The Moderating Role of COVID-19 Perceived Risk between Health Concern and Psychological Well-Being of Active Senior Campers Using PROCESS Macro Model.

This study attempts to contribute to improving the life of the elderly by empirically analyzing the factors affecting the psychological well-being of active seniors in the 'With COVID-19' era. To this end, the relationship between psychological well-being, health concern, and perceived risk of COVID-19 was verified for active seniors in Korea who enjoy camping. Two hundred and sixty-four valid questionnaires collected from active senior participants of "The Korea Camping Fair 2022" held in EXCO, Daegu, Korea, from 29 April to 1 May 2022, were analyzed. The main results were as follows. The health and psychological well-being of active seniors were higher than the normal level, and the perceived risk of COVID-19 was lower than the normal level. It was found that the health of active seniors had a positive effect on their psychological well-being. The perceived risk of COVID-19 was found to moderate the effect of health concern on psychological well-being. In conclusion, in order to improve the psychological well-being of active seniors, active leisure activities of the elderly are essential even in the COVID-19 situation, and various measures are needed to increase health. Not only this, but also, importantly, accurate information sharing on COVID-19 should be premised.

Will Perceived Risk of COVID-19 Move Exhibition Visitors from On-Site to Virtual? Focusing on Exhibition Quarantine Service Quality and Switching Intention.

COVID-19 has shifted people's activities from the real world to the virtual world in many fields, such as conferences, shopping, education, and more. In the field of MICE, however, exhibitions have been held steadily since the second half of 2020 in the form of on-site exhibitions. The exhibition organizers and related authorities have tried to attract exhibitors and visitors to the exhibition hall by providing exhibition quarantine services. Moreover, despite various perceived risks during the COVID-19 period, exhibition visitors continue to visit the exhibition. This study, therefore, paid attention to the psychological factors of visitors who consistently visit on-site exhibitions even during the pandemic. In addition to the perceived risks, this study tried to examine the quality of exhibition quarantine services and switching intention of visitors, and to analyze the relationships between them. A survey of 167 people who visited the camping exhibition and well-food exhibition held in June 2021 found that they would not visit the exhibition due to the functional and financial risk of the exhibition rather than the risk of the virus. On the other hand, it was found that visitors who felt the social risk of COVID-19 valued the quality of exhibition quarantine service. Furthermore, the study found that the quarantine service quality lowered switching intention. Therefore, the study suggests that exhibition organizers should think about ways to strengthen the most essential characteristics of on-site exhibitions along with appropriate quarantine measures to induce steady visits even during the pandemic.

How Will Video Conference Fatigue Affect Participants of MICE in the With-COVID-19 Era? Focusing on Video Conference Quality, Social Presence Theory, and Flow.

Is our mental health at risk due to spending a significant amount of time online due to the COVID-19 pandemic? In the new era that we are living in, where we live a life that coexists with the virus, we are participating in video conferences held online rather than on-site in order to slow the spread of the virus. Video conferencing has become our necessity since March 2020, and is becoming a new standard, especially in the MICE industry. Recently, however, people who have excessively used video conference platforms are complaining of video conference fatigue, which is a new negative emotion such as stress, anxiety, and worry as well as general work fatigue. Therefore, this study focused on the mechanism of video conferencing in MICE, which is rapidly digitally converted by the virus, and the digital psychological factors of the participants. This study derived the quality attributes of video conferencing in MICE and empirically analyzed the relationship with digital psychological factors of the video conference participants, such as video conference fatigue, social presence, and flow. One hundred and thirty-eight valid questionnaires collected from participants of several international academic conferences held in EXCO, Daegu, Korea, from 23 to 28 May 2021, were analyzed. The main results are as follows. First, unlike general video conference fatigue, MICE video conference fatigue was not found to be related to the preceding and following variables. This is due to the characteristics of the MICE video conference and the expertise of the participants. Second, social presence was identified as an important variable in MICE video conferencing. Although media-mediated, the feeling of being present with the presenter and participants was found to affect the participants' flow in the video conference. Third, in this study, the fun factor was identified as the most important video conference quality that can enhance the social presence of the video conference participants of MICEs.

Immunological measurement of aspartate/alanine aminotransferase in predicting liver fibrosis and inflammation.

BACKGROUND/AIMS: Enzymatic analysis of aspartate/alanine aminotransferase (AST/ALT) does not exactly represent the progression of liver fibrosis or inflammation. Immunoassay for AST (cytoplasmic [c] AST/mitochondrial [m] AST) and ALT (ALT1/ALT2) has been suggested as one alternatives for enzymatic analysis. The objective of this study was to evaluate the efficacy of immunoassay in predicting liver fibrosis and inflammation. METHODS: A total of 219 patients with chronic hepatitis B (CHB) who underwent hepatic venous pressure gradient (HVPG) and liver biopsy before antiviral therapy were recruited. Serum samples were prepared from blood during HVPG. Results of biochemical parameters including enzymatic AST/ALT and immunological assays of cAST, mAST, ALT1, and ALT2 through sandwich enzyme-linked immunosorbent assay (ELISA) immunoassay with fluorescence labeled monoclonal antibodies were compared with the results of METAVIR stage of live fibrosis and the Knodell grade of inflammation. RESULTS: METAVIR fibrosis stages were as follows: F0, six (3%); F1, 52 (24%); F2, 88 (40%); F3, 45 (20%); and F4, 28 patients (13%). Mean levels of AST and ALT were 121 ± 157 and 210 ± 279 IU/L, respectively. Mean HVPG score of all patients was 4.7 ± 2.5 mmHg. According to the stage of liver fibrosis, HVPG score (p < 0.001, r = 0.439) and ALT1 level (p < 0.001, r = 0.283) were significantly increased in all samples from patients with CHB. ALT (p < 0.001, r = 0.310), ALT1 (p < 0.001, r = 0.369), and AST (p < 0.001, r = 0.374) levels were positively correlated with Knodell grade of inflammation. CONCLUSION: ALT1 measurement by utilizing sandwich ELISA immunoassay can be useful method for predicting inf lammation grade and fibrosis stage in patients with CHB.

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.

Comparison of four serological tests for detecting antibodies to Japanese encephalitis virus after vaccination in children.

OBJECTIVES: Several different methods are currently used to detect antibodies to Japanese encephalitis virus (JEV) in serum samples or cerebrospinal fluid. These methods include the plaque reduction neutralization test (PRNT), the hemagglutination inhibition (HI) test, indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). The purpose of this study was to compare the performance of each method in detecting vaccine-induced antibodies to JEV. METHODS: The study included 29 children who had completed a primary immunization schedule with an inactivated vaccine against JEV derived from mouse brain (n = 15) or a live attenuated SA14-14-2 vaccine (n = 14). Serum samples were collected between 3 months and 47 months after the last immunization. The serum samples were tested by performing the PRNT, HI test, in-house IFA, and commercial ELISA. The antibody detection rates were compared between tests. RESULTS: All 29 serum samples were positive with the PRNT, showing antibody titers from 1:20 to 1:2560. The HI test showed positive rates of 86.7% (13/15) and 71.4% (10/14) in the inactivated and live attenuated vaccine groups, respectively. The results of the IFA for immunoglobulin (Ig)G were positive in 53.3% (8/15) of children in the inactivated vaccine group and 35.7% (5/14) in the live attenuated vaccine group. Neither the IFA nor ELISA detected JEV IgM antibodies in any of the 29 children. CONCLUSION: These results show that detection rates of vaccine-induced antibodies to JEV have a wide range (0-100%) depending on the testing method as well as the time since immunization and individual differences between children. These findings are helpful in interpreting serological test results for the diagnosis of Japanese encephalitis in situations where vaccines are widely administered.

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.

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 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.

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.

Could HTR2A T102C and DRD3 Ser9Gly predict clinical improvement in patients with acutely exacerbated schizophrenia? Results from treatment responses to risperidone in a naturalistic setting.

OBJECTIVE: This study seeks to replicate previous results indicating that T102C in the serotonin 2A receptor (HTR2A) and Ser9Gly in the dopamine D3 receptor (DRD3) were associated with a risperidone response to acutely exacerbated schizophrenia, and to determine whether possession of these alleles predicts clinical improvement in a naturalistic setting. METHODS: We consecutively recruited 100 schizophrenia patients and assessed clinical improvement after 4 weeks of risperidone treatment. RESULTS: The patients with T/T in the HTR2A gene showed less clinical improvement than did those with T/C or C/C (p = 0.044). In the case of the DRD3 gene, we did not find statically significant association with clinical improvement (p = 0.061). When patients were categorized into responders and nonresponders, the C allele was more frequent in responders (OR = 2.28, 95%CI = 1.06-4.91, p = 0.039). When combinations of the two polymorphisms were considered, patients who had T/T in the HTR2A gene and encoded Ser/Ser or Ser/Gly from DRD3 gene had a higher propensity to non-responsiveness compared to other subjects (OR = 3.57, 95%CI = 1.10-11.62, p = 0.039). CONCLUSIONS: Our findings suggest that the HTR2A T102C could be a potential indicator of clinical improvement after risperidone treatment.

Role of p38 MAPK on the down-regulation of matrix metalloproteinase-9 expression in rat astrocytes.

In spite of their pathophysiological importance in neuro-inflammatory diseases, little is known about the signal transduction pathways that lead to the induction of matrix metalloproteinases (MMPs) in the central nervous system. We reported previously that lipopolysaccharide (LPS) induced MMP-9 expression through ERK1/2 pathway in rat primary astrocytes (Glia 41:15-24, 2003). Here, we investigated the role of other MAPK pathways, including p38 and JNK/SAPK, on the regulation of MMP-9 expression in LPS-stimulated rat primary astrocytes. LPS activated both p38 and JNK in astrocytes. Treatment with a specific p38 MAPK inhibitor SB203580, but not JNK inhibitor SP600125, increased the LPS-stimulated MMP-9 expression in a concentration-dependent manner. Anti-inflammatory cytokines, including IFN-gamma and IL-4, activated p38 MAPK and decreased MMP-9 production in LPS-stimulated astrocytes. When p38 MAPK activation was blocked by SB203580, the inhibitory effects of these cytokines on MMP-9 induction were abolished. Finally, direct injection of SB203580 into the lateral ventricle of rat brain increased the LPS-induced MMP-9 activity in cerebral cortex. Altogether, these results suggest that p38 activation down-regulates the inflammatory stimulation-induced overexpression of MMP-9, both in primary astrocytes and in cerebral cortex. The elaborate interplay between ERK1/2 and p38 pathways provides a more sophisticated mechanism for regulating MMP-9 activity in neuroinflammatory diseases.

Analysis of C-reactive protein on amide-linked N-hydroxysuccinimide-dextran arrays with a spectral surface plasmon resonance biosensor for serodiagnosis.

A new label-free array system using amide-linked (AL) NHS-dextran and a spectral SPR biosensor are presented for the high-throughput analysis of C-reactive protein (CRP) in human sera. The AL NHS-dextran layer on the surface of gold arrays was composed of an amide linkage between NHS-modified carboxymethyl-dextran and amine-modified 11-mercaptoundecanoic acid. The topology of the AL NHS-dextran layer was analyzed by atomic force microscopy, and it was found to be superior to the previously used epoxide-linked carboxymethyl-dextran layer in its immobilization of proteins. Specific immunoreactions and a dose-dependent increase of SPR signals were demonstrated on the AL NHS-dextran layer. Then, the label-free array system was successfully applied to the rapid analysis of CRP in 120 human sera. CRP levels in human sera determined by the array-based spectral SPR biosensor showed a good correlation with those determined by the latex-enhanced turbidimetry immunoassay (n = 120, r = 0.945, p < 0.0001). Thus, the array-based spectral SPR biosensor based on the AL NHS-dextran surface is a potential system for rapid and label-free serodiagnosis of human diseases.

