Tadahiro Kajita

Detection of hepatitis C virus specific core protein in serum of patients by a sensitive fluorescence enzyme immunoassay (FEIA)

A protein-capture fluorescence enzyme immunoassay (FEIA) was developed using monoclonal antibodies (mAbs) against recombinant hepatitis C virus (HCV) core protein. Four hybridoma cell lines (5E3, 5F11, 515S, 1080S) were established and characterized. These monoclonal antibodies (mAbs) each had IgG1 and OgG2 isotypes, and recognized major B cell epitopes within the immunodominant nucleoprotein amino terminal subregion. Using mAb 5F11 as the first antibody to the solid phase and beta-D-galactosidase-conjugated mAb 5E3 as the second antibody to the protein, we established a specific HCV core protein capturing FEIA capable of detecting as little as 20 pg/ml of recombinant HCV core protein. HCV core protein in serum was detectable after treatment with 4.0% polyethyleneglycol, 0.5 NaOH, and 5% Triton X-100. The results of a peptide inhibition assay indicated that this FEIA is specific for HCV RNA positive sera. The quantity of HCV core protein detected in serum was significantly correlated to the level of HCV RNA. The detection limit for HCV core proteins was an HCV RNA per titer of approximately 10(4)/ml. Using this FEIA system, the detection ratio of HCV core protein in patients with chronic HCV infection was 92.3% (70/76).

Measurement of plasma annexin V by ELISA in the early detection of acute myocardial infarction

Annexin V is a calcium binding protein which is widely present in various cells and tissues. Using annexin V which we isolated and purified from human cardiac muscle, we prepared an anti-human cardiac annexin V monoclonal antibody. Identification of annexin V was made by means of partial amino acid sequences. An enzyme-linked immunosorbent assay (ELISA) was developed using this monoclonal antibody and anti-canine cardiac annexin V polyclonal antibody. With this ELISA, plasma annexin V concentration was measured in 196 normal healthy individuals, 23 acute myocardial infarction (AMI) patients who were hospitalized within 6 h after the onset of chest pain, and 130 patients with other diseases, including lung, liver and kidney disease. The plasma annexin V concentration in normal healthy individuals was 1.7 +/- 0.6 ng/ml (mean +/- S.D.), while that in AMI patients was elevated to 13.2 +/- 6.8 ng/ml (P < 0.0001) at the time of initial blood drawing, 3.2 +/- 1.5 h after onset of pain, and these values were higher than normal in 21 out of 23 cases (91.3%) of AMI. In all cases excepting 3, annexin V concentration immediately decreased after the onset of pain. The annexin V concentration in patients with old myocardial infarction, chest pain syndrome, valvular heart disease, lung disease and kidney disease was 1.8 +/- 0.8, 2.0 +/- 0.7, 1.7 +/- 1.1, 2.3 +/- 1.4 and 2.1 +/- 1.2 ng/ml, respectively, being within normal limits. The values in liver disease patients and trauma patients were 3.7 +/- 2.7 (P < 0.05) and 3.3 +/- 2.4 (P < 0.05) ng/ml, respectively, being slightly higher than that in normal healthy individuals.