line on the strip were obtained and converted in a laser FL scanner to determine the concentration of biomarker. The analytical performance of immunoassay system was evaluated by linearity and imprecision tests. The comparability of the FL immunoassay method was examined with a reference method. RESULTS: FL intensities and the levels of myoglobin, CK-MB, and cTnI displayed good linearity and high correlations (r = 0.999, 0.998, and 0.989, respectively). The coefficient of variations (CVs) of imprecision for all cardiac biomarkers were less than 8% in both intra- and interassays. When the results from the developed method and bioMerieux VIDAS assay were analyzed by Bland-Altman plot and Passing-Bablok plot, the two assay methods were in good agreement. CONCLUSION: The FL immunoassay system can provide a platform for point-of-care testing (POCT), and it is an easy, fast, and reliable method for the quantification of cardiac biomarkers.

Comparing the Efficacy of Combined Treatment with Medial Branch Block and Facet Joint Injection in Axial Low Back Pain.

Introduction: Medial branch nerve block (MBB) and facet joint injections (FJIs) can be used to manage axial low back pain. Although there have been studies comparing the MBB and FJI effects, a few studies have compared the therapeutic effects of both interventions combined with each separate intervention. This study aimed to compare the pain relief effect of MBB, FJI, and combined treatment with MBB and FJI in patients with axial low back pain. Methods: We conducted a retrospective review of patients with axial low back pain who had chart records of the Numeric Rating Scale (NRS) and Oswestry Disability Index (ODI) scores measured before treatment and within 6 weeks after treatment. The proportion of patients with successful responses (>30%) was calculated and is presented with Wald confidence intervals. Results: We included 66 patients (33, 17, and 16 patients in the MBB, FJI, and combined treatment with MBB and FJI groups). All the patient groups showed significant posttreatment improvements in the NRS [(proportion >30% decrease: MBB 24.2% (9.6-38.9), FJI 29.4% (7.8-51.1), and MBB + FJI 25.0% (3.8-46.2)] scores and the ODI [proportion >30% decrease: MBB 39.4% (22.7-56.1), FJI 23.5% (3.4-43.7), and MBB + FJI 37.5% (13.8-61.2)] scores. Furthermore, there was no significant among-group difference in the ODI and NRS scores. Conclusion: MBB, FJI, and combined treatment with MBB and FJI can reduce axial low back pain and improve secondary functional degradation. Although combined treatment with MBB and FJI required a longer intervention time, it did not have a pain relief effect superior to that of MBB or FJI alone.

Production and Characterization of Monoclonal Antibodies Against Primo Vascular System of Rat.

BACKGROUND: The primo vascular system (PVS) has been difficult to detect due to its small diameter and translucent features of the threadlike network. Thus, contrast-enhancing dyes including Alcian blue, Trypan blue and Janus green B had to be used for finding and taking out PVS from rat and mouse. OBJECTIVE: Generation of monoclonal antibodies (mAbs) against PVS of rat was intended to use as a detector for PVS and a biological tool for functional study of PVS. MATERIALS AND METHODS: Primo vessel (PV) and Primo node (PN) were isolated from organ surfaces of rat and then their proteins were isolated and injected into mouse as an immunogen. The classical traditional method was applied for production of mAbs against PVS. The various techniques, such as cell fusion, screening of hybridoma, ELISA, Western blotting (WB), immunofluorescence microscopy (IF), and limiting dilution, were used to generate mAbs against PVS. RESULTS: Among 16 mAbs generated, 4 representative mAbs were characterized with their specificities in ELISA, WB, and IF. α-rPVS-m1-1 and α-rPVS-m4-6 had strong binding affinities to PVS in both ELISA and WB but did not show specificities in IF at all. On the contrary, α-rPVS-m3-2 and α-rPVS-m3-4 almost did not respond in WB but had strong binding affinities in ELISA and specificities in IF. Two mAbs stained predominantly at extra cellular matrix and cell membrane of PVS of rat in IF, and they were able to discriminate PVS from blood vessel (BV) and lymphatic vessel (LV). CONCLUSIONS: 4 representative mAbs against PVS of rat were characterized by ELISA, WB, and IF. α-rPVS-m3-2 and α-rPVS-m3-4, which had strong specificities in IF, can be used as a tool in discriminating PVS from other similar tissues and in elucidate biological function of PVS.

