Sensitivity and specificity of newly generated monoclonal antibodies to detect novel antigens of Mycobacterium tuberculosis for the diagnosis of all forms of tuberculosis

Recombinant antigens preparation

The selected three novel recombinant antigens of M. tuberculosis were prepared as previously described by Singh et al.^[27] Briefly, clones were cultured at 37°C in Luria Bertani broth containing 100 μg/mL ampicillin and 50 μg/mL kanamycin antibiotics. The protein expression was induced with 1 mM isopropyl-b-D-1-thiogalactopyranoside (IPTG)/mL. Further, bacterial cells were harvested by centrifugation at 10,000× g for 10 min and lysed by sonication method. The recombinant proteins, SS-1 (KC147004.1), SS-4 (KC147003.1), and SS-5 (KC147008.1), were purified by Ni²+ Nitrilo Tri Acetic Acid (Ni²+NTA). metal-ion-affinity chromatography using the manufacturer’s instructions (Qiagen, Germany). Subsequently, purity of each recombinant protein was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Immunization of BALB/c mice

Six to 8 weeks old male BALB/c mice weighing 20–25 g, supplied by National Institute of Nutrition, Hyderabad, was used as an experimental host. The mice were housed, handled, and immunized, and other experimental procedures were performed as per institute guidelines (Ref No-748/IAEC/13). A total of 54 BALB/c mice were used in this study [Table 1]. The mice were injected subcutaneously with 30 μg of purified recombinant antigens (SS-1, SS-4, or SS-5) mixed with Incomplete Freund’s Adjuvant (Sigma Aldrich, USA) in a 1:1 volumetric ratio. Subsequently, three booster injections were given to each mouse at 3 weeks of intervals. Post 1 week of following the booster injection, the serum antibody titer was determined by indirect ELISA. The terminal booster dose (50 μg recombinant antigen/mice without adjuvant) was given intraperitoneally 3 days before fusion.

Myeloma cell line

Mouse Sp2/0-Ag14 myeloma cell line was received as a kind gift from Dr. Girish Varshney, Chief Scientist, Institute of Microbial Technology, Chandigarh India. The cells were cultured in the 25 cm² tissue culture (TC) flasks (BD Falcon, San Jose, CA) containing Roswell Park Memorial Institute (RPMI)-1640 medium (Hiclone Laboratories, UK) with L-glutamine (2.01 mM) supplemented with heat-inactivated fetal bovine serum 10% (vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), and gentamycin (100 μg/mL) (Sigma Aldrich, USA) denoted here as complete RPMI medium (cRPMI) medium in a humidified atmosphere at 37°C with 5% CO₂ in the incubator (Nuaire, UK). Actively growing Sp2/0-Ag14 myeloma cells were treated with 8-Azaguanine (Sigma Aldrich, USA) for sensitization of cell before fusion.

Establishment of hybridomas

Three days after the terminal booster dose, mice were sacrificed via compressed CO₂ inhalation method and removed spleen aseptically with a sterile surgical blade. Later, the spleen was washed with RPMI-1640 medium twice and cut into small pieces using a sterile surgical blade. Subsequently, splenocytes were collected, washed thricely with RPMI medium, and centrifuged at 900× g for 10 min. The standard cell fusion protocol was used as described by Köhler and Milstein^[28] with some modifications. Briefly, splenocytes were mixed with Sp2/0-Ag14 myeloma cells in a 5:1 ratio and fused by polyethylene glycol (PEG)-1500. The cells were incubated for 5 min at 37°C, followed by a drop-by-drop addition of 20 mL RPMI and centrifugation at 700× g for 10 min. The fused cells were resuspended in cRPMI-1640 medium with 1× hypoxanthine-aminopterinthymidine (HAT) (Sigma Aldrich, USA). The 100 μL medium per well (96-well TC plates) was dispensed and kept for incubation in a humidified atmosphere at 37°C with 5% CO₂ in an incubator for 2 weeks.^[29]

Screening and isolation of monoclonal antibodies producing hybridomas

The hybridoma clone screening was started after 2 weeks of fusion, hybridomas medium were tested with individual recombinant antigen by indirect ELISA assay. Succinctly, the high protein binding ELISA plates (Corning, Merck, USA) were used for the ELISA assay. The plates were coated overnight at 4°C with respective protein (1 μg/mL in bicarbonate buffer, pH-9.6). The plates were then blocked using 250 μL/well of blocking buffer (1% Bovine serum albumin (BSA) in 1× Phosphate Buffer Saline [PBS] for 2 h at 37°C. After washing with PBS 0.05% Tween 20 [PBS-T], 100 μL/well of hybridoma medium was added and incubated for 2 h at 37°C. The polyclonal antibody produced against each recombinant antigen was used as a positive control, and medium of myeloma cells was used as a negative control. The unbounded antibodies were removed by washing with washing buffer (PBS-T). Subsequently, 100 μL/well of anti-mouse immunoglobulin (Ig)G Horse Reddish Peroxidase (HRP) conjugate diluted in 1: 10,000 ratios with dilution buffer was added and incubated at 37°C for 1 h. After washing with PBS-T, 100 μL of 3,3’,5,5’-Tetramethylbenzidine (TMB)/well was added and incubated at 37°C for 15 min. Enzyme reaction was then stopped by adding 50 μL of 2N H₂SO₄ per well. The optical density (O.D.) at 450 nm of ELISA plate was measured by ELISA plate reader (Elx-808, Biotek).

The ELISA-positive wells were selected and observed for the presence of hybridomas colonies with an inverted microscope. Only hybridomas positive wells were selected for further experiment. The limiting dilution method was used for the isolation of single hybridomas. Thereafter, positive hybridomas (secreting monoclonal antibodies) were allowed to be adopted in continuous culture and subsequently cryopreserved in liquid nitrogen.

Purification of monoclonal antibodies

The mAb was purified using protein-A resin-mediated affinity chromatography as per instruction of the manufacturer (Abcam, USA). Briefly, protein-A column was equilibrated using five column volumes of 1× binding buffer (1× PBS buffer, pH-7.2). The cell culture medium of hybridomas was diluted with an equal volume of equilibration buffer, loaded on the protein-A column, and incubated at room temperature (RT) for 2 h. Then, the column was washed with ten column volumes of washing buffer. The mAb was eluted using 1 mL of elution buffer (0.1 M glycine buffer, pH-2.5) that was immediately neutralized by 100 μL of neutralization buffer (1M Tris-Cl, pH-8.0) per mL of mAb. The column was regenerated by washing with 1× PBS and stored column in the 20% (v/v) ethanol at 4°C. The concentration of the purified mAb was determined by Bradford proteins assay.^[30] The purity of mAbs was analyzed by SDS-PAGE.^[31]

Western blot

The purified recombinant proteins were resolved in the 12% resolving gel (SDS-PAGE) and transferred onto nitrocellulose membranes (Millipore, MA, USA) using a semi-dry blotting apparatus (Bio-Rad^®, USA) following the manufacturer’s protocol. The membrane was then blocked with 5% non-fat skimmed milk, washed four times with PBS-T for 10 min each, and incubated thereafter with diluted purified mAbs (100ng/10mL in dilution buffer, [PBS+1% BSA]) for 2 h at 37°C. Subsequently, membrane was washed thricely with PBS-T and incubated for 1 h at 37°C with 1:10,000 dilution of Horseradish peroxidase (HRP)-conjugated anti-mice IgG (PBS+1% BSA, pH 7.0). After washing, the blot was developed using Diaminobenzidine (DAB) (Sigma, USA) and 0.1% hydrogen peroxide (H₂O₂) was used as a substrate.

