A 16-mer synthetic peptide, sequencing NPGNDSLLSTTDNNIA, from constant part of P110 protein of M.genitalium was selected as immunogen. A cysteine residue was added to the C-terminus end of peptide to facilitate the conjugation to carrier protein. Immunograde peptide was purchased from Thermo Electron Corporation (GmbH, Ulm, Germany) and was conjugated to Keyhole Limpet Hemocyanin (KLH) and Bovine Serum Albumin (BSA), separately as described elsewhere (14). The peptide-KLH and peptide-BSA conjugates were used for immunization and conjugation assessment, respectively.

To check the efficacy of conjugation, 10 µg of peptide-BSA conjugate was mixed with 10 µl of sample buffer and boiled for 5 min. Electrophoresis was performed on 10% SDS-PAGE condition using mini-PROTEAN electrophoresis instrument (Bio-Rad laboratories, Philadelphia, PA) with 100 mA for 1 hr. The gel was stained with Coomassie Blue R-250 (Sigma, St. Louis, MO). The change in mobility shift of conjugated BSA represented the efficiency of conjugation (Figure 1).

A female white New Zealand rabbit was immunized 3 times with one-week interval for each injection.

In first immunization, 100 µg KLH-peptide conjugate and 250 µl IMMACCEL (Pick cell Laboratories, Netherlands) was mixed with an equal volume of Freund's complete adjuvant (Sigma), and injected subcutaneously in 4-6 regions. For the subsequent immunizations, 500µg peptide-KLH and 250 µl IMMACCEL were admixed and injected with Freund's incomplete adjuvant (Sigma).

The last immunization was perfumed using 1000 µg peptide-KLH together with 250 µl IMMACCEL and Freund's incomplete adjuvant. The IMMACCEL reduces the antibody production time in rabbit from standard 80-day protocol to 28 day without any difference in affinity or specificity (15).

Before each immunization and 7 and 14 days after the last immunization, blood was drawn by venipuncture of the rabbit ear and allowed to clot for periods of 2 to 3 hr at room temperature before preparation of serum. Titration of the specific polyclonal antibody was then performed as follow: A96-well ELISA plate was coated with 100 µl of the immunizing peptide (20 mg/ml in PBS) at 37 °C for one hr followed by overnight incubation at 4 °C. The plate was washed 3 times with PBS containing 0.05% Tween 20 (PBS-T) for 5 min. The wells were then blocked with 2.5% BSA at 37 °C for 1.5 hr. Wells were washed 3 times as above and rabbit serum was added to the wells in two fold serial dilutions starting from 1:100. The plate was incubated at 37 °C for 1.5 hr and washed again with PBS-T.

At the next step, 100 µl of 1:1000 dilution of HRP-conjugated sheep anti-rabbit immuneglobulin (Avicenna Research Institute, Tehran, Iran) was added to the wells and incubation was continued for 1 hr, at 37 °C. After washing, 100 µl of Tetramethylbenzidine (TMB) chromogen was added to each well and the plate was incubated at room temperature in a dark place. After 15 min, the reaction was stopped by adding 30 µl of stopping solution (0.16 M H₂So₄) to each well. The Optical Density (OD) of the reaction was measured at 450 nm by an ELISA reader. Negative controls included omission of coating layer, serum (as primary antibody) or combination of both (Figure 2).

Rabbit serum was filtered through 0.45 µm filter and antibody was purified by affinity chromatography column prepared by coupling immunogenic peptide to SulfoLink Coupling Resin (Thermo Scientific). The elution was performed using 0.1M glycine. HCl (pH = 2.6). The pH of eluted antibody was adjusted to 7.0 with 1 MTris.HCl pH = 9.0. The eluted anti-body was dialyzed overnight against PBS pH = 7.5. The reactivity of the antibody was measured by ELISA and its purity evaluated by SDS-PAGE (Figures 3 and 4).

M.genitalium G-37 (a gift from Dr. Jaume Piñol, Institut de Biotecnología i Biomedicina Universitat Autònoma de Barcelona) was cultured in SP-4 medium (16). The color changing (reddish yellow) in medium after 48-72 hr was evident for mycoplasma growth. The surface-attached mycoplasmas were harvested by scraping off in PBS. The bacterial cell lysate was prepared by sonication in 500 µl of lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl, pH = 7.4, 150 mM NaCl, 5 mM EDTA, 1% protease inhibitor cocktail (Sigma) and 10% phosphatase inhibitor (Roche Diagnostics GmbH, Penzberg, Germany) on ice. The protein concentration of lysate was measured by BCA protein assay kit (Thermo Scientific).

