Hrp conjugated polyclonal goat anti rabbit igg

To detect Mfa1 fimbriae expressed on the cell surface, filtration ELISA was performed as previously described [13 (link)] using intact cells as the antigens. A 100 μL cell suspension containing 1 × 10⁷ cells was applied over filters in a 96-well MultiScreen-GV filtration plate with a pore size of 0.22 μm (MilliporeSigma, Burlington, MA, USA). Subsequently, the cells were washed with Tris-buffered saline containing 20 mM Tris pH 7.5, 150 mM NaCl, and 0.05% Tween 20 (TBST) and blocked with 3% bovine serum albumin in TBST. The cells were then probed with antiserum against each Mfa1 fimbriae type, washed, and labeled with HRP-conjugated polyclonal goat anti-rabbit IgG (Dako, Glostrup, Denmark). Subsequently, o-phenylenediamine and H₂O₂ in citrate buffer (pH 5.0) were added as the substrates. The reactions were terminated using 1 M H₂SO₄ and the absorbance at 490 nm (OD490) was measured. SMF1 was included as a negative control and the SMF1 value was subtracted from each measurement of the strains.

To detect Mfa1 fimbriae exposed on the cell surface, filtration ELISA was performed using intact cells as antigen [30 (link)]. A 100 μL cell suspension containing 5×10⁶ cells was applied over filters in a 96-well MultiScreen-GV filtration plate with pore size 0.22 μm (EMD Millipore, MA). Subsequently, wells were washed with a buffer consisting of 20 mM Tris pH 7.5, 150 mM NaCl, and 0.05% Tween 20 (TBST), and blocked with 3% bovine serum albumin in TBST. Wells were then probed with a 1,000-fold dilution of antiserum against Mfa1-only fimbriae, washed, and then labeled with HRP-conjugated polyclonal goat anti-rabbit IgG (Dako, Glostrup, Denmark). Subsequently, o-phenylenediamine and H₂O₂ in citrate buffer pH 5.0 were added as substrate. Reactions were terminated with 1 M H₂SO₄ and absorbance at 490 nm (OD₄₉₀) was measured. To normalize Mfa1 expression to the amount of intact cells, filtration ELISA was also performed using antibodies raised against P. gingivalis whole cells [31 (link)].