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

A Preliminary Study on the Development of a Fluorescence Immunochromatographic Assay for the Rapid Quantification of the Thyroid Stimulating Hormone in Serum Sample / 대한진단검사의학회지

BACKGROUND: Since the first introduction of radioimmunoassay for the quantification of the thyroidstimulating hormone (TSH), more advanced analytical methods have been developed and used in laboratories. However, they are still inconvenient in that they require time-consuming procedures, special safety in handling isotopes, expensive equipment, and a highly qualified expert. METHODS: As an immunoassay system for the rapid measurement of TSH in serum, we have developed a new analytical system based on immunochromatographic assay with fluorescencelabeled anti-TSH monoclonal antibodies. The assay system is composed of a test strip housed within a cartridge and a laser-fluorescence scanner for quantification. The strip contains a sample pad, an absorption pad, and a nitrocellulose membrane where a captured antibody is immobilized and antigen-antibody reaction occurs. Fifty microL of serum was added to 50 microL of a detector solution and the mixture was loaded onto the well of the sample pad on the cartridge. After incubation for 12 min, the cartridge was quantified with the laser-fluorescence scanner. RESULTS: The calibration curve displayed linearity (R=0.95) at concentrations of 1-40 mIU/L. Intraand inter-assay imprecisions were determined to be CVs within 10%. Analytical recovery was 93.9% at 3 different concentrations and the detection limit was 0.868 mIU/L of TSH. The new assay system correlated well with an Abbott AxSYM for quantification of TSH (R=0.97, slope 0.94, N=20). CONCLUSIONS: The TSH measurement system developed in this study showed good reproducibility. However, our TSH quantification system needs some improvement to be used in the medical field because of its low analytical sensitivity. With enhanced performance in analytical sensitivity, introduction of a whole-blood type strip, and a more miniaturized fluorescence scanner, we expect the TSH analytical system to be used for point-of-care testing in the near future.

Development a Fluorescence Immunochromatographic Assay for Rapid Quantification of beta-hCG in Serum Sample / 대한산부인과학회잡지

OBJECTIVE: To develop an immunoassay system for the rapid measurement of beta-hCG in serum sample, we designed an immunochromatographic assay-based one-step assay that uses two monoclonal antibodies to beta-hCG as a sandwich pair. METHODS: The assay system is composed of a test strip housed within a cartridge for sample application and a laser-fluorescence scanner for quantification. The strip contains a sample pad, an absorption pad, and a nitrocellulose membrane where a capture antibody is immobilized and antigen-antibody reaction occurs. Ten L of serum was added to 60 L of detector solution, and the mixture was loaded onto the well of the sample pad on the cartridge. After incubation for 12 min, the cartridge was scanned for quantification with the laser-fluorescence scanner. RESULTS: No cross-reactivity was observed between beta hCG antibodies and other pituitary hormones. The calibration curve displayed linearity (R2=1) at concentrations of 0-1,000 IU/L. Intra- and interassay imprecisions were determined with serum samples to be their CVs within <6% and <7%, respectively. Analytical recovery was 103-108.4% in serum samples at three different concentrations. There was high correlation between beta-hCG concentrations measured by Boditech system and those measured by Abbot AxSYM system. CONCLUSION: The new fluorescence assay system is a convenient and fast method and can be used as a good tool for detection and quantification of beta-hCG in serum samples. Furthermore, the portable system allows us to perform the tests on a sampling site in any places without bringing the samples to a laboratory.

Development of a Method for the Immunological Measurement of Aspartate Aminotransferase with Monoclonal Antibodies / 대한간학회지

BACKGROUND/AIMS: For laboratory diagnostics in liver diseases, many enzymes have been used for the assessment of hepatocellular function. Among them, two transaminases, alanine and aspartate aminotransferase, have been regarded as the most sensitive indicators of hepatocellular damage. However, the enhanced enzyme activities of the enzymes do not exactly indicate or represent the cause and progression of diseases in the patients with liver disease. To overcome such limitations, immunological methods have been suggested as one of the alternatives for the replacement or supplement of the conventional enzymatic analysis. METHODS: In the hope of developing a new assay system for measuring the AST concentration rather than its activity, we have developed a new assay using fluorescence labeled anti-AST monoclonal antibodies. Blood was obtained from a normal population of 234 patients and 43 liver disease patients. The linearity, limit of detection, and performance of the new assay system were tested and evaluated. The comparability of assay was examined with an ELISA and biochemical assays. RESULTS: The linearity fell in the range of 0-1 mg/L of AST (R=0.995), and the analytical detection limit was 12 microgram/L of AST. The mean recovery of the control was 102.4 % in a working range. The precision of the intra- and inter-assay in a range of 50-800 microgram/L was CVs < 7% and CVs < 6%, respectively. In the normal population, the mean AST concentration was 35.5 microgram/L. The mean AST concentration in patients with liver disease was 266.5 microgram/L. The new assay system correlated well with an ELISA and biochemical assay for quantification of AST concentration (R=0.92 and 0.88, respectively; N=43). CONCLUSIONS: We have developed a new immunological assay using generated monoclonal antibodies to human cytosolic AST and used them for the development of a fluorescent assay measuring the enzyme mass. Cytosolic AST mass in sera could be measured reproducibly by the immunological method. In conclusion, this study has provided us with a new type of tool for an accurate measurement of the enzyme amount in circulation.

