Plaque reduction test: an alternative method to assess specific antibody response to pIII-displayed peptide of filamentous phage M13.
Phage-displayed peptide systems have been used to identify the immunogenic epitopes and to develop the design of peptide-based or peptide-displaying phages themselves as vaccine candidates. To estimate the humoral immunity of phage-based vaccine, it is necessary to evaluate the antibody response specifically directed at the displayed peptide. Enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis are commonly used for this purpose. However, using these methods, it is not easy to distinguish the antibody response against phage coat protein or the antibody response specific to the displayed peptide. The purified anti-Mycoplasma hyopneumoniae IgG was used to screen heptapeptides displaying on the pIII coat protein of M13 phage. Four selected phage clones were chosen to immunize mice. In order to evaluate the specific antibody response that is directed against heptapeptides, advantage was taken of the natural property of M13 phage to infect Escherichia coli, which is mediated by the pIII coat protein binding with the F pili of E. coli, and plaque reduction tests were performed to assess the specificity of antibody response. By comparing the number of plaques produced by the different phages (which are the same except for the displayed peptides) neutralized by the antiserum, we could demonstrate that the specificity of antibody response is directed against the peptide displayed on pIII coat protein. The results described here indicate that plaque reduction test is a convenient and more precise method to detect the antibody against the phage-displayed peptide.
Yang
Wen-Jen
WJ
Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan, ROC. shiuan@mail.ndhu.edu.tw
Shiuan
David
D
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000235:genetics / Q000254:growth & development*; D036022:Capsid Proteins; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000821:virology; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D009174:Mycoplasma / Q000276:immunology; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology; D016415:Sequence Alignment; D014760:Viral Fusion Proteins / Q000235:genetics / Q000276:immunology*; D010948:Viral Plaque Assay / Q000379:methods*
D000907:Antibodies, Bacterial; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D009842:Oligopeptides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins