Tuberculosis (TB) is a major health problem with a high mortality rate worldwide. The standard diagnosis methods of TB include clinical examination, direct sputum microscopy, and bacterial culture. Limitations of these methods include low sensitivity of acid-fast smears (only 40%-50% of patients with pulmonary TB are smear-positive using Ziehl-Neelsen direct microscopy), a lengthy period of bacterial cultivation (approximately eight weeks) with undetectable growth in 10% to 20% of cases (Chakravorty et al., 2005; El-Masry et al., 2008). However, rapid testing methods, such as PCR, though sensitive, are expensive and scarce in most diagnostic or health care centers (Beck et al., 2005a; Chakravorty et al., 2005).
The tuberculin skin test (TST) using purified protein derivative (PPD) has limitations in differentiating between active disease and latent TB infection due to cross-reactions with Bacillus Calmette-Guérin (BCG) vaccine substrain or infection with environmental nontuberculous mycobacteria (NTM) (Beck et al., 2005b). Moreover, 10%-25% of TB patients have a negative TST result, and this percentage increases up to 50% in patients with advanced disease or people infected with human immunodeficiency virus (HIV) (Beck et al., 2005a; Getahun et al., 2010). Therefore, the development of an easy, rapid, and inexpensive diagnostic tool for TB is an important goal, particularly in view of a global increase in the number of cases of active TB.
In recent years, much effort has been devoted to identify immunologically important antigens of Mtb that could react specifically with antibodies from TB patients’ sera. Antigens secreted into the extracellular environment by Mtb are the usual target of immune response in the infected host (Arend et al., 2000). Many of these antigens have been studied, purified, and characterized.
For a number of years, the components of culture filtrate have been investigated using narrow-molecular-mass fractions as a guide to identifying immunologically active single molecules. Low molecular proteins demonstrated to be strongly recognized by T cells isolated from human TB patients and those of experimentally infected mice and cattle.
A large number of mycobacterial antigens have been identified and purified which include Ag85A, Ag85B, MTB81 (malate synthase), MTB48, MPT 51, MPT 32, crystalline, 38-kDa antigen, CFP-29 (29 kDa culture filtrate protein), MTC28 (28 kDa Mtb complex), CFP-10 (10 kDa culture filtrate protein), ESAT-6 (6 kDa early secretory antigenic target), thiol peroxidase, ICD I (Isocitrate dehydrogenase 1) and ICD II (Young et al., 1992; Beck et al., 2005b; Wu et al., 2010). The 38-kDa protein is an immunodominant antigen isolated by gel chromatography and specific only for the Mtb complex. This antigen has the potential to be used in Enzyme-Linked Immunosorbent Assay (ELISA) and detects up to 85% of smear-positive cases (Young et al., 1986). The 30/32-kDa complex of major secretory proteins is among the abundant proteins produced by Mtb growing either extracellularly in broth culture or in human mononuclear phagocytes. G. Harth et al. (1996) purified the 30/32-kDa proteins from the culture filtrate of Mtb by gel filtration chromatography. Their immunocytochemical analyses conclusively demonstrated that the 30/32-kDa proteins were abundantly expressed by intracellular bacteria within infected human monocytes.
CFP-29 is present in both the culture filtrate and the membrane fraction from Mtb, suggesting that this antigen is released from the envelope to culture filtrate during the growth (Young et al., 1992; Weldingh et al., 2005; Sonawane et al., 2012).
The ESAT-6 and CFP-10 are present in form of a complex in the cytosol and the culture filtrate of tubercle bacilli, before and after secretion, respectively (De Jonge MI et al., 2007). A combination of these two molecules provides specific and sensitive targets for the detection of MTC infection and has been used for vaccines, skin tests, and the gamma interferon release assay (IGRA) (Shen et al., 2011).
In this study, low molecular weight antigens were purified from the culture filtrate of Mtb strain C (ATCC 35808 TMC 116) by ammonium sulphate precipitations and gel chromatography and later identified the antigens using Dot-ELIZA and Western Blotting assay and sera from pulmonary tuberculosis patients.
2. Material and Methods
2.1. Mycobacterial Cultures and Growth Conditions
Mtb standard strain C (ATCC 35808 TMC 116), provided by the Tuberculin and Mallein Production and Research Department in Razi Vaccine and Serum Research Institute, Alborz, Karaj, Iran, were grown on Lowenstein-Jensen medium with glycerol at 37°C for eight weeks. Bacteria grown on the Lowenstein-Jensen medium were inoculated in a biphasic medium of Potato-Dorset-Henley.
After incubation at 37°C for 6-8 weeks in biphasic medium, the bacterial suspension was transferred to a 1-liter flask containing 250 ml of Dorset-Henly liquid medium and incubated at 37ºC for up to 6 weeks, without shaking.
2.2. Inactivation of Bacteria and Filtration
Six-week-old culture filtrates from Mtb were inactivated at 68 °C for 1 h6. After the completion of inactivation, the bacterial mass was removed from the culture filtrate by filtration under pressure on filter membrane type 0.5 µm and 0.22 µm, first passing the cultures through 0.5µm filter, followed by 0.22 µm filter and culture supernatant was harvested.
2.3. Protein Precipitation by Ammonium Sulphate
For the isolation of low molecular weight proteins, the obtained culture supernatant was twice subjected to ammonium sulphate precipitation (20% saturation) at 4ºC overnight. Subsequently, the precipitated proteins were sediment by centrifugation at 3000 rpm for 20 min. The collected precipitates were dissolved in 200 µl phosphate-buffered saline (PBS, 0.01 M), and stored at 4ºC until further use. The proteins were dialyzed using Dialysis Cassettes with a molecular weight cut off (MWCO) of 6,000 Da to remove the salt. The protein solution was dialyzed against 0.01 M PBS and left with stirring at 4 ºC for 72 hours. The PBS was changed thrice at regular intervals.
2.4. Purifications of Culture Filtrate Protein (CFP) by Gel Chromatography
The protein solution was purified by gel filtrations on Sephadex G-50 columns (with the height and diameter of 150 cm and 2 cm, respectively). The final volume of concentrated protein solution after dialysis was 5 ml with 1.6 mg/ml density. The chromatography was monitored at 280 nm and antigen fractions were separated. Additionally, the protein concentrations were determined using the Lowry method at 750 nm.
2.5. Protein Estimation and Molecular Weight Determination
Protein concentrations were determined using the Lowry method and bovine serum albumin as the standard. Calibration curves were prepared for the absorptions measurements and the protein content of the samples (unknown model) was calculated using this curve. Purified protein fractions obtained during the gel filtration were separated using 12.5% Sodium Dodecyl Sulfate-Polyacrylamide Gel (SDS-PAGE) according to Laemmli SDS PAGE procedure (1970). Prestained protein markers, ranging from 10kDa to 250kDa (Sinaclone) were used in this study. The SDS-PAGE was conducted at 80 V for 2 h. Afterward, the components in the fractions were visualized by silver staining assay.
2.6. Dot Blot Immunoassay
The qualitative dot-blot immunoassay was carried out to detect the low molecular weight antigens fractions of Mtb. Samples were applied as a spot to strips of pre-treatment Polyvinylidene fluoride (PVDF) membranes and were allowed to dry. Pre-treatment involved submersion in 100% methanol for five min or until the membrane was translucent, followed by equilibration in the transfer buffer (3.03 gr of Tris base, 14.25 gr Glycine, 200 ml Methanol, and 5 ml SDS 20 %) until the membrane no longer floated on the surface. After 1 h, the PVDF membrane was blocked with 3% Bovine Serum Albumin (BSA) in 1X Tris Buffered Saline (TBS) with 10% Tween 20 (TBST) pH 7.6. The membrane strips were washed thrice with 1X TBST, and incubated with serum samples from Mtb infected patients and healthy individuals (diluted 1/50 in TBST containing 3% BSA) or 1 h at 4ºC as a primary antibody. Following another wash cycle, the strips were immersed in diluted 1/5000 horseradish peroxidase (HRP) goat anti-human (Abcam 102420), as a secondary antibody at room temperature with shaking for 1 h. The membrane strips were washed again thrice with 1X TBST and the blots were developed by addition of the 0.005 gr diaminobenzidine (DAB: Sigma D8001) as substrate, in 10 ml PBS and 500 µl H2O2 (30%) and incubated for 5-15 min in the dark. The reaction was stopped by washing the blot in distilled water. All incubations were carried out under constant shaking.
2.7. Western Blot Immunoassay
Western blotting of Mtb culture filtrate antigen was performed with serum samples from both Mtb patients and healthy individual human. In brief, 20 µl of the protein samples were transferred electrophoretically onto PVDF membranes (400 mA for 2 h). The purified chromatographic fractions were separated on SDS-poly-acrylamide 12.5% gels and transferred onto PVDF membrane. Transfer efficiency was monitored by checking for the presence of prestained low molecular weight marker bands on the membrane.
Sinaclone prestained protein marker ranging from 10 kDa to 250 kDa was used in this study. After transfer, the detection method was carried out the same as dot blot assay detection. Primary and secondary antibody dilutions were prepared from the dot-blot assay.
3.1. Protein Estimation by Lowry Method
After precipitating the proteins in the culture filtrate medium of Mtb, protein concentration in the precipitated solution was approximately 1.05 mg/ml (estimated by Lowry assay). However, this concentration was slightly increased after dialysis and was shown to be about 1.662 mg/ml.
3.2. Gel Chromatography
The precipitated protein solution was loaded on a Sephadex G-50 column for gel chromatography. On Sephadex G-50, the proteins were eluted with phosphate buffer saline 0.5 molars (pH: 7.4). Elution was followed by recording the absorbance at 280 nm.
At this stage, three fractions related to the obtained peaks were collected. Figure 1 shows the elution profile after the chromatography of the protein on the Sephadex G-50 column.
The protein concentrations in the collected fractions F1, F2, and F3 were approximately 0.121, 0.6128, and 0.063 mg/ml, respectively.
3.3. Molecular Weight Determination
Figure 2 shows the SDS-PAGE of the fractions obtained after chromatography. Based on these results, a series of proteins in the approximate range of 14-41 kDa were observed in F2 fraction by SDS-PAGE analysis. However, F1 and F3 showed no significant protein bands and were omitted from the study.
3.4. Dot Blot Analysis
Defined antigens present in the filtrate were identified by immunoblot analysis using polyclonal antibodies. Results of dot blot analysis are shown in Figure 3. The proteins in the F2 fraction were recognized by the used TB patients’ polyclonal sera and confirmed the presence of the antigenic proteins in this fraction (A). No spots were detected on membranes treated with healthy patients’ sera which indicated the specificity of the test. The tuberculin-negative control did not respond significantly to any of the fractions (B).
3.5. Western Blot Analysis
Figures 4 and 5 show the Western blotting results of F2 fraction proteins with the polyclonal antibodies in the serum samples collected from both Mtb patients and healthy individuals. The TB patients’ serum predominantly recognized antigen fractions with a molecular weight between 30kDa and 41kDa on PVDF membrane (Figure 4), while, serum from healthy individuals had negative results.
This study aimed to isolate and purify low molecular weight antigens secreted from Mtb and use them for diagnostic purposes. The results demonstrated that these purified proteins showed significant protein bands approximately in the range of 14kDa -41 kDa on SDS-PAGE (12.5 %) and in Western blotting assay using TB patients’ sera as a primary antibody. Bands in the range of 30-41 kDa were identified.
In the present study, the immunoblotting assay was used to detect the purified low molecular weight antigen fractions of Mtb in TB patients’ sera. Obtained results showed that positive patients’ sera identified bands at the approximate range of 30 to 41kDa. Therefore, the results support this hypothesis that the low molecular weight secreted antigens in culture filtrates of Mtb could make the protein complex at 30-41 kDa regions and that it is responsible for immune responses in tuberculosis patients. The 30-kDa protein, the major secretory protein of Mtb accounted for almost a quarter of the total extracellular protein in the culture filtrate (Harth et al., 1996; Horwitz et al., 1996). Moreover, some studies have shown that 38-kDa Mtb protein is one of the major secretory proteins of mycobacteria (Abebe et al., 2018).
Applied methods were employed in this study to purify the natural proteins from the Dorset-Henley liquid medium and decrease the purification stages. Accordingly, in this study, the bacteria were cultured in a high volume of Dorset-Henley liquid medium, and as described in the Methods section Mtb standard strain C were grown on dedicated culture media.
The secreted low molecular weight antigens from Mtb were precipitated by ammonium sulfate with 20% saturation and were purified further by Gel filtration (GF) chromatography on Sephadex-G50.
There are many different methods for protein precipitation. Protein samples should have an appropriate concentration after precipitation to be suitable for electrophoresis (Fountoulakis and Takcs, 2002). Horwitz et al. (1996), reported that the antigenic proteins from the culture supernatant could be harvested by filtration and saturation with 10-60 % ammonium sulfate. However, during gradient analysis, maximum precipitation of the proteins was achieved using 20 % ammonium sulphate.
The GF chromatography separates proteins solely on the basis of molecular size and proteins are eluted from the GF column in descending size order (Babaie M., et al., 2013). In this study, three protein fractions were obtained using Sephadex-G50 gel chromatography.
Both HPLC (High-performance liquid chromatography) and GF chromatography methods are used to isolate proteins. The HPLC method is recommended for the separation of proteins due to the higher quality of the proteins separated by HPLC, compared to those separated by GF chromatography and regarding higher sensitivity of this method (Fong and Lam, 1991).
In this study, the Lowry method was used for the estimation of protein fraction concentrations, and molecular weight was determined by SDS-PAGE. Based on the results, protein concentration in the collected fraction F2 was at maximum (approximately 0.7 mg/ml). The purified protein fraction F2 appeared at regions with macular weight range of 14-41 kDa on silver staining SDS-PAGE, and immunoblotting assay showed that these regions contained antigens recognized strongly by TB patient sera.
Several physical and chemical methods, such as sonication and high temperatures have been employed for the inactivation of bacterial mass; however, these methods could destroy the existing proteins.
Normally, PPD is produced by inactivation of the bacterial mass by subjecting the bacterial culture to high temperatures for 3 h. The early work demonstrated that subjection of the mycobacterial culture filtrates to high temperatures led to a significant loss by denaturation of the antigenic protein; therefore, the PPD test had a low specificity for making a distinction between Mtb and other non-tuberculous mycobacteria (Yang et al., 2012). Therefore, in this study, the protein from the bacterium was extracted using moderate heat shock, i.e., subjection of live bacterial Mtb cultures to a temperature of 68 °C for 1 h.
Based on the results, TB patient sera recognized antigen fractions with a molecular size between 30 kDa and 41 kDa on PVDF membrane. Various parameters are effective on the quality of immunoblotting after the transfer of antigen to the membrane, which includes optimal blocking conditions, antibody dilution, and washing buffers. It should be noted that not only is the affinity of the primary antibody for the antigen important, but primary and secondary antibody concentrations also have a profound effect on signal intensity. Accordingly, too much HRP on the blot can be caused by either primary and secondary antibody concentrations or both. The minimal primary antibody is advantageous since it promotes target-specific binding and low background (Alegria-Schaffer et al., 2009). Therefore, the Dot-ELISA assay was used to optimize the primary and secondary antibody dilutions for use in Western blotting.
In the present study, TB patients’ sera were used as the primary polyclonal antibody. Although the monoclonal antibody has a high specificity to a single epitope, the polyclonal antibody is both inexpensive and has the high ability to capture the target protein (it is recommended as the capture antibody in a sandwich ELISA).
The results obtained in the present study were compatible with those obtained in the previous studies. Until now many low molecular weights Mtb antigens have been identified, including 71, 65, 38, 23, 19, 16, 14, and 12-kDa proteins (El-Masry et al., 2008). The 38-kDa protein is an immunodominant antigen isolated by affinity chromatography and is specific only to the Mtb complex (Beck et al., 2005b). The study conducted by E. Rovatti et al., in 1996 showed that a semi-quantitative western blot test against the 38, 28, 24, and 19 kDa protein fractions can identify individuals with mycobacterial infection (Rovatti et al., 1996).
The 22- to 32-kDa antigen has been reported to be the major component of culture filtrate when bacteria are grown in cultures without shaking. C. Espitia et al. (1989), purified culture filtrate antigens in the weight range of 122-14 kDa from MTB. Immunoblot analysis of the antibody response in patients with pulmonary TB and in healthy individuals with these antigens showed that 32-31 kDa band was found reacting with 90% of TB sera. Another antigen band with similar reactivity was a 60 kDa antigen which was recognized by 82% of pathologic sera.
The 16-kDa antigen is an immunodominant antigen, frequently called 14 kDa, related to the family of low molecular weight heat-shock proteins. This antigen contains B-cell epitopes specific to the Mtb complex. In another study conducted by Senol et al. (2007), Western blot analysis showed that 30 (58%), 26 (50%), and 31 (60%) of the patients produced specific IgG antibodies against antigens in the 6 kDa, 16 kDa, and 38 kDa ranges, respectively. The important point is the low expression of these proteins under in vitro conditions. Therefore, cloning methods and gene expression should be employed to obtain these proteins. However, the recombinant proteins do not have natural protein abilities (Berthet et al., 1998).
During TB infection, several antigenic proteins are produced by Mtb. Many of these proteins have been studied, purified, and characterized for their role in serological and cellular immune responses. New serological tests are used to detect the host antibody response to low molecular weight mycobacterial antigens (Min et al., 2015). The key to success in controlling the wide spread of TB is the development of novel and more effective methods to detect TB. Improved detection of the latent bacilli will lead to early intervention strategies, and will likely reduce disease morbidity and break the cycle of disease transmission. Taken together, the results of this study indicated that the protein antigens detected in the range of 30-41 kDa could discriminate NTM and MTBC infections. However, further in vivo studies are required to confirm these results.
Study concept and design: N. M.
Acquisition of data: N. M.
Analysis and interpretation of data: N. M.
Drafting of the manuscript: N. M.
Critical revision of the manuscript for important intellectual content: N. M.
Statistical analysis: N. M.
Administrative, technical, and material support: N. M.
We hereby declare all ethical standards have been respected in preparation of the submitted article.
Conflict of Interest
The authors declare that they have no conflict of interest.
This study supported by Razi Vaccine and Serum Research Institute.
- Abebe F, Belay M, Legesse M. IFN-γ against the 38-kDa antigen of Mycobacterium tuberculosis discriminates pulmonary tuberculosis from infection and infection from exposure: evidence from a study of human population in a high endemic setting. APMIS. 2018; 126: 135-142.
- Alegria-Schaffer A, Lodge A, Vattem K. Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods Enzymol. 2009; 463: 573-599.
- Arend SM, Andersen P, van Meijgaarden KE, Skjøt RLV, Subronto YW, van Dissel JT, et al. Detection of Active Tuberculosis Infection by T Cell Responses to Early-Secreted Antigenic Target 6-kDa Protein and Culture Filtrate Protein 10. J Infect Dis. 2000; 181: 1850-1854.
- Babaie M, Salmanizadeh H, Zolfagharian H. Blood Coagulation induced by Iranian saw-scaled viper (Echis Carinatus) venom: Identification, purification and characterization of a prothrombin activator. Iran J Basic Med Sci. 2013; 16(11): 1145-1150.
- Beck ST, Leite OM, Arruda RS, Ferreira AW. Combined use of Western blot/ELISA to improve the serological diagnosis of human tuberculosis. Braz J Infect Dis. 2005a; 9: 35-43.
- Beck ST, Leite OM, Arruda RS, Ferreira AW. Humoral response to low molecular weight antigens of Mycobacterium tuberculosis by tuberculosis patients and contacts. Braz J Med Biol Res. 2005b; 38: 587-596.
- Berthet F-X, Rasmussen PB, Rosenkrands I, Andersen P, Gicquel B. A Mycobacterium tuberculosis operon encoding ESAT=6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiology. 1998; 144: 3195-3203.
- Chakravorty S, Sen MK, Tyagi JS. Diagnosis of extrapulmonary tuberculosis by smear, culture, and PCR using universal sample processing technology. J Clin Microbiol. 2005; 43: 4357-4362.
- De Jonge MI, Pehau-Arnaudet G, Fretz Mm, Romain F, Bottai D, Brodin P, et al. ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity. J Bacteriol. 2007; 189: 6028-6034.
- El-Masry S, El-Kady I, Zaghloul MH, Al-Badrawey MK. Rapid and simple detection of a mycobacterium circulating antigen in serum of pulmonary tuberculosis patients by using a monoclonal antibody and Fast-Dot-ELISA. Clin Biochem. 2008; 41: 145-151.
- Espitia C, Cervera I, González R, Mancilla R. A 38-kD Mycobacterium tuberculosis antigen associated with infection. Its isolation and serologic evaluation. Clin Exp Immunol. 1989; 77: 373-377.
- Fong GW, Lam SK. HPLC in the Pharmaceutical Industry, CRC Press, Boca Raton, Florida; 1991.
- Fountoulakis M, Takcs B. Enrichment and proteomic analysis of low-abundance bacterial proteins. Methods Enzymol. 2002; 358: 288-306.
- Getahun H, Gunneberg C, Granich R, Nunn P. HIV Infection–Associated Tuberculosis: The Epidemiology and the Response. Clin Infect Dis. 2010; 50: S201-S207.
- Harth G, Lee BY, Wang J, Clemens DL, Horwitz MA. Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton major extracellular protein of Mycobacterium tuberculosis. Infect Immun. 1996; 64: 3038-3047.
- Horwitz MA, Lee BW, Dillon BJ, Harth G. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci. 1996; 92: 1530-1534.
- Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970; 227: 680-685.
- Min F, Pan J, Wu R, Chen M, Kuang H, Zhao W. Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria. Exp Anim. 2015; 65: 11-16.
- Rovatti E, Corradi MP, Amicosante M, Tartoni PL, Panini W, Ancora A, et al. Evaluation of a western blot serum test for the diagnosis of Mycobacterium tuberculosis infection. Eur Respir J. 1996; 9: 288-292.
- Senol G, Erer OF, Yalcin YA, Coskun M, Gndz AT, Bimen C, et al. Humoral immune response against 38-kDa and 16-kDa mycobacterial antigens in tubercolosis. Eur Respir J. 2007; 29: 143-148.
- Shen GH, Chiou CS, Hu ST, Wu KM, Chen JH. Rapid identification of the Mycobacterium tuberculosis complex by combining the ESAT-6/CFP-10 immunochromatographic assay and smear morphology. J Clin Microbiol. 2011; 49: 902-907.
- Sonawane A, Mohanty S, Jagannathan L, Bekolay A, Banerjee S. Role of glycans and glycoproteins in disease development by Mycobacterium tuberculosis. Crit Rev Microbiol. 2012; 38: 250-266.
- Weldingh K, Rosenkrands I, Okkels LM, Doherty TM, Andersen P. Assessing the Serodiagnostic Potential of 35 Mycobacterium tuberculosis Proteins and Identification of Four Novel Serological Antigens. J Clin Microbiol. 2005; 43: 57-65.
- Wu X, Yang Y, Zhang J, Li B, Liang Y, Zhang C, et al. Comparison of antibody responses to seventeen antigens from Mycobacterium tuberculosis. Clinica Chimica Acta. 2010; 411: 1520 - 1528.
- Yang H, Kruh-Garcia NA, Dobos KM. Purified protein derivatives of tuberculin - past, present, and future. FEMS Immunol Med Microbiol. 2012; 66: 273-280.
- Young D, Kent L, Rees A, Lamb J, Ivanyi J. Immunological activity of a 38-kilodalton protein purified from Mycobacterium tuberculosis. Infect Immun. 1986; 54: 177-183.
- Young DB, Kaufmann SHE, Hermans PWM, Thole JER. Mycobacterial protein antigens: a compilation. Mol Microbiol. 1992; 6: 133-145.