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).
Kashiwakuma
T
T
Tonen Corporation, Saitama, Japan.
Hasegawa
A
A
Kajita
T
T
Takata
A
A
Mori
H
H
Ohta
Y
Y
Tanaka
E
E
Kiyosawa
K
K
Tanaka
T
T
Tanaka
S
S
Hattori
N
N
Kohara
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000915:Antibody Affinity; D000918:Antibody Specificity; D018604:Epitope Mapping; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D016174:Hepacivirus / Q000737:chemistry / Q000276:immunology*; D006508:Hepatitis Antibodies / Q000276:immunology; D018963:Hepatitis Antigens / Q000032:analysis*; D006526:Hepatitis C / Q000175:diagnosis*; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D013050:Spectrometry, Fluorescence; D014758:Viral Core Proteins / Q000097:blood*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D006508:Hepatitis Antibodies; D018963:Hepatitis Antigens; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus