Sang Wook Oh

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.

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.

Sandwich ELISA for Measurement of Cytosolic Aspartate Aminotransferase in Sera from Patients with Liver Diseases

With the introduction of modern biochemical techniques, more than 50 enzymes have been developed for diagnostics in liver tests. Among the enzymes, aspartate aminotransferase (AST; EC 2.6.1.1; formerly known as GOT) was introduced in the mid-1950s and has remained the mainstay of enzyme diagnosis for liver disease for more than half a century (1). AST is widely distributed among human organs as cytosolic and mitochondrial isotypes (2). Increased activity of this enzyme is considered one of the most sensitive indicators of hepatocellular damage (3). Previously, several attempts were made to develop an immunoassay procedure for measuring AST mass rather than enzyme activity, but they are currently not used for the diagnosis of liver diseases (4)(5)(6)(7)(8). In this study, we generated monoclonal antibodies (mAbs) to AST and used them to develop a sandwich ELISA to measure the enzyme mass in sera. Because the amino acid sequences of the human and porcine enzymes are similar, we used porcine AST as an immunogen for the production of mAbs (9). The porcine enzyme is commercially available as a highly purified form in large quantities. Twenty hybridoma cells to porcine AST were initially screened by ELISA from several fusions, and four clones were selected for further characterization. To confirm the specificity of the mAbs to the human enzyme, we immunoblotted partially purified human enzyme and total proteins extracted from a human liver with the mAbs. The antibodies specifically recognized a single protein band of 45 kDa, which coincided well with the expected size of AST. To evaluate whether the mAbs recognized a cytosolic or a mitochondrial form of AST in human tissues, we prepared cytosolic …

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.

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

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.

A rapid and sensitive detection of HPV by combined assay of PCR and fluorescence DNA chip

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.

Fluorescence immunochip assay for thyroid stimulating hormone 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<0.001). The developed FL immunochip assay is the only method that quantifies TSH concentration in whole blood and meets the criteria of POCT including affordable cost, a disposable device, and requiring minimum maintenance to perform test.

Common Salivary Protein 1 in Serum of Diabetes Patients.

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.

An universal biolinker for immobilization of protein and oligoDNA on a glass slide chip

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.

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.