Figure 1.
Comparison of colorimetric focus-forming assays using various secondary antibodies and chromogenic substrates.
(A) Huh-7.5 cells inoculated with serial dilutions of HCV were immunostained with monoclonal anti-HCV core antibody and secondary antibodies conjugated with different enzymes, as indicated, followed by chromogenic development using various substrates, and image scanning by ELISpot reader. (B) A magnified view of the scanned image of the colorimetric focus-forming assay revealed that alkaline phosphatase-conjugated secondary antibody with BCIP/NBT yielded the best results, considering background and distinctness of the foci. Biotin-2°Ab, biotin-conjugated secondary antibody; SA-AP, streptavidin-conjugated alkaline phosphatase; AP-2°Ab, alkaline phosphatase-conjugated secondary antibody; HRP-2°Ab, horseradish peroxidase-conjugated secondary antibody; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; DAB, 3,3′-diaminobenzidine; TMB, 3,3′,5,5′-tetramethylbenzidine.
Figure 2.
Microscopic images of HCV foci, developed using various secondary antibodies and chromogenic substrates.
(A) Biotin-conjugated secondary antibody in conjunction with streptavidin-conjugated alkaline phosphatase and BCIP/NBT. (B) Alkaline phosphatase-conjugated secondary antibody and BCIP/NBT. (C) Horseradish peroxidase-conjugated secondary antibody and DAB. (D) Horseradish peroxidase-conjugated secondary antibody and DAB. (E) Fluorescence-conjugated secondary antibody. Magnification, 100×.
Figure 3.
Prevention of cell detachment by type I collagen pre-coating.
(A) The scanned image of a colorimetric focus-forming assay emphasizes the importance of pre-coating the plate with type I collagen for preventing cell detachment during the vigorous washing steps in the assay. (B) Magnified view of boxed images in (A). (C) The culture plate was pre-coated with either poly-D-lysine 4 μg/cm2 or type I collagen 5 μg/cm2, and the colorimetric focus-forming assay was performed. Type I collagen coating was superior to poly-D-lysine coating in terms of prevention of cell detachment.
Figure 4.
Automated quantification of HCV foci by image analysis.
(A) Several parameters, including spot size, sensitivity, background balance, and others, were adjusted to define the foci for automated focus counting. (B) The defined foci are encircled in red, and the number of counted foci is denoted in the left upper corner. (C) A histogram depicting the distribution of foci size in a well.
Figure 5.
Comparison of colorimetric assay with immunofluorescence-based focus-forming assay.
(A) The number of foci in each well determined by colorimetric focus-forming assay with image analysis strongly correlated with that determined by immunofluorescence-based focus-forming assay and manual counting. (B) The virus titers, calculated using the foci numbers obtained by the two different methods, also showed significant correlation. (C) The determined viral titers in six different batches of HCVcc indicated no significant differences between the results of the two types of focus-forming assay.