Korean Version of the Stroke Rehabilitation Motivation Scale: Reliability and Validity Evaluation.

OBJECTIVE: To translate the Stroke Rehabilitation Motivation Scale (SRMS), developed to evaluate the motivation level of stroke patients during rehabilitation, into the Korean language and to verify the reliability and validity of the Korean version of SRMS (K-SRMS). METHODS: The K-SRMS was developed following a structured process that included translation, verification, compromise assessment, reverse translation, feedback, and final correction. K-SRMS reliability was evaluated by performing internal consistency and test-retest analyses. The reliability test was conducted in 50 stroke patients. Its validity was assessed by comparing the K-SRMS with the scale and performing exploratory factor analysis. The validity test was conducted in 102 stroke patients. RESULTS: The test-retest analysis showed good reliability, and the internal consistency of the K-SRMS was similar to that of the original version for all, except 4, items. Thus, these 4 items were excluded, and then the validity test was conducted. Pearson correlation analysis demonstrated that the K-SRMS score was significantly correlated with the BAS total score (Pearson r=0.207, p<0.05). In the exploratory factor analysis, K-SRMS items were categorized into 7 groups (factors), and factors 1 and 4 showed mutual concordance with K-SRMS subscales, including intrinsic motivation factors and amotivation, respectively. CONCLUSION: The newly developed K-SRMS showed good reliability and validity. It could also be used as a tool to objectify the degree of motivation for rehabilitation among stroke patients in clinical care and research.

Changes in the Trend in Bladder Emptying Methods in Patients With Spinal Cord Injury: A 20-Year Single-Center Retrospective Study.

OBJECTIVE: To review trends in bladder emptying methods over a 20-year period in patients with spinal cord injury (SCI) by severity according to the American Spinal Injury Association impairment scale (AIS). METHODS: Medical records of patients with SCI from 1994 to 1998 (group 1) and from 2012 to 2016 (group 2) were retrospectively reviewed. We classified bladder emptying methods according to the International Spinal Cord dataset. We grouped patients with normal voiding, bladder reflex triggering, and bladder expression as those using voiding without catheter. RESULTS: A total of 667 patients were included in the analysis. The proportion of patients using voiding without catheter and intermittent catheterization decreased from 67.0% to 30.0% and increased from 26.8% to 54.8%, respectively. In patients with AIS-A and AIS-B, the proportion of patients with intermittent catheterization increased from 32.8% to 73.3%. In patients with AIS-D, the proportion of patients using voiding without catheter and intermittent catheterization decreased from 88.5% to 68.9% and increased from 11.5% to 26.8%, respectively. In group 2, among 111 patients with AIS-D using voiding without catheter at admission, 8 (7.2%) switched to intermittent catheterization at discharge due to decreased bladder volume, increased post-voiding residual urine, or incontinence. CONCLUSION: Over the past 20 years, trends in bladder emptying methods in patients with SCI changed from voiding without catheter to intermittent catheterization in Korea. This was especially prominent in patients with AIS-A, AIS-B, and AIS-C. Even in patients with AIS-D, the use of intermittent catheterization at hospital discharge increased.

Energy Efficiency and Patient Satisfaction of Gait With Knee-Ankle-Foot Orthosis and Robot (ReWalk)-Assisted Gait in Patients With Spinal Cord Injury.

OBJECTIVE: To compare the energy efficiency of gait with knee-ankle-foot orthosis (KAFO) and robot-assisted gait and to develop a usability questionnaire to evaluate the satisfaction of walking devices in paraplegic patients with spinal cord injuries. METHODS: Thirteen patients with complete paraplegia participated and 10 completed the evaluation. They were trained to walk with KAFO (KAFO-gait) or a ReWalk robot (ReWalk-gait) for 4 weeks (20 sessions). After a 2-week wash-out period, they switched walking devices and underwent 4 additional weeks of training. Two evaluations were performed (after 2 and 4 weeks) following the training periods for each walking device, using the 6-minute walking test (6MWT) and 30-minute walking test (30MWT). The spatiotemporal variables (walking distance, velocity, and cadence) and energy expenditure (heart rate, maximal heart rate, the physiologic cost index, oxygen consumption, metabolic equivalents, and energy efficiency) were evaluated duringthe 6MWT and 30MWT. A usability evaluation questionnaire for walking devices was developed based on the International Organization for Standardization/International Electrotechnical Commission guidelines through expert consultation. RESULTS: The ReWalk-gait presented significant advantages in energy efficiency compared to KAFO-gait in the 6MWT and 30MWT; however, there were no differences in walking distance or speed in the 30MWT between ReWalk-gait and KAFOgait. The usability test demonstrated that ReWalk-gait was not superior to KAFO-gait in terms of safety, efficacy, efficiency, or patient satisfaction. CONCLUSION: The robot (ReWalk) enabled patients with paraplegia to walk with lower energy consumption compared to KAFO, but the ReWalk-gait was not superior to KAFO-gaitin terms of patient satisfaction.

