Cell-based panning as a means to isolate phage display Fabs specific for a bacterial surface protein

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model

“…In both cases, reactive antibodies were isolated by screening against adhesive (+ fimbriae) and non‐adherent (– fimbriae) strains of S. parasanguis FW213. The specificity of MAbs F51, Ph5, and Ph12 for the Fap1 protein was demonstrated previously (Stephenson et al , 1998; Wu et al , 1998). In this study, the specificity of MAbs D10, A6, B20, and E42 for the Fap1 protein was demonstrated.…”

“…The antibodies used in this study are summarized in Table 1. Three monospecific antibodies (F51, Ph5 and Ph12) had been characterized previously with specificity for the Fap1 protein that runs at an apparent molecular weight of 200 kDa on SDS–PAGE (Stephenson et al , 1998; Wu et al , 1998). Western blot analysis revealed that the four remaining MAbs (D10, B20, E42 and A6) also reacted with the 200 kDa protein, thus demonstrating specificity for the Fap1 protein (data not shown).…”

“…The antibodies used in this study were produced using two techniques: phage display technology (Barbas et al , 1991) and standard hybridoma methodology (Kohler and Milstein, 1975). Two phage display Fabs, Ph5 and Ph12, were derived from mice immunized with purified Fap1 protein (Stephenson et al , 1998). Five hybridoma monoclonal antibodies (A6, B20, D10, E42 and F51) were derived from mice immunized with whole FW213 cells (Elder and Fives‐Taylor, 1986).…”

“…Desired cell densities were determined based on a growth curve generated using an absorbance of 470 nm. Escherichia coli XL1‐Blue containing pCOMBIII plasmids were grown on Superbroth (SB) agar plates or liquid cultures containing selective antibiotics as described previously (Stephenson et al. , 1998).…”

“…In addition, five monoclonal antibodies (MAb) and two monospecific Fabs (fragment antigen binding) are reactive with the adhesive, fimbriated FW213 but fail to react with non‐adhesive, non‐fimbriated mutants (Fachon‐Kalweit et al. , 1985; Stephenson et al , 1998).…”