Detection and quantitation of HCV core protein in single hepatocytes by means of laser capture microdissection and enzyme-linked immunosorbent assay.
Immunohistochemistry provides valuable information concerning the localization and distribution of hepatitis C virus (HCV)-related proteins in histological sections of liver tissue, but does not readily permit their quantitation in individual cells and the staining intensity of cell immunodeposits cannot be calibrated with the current number of antigen molecules. We specifically detected and quantitated HCV core protein in single hepatocytes by coupling laser capture microdissection (LCM) with a sensitive enzyme-linked immunosorbent assay (ELISA). Quantitation of HCV core protein per cell was carried out on liver tissue cells obtained by LCM from fixed and stained frozen sections of 10 HCV-positive patients with chronic active hepatitis (CAH). Macromolecules from captured cells were solubilized in an extraction buffer and directly assayed for core protein using a sandwich ELISA. Calibration was achieved by developing a standard curve based on known concentrations of HCV core protein. Precision, linearity and sensitivity were verified for known numbers of microdissected tissue cells. In this study, the concentration of HCV core protein in single hepatocytes ranged from 7 to 56 pg/cell. Specificity was verified on 10 replicates of 10 HCV-negative liver tissues. Immunohistochemical staining of HCV core protein was compared with the results of the soluble immunoassay for the adjacent liver tissue sections. Independent scoring of HCV immunostaining failed to parallel the LCM quantitative immunoassay. LCM-based immunoassay significantly expands our ability to investigate function-related antigens in apparently pure cell populations in HCV infection.
Sansonno
D
D
Section of Internal Medicine and Clinical Oncology, Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy.
Lauletta
G
G
Dammacco
F
F
D000368:Aged; D001706:Biopsy; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D016174:Hepacivirus; D019698:Hepatitis C, Chronic / Q000473:pathology / Q000821:virology*; D022781:Hepatocytes / Q000737:chemistry* / Q000821:virology*; D006801:Humans; D007834:Lasers; D008099:Liver / Q000473:pathology / Q000821:virology; D008297:Male; D042282:Microdissection / Q000379:methods*; D008875:Middle Aged; D012367:RNA, Viral / Q000097:blood; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014758:Viral Core Proteins / Q000032:analysis* / Q000276:immunology; D014766:Viremia
D012367:RNA, Viral; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus