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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. Conclusions ALT1 measurement by utilizing sandwich ELISA immunoassay can be useful method for predicting inf lammation grade and fibrosis stage in patients with CHB.

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.

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.

A personal watercraft (PWC) is a vessel that uses an inboard motor powering a water jet pump as a source of power and is operated by a person sitting, standing, or kneeling. Maneuvering a PWC is different from operating a motor vehicle or boat. An obstacle cannot be avoided by slowing down and turning the watercraft, throttle power is required to turn or maneuver the PWC. The watercraft stops only by drifting or turning sharply. The study examined sixty court decisions published in LexisNexis databases of the United States over the last decade. Cases included individuals injured while operating a PWC as a driver, passenger, or as a result of contact with a watercraft. A content analysis identified items to be used in the study. Crosstab and logistic regression analyses were used to identify demographic information and the characteristics of those who succeeded in a court of law. One-third of the cases were successful, adults, males, and the party who sustained a severe injury were more successful in a court of law with the exception of the statistically significant factors (high risk maneuvers and sharp turns). Among the additional results, we should be aware that insurance companies may not pay, additionally, it is unwise to loan a PWC to a female who has no experience.

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.

We investigated the influence of a revised marketing mix model that included the factors of human service, cost, communication, and convenience of facility on expected and experienced satisfaction of 227 users of various martial arts centers in Korea. By employing descriptive statistics and multiple regression analyses, the following findings resulted. First, expected satisfaction scored slightly higher than experienced satisfaction. Second, human service and communication positively influenced expected satisfaction. Third, human service and convenience of facility positively affected experienced satisfaction. In terms of the effect of the factors of the marketing mix on both expected and experienced satisfaction, human service was identified as a significant factor influencing sports consumer behavior. Our findings highlight the importance of human service in areas that demand direct relationships between service users and providers.

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

This study was performed to produce a strong antibody against repeated peptides for a specific region of an antigen. Nine amino acids at C-terminal from human brain β-tubulin III were selected as a target sequence since it is well known for a high conservancy of sequence and resistance to antibody production. The synthetic oligonucleotides had six bases (5′-CGAC—GT-3′, 5′-TC—CAGC-3′) appended at each end and were repeatedly linked and directly joined by ligase in the presence of restriction enzymes (AccI, AvaI) to allow for the construction of head-to-tail repeats. Ligated oligonucleotides containing more than 10-unit repeats were eluted from agarose gels and cloned into the AccI site of a modified pGEX expression vector. The cloned repeated target sequences were subsequently expressed in a bacterial system. A large amount of glutathione-S-transferase-fusion protein containing the unit repeats was purified by affinity chromatography on glutathione-agarose beads and injected into Balb/c mice to produce pol...

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.

A human papilloma virus (HPV) test can be used to identify women that are at risk for cervical cancer. Hybrid Capture (HC2) and PCR are the current gold standard assays and are used globally to detect HPV infection. We present a combined assay system that uses PCR and fluorescence (FL) DNA chip for simple, rapid, and sensitive HPV DNA detection. In the developed assay system, PCR-amplified DNA produced by biotin-labeled consensus GP5+/GP6+ primers was denatured, neutralized, mixed with FL-streptavidin detector solution and then the mixture was loaded onto the HPV DNA chip for DNA hybridization. The DNA chip was inserted into the laser FL scanner and the FL signal was measured. MgCl2 of PCR mixture completely nullified FL signal in DNA chip assay but addition of 50mM EDTA to detector solution recovered FL signal. When PCR product of HPV 16 or 18 infected patient’s sample was subjected to DNA chip assay, FL signal was observed only from its corresponding HPV type DNA chip, demonstrating the HPV assay system differentiate different HPV genotypes. In rapidity and sensitivity test, the FL signal was illuminated from a PCR product produced by 5 cycles, which did not resolve as a DNA band in gel electrophoresis, suggesting that the combined assay system is more sensitive than an HPV PCR assay. Additionally, this test can be completed within 30 minutes, which will allow patients to be evaluated for infection.

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.

The determination of blood level of thyroid stimulating hormone (TSH) is an important biomarker for the clinical assessment of thyroid status. Here, we presented a new fluorescence (FL) immunochip TSH assay system, which was developed with a platform of point-of-care test (POCT) for clinical applications. The assay system adopted a lateral-flow immunochromatographic technology and consisted of anti-TSH-mAb coated strip in a disposable chip, a detection buffer containing FL-labeled anti-TSH-pAb, a calibration chip, and a laser FL scanner. The analytical performance of FL immunochip TSH assay system was evaluated by linearity, interference, recovery, and imprecision tests. The comparability of the developed assay was examined with an automated reference assay. The developed assay system exhibited an excellent linearity in working range of 1–100 μIU/mL and was affected neither a large amount of various serum interference substances nor similar structure biomolecules to TSH. The analytical mean recovery of control was 97.6% in a dynamic working range and the imprecision of intra- and inter-assay of CVs was less than 10%. There was a significant correlation between the developed TSH assay and the Beckman Coulter Access 2 TSH assay (r=0.989, p

This paper presents a new air-bubble free microfluidic blood cuvette for the measurement of hemoglobin concentration. The microfluidic blood cuvette was filled with blood samples by capillary force, and hemoglobin levels in the blood were determined by measuring absorbance at the wavelength of 530 nm. Two different microfluidic blood cuvettes with dual and single sidewall microchannels were investigated. The microfluidic blood cuvette was fabricated using a polymethyl methacrylate substrate and a dry film photoresist. During the blood-filling process, air was trapped in the dual-sided wall-type cuvettes, while no air trapping occurred in the single sidewall-type cuvettes. The sensitivity of the hemoglobin measurements was more linear in a 105 μm deep microchannel than in a 35 μm deep microchannel.

Binding affinity between calix[4]crown-5-ether and amino acids have been compared by studying the complexation association constant, and the best value has been obtained from complex of calix[4]crown-5 ether, and HrSOD tagged with Arg and Lys were tested to investigate the effects of specific residues in protein immobilization on calix[4]crown-5-ether. The protein tagged with 9Args has been shown to have much better immobilization potential. Taking advantage of the similar structure of a moiety of guanine base to that of Arg side chain, different homo-oligonucleotides have been immobilized, and it was found that calix[4]crown-5-ether is an appropriate agent in the immobilization of dGTP homo-oligonucleotides. The results demonstrate that calix[4]crown-5-ether on glass slide chip could be applied as an excellently oriented immobilization agent for protein or for DNA microarray designing. It has the ability of single base differentiation in SNP sequence detection.

We have previously shown that a Calixarene derivative was an excellent agent for protein immobilization on a solid phase. We further characterized the recognition mechanism between the ‘host’ Calixarene derivative and ‘guest’ molecules in this study. The rate of complex formation of Calixcrown with amino acids informed that Calixcrown had the strongest interaction with Arg among the tested amino acids. In order to confirm that Arg is an effective residue in immobilization of protein on Calixcrown-coated glass, several human recombinant superoxide dismutase tagged with different numbers of Arg or Lys were generated. The recombinant protein tagged with longer Arg was immobilized better than the ones tagged with shorter Arg or Lys. We also tested if Calixcrown can immobilize oligoDNA as efficiently as it does protein. Calixcrown most strongly captured homo-dGTP oligoDNA among the four homo-oligoDNAs. When Calixcrown chip was applied to detect single nucleotide polymorphism, it differentiated one base pair mismatch of the double helix oligoDNA. Thus, Calixarene derivative linker molecule can be used as an efficient immobilization agent for both protein and oligoDNA with the right orientation on a solid phase.

The developments of immunosensors with a variety of formats are increasingly finding applications in clinical diagnostics and biological researches. A strategy for the immunoassay and preparation of Calixcrownchips is proposed. This strategy is based on the immobilization of antigens or antibodies on the surface of Calixcrown and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to an antibody or antigen, with its activity determined by using tetramethylbenzidine (TMB) as an electrochemical substrate. The present study includes general considerations of the competitive immunoreaction protocols. Alanine aminotransferase (ALT) monoclonal antibody (anti-ALT-mAb) was successfully immobilized on thiol derivative of Calixcrown fixed to a gold surface. ALT antigen was detected by competitive immunoreactions based on microarrays of anti-ALT-mAb or antigen immobilized on the surface of the Calixcrownchip. For the anti-ALT-mAb immobilized microarray the dynamic range is 0.05 ng/mL–10 μg/mL, the detection limit is 0.05 ng/mL and the sensitivity is 10 nA/(ng/mL) respectively. The Calixcrownchip immunosensor microarray provided much better technical performance than a comparable enzyme sensor with immobilized-anti-ALT-mAb. To investigate the complexation site, the structures of the complexes formed between the crown-5-ether moiety of Calix[4]arene and protonated Arginine and Lysine were determined by minimizing the complex formation energies. The complex stability depends on the number of amine groups in the alkyl chain of the amino acid and also the number of methylene groups between the amine groups of the amino acid.

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 A450, 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.

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.

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.

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).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.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 CVs3% and5% 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).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.