Abundance of immunologically active alanine aminotransferase in sera of liver cirrhosis and hepatocellular carcinoma patients.

BACKGROUND: Although alanine aminotransferase (ALT) is a widely used indicator of liver function, ALT enzymatic activity may not always reflect the degree of liver damage. Improved methods or approaches would be useful. METHODS: Monoclonal antibodies (mAbs) to ALT were generated to develop a sandwich enzyme immunoassay system. We used an immunoassay to measure ALT mass concentration and a common biochemical analyzer to assay ALT enzymatic activity in serum samples from patients with liver diseases and healthy individuals. The results from the 2 methods were compared and analyzed by ROC curve analysis. RESULTS: The ALT sandwich enzyme immunoassay system demonstrated reliable performance in linearity, recovery, and imprecision studies. The ALT activity assay exhibited a higher diagnostic accuracy in acute hepatitis (AH) patients, but the ALT immunoassay exhibited higher sensitivity and specificity in patients with chronic liver diseases. The areas under the ROC curve for ALT mass and enzymatic activity were 0.82 and 0.98, respectively, in AH, 0.99 and 0.52 in hepatocellular carcinoma (HCC), and 0.94 and 0.45 in liver cirrhosis (LC). Serum samples from HCC and LC patients had higher amounts of ALT-immunoglobulin complexes [median A(450), 1.7 (interquartile range, 1.4-1.9)] than the other groups [1.3 (interquartile range, 0.9-1.6)]. CONCLUSIONS: Our analysis of sera from the HCC and LC patient groups revealed considerable amounts of immunologically active but catalytically inactive ALT. The amount of the ALT-immunoglobulin complex increased with the severity of the liver disease. The 2-site immunoassay method may be useful in the differential diagnosis of some causes of liver disease.

Validity of the Budapest Criteria For Poststroke Complex Regional Pain Syndrome.

OBJECTIVES: Complex regional pain syndrome-1 is a chronic neuropathic disorder, and poststroke complex regional pain syndrome (PS-CRPS) is not a rare complication. There is a lack of study implementing the Budapest criteria for PS-CRPS diagnosis. Thus, the present study investigated the validity of the Budapest criteria for PS-CRPS diagnosis and assessed the PS-CRPS-related factors in stroke patients with an affected upper extremity. METHODS: The study included 72 patients with first-ever stroke resulting in hemiplegia. The prevalence of PS-CRPS and diagnostic validity were compared among the Budapest clinical criteria, Budapest research criteria, modified Budapest criteria (removal of the motor factor from the motor/trophic category), and International Association for the Study of Pain (IASP) criteria in patients diagnosed with PS-CRPS according to the Budapest clinical criteria. RESULTS: PS-CRPS was diagnosed in 6 (8.3%), 1 (1.4%), 6 (8.3%), and 11 patients (15.3%) according to the Budapest clinical criteria, Budapest research criteria, modified Budapest criteria, and IASP criteria, respectively. The Budapest criteria and IASP criteria had sensitivities of 0.99 and 1.00, respectively, and specificities of 0.68 and 0.41, respectively, for PS-CRPS diagnosis. There were no differences in risk factors between PS-CRPS patients and non-PS-CRPS patients when the diagnosis was based on the Budapest clinical criteria. However, there were differences in muscle strength and Brunnstrom stage between PS-CRPS patients and non-PS-CRPS patients when the diagnosis was based on the IASP criteria. DISCUSSION: Our findings indicate that the diagnostic validity of the current Budapest clinical criteria for PS-CRPS is low. Thus, the current Budapest criteria might not be appropriate for PS-CRPS diagnosis.