Usefulness of Rapid PSA Kit(R) / 대한비뇨기과학회지

PURPOSE: The incidence rate of prostate cancer has increased remarkably in Korea. The serum prostate specific antigen (PSA) value has been used for screening, although its clinical significance in prostate cancer screening is still inconclusive. However, if the measurement time was short and the cost was low, such an assay kit should be sufficient for prostate cancer screening. MATERIALS AND METHODS: We developed pared monoclonal antibodies against PSA which could be used in assay kits for PSA. The Rapid PSA Kit(R) used an immunochromatographic method to qualitatively judge a positive or negative result. Serum specimens from 78 men with benign prostate hyperplasia or prostate cancer were tested using the kit. RESULTS: The sensitivity of the kit was determined to be 4ng/ml. 33 samples had a value of greater than 5ng/ml, so were considered positive. 5 samples had values between 4ng/ml and 5ng/ml, of which 3 were positive. The other 40 samples had values less than 4ng/ml, and 11 of these were judged positive. These results indicated that the sensitivity and specificity of the Rapid PSA Kit(R) were 94.7 and 72.5%, respectively. CONCLUSIONS: Tests using the Rapid PSA Kit(R) can be easily performed at outpatient clinics or elsewhere. This kit is useful in the initial screening of prostate cancer as the results can be obtained within 15 minutes and the cost is lower than with ordinary serum PSA tests.

Usefulness of Rapid PSA Kit(R) / 대한비뇨기과학회지

PURPOSE: The incidence rate of prostate cancer has increased remarkably in Korea. The serum prostate specific antigen (PSA) value has been used for screening, although its clinical significance in prostate cancer screening is still inconclusive. However, if the measurement time was short and the cost was low, such an assay kit should be sufficient for prostate cancer screening. MATERIALS AND METHODS: We developed pared monoclonal antibodies against PSA which could be used in assay kits for PSA. The Rapid PSA Kit(R) used an immunochromatographic method to qualitatively judge a positive or negative result. Serum specimens from 78 men with benign prostate hyperplasia or prostate cancer were tested using the kit. RESULTS: The sensitivity of the kit was determined to be 4ng/ml. 33 samples had a value of greater than 5ng/ml, so were considered positive. 5 samples had values between 4ng/ml and 5ng/ml, of which 3 were positive. The other 40 samples had values less than 4ng/ml, and 11 of these were judged positive. These results indicated that the sensitivity and specificity of the Rapid PSA Kit(R) were 94.7 and 72.5%, respectively. CONCLUSIONS: Tests using the Rapid PSA Kit(R) can be easily performed at outpatient clinics or elsewhere. This kit is useful in the initial screening of prostate cancer as the results can be obtained within 15 minutes and the cost is lower than with ordinary serum PSA tests.

Purification and Characterization of Transforming growth factor - beta1 from Human Platelets / 대한면역학회지

Transforming growth factor-j31 (TGF-p1) has potential for therapeutic use in common clinical conditions for which there are no adequate pharmacological agents. However, in vivo studies using TGF-p1 were hindered by high price of this cytokine. As a first step towards large scale purification of TGF-p1, it was purified in a small scale (10 unit platelets) from human platelets by four purification steps: platelet extraction, gel filtration, cation exchange chromatography, and reversed phase high performance liquid chromatography (HPLC). A single protein band with a molecular weight of 25 Kd corresponding to purchased TGF-p1 (R8D Systems) was confirmed by silver staining after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of eluant from reversed phase HPLC. Recovery (%) of each step was about 50-60%, resulting in the final recovery of 20% based on the detection by a sandwich ELISA. Approximately, 3.7 p,g of purified TGF-p1 was obtained from 18 pg of platelet extracts. This result was confirmed by receptor (TGF-j31 type II) ELISA and bioassay using a mink lung epithelial'cell line (MV1LU). Further, in vitro characterization study showed that purified TGF-p1 inhibits G1/S transition of LPS-activated murine spleen B cells and increases surface IgA expression by the same cell population, which are typical activities of TGF-p1 in B cell differentiation. Taken together, the results from the present study reveals that purified TGF-p1 is fully biologically active and our purification methodology could be usbful to obtain a large scale of recombinant TGF-p1 in the future.

Erythrocyte Band 7 Integral Protein Defect in Congenital Hemolytic Anemia: Hereditary Stomatocytosis

Hereditary stomatocytosis is a rare congenital hemolytic anemia, named after mouth shaped or stomatocytic erythrocyte morphology. In this report, we present a case of a hereditary stomatocytosis in a 1 month old boy. During the initial identification process, we overlooked the morphology of the RBC in peripheral blood smear and tentatively diagnosed it to be a hereditary spherocytosis case. In order to study further, we isolated RBC membranes from the patient, separated membrane proteins by SDS polyacrylamide gel electrophoresis, and found that a protein band, band 7, was missing in the patient. We suggest that erythrocyte morphology as well as erythrocyte membrane protein analysis is an important criterion in the diagnosis of hereditary hemolytic anemia.