Microalbuminuria (MAU), defined as a urinary albumin excretion of 30–300 mg/day, indicates a high probability of renal damage and is an accepted predictor for the early diagnosis of nephropathy in diabetic patients (1)(2). In addition, MAU has diagnostic implications in pregnancy as a predictive marker of preeclampsia (3)(4) and may play a role in identifying high risk of developing complications from cardiovascular diseases even in nondiabetic patients (5)(6)(7). Dye-binding assays can measure serum albumin but are too insensitive for MAU testing, making immunochemical assays the most widely used MAU methods (8). These immunoassays include immunoturbidimetry, immunofluorescence, ELISA, RIA, and zone immunoelectrophoresis. Recently, Kessler and coworkers (9)(10) introduced a laser-induced fluorescence system coupled to an automated centrifugal analyzer as a nonimmunologic assay for urinary albumin. Their system was based on the probe Albumin Blue 670/580, which becomes highly fluorescent on binding to albumin. We report a fluorescence immunochromatography assay (ICA) for quantitative determination of albumin in urine. The assay system consists of an ICA test strip in a disposable cartridge, a fluorescently labeled detector, and a laser fluorescence reader. Basically, the assay system adopts the inherent simplicity of a lateral-flow ICA and uses a competitive immunoassay mode with a simple, one-step operation (11). Briefly, fluorescently labeled albumin in the detector buffer competes with albumin in the sample for binding to an anti-albumin antibody immobilized on the test strip matrix. The more albumin is in the sample, the less the fluorescently labeled albumin reacts with the anti-albumin antibody and, thus, the lower the accumulation of fluorescence in the test line of a test strip. We generated a monoclonal antibody against human albumin (Sigma A8763) as an immunogen and conducted immunizations, cell fusion, and screening of hybridoma cells according to a standard …

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.

Immunoassays have become indispensable tools and achieved great importance in scientific and medical research. However, typical immunoassays are time‐consuming and use complex, multi‐step procedures. In this study, we introduce a new immunoassay system for the quantification of several analytes at a time without any washing steps. It is comprised of a detector solution with fluorescence‐labeled antibodies and a test strip with immobilized capture antibodies. Using a micro‐array scanner, the antigen‐anti body complex was quantitatively determined by measuring the intensities of fluorescence on the capture lines or dots of nitrocellulose membrane. This method demonstrated its rapid quantitative determination of analytes without many processing steps as well as specific identification of multiple analytes in biological specimens.

Airway mucin plays crucial role in host-defense and has been implicated in pathophysiology of various airway diseases including asthma and cystic fibrosis. The analysis of airway mucin has been hampered mostly by the lack of specific and efficient methods for the detection of mucin. Recent production of antibodies against airway mucin from several species and also the development of immunoassay procedures make it more efficient to study the airway mucin. However, the cross-species immunoreactivity of antibodies against airway mucin has not been clearly demonstrated and this prompted us to investigate the cross-species immunoreactivity of monoclonal antibodies against human (HM02), hamster (HTA), and rat airway mucin (RT03), which is three most widely used species in the study of mucin. All the monoclonal antibodies (MAbs) used in this study is IgM isotype and recognizes N-acetyl-galactosamine-linked carbohydrate core or backbone portion of airway mucin. In enzyme-linked immunoadsorbent assay (ELISA), Western blot, immunoprecipitation, and immunohistochemical staining experiments, it was demonstrated that human and hamster airway mucin showed strong cross-species immunoreactivity. However, rat airway mucin did not show any cross-species immunoreactivity against human and hamster airway mucin. Endotoxin-induced secretory cell metaplasia and hence the increase in mucin release from hamster airway mucin could be detected with antibodies against hamster and human airway mucin in vivo and in vitro. However, the same increase from rat airway could only be detected with antibody against rat airway mucin but not with antibodies against human and hamster airway mucin. In addition, the increase in mucin release from asthmatic patients could be detected with antibodies against human and hamster airway mucin but not with the antibody against rat airway mucin. The data from the present study implicates that the carbohydrate chain of human and hamster airway mucin, but not that of rat airway mucin, share common antigenic structure. In case of the interspecies use of the antibodies against airway mucin, it would be more desirable to clearly identify the cross-species immunoreactivity otherwise might lead to erroneous results.

To investigate whether the squamous differentiation of primary hamster tracheal epithelial cell, which is induced by retinoic acid deficiency or chronic PMA treatment, regulates Muc1 expression, we first produced and characterized a monoclonal antibody against hamster tracheal Muc1 mucin using pGEX-Muc1 fusion protein as an antigen and the changes of Muc1 mucin expression was determined by Western blot. Squamous differentiation downregulated the expression of Muc1 mucin from HTSE cells. The decrease in the immunoreactivity of Muc1 mucin was parallel to the decrease in the immunoreactivity of high molecular weight mucin, which is secreted from HTSE cells. The data from the present study implicate a possible role of Muc1 mucin in squamous differentiation of HTSE cells.