Study subjects and diagnosis
The study was carried out with the approval of the Institute's human & animal ethics committee (Ref. 51/7.1.2004/AIIMS). Blood Samples were collected over a period of two and a half years i.e Sep.05 through March 07 from 62 patients between 1 m to 70 yrs of age, admitted in the wards and intensive care units of AIIMS hospital. Each patient's medical history was recorded in a specific format and written informed consent was obtained.
Commercially available Serion ELISA classic IgM (Virion-Serion, GmbH, Germany) kit, which uses mixture of undefined M. pneumoniae antigens, was used as screening test as well as reference test for comparison. This kit was selected, because of its availability and its use in various studies [17, 26, 27].
Bacterial strains and culture conditions
M. pneumoniae standard strain FH (NCTC 10119), was grown in Edward Hayflick medium containing PPLO basal broth with supplements including glucose(1%; Difco, USA) and phenol red (0.0002%) as indicator in glass tubes. The culture was grown at 37°C, aerobically until the colour of the medium changed (red-orange). Mycoplasma cells were harvested at this stage by centrifugation at 15,000 rpm for 15 min, washed twice with PBS (pH 7.2) and were stored at -70°C until use. To confirm that the colour change of culture medium is due to M. pneumoniae growth, the broth culture was plated on PPLO agar plates and the plates were incubated at 37°C in a CO2 incubator. The plates were examined microscopically once in five days with X10 magnification. The suspected colonies were stained with Diene's stain and checked under light microscopy. Further confirmation of the colonies was carried out by growth inhibition assay using M. pneumoniae polyclonal antisera (NCTC).
PCR amplification and cloning of P116-609 bp fragment
To clone 609 bp fragment of M. pneumoniae
P116 gene that codes for a protein of 203 amino acids, primers were designed using published sequence (accession no. Z71425
]. M. pneumoniae
genomic DNA was extracted as per the method of Stauffer et al [29
]. Following set of primers with underlined introduced restriction sites (Microsynth, Switzerland) were used for the gene amplification:
The F1 was positioned at 786 nt whereas R1 at 1375 nt within the gene coding for P116 protein. PCR amplification was performed in a reaction volume of 50 μl containing 1× PCR buffer(100 mM Tris-HCl, pH 9.0, 500 mM KCl, 15 mM MgCl2 and 0.1% gelatin), 200 μm dNTP's, 20 pmol of each primer, 1U Taq Polymerase (5U/μl, MBI, Fermentas) and template DNA (50 ng) in a GeneAMP PCR system 9700 (Applied Biosystems, Switzerland). The PCR conditions were-initial denaturation at 94°C for 5 min followed by 30 cycles of amplification (each of 94°C for 30 s, 46°C for 30 s and 72°C for 1 min) with final extension at 72°C for 5 min. The PCR product was analysed by 1% agarose gel electrophoresis in 0.5% Tris-borate EDTA buffer and purified by gel extraction kit (Qiagen, Germany).
Cloning and Expression of N-terminal fragment of P116 protein of M. pneumoniae in a prokaryotic expression system
The purified P116 gene fragment was ligated into pGEMT-easy vector and the ligation mixture was used to transform DH5-α E. coli cells. Colonies were selected on Luria Bertani (LB) agar plates containing 100 μg/ml of ampicillin, 20 mg of X-gal and 200 mg/ml of IPTG (Sigma-Aldrich, USA). The selected white colonies were further analyzed for the presence of P116 gene fragment. Recombinant plasmids were extracted using Miniprep plasmid extraction kit (Qiagen).
For the expression and purification of P116 protein fragment, the 609 bp fragment was cloned in pQE-30 vector with Bam HI and Pst I restriction sites. The ligated plasmid DNA was used to transform competent M-15 cells. Transformants were selected on ampicillin (100 μg/ml) and kanamycin (25 μg/ml) plate. M-15 cells containing the recombinant plasmids were cultivated in 5 ml of LB broth and the protein expression was induced by the addition of 1 mM IPTG final concentration, followed by 3 hrs of shaking at 37°C. Bacterial pellet was subjected to SDS-PAGE and western blotting using anti-His antibody to analyze the expression of recombinant protein.
SDS-PAGE and western blotting
To analyse the expression of the (P116(N-27)) recombinant protein, induced and un-induced E. coli pellets from 1 ml of grown cultures were resuspended in 100 μl of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2.3% w/v SDS, 5% v/v β-mercaptoethanol and 0.05% w/v bromophenol blue) and boiled for 5 min. The proteins were resolved on 10% SDS-PAGE gel and stained with Coomassie brilliant blue R-250. For immunoblotting, after separating proteins on gel, the resolved proteins were transferred onto a nitrocellulose membrane (Sigma-Aldrich) in a transblot apparatus (Mini-PROTEAN III, Bio-Rad, USA). The membrane was blocked in blocking buffer (5% skimmed milk in PBS-T) for 2 h. The blots were washed and incubated with anti-His primary antibody (1:10,000 dilution) or anti-M. pneumoniae antibody (1:5,000 dilution) or sera from M. pneumoniae infected patients (1:50) for 1 h. Later the blots after washing, were incubated with secondary antibody (1:2000 dilution of anti-mouse, anti-rabbit or with 1:5000 dilution of anti-human antibody conjugated with horse-raddish peroxidase. The blots then developed with 3, 3'-diaminobenzidine tetrabenzidine hydrochloride (DAB)-H2O2 (Sigma-Aldrich).
Purification and Characterization of N-terminal fragment of P116 protein
Sub-cellular localization studies were carried out to analyse the expression of P116 protein fragment in E. coli cells and the protein was found to be expressed in inclusion bodies. For the preparation of inclusion bodies E. coli. cells were disrupted by sonication in buffer ((0.05 M Tris (pH 8.0), and 0.3 M NaCl) with 1-min pulses at 1-min intervals 10 times using mini probe (Torbeo, ultrasonic processor 36800-series). The soluble and insoluble fractions were separated by centrifugation at 14,000 rpm at 4°C for 30 min and were analyzed by SDS-PAGE. To purify the protein from the inclusion bodies, E. coli pellet from 200 ml of culture was suspended in 1/50 original volume of lysis buffer (0.05 M Tris (pH 8.0), 0.3 M NaCl and 8 M Urea). The lysate was loaded onto a Ni-NTA column (Qiagen) and the bound protein was eluted under denaturing conditions in a buffer containing 0.05 M Tris (pH 8.0), 0.3 M NaCl, 0.3 M imidazole and 8 M Urea. The denatured protein was refolded by dialysis using buffers containing 0.05 M Tris (pH 8.0), 0.3 M NaCl and decreasing concentrations of Urea, 6M-0.5M. The refolded protein was stored in buffer containing 0.05 M Tris (pH 8.0), 0.15 M NaCl, 0.5 M Urea and 5% glycerol. Purified recombinant P116 protein fragment (P116(N-27)) was analysed for its reactivity with positive anti-M. pneumoniae human sera in western blot assay.
Expression and purification of the C-terminal recombinant P1 protein fragment P1(C-40) of M. pneumoniae
Expression and purification of P1 protein fragment was carried out by a protocol described by Chaudhry et al .
Immunization of Rabbits
The protein concentration of both the antigens preparation was determined by Bradford assay. To check the immunogenicity of the two recombinant proteins-P116 and P1, four White New Zealand rabbits (two tests and two controls) were selected. Each one of the two test rabbits was immunized with 300 μl (250 μg) of purified recombinant P116(N-27) or P1(C-40) protein emulsified in 300 μl Complete Freund adjuvant (CFA, Sigma-Aldrich) intramuscularly (i.m.). Rabbits were subsequently boosted with 300 μl (250 μg) of the same protein in 300 μl Incomplete Freund adjuvant (IFA, Sigma-Aldrich), through the same route on the 21st and 42nd days. Control rabbits were injected with complete and incomplete Freund's adjuvant in normal saline according to the immunization schedule. Blood samples were obtained by ear vein puncture on 0th, 14th, 21st, 28th, 35th, 49th and 56th days. IgM and IgG antibody responses against the two purified recombinant proteins were analysed by ELISA and end point titres were determined. In order to confirm the specificity of the antisera western blotting with whole M. pneumoniae as antigen was also performed.
Comparative ELISA with purified recombinant P116(N-27), P1(C-40) and commercial IgM assay
Sera obtained from sixty two patients suffering from respiratory tract infections were analysed for the presence of anti-M. pneumoniae IgM antibody (which is an indicator of the acute infection), using Serion ELISA kit. Among the sixty two sera samples tested, thirty one samples were found positive for the anti-M. pneumoniae IgM antibodies. We next compared the antibody response seen with Serion kit with the response seen with recombinant P116(N-27), P1(C-40) or P116(N-27) + P1(C-40) together for IgM antibodies. The experiment was done in duplicate. Briefly, 100 ng of P116(N-27) or P1(C-40) was added to each well of 96-well microtiter plates. In case of P116(N-27) + P1(C-40) assay, P116(N-27) and P1(C-40) antigens were mixed in 1:1 ratio. The plates were incubated overnight at 4°C and further for one hour at 37°C next day. The plates were washed with PBS-T and blocked with 5% BSA in PBS for 2 h. The plates were subsequently washed twice with PBS-T, once with PBS and incubated with 1:50 diluted patient sera at 37°C for 1 h. The positive control (individual serum, tested positive by the commercial kit) and negative control (individual serum, tested negative by the commercial kit) were included in each ELISA assay and they were also diluted to the same extent. The wells were washed and incubated with HRP-conjugated goat anti-human IgM (Sigma-Aldrich) diluted 1:3000 in PBS-T, for 1 h. The enzyme reaction was developed by the addition of the substrate ortho-phenylenediamine (Sigma) (1 mg/ml) diluted in phosphate-citrate buffer (pH 5.0) containing 0.03% (v/v) hydrogen peroxide. The enzymatic reaction was stopped with 100 μl of 2N H2SO4 and absorbance was read at 490nm with ELISA (Bio-Tek Microplate) reader.
Comparative statistical analysis of ELISA assays for P116 and P1 proteins was done taking Serion IgM kit (100% sensitive and 75% specific as per the manufacturer's claim) as reference test. Analysis was performed with the SPSS version 15.0(Chicago, USA). The parameters of in house ELISA assays were calculated by using Epitable module of Epiinfo (6.04D) software. Cohen's Kappa test was used to find the agreement between two different modalities. The chi-square test of proportions was applied to compare two proportions with the p value < 0.05 considered statistically significant. Cut-off values were calculated by taking median values of the controls.