Cross-genotypic polyclonal anti-HCV antibodies from human ascitic fluid

Abstract

Many anti-HCV antibodies are available, but more are needed for research and clinical applications. This study examines whether ascitic fluid from cirrhotic patients could be a source of reagent-grade antibodies. Ascitic fluid from 29 HCV patients was screened by ELISA for anti-HCV antibodies against three viral proteins: core, NS4B, and NS5A. Significant patient-to-patient variability in anti-HCV antibody titers was observed. Total ascitic fluid IgG purified by Protein-A chromatography reacted with HCV proteins in immunoblots, cell extracts, and replicon-expressing cells. Affinity-purification using synthetic peptides as bait allowed the preparation of cross-genotypic antibodies directed against pre-selected regions of HCV core, NS4B, and NS5A proteins. The performance of the polyclonal antibodies was comparable to that of monoclonal antibodies. Anti-NS4B antibody preparations reacted with genotype 1a, 1b, and 2a NS4B proteins in immunoblots and allowed NS4B to be localized in replicon-expressing cells. Ascitic fluid is an abundant source of human polyclonal cross-genotypic antibodies that can be used as an alternative to blood. This study shows the utility of selectively purifying human polyclonal antibodies from ascitic fluid. Affinity purification allows antibodies to be selected that are comparable to monoclonal antibodies in their ability to react with targeted regions of viral proteins.

Figure 1. Anti-HCV antibodies in ascitic fluid react with HCV peptides in ELISAs

Panels A–C. The ascitic fluid of 28 patients with HCV mono-infection and one patient with HIV/HCV (#29) infection were tested in an indirect antibody ELISAs using the Core-2 peptide (amino acids 7–25), NS4B (amino acids 1901–1922), and NS5A (amino acids 2297–2314). Normalized values greater than 1.0 were considered positive.

Figure 2. IgG fractions from ascitic fluid produce strong and specific signals from viral proteins in immunoblots and immunofluorescence assays of Huh-7.5 cells harboring HCV replicons

Panels A–E: Immunoblots reacted with total IgG populations purified from ascitic fluid are presented in Panels A–C. IgG preparations from three different subjects were reacted with proteins extracted from Huh-7.5 control cells (lane a), or cells producing HCV proteins (lanes b and c). Specifically, Huh-7.5 cells with SG JFH-1 replicons (lane b) and cells with FL J6/JFH replicons (lane c) are shown. Size markers are shown on the left. Panel A shows the protein bands that react with ascitic fluid IgG from subject #7; Panel B shows the proteins that react with ascitic fluid IgG from subject #8; and Panel C shows the proteins that react with ascitic fluid IgG from subject #2. The p21 core protein is identified by an arrowhead; the core-neomycin fusion protein is marked by an arrow; the NS5A protein is designated by a closed circle, and the E2 envelope protein is denoted by a star. A blot reacted with a mixture of anti-core and anti-NS5A mouse monoclonal antibodies is shown in Panel D, and a blot reacted with an anti-E2 rat monoclonal is shown in Panel E. Panels F–K: The IF signals produced by IgG preparations reacted with Huh-7.5 cells harboring FL H77 bicistronic replicons (Panels F–H) or Huh-7.5 cells without replicons (Panels I–K) are shown. Prior to IF microscopy, cells were incubated with goat-anti-human IgG conjugated to Alexa Fluor 594 and with DAPI, which stains nuclei blue. Cells exposed to IgG #7 are shown in Panels F and I; cells exposed to IgG #8 are shown in Panels G and J; and cells exposed to IgG #2 are shown in Panels H and K.

Figure 3. Enrichment of anti-Core-2 antibody

Levels of anti-Core-2 and anti-Core-5 antibodies in the total IgG population of ascitic fluid from subject #8 are compared in Panel A. The total IgG population was then exposed to a Core-2 peptide column and the flow-through was collected. Depleted levels of anti-Core-2 antibodies were noted compared to anti-Core-5 (Panel B). The peak elution from the Core-2 column contained a 534-fold enrichment of anti-Core-2 antibodies and undetectable levels of anti-Core-5 antibodies (Panel C).

Figure 4. Affinity-purified anti-Core, anti-NS4B, and anti-NS5A antibodies from ascitic fluid produce clear immunoblot and immunofluorescent signals

Panel A–D show proteins from Huh-7.5 cells harboring either FL J6/JFH replicons (lane a), SG JFH replicons (lane b), or no replicons (lane c). Proteins were reacted with the total IgG of subject #29 (Panel A). Antibodies were affinity-purified from IgG #29 using the Core 2 peptide (amino acids 7–25; Panel B), and Core 3 peptide (amino acids 28–41; Panel C). An arrow indicates the position of the core-neomycin fusion protein and an arrowhead marks the position of the p21 core protein. Panel D shows that antibodies affinity-purified using the NS5A peptide (amino acids 2297–2341) reacted with NS5A protein expressed by SG JFH (lane a) and by FL J6/JFH (lane b). These antibodies did not react with proteins of Huh-7.5 control cells (lane c). Panel E indicates the positions of the NS5A and p21 core proteins expressed by SG JFH (lane a) and FL J6/JFH (lane b) in blots reacted with mouse monoclonal antibodies. A closed circle marks the position of the NS5A protein and an arrowhead identifies the position of the p21 core protein. Size markers are presented on the left hand side of the figure.

Figure 5. Detection of NS4B protein using affinity purified anti-NS4B polyclonal antibodies

In Panel A–E antibodies affinity-purified from IgG #2 are used. Panel A shows that human polyclonal anti-NS4B antibodies react with NS4B of genotype 1a (lane a), genotype 1b (lane b), and genotype 2a (lane c) proteins in Western blots, but did not react with proteins from Huh-7.5 control cells (lane d). A closed square shows the position of the NS4B protein. Panels B–E show that affinity-purified anti-NS4B antibodies produced an intense IF signal in Huh-7.5 cells harboring replicons (Panel B). This signal was blocked by incubation with 1 μg/ml of the NS4B peptide (Panel C), but it was not blocked by incubation with10 μg/ml of PDC-E2, an irrelevant peptide (Panel E). No signal was produced when the anti-NS4B antibodies were incubated with control Huh-7.5 cells (Panel D). The NS4B protein in panels B and C is from H77 HCV (genotype 1a) and the NS4B protein in panel E is from Con1 (genotype 1b). DAPI-stained nuclei are blue.