Calixarene derivative as a tool for highly sensitive detection and oriented immobilization of proteins in a microarray format through noncovalent molecular interaction.

One important factor in fabricating protein microarray is to immobilize proteins without losing their activity on a solid phase. To keep them functional, it is necessary to immobilize proteins in a way that preserve their folded structural integrity. In a previous study, we developed novel Calixarene derivatives for the immobilization of proteins on the surface of a glass slide (1). In this study, we compared the sensitivity and the specificity of the linker molecules with those of five other protein attachment agents on glass slides using a prostate-specific antigen and its antibodies as a model system. The Calixcrown-coated protein chip showed a superior sensitivity and a much lower detection limit than those chips prepared by other methods. When we tested the capability of Calixcrown to immobilize antibody molecules, it appeared that Calixcrown makes arrangement of antibody be more regular with the vertical orientation than the covalent-bond agent. We also observed that the Calixcrown chip could be used for the diagnostic application with clinical samples from prostate cancer and HIV patients. Finally, we applied the Calixcrown chip using an antibody microarray to identify up- or down-regulated proteins in specific tissue and detected several up- or down-regulated proteins from a rat liver by administering toxin. Thus, the Calixcrown chip can be used as a powerful tool with a wide range of applications, including protein-protein interaction, protein-DNA interaction, and an enzyme activity assay.

Levels of common salivary protein 1 in healthy subjects and periodontal patients.

PURPOSE: Human saliva, as a vital part of the immune defense system, contains a number of distinct proteins and peptides. Recently human common salivary protein 1 (CSP1) has been identified as an abundant salivary protein and may play a role in promoting the binding of cariogenic bacteria to salivary pellicles. However, nothing else is known regarding the role of CSP1 in periodontology. The aim of this study was to quantify and compare CSP1 levels between healthy subjects and periodontal patients. METHODS: This controlled clinical study was conducted in periodontally healthy individuals and patients with chronic periodontitis Chonbuk National University Hospital, with Institutional Review Board approval. Whole saliva samples were collected from 36 healthy subjects and 33 chronic periodontitis patients and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immune blotting were conducted to ensure that anti-CSP1 monoclonal antibody (mAb) binds to CSP1 in human saliva. A sandwich enzyme-linked immunosorbent assay (ELISA) system was house-fabricated using mAb-hCSP1#14 and mAb-hCSP1#4 as a capture and a detector mAb, respectively. The CSP1 concentrations in saliva from 36 healthy subjects and 33 periodontal patients were quantified using the CSP1 sandwich ELISA system, and the results were analyzed using the Student's t-test. RESULTS: Immunoblot analysis using mAb-hCSP1 as a probe confirmed that CSP1 in human saliva existed as a single band with a molecular weight of approximately 27-kDa. The quantification of CSP1 concentrations by CSP1 ELISA showed that the median values (25th to 75th percentiles) of periodontal patients and healthy subjects were 9,474 ng/mL (range, 8,434-10,139 ng/mL) and 8,598 ng/mL (range, 7,421-9,877 ng/mL), respectively. The Student's t-test indicated the presence of a statistically significant difference between the 2 groups (P=0.024). CONCLUSIONS: The presence of a significant difference in CSP1 levels between healthy subjects and periodontal patients suggests that CSP1 may be a potential biomarker for the detection or screening of periodontitis patients.

Evaluation of fluorescence hs-CRP immunoassay for point-of-care testing.

BACKGROUND: C-reactive protein (CRP) is one of acute phase respondents that has been used to monitor infection and inflammation episodes. Recent studies have shown that high-sensitivity C-reactive protein (hs-CRP) is a potential risk predictor for future atherosclerosis and cardiovascular diseases (CVD). METHODS: We previously developed a fluorescence-based immunochromatographic method for measuring hs-CRP concentrations (i-CHROMAtrade mark hs-CRP assay) in blood. Whole blood was mixed with detector buffer, and then loaded onto a test cartridge. After 10 min of incubation, the test cartridge was inserted and scanned for acquisition of fluorescence intensity in a laser fluorescence reader (i-CHROMAtrade mark reader). The fluorescence intensity was microprocessed and converted into the concentration of CRP in blood. The test result of 150 samples by the i-CHROMAtrade mark hs-CRP assay method was compared and evaluated with those by TBA 200FR turbidimetry and BN II nephelometry method. The Deming regression and the Bland-Altman difference plot analysis were used for comparison of hs-CRP test result. RESULTS: The i-CHROMAtrade mark hs-CRP assay system exhibited a good linearity with in the whole measuring range (R=0.997). The imprecision of intra- and the inter-assay CVs (coefficient of variation) of assay system were CVs< 3% and < 5% in the range of 0.5-20 mg/l, respectively. The i-CHROMAtrade mark hs-CRP assay method correlated well with TBA 200FR turbidimetry and BN II nephelometry assay method (R=0.988, N=143 and R=0.989, N=143). CONCLUSION: The i-CHROMAtrade mark hs-CRP assay system is comparable to those of other well-known fully automated hs-CRP assay and is suitable for point-of-care testing (POCT) in detection and quantification of hs-CRP.

Development of an electrochemical immunosensor for alanine aminotransferase.

Alanine aminotransferase (ALT) has been regarded as one of the most sensitive indicators of hepatocellular damage. While ALT is widely used in the practice of medicine, few attempts have been made to develop biosensors applicable to the on-site diagnosis of liver diseases. In the hope of developing an immunosensor for measurement of ALT activity, we have generated monoclonal antibodies to human recombinant ALT and fabricated them for use in a sensor. The ALT immunosensor was composed of the followings: (1) anti-ALT antibody-immobilized outer membrane; (2) pyruvate oxidase-absorbed inner membrane; (3) a self assembled monolayer mediator-coated gold working electrode and an Ag/AgCl reference electrode. The chronoamperometric measurement of the immunosensor was performed with 40 microl of PBS containing substrates and ALT without a washing step in less than 5 min. The dynamic range of ALT immunosensor was presented as five orders of magnitude, ranging between 10 pg/ml and 1 microg/ml. The detection limit and the sensitivity were 10 pg/ml and 26.3 nA/(ng/ml), respectively. In the meantime, the enzyme sensor fabricated without anti-ALT antibody showed much poorer analytical values. The dynamic range, the detection limit, and the sensitivity were 10 ng/ml-100 microg/ml, 10 ng/ml and 11.4 nA/(ng/ml), respectively. The presented results indicated that the immunosensor system provided much better technical performance in all of the aspects evaluated than did the enzyme sensor without the immobilized-antibody.

A rapid, simple measurement of human albumin in whole blood using a fluorescence immunoassay (I).

BACKGROUND: Human serum albumin (HSA) is the most abundant plasma protein and plays key a role in metabolism. The variation in albumin concentration provides valuable information related to metabolic diseases and diagnostic application. METHODS: We constructed two assay systems to quantify the albumin concentration. The immunoassay used a fluorescence (FL) dye to detect albumin in samples and employed the conventional chromatography as a separation system. The assay system consists of an anti-HSA-mAb or an HSA immobilized test strip in a disposable cartridge, a fluorescence-labeled detector buffer and a laser-fluorescence scanner. We mixed the sample with detector, loaded it onto a cartridge, incubated it for 10 min and measured the concentration of albumin in a laser-fluorescence scanner. We examined the comparability of assay with an automated BCG dye binding method using a Hitachi 747 biochemical analyzer. RESULTS: The correlation of coefficient between AT/AC as converted from the relative fluorescence units (RFU) and albumin concentration displayed reasonable reliability in both the competition and the inhibition assay systems (r = 0.998). Using the Bland-Altman difference plot analysis, we observed an acceptable agreement between two methods, the fluorescence immunochromatography assay (FL-ICA) and the automated BCG dye-binding method of a Hitachi biochemical analyzer, over the clinical relevant range of HSA concentrations. The coefficient of variation (CV) of within- and between-run variation in the immunoassay system was < 8% and the recovery fell within 5% in each control sample. In addition to its reliable analytical performance, the assay with whole blood can be completed in 12 min using a one-step operation without any pretreatment. CONCLUSION: The developed immunoassay system using fluorescence dye and lateral-flow chromatography is a simple, fast and reliable method for quantifying the albumin concentration in whole blood.

