2.1. Synthesis of SMAP28
SMAP28 (RGLRRLGRKIAHGVKKYGPTVLRIIRIA-(NH2)) was synthesised by NeoMPS, Inc. (San Diego, CA) and suspended in 0.01 M sodium phosphate buffer (pH 7.2) with 145 mM NaCl (0.01 M phosphate-buffered saline (PBS) (pH 7.2)). Its purity was confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) (High Resolution Mass Spectrometry Facility, The University of Iowa, Iowa City, IA) and reversed-phase high-performance liquid chromatography (HPLC).
2.2. Bacterial species and growth conditions
strain 381 (obtained from Ann Progulske-Fox, Department of Oral Biology, University of Florida, Gainesville, FL), A. actinomycetemcomitans
FDC-Y4 and P. micros
were used. All three organisms were grown as previously described at 37 °C in an atmosphere containing 85% N2
, 10% H2
and 5% CO2
]. Porphyromonas gingivalis
strain 381 was grown in tryptic soy broth (Difco Laboratories, Detroit, MI) supplemented with 5 μg/mL hemin (Sigma, St Louis, MO) and vitamin K (Sigma). Aggregatibacter actinomycetemcomitans
was grown in tryptic soy broth supplemented with 0.6% yeast extract (Difco Laboratories). Peptostreptococcus micros
was grown in brain–heart infusion (Difco Laboratories) supplemented with 0.5% neopeptone and 5 μg/mL hemin.
2.3. Preparation of antiserum and isolation of P. gingivalis-specific IgG antibody
A water-in-oil emulsified, whole-cell bacterin was prepared. Each immunising 1 mL dose contained 1.0 × 108 colony-forming units (CFU) of P. gingivalis strain 381 and 25 μg of MDP in 140 mM NaCl with 0.3% formalin in a 50% oil emulsion of Freund’s incomplete adjuvant. Two rabbits were immunised seven times over a 13-week period (IMGENEX Corp., San Diego, CA). Pre-bleed serum samples were collected before immunisation and antiserum samples were collected at 9, 11 and 13 weeks.
Rabbit IgG antibody to P. gingivalis cell surface antigens was isolated by affinity chromatography using the Pierce AminoLink® Plus Immobilization Kit (Nos. 44894 and 20394; Pierce, Rockford, IL) to link formalin-fixed whole cells of P. gingivalis to the gel matrix. The coupling efficiency was estimated to be 33.9% for P. gingivalis whole cells. Porphyromonas gingivalis-specific IgG antibodies were isolated from the affinity column-eluted fractions concentrated using a HiTrap Protein G HP Cartridge (No. 17-0405-01; Pierce) and dialysed overnight against 0.01 M PBS (pH 7.2). Seven millilitres of 0.10 mg/mL (total protein 0.7 mg) of affinity-purified P. gingivalis IgG antibody was obtained. IgG isolated from pre-immune rabbit serum (IgGAb control) and IgG to P. gingivalis isolated from immune rabbit serum (PgAb control) were used as control solutions.
2.4. Whole cell enzyme-linked immunosorbent assay (ELISA) analysis
A whole-cell ELISA was performed at 26 °C as described by Zagursky et al. [24
]. Briefly, a suspension of P. gingivalis
in 140 mM NaCl with 0.3% formalin was adjusted to contain 1.0 × 108
CFU/mL in the spectrophotometer (0.108 optical density (O.D.) at 600 nm) and then 100 μL/well of this suspension was put into Immulon 1 microtitre wells and dried overnight. Non-specific antibody binding sites were blocked with 0.01 M Tris buffer containing 0.145 M NaCl, 1.0% fish gelatin and 0.05% blocking buffer and then two-fold dilutions of rabbit antiserum were added. After 60 min, all wells were washed twice with blocking buffer and bound rabbit IgG was detected with peroxidase-labelled goat anti-rabbit IgG antibody (1 μg/mL) (No. 074-1506; KPL, Inc., Gaithersburg, MD) using tetramethylbenzidine (TMB) peroxidase developing reagent (No. 50-85-05; KPL, Inc.) and TMB stop reagent (No. 50-85-05; KPL, Inc.). The O.D. values of the wells in the plate were determined at 450 nm (PowerWaveX; Bio-Tek Instruments, Inc., Winooski, VT) and values of the blank wells were subtracted from the test values. Linear regression of the log2
of dilution (x
-axis) was plotted against the O.D. of the dilution (y
-axis) to determine the slope of the line. Antibody titre was calculated as the log2
dilution value at the 0.1 O.D. intercept [25
2.5. SMAP28 and IgG conjugation
SMAP28 was conjugated to affinity-purified IgG antibody using the Controlled Protein–Protein Cross-Linking Kit (No. 23456; Pierce) as outlined in . This kit uses maleimide to link the amine groups of SMAP28 to the sulfhydryl groups of the IgG antibody. SMAP28 contains 11 free amine groups, of which 4 are available for linker attachment, 1 at each lysine or arginine residue.
SMAP28 (1.0 mg/mL of 0.01 M PBS (pH 7.2)) was mixed with Sulfo-SMCC and incubated for 30 min at 26 °C. Unreacted maleimide reagents were removed using a Dextran Desalting Column. Peptide concentrations in eluted fractions were determined and those containing SMAP28 with maleimide linkers were pooled. MALDI-TOF-MS was used to confirm that maleimide linkers attached to SMAP28 ().
Fig. 2 Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) showing that the maleimide linkers were successfully attached to sheep myeloid antimicrobial peptide (SMAP) 28. Up to four distinct peaks were seen. Each peak (more ...)
