Production of Monoclonal Antibodies Specific to FimA of Porphyromonas gingivalis and Their Inhibitory Activity on Bacterial Binding

Purification of fimbriae protein from P. gingivalis

Fimbriae of P. gingivalis 2561 was purified according to the method described by Lee et al (16). Briefly, after harvesting cells of P. gingivalis strain 2561, the bacterial cells were subjected to mild ultrasonication (Vibra Cell, Model VC-600, Sonic and Materials Inc., Danbury, CT). After ultrasonication, crude fimbriae of the sonic extract were obtained by centrifugation. The fimbriae-containing supernatant was brought to 40% saturation by stepwise addition of solid ammonium sulfate and stirred at 4℃ overnight. The precipitated crude fimbrial extract was dialyzed and clarified by centrifugation. The clarified crude fimbrial extract was mixed with guanidine HCl (UltraPURE, enzyme grade; BRL, Gaithersburg, MD) and subjected to gel filtration on a Sepharose CL-6B (Pharmacia Biotech, Sweden) column equilibrated with 6 M guanidine HCl. The fimbriae were purified by repeated Sepharose column chromatography. Homogeneity of the purified fimbrial protein was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Preparation of B hybridoma clones

Syngeneic BALB/c mice were purchased from Orient Bio (Sungnam, Korea) and immunized with 20 g/mouse FimA protein emulsified with complete Freund's adjuvant. The same mice were boosted with the same antigen emulsified with incomplete Freund's adjuvant. The spleen cells of immunized mice were fused with SP2/0 myeloma cells (ATCC, Manassas, VA) at a ratio 1:1 using PEG 1500 (Roche Diagnostics GmbH., Mannheim, Germany). The fused cells were spread onto 96-well culture plates in Dulbecco's modified Eagle's medium (Hyclone Laboratories, Logan, UT) supplemented with HAT and 20% FBS (PAA, Etobicoke, Ontario, Canada). Generated hybridoma clones were subcloned three times by limiting dilution procedure and the binding specificity of the antibodies produced by the hybridoma clones were determined through ELISA and Western blot analyses.

Determination of antibody specificity and isotypes

Specificity of antibodies produced from established hybridoma clones was determined by ELISA and Western blot analyses. For ELISA, briefly, 96-well plates were coated with native FimA protein (65 ng/well) and blocked with 5% non-fat dried milk. After the blocking, culture supernatants of hybridoma clones were added. Following incubation, plates were washed and AP-conjugated anti-mouse antibodies were added to wells. Finally, plates were incubated with the substrate of p-nitrophenyl phosphate and absorbance at 405 nm was measured using ELISA reader (Packard Instrument, Downers Grove, IL). The isotype of antibodies produced from stabilized hybridoma clones was determined using isotyping kit according to the protocol suggested by the supplier.

Western blot analysis was conducted using two different conditions of antigen preparation were used to determine the specificity of the antibodies. In order to prepare monomeric FimA, FimA protein was incubation at 100℃ for 10 min in a sample buffer containing 60 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, glycerol and 2-mercaptoethanol. In order to prepare partially depolymerized FimA, FimA protein was incubated at 80℃ for 5 min in a sample buffer without 2- mercaptoethanol. Two micrograms per well of prepared FimA proteins were loaded and separated in 10% SDS PAGE. The resolved proteins were transferred to nitrocellulose membranes (Whatman, Dassel, Germany) and blocked with 5% skim milk. The blotted membranes were incubated with hybridoma culture supernatants or diluted-polysera, followed by incubation with AP-conjugated anti-mouse secondary antibodies.

Ascites fluid preparation

Three 12-week old male BALB/c mice were inoculated with 0.5 ml of pristine through an intra-peritoneal injection. After 7 days, 1×10⁶ hybridoma cell producing monoclonal antibodies, which had been harvested and washed in PBS for twice, were injected peritoneally to induce ascites production.

Inhibition assay for bacterial attachment

Antibody-mediated inhibition of bacterial binding assay was performed with saliva-coated hydroxyapatite (SHA) beads as described in a previous study (17). Briefly, P. gingivalis 2561 grown overnight was washed in buffered KCl twice and adjusted to OD₆₀₀=0.1, which is equivalent to 1×10⁸ CFU/ml. The bacterial suspension (100 µl) was added to a series of tubes containing 2 mg SHA beads and brought to a final volume of 400 µl with buffered KCl. Simultaneously, antibody was added to the tubes in a final dilution of 1:100, 1:200 and 1:400. This mixture was incubated at room temperature. After an incubation time, the reaction mixture was layered on 1 ml of 100% Percoll in a new siliconized borosilicate tube to separate cells which were free from those bound to the SHA beads. Unbound, free P. gingivalis cells floating on the Percoll layer were removed and the beads with bound cells and the wall of the tube were then washed.

Quantitative real-time PCR

Quantitative real-time PCR (QRT-PCR) of bacterial cells bound to the SHA beads was performed as described previously (18). Genomic DNA was extracted from the bound bacteria in each test tube using DOKDO-prep™ Bacterial Genomic DNA Purification Kit (ELPIS-Biotech Inc., Korea) and used as template. To obtain standard curves for QRT-PCR that relates bacterial concentration to Ct, the turbidity of bacterial suspension of P. gingivalis 2561 grown overnight was adjusted to OD₆₀₀=0.1 (1×10⁸ CFU/ml). For amplification of P. gingivalis 16S rRNA gene, 3 µl of each template was added to the reaction mixture containing 10 µl of iQ™ SYBR® Green SuperMix (Bio-Rad, Hercules, CA) and 250 nM of each primer, Pg16S-F (TGT TAC AAT GGG AGG GAC AAA GGG) and Pg16S-R (TTA CTA GCG AAT CCA GCT TCA CGG), which were prepared based on the primer sequence published previously (19), in a final volume of 20 µl. QRT-PCR was carried out using MiniOpticon™ Real-Time PCR Detection System (Bio-Rad). The reaction was cycled with preliminary denaturation for 5 min at 95℃, followed by 40 cycles of denaturation at 95℃ for 10 sec, annealing at 61℃ for 20 sec and primer extension at 72℃ for 30 sec. To confirm that a single PCR product was amplified, melting curve analysis was performed with the following condition: 65℃ to 95℃ with a heating rate of 0.2℃ per 1 sec. The amount of bacterial DNA in each sample was quantified by interpolation of the corresponding Ct values in the standard curves. Inhibitory efficiency of monoclonal antibodies was measured by determining relative percentage of DNA from the cells bound to SHA beads in the presence of antibody to DNA from the bound cells in the absence of antibody.

Statistics

The significance of differences between means of groups was determined using the Student's t-test using Sigmaplot™ (SPSS, San Rafael, CA) and a p-value<0.05 was considered significant.