Development of a point-of-care assay system for high-sensitivity C-reactive protein in whole blood.

BACKGROUND: C-reactive protein (CRP) is emerging as a potential risk predictor for future cardiovascular diseases (CVD). High sensitivity assays have been developed and applied for clinical purposes. METHODS: The fluorescence immunochromatographic assay was employed to detect and quantify CRP in whole blood. It consisted of a fluorescence (FL) antibody detector buffer, a test strip housed in a disposable cartridge, and a laser fluorescence scanner. Whole blood sample was mixed with detector, loaded onto a cartridge, incubated for 10 min, and the concentration of CRP was measured in a laser fluorescence scanner. The linearity, limit of detection (LOD), and performance of new assay system was tested and evaluated. The comparability of assay was examined with an automated reference method. RESULTS: With the new assay system, a reliable correlation of coefficient (r) was obtained between the ratio value (A(T)/A(C)) and a concentration of CRP in samples. The linearity fell in the range of 0-10 mg/l of CRP, and the analytical detection limit was 0.133 mg/l of CRP. The mean recovery of the control was 105.2% in a working range. The precision of the intra- and inter-assay in a range of 0.5-6 mg/l was CVs <6% and <8%, respectively. The new fluorescence immunochromatographic assay system correlated well with a traditional immunoturbidimetric assay for quantification of CRP concentration (r=0.955, N=90). CONCLUSION: The fluorescence immunochromatographic assay is fast, reliable, and a reproducible platform for point-of-care testing (POCT) of high-sensitive (hs)-CRP in whole blood.

Loss of function of endothelin-2 leads to reduced ovulation and CL formation.

Endothelin-2 (EDN2), a potent vasoconstrictive peptide, is transiently produced by periovulatory follicles at the time of ovulation when corpus luteum (CL) formation begins. EDN2 induces contraction of ovarian smooth muscles ex vivo via an endothelin receptor A-mediated pathway. In this study, we aimed to determine if EDN2 is required for normal ovulation and subsequent CL formation in?vivo. In the ovaries of a mouse model that globally lacks the Edn2 gene (Edn2 knockout mouse; Edn2KO), histology showed that post-pubertal Edn2KO mice possess follicles of all developmental stages, but no corpora lutea. When exogenous gonadotropins were injected to induce super-ovulation, Edn2KO mice exhibited significantly impaired ovulation and CL formation compared to control littermates. Edn2KO ovaries that did ovulate in response to gonadotropins did not contain histologically and functionally identifiable CL. Intra-ovarian injection of EDN2 peptide results suggest partial induction of ovulation in Edn2KO mice. Endothelin receptor antagonism in wild type mice similarly disrupted ovulation, CL formation, and progesterone secretion. Overall, this study suggests that EDN2 is necessary for normal ovulation and CL formation.

Point-of-care fluorescence immunoassay for prostate specific antigen.

BACKGROUND: Prostate specific antigen (PSA) is widely used as a clinical marker for diagnosis, screening, and prognosis of prostate cancer. A fluorescence (FL) dye-incorporated immunochromatographic assay (ICA) was developed for detection of PSA concentration in whole blood or serum. METHODS: Whole blood or serum mixed with FL-conjugated detector was loaded onto a cartridge and incubated for 12 min. The FL intensity of the cartridge was then measured in a laser FL scanner. The analytical performance of the FL-ICA system was evaluated by precision and recovery tests. The comparability of the new method was examined with an automated analyzer. RESULTS: A reliable correlation between area ratio (AT/AC), reflecting FL intensity of test/control line, and PSA concentration was observed (r=0.998). The CVs of intra- and inter-assay precision in a range of 2.5-8 microg/l were <6.0% and 4.5%, respectively, and analytical recovery of the FL-ICA system fell within 6.5% at the tested samples. When the FL-ICA method was compared with Abbott AxSYM and Bayer Centaur analyzers, there were strong correlations (r=0.993 and r=0.992, p<0.0001). CONCLUSION: The FL-ICA system with point-of-care-testing (POCT) appeared to be an easy, fast and suitable method for measurement of PSA concentration in whole blood, and needs no accessory equipment to separate serum.