Down-regulation of matrix metalloproteinase-9 expression by nitric oxide in lipopolysaccharide-stimulated rat primary astrocytes.

Immunologically activated astrocytes over-express matrix metalloproteinase-9 (MMP-9) and nitric oxide (NO). Because they have both beneficial and detrimental effects on the pathophyiological outcomes of several neurological diseases, their expression should be tightly regulated in the CNS. NO can modify the activity of other proteins either by directly modifying protein structure or regulating the expression of target proteins. In this study, we investigated the role of NO on the expression of MMPs in rat primary astrocytes. Rat primary astrocytes were stimulated with lipopolysaccharide (LPS), resulting in the over-expression of both MMP-9 and NO. Inhibition of NO production using nitric oxide synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), further increased MMP-9 expression, suggesting NO inhibits MMP-9 expression. In line with this observation, exogenous addition of NO donor, sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP), inhibited MMP-9 expression in astrocytes. The inhibitory effect of NO was mediated by the down-regulation of mRNA and protein levels of MMP-9 but not by the direct modification of the enzymatic activity of MMP-9. The effect of NO on MMP-9 expression was mimicked by dibutyryl-cGMP and inhibited by PKG inhibitor KT5823, suggesting NO regulates MMP-9 expression via guanylate cyclase-PKG pathway. Finally, SNP or dibutyryl-cGMP inhibited the activation of ERK1/2 in LPS-stimulated astrocytes, which is an essential regulator of MMP-9 expression in astrocytes. The regulation of MMP-9 expression by NO may confer additional levels of fine-tuning of the level of MMP-9 during brain inflammatory conditions.

Fine epitope mapping of monoclonal antibodies specific to human alpha-synuclein.

The neuronal phosphoprotein alpha-synuclein has been increasingly implicated in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative diseases; however, the exact function of alpha-synuclein still remains illusive. Suitable antibodies (Abs) specific for the gene of interest are indispensable for studying biological and immunological properties of the target gene. Here, we report not only the generation and characterization of monoclonal Abs, Syn-1 and Syn-17, against human alpha-synuclein, but also the epitope mapping by using recombinant synuclein family proteins and various GST fusion proteins of human alpha-synuclein domains. Syn-17 recognizes human and rodent alpha-synuclein, and its epitope is localized within residues 97-99 and 101 of alpha-synuclein. In contrast, the Syn-1 epitope is localized in residues 121 and 122 of human alpha-synuclein, and Syn-1 recognizes only human but not rodent alpha-synuclein, indicating that it can be utilized as a useful reagent for studying human alpha-synuclein transgenic mouse and zebrafish lines.

Identification and characterization of a lysosomal membrane protein of Dictyostelium discoideum with monoclonal antibodies.

Lysosomes are responsible for the degradation of macromolecules derived from the cell exterior by endocytosis, or from within the cell by autophagy. While our knowledge of the biosynthesis and targeting of lysosomal hydrolases is considerable, much less is known about the lysosomal membrane itself. To identify the lysosomal membrane proteins that mediate these functions, we have isolated lysosomes from amebae and injected them into mice to produce monoclonal antibodies (MAbs). We produced nine MAbs against Dictyostelium lysosomes from the batches of fused cells. Among them, three MAbs were specific to lysosomal membrane and gave a strong signal, and thus used in this study. The MAbs specifically reacted with a single protein band of 27 kd and stained a lysosome-like structure by immunofluorescence microscopy. To identify the antigen that the MAbs recognize, we processed differential centrifugation with whole-cell extract of Dictyostelium and traced p27 protein by activity assay of organelle marker enzyme. We showed that p27 is one component of the lysosomal system on the basis of comigration with a lysosomal marker enzyme. We also demonstrated that the 27-kd lysosomal protein is a tightly bound integral membrane protein by using a phase separation method of Triton X-114.

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.

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.

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.