Two options (IIA and IIB) were used to prepare sulfhydryl-modified IgG antibody. In option IIA, the IgG antibody was suspended in 0.01 M PBS (pH 7.2), mixed with the kit conjugation/activation buffer and activated kit immobilised-reductant and incubated for 60 min at 37 °C. The mixture was centrifuged to pellet the kit immobilised-reductant. The supernatant contained the sulfhydryl-modified IgG antibody for the protein–protein cross-linking procedure with the SMAP28 with maleimide linkers.
In option IIB, sulfhydryl groups were artificially added by reaction with N-succinimidyl-S-acetylthioacetate (SATA). The IgG antibody was suspended in 0.01 M PBS (pH 7.2) (1.0 mL) and a 10 molar excess of SATA solution (SATA/dimethylformamide) was added and incubated for 20 min at 26 °C. Then, 100 μL of hydroxylamine HCl solution was added to the SATA-modified IgG antibody and the mixture was incubated for 120 min at 26 °C. Unbound reagents were removed by passing the de-protected sulfhydryl-IgG antibody solution over a desalting column. PBS–ethylene diamine tetra-acetic acid (EDTA) was used to elute sulfhydryl-IgG antibody and 1.0 mL fractions were collected. Protein concentrations were determined at A280 and fractions containing most of the sulfhydryl-IgG antibody were pooled. SMAP28 with maleimide linkers and sulfhydryl-IgG antibody were mixed in approximately equal molar amounts and incubated for 60 min at 26 °C.
Preliminary work showed that conjugation by option IIB was more effective and a P. gingivalis IgG–SMAP28 conjugate developed this way was more specific to P. gingivalis. Thereafter, the conjugate was prepared using option IIB and was called the P. gingivalis IgG–SMAP28 conjugate.
SMAP28 and the SMAP28 with maleimide linkers were separated on an Acclaim 300, C18, 3 μm analytical column (Dionex Corp., San Francisco, CA) using a Summit HPLC system (Dionex) with Chromeleon software and eluted with a gradient of acetonitrile (0–100%) in 0.1% trifluoroacetic acid. Fractions were collected in a FC 144 fraction collector, pooled with similar fractions of previous runs and dried overnight by rotary evaporation under vacuum at room temperature.
2.7. Spot blot
A dot-blot assay was used to confirm the presence both of the affinity-purified rabbit IgG antibody and of SMAP28 in the P. gingivalis
IgG–SMAP28 conjugate. Briefly, 1.0 μL of the P. gingivalis
IgG–SMAP28 conjugate and P. gingivalis
IgG were placed on Immobilon-P transfer membranes, allowed to dry and soaked overnight at 26 °C in blocking buffer. Rabbit IgG antibody in the P. gingivalis
IgG–SMAP28 conjugate was detected with peroxidase-labelled goat anti-rabbit antibody (1.0 μg/mL) (No. 074-1506; KPL, Inc.) using CN peroxidase developing reagent (No. 50-73-014; KPL, Inc.). SMAP28 in the P. gingivalis
IgG–SMAP28 conjugate was detected first using goat anti-SMAP28 antibody (1:20 dilution; courtesy of Brian Tack) [25
] and then peroxidase-labelled rabbit anti-goat antibody (1.0 μg/mL) (No. 14-13-06, KPL, Inc.) using 4CN peroxidase developing reagent (No. 50-73-01; KPL, Inc.).
2.8. Radial diffusion antimicrobial assay
The MICs of SMAP28 and SMAP28 with maleimide linkers were determined by the radial diffusion assay as previously described in our laboratory [23
]. After incubation for 24–48 h, zones of inhibition were recorded with a Boley gauge as radial diffusion units (zone of inhibition–well diameter × 10). The x
-intercept was obtained from the relationship between radial diffusion units versus log10
peptide concentration as determined after regression [23
2.9. Porphyromonas gingivalis IgG–SMAP28 conjugate-specific killing in a mixed culture suspension
The specificity and activity of the P. gingivalis IgG–SMAP28 conjugate was determined in an artificially generated microbial community containing P. micros, A. actinomycetemcomitans and P. gingivalis. These organisms were grown for 48 h in their respective media at 37 °C under anaerobic conditions and diluted in reduced saline to a density containing ca. 1 × 108 CFU/mL (0.108 O.D. at 600 nm) (Spectronic 20D+; Thermo Fisher Scientific, Inc., Waltham, MA). Each culture was diluted 10-fold to contain 104 CFU/mL. Then, 2.0, 0.25 and 2.0 mL of P. gingivalis, A. actinomycetemcomitans and P. micros, respectively, was added to 5.75 mL of reduced anaerobic saline (e.g. constituting the 10−5 dilution of each) and diluted two-fold with the P. gingivalis IgG–SMAP28 conjugate and two control solutions, IgGAb control and PgAb control. At 0, 10, 20 and 30 min, 100 μL was removed and plated onto anaerobic blood agar (CDC formulation; Remel, Lenexa, KS) and incubated at 37 °C. After 9 days, bacterial colonies of differing morphologies, sizes and colour were counted by three different observers.
2.10. Statistical analyses
The experiment was divided into treatments at different time points: P. gingivalis IgG–SMAP28 conjugate, P. gingivalis antibody control (PgAb control) and pre-immune IgG antibody control (IgGAb control) at 0, 10, 20 and 30 min, defining 12 total treatments for the study. Each treatment had three plates and the experiments were performed on two different days using two different freshly prepared conjugates. Since day-to-day differences could affect the assays, each day was considered a block.
A multifactorial analysis of variance (ANOVA) model was used to analyse the effect of treatments, time and day on the number of CFU as well as to assess two- and three-way interactions. Logarithm transformations of the form log10 (CFU+1) were used as needed to conform to model assumptions such as normality and homoscedasticity. Adjustments for multiple comparisons utilised Tukey’s studentized range.