Twenty µg of M.genitalium lysate was run on a 10% SDS-PAGE (100 V for 2 hr). After electrophoresis, resolved proteins were transferred onto Immobilon-PVDF membrane (Millipore Corporation, USA). The membrane was blocked for overnight at 4 °C with 5% non-fat milk in PBS plus 0.05% Tween 20 (PBS-T). All antibody incubations were performed in PBS-T containing 3% non-fat milk. Filter was incubated with 10 µg/ml of anti-body 1.5 hr at room temperature. After extensive washing with PBS-T, the filter was incubated with peroxidase-conjugated sheep anti-rabbit immunoglobulin (Avicenna Research Institute) for 1 hr at room temperature followed by washing and developing with ECL chemiluminescence detection system (GE Healthcare, Uppsala, Sweden) (Figure 5).

The Peripheral Blood Mononuclear Cells (PBMC) from a healthy donor were separated by ficoll density gradient centrifugation (17). Freshly-prepared PMBC were cultured (2-3×10⁴ cells/well) on an 8-well laminated glass slides (Paul Marienfeld GmbH & Co. KG, LaudaKönigshofen, Germany) in RPMI 1640 contain 10% FBS (Invitrogen, Carlsbad, CA) with subsequent incubation in moisturized and 5% CO2 conditions for overnight. The cultured and harvested M.genitalium was dispersed through 25-gauge needle and passed through 0.45 µm filter (18) followed by infecting the isolated PBMC. The infected cells were seeded on glass slide and incubated at 37 °C for 3 hr. The slides were washed by Tris-Buffered Saline (TBS), pH = 7.4, air-dried at room temperature for 15 min, followed by permeabilization by acetone (at -20 °C) for 2 min. The slides were kept at 4 °C for 30 min for drying. As negative control, few PBMC-cultured slides, were prepared without infecting by M.genitalium.

The uninfected and M.genitalium-infected slides were washed with TBS containing 0.1% bovine serum albumin (TBS-BSA) for three times, each 3 min. Slides were then blocked with 5% sheep serum for 10 min at room temperature. The primary anti-P110 antibody was diluted in TBS-BSA to a final concentration of 10 µg/ml and added to the slides. Slides were incubated at room temperature for 60 min followed by three times (3×3 min) washing steps with TBS-BSA. Slides were incubated at room temperature with Fluorescein Isothiocyanate (FITC)-conjugated sheep anti-rabbit polyclonal antibody (1:100 TBS-BSA) (Avicenna Research Institute) for 45 min. After washing with TBS-BSA, the nuclei were counterstained by 4’,6-diamidino-2-phenylindole dihydrochloride (D API) (Sigma) at concentration of 1 µg/ml for 5 min, then the slides were washed, mounted in TBS-glycerol 80% and examined under a fluorescence microscope (Olympus, Tokyo, Japan) (Figure 6).

Proper conjugation of peptide to carrier protein was assessed by SDS-PAGE electrophoresis. The change in mobility shift of peptide-BSA conjugate in SDS-PAGE gel represented the efficiency of conjugation (Figure 1). The presence of antibody against immunizing peptide in rabbit serum was evaluated by ELISA before and after immunization. Results indicated the presence of anti-body against immunizing peptide in rabbit serum which was upraised considerably and reached to the plateau at day 28 post immunization (Figure 2).

SDS-PAGE analysis of the purified anti-body revealed the presence of a single band of about 150 kDa indicative of the desired purity (Figure 3). Purified antibody exhibited excellent immunoreactivity with immunizing peptide in ELISA assay as well as suggesting its functionality (Figure 4).

The reactivity of the purified antibody to its corresponding native protein was determined by Western blot. The produced antibody recognized a single band of 110 kDa representtative of P110 protein of M.genitalium G-37 both at reducing and non-reducing conditions (Figure 5).

To analyse the capability of produced anti-body to recognize M.genitalium in infected cells, indirect immunofluorescent assay was performed. Infected cells showed a bright speckled fluorescent signal located mainly in the cytoplasm of the cells 3 hr after infection (Figure 6).