Study population

The study was conducted between the year 2014 and 2017. The Institutional Ethics Committee (IEC) of All India Institute of Medical Sciences (AIIMS), New Delhi, approved this study (Ref No. 48/03.03.2014). Participants referred from the various clinics of our institute (AIIMS, Delhi) and other hospitals in Delhi for routine diagnosis of TB were included in this study. The participants were included only after the informed written consent of the study. A total of 384 (n = 141 PTB; n = 68 EPTB; healthy control [HC] participants = 125; disease control [DC] participants = 50) participants were included in this study. The diseased groups PTB and EPTB were selected based on culture-confirmed TB and standard clinic-radiological findings as mentioned in the Centers for Disease Control and Prevention (CDC) and anti-tetanus serum (ATS) guidelines.^[4,27] TB case definition adopted for this study was defined as “a patient with M. tuberculosis identified from a clinical specimen by culture or molecular methods.” The inclusion criteria for PTB participants (n = 141) were based on the presence of two or more of the following clinical symptoms: A cough for more than two weeks, fever, weight loss, night sweats, hemoptysis, and anorexia with or without cavitary lesions in the lung fields on radiological examination. The inclusion criteria for EPTB cases were made on the basis of symptoms presented by the patients, along with clinical and radiological examination. This included anorexia, malaise, fever, and weight loss with pleuritic or organ-specific pain. In addition, extensive exudative pleural effusion, peripheral (superficial) tuberculous lymphadenitis of cervical and axillary regions on X-ray, or other radiological examination suggestive of lytic lesions were included in the criteria for EPTB cases.^[4] The MDR-TB is defined as resistant to rifampicin (RIF) and isoniazid (INH), with or without resistance to other first-line drugs. Those isolates that have shown resistance to INH and RIF were classified as MDR-TB cases. In the other diseased control group, only three categories, namely, HIV, toxoplasmosis, and leishmaniasis (confirmed by commercial immune-chromatography test /enzyme-linked immunosorbent assay (ICTs/ELISA)-based test) of patients were included in the study. The status of TB in other disease control groups was confirmed by liquid culture.

The definition of a healthy participant was defined as “a person who does not have a disorder or disease being studied.” The friends and laboratory staff (other than TB laboratory) were selected as HC for this study. The inclusion criteria for a healthy participant were a person, who does not present TB symptoms and must have a negative result of either TST or IGRA. The TST was done by a trained phlebotomist. The procedure was 5 TU/0.1 mL tuberculin (Span Diagnostics, Surat, India) administered intradermally on the volar part of the forearm and read after 48–72 h, and induration ≥10 mm was defined as positive. The IGRA test was performed as per the instruction of the manufacturer (Qiagen, Germany). Mantoux/IGRA test was not done in patients or healthy subjects who did not provide consent or refused the test. The venous blood (~5 mL) was collected from the confirmed TB patients as well as from control subjects, and then, the serum was separated. The sera were stored in a minimum of three aliquots at −80°C. The other details of participants, that is, age, gender, and BCG vaccination status, are mentioned in Table 2.

Sandwich ELISA

The checkerboard titration method was used to detect the optimum concentration of antigen, antibody, and sera dilution for ELISA. The 96 well ELISA plates were coated with 100 μL/well of each purified mAb in 0.1M bicarbonate buffer (pH 9.6) and incubated at 4°C overnight. The dilution buffer was taken as plate control (Blank). Plates were washed with 1× PBS-T and blocked by blocking buffer (5% skimmed milk in PBS) for 2 h at 37°C. Subsequently, plates were washed five times with 1× PBS-T, added 100 μL/well of diluted antigen (200 ng/mL), and incubated for 1 h at 37°C. The unbounded antigens were removed by washing, and then, after 100 μL/well of diluted serum samples at a 1:100 ratio in dilution buffer (0.1% skimmed milk powder in 1× PBS) was added to each well. Plates were again incubated for 1.5 h at 37°C followed by washing with PBS-T. After that, 100 μL of anti-human IgG whole antibody conjugated with HRP (Sigma Aldrich, USA) diluted at 1:15000 ratio in dilution buffer was added to each well and incubated for 1 h at 37°C. Post washes with 1× PBS-T, enzyme activity was observed by incubating plates for 15 min at 37°C with 100 μL of TMB per well. Later, 50 μL of 2N H₂SO₄ was added to each well as a stop reagent, and measured O.D. was taken at 450 nm using a spectrophotometer (ELx808 absorbance reader, BioTek, USA). All sera included in this study were tested in duplicate wells and repeated at least thricely to verify the reproducibility of results.

Data management

Data were presented as means and standard deviation (mean ± standard deviation [SD]). The test cutoff value was determined by mean O.D. of the healthy subject plus 2SD. The individual sample was considered positive if the O.D. value was equal to or above the cutoff. Sensitivity and specificity were calculated by an online available tool (OpenEpi), Graph plot and receiver operative characteristic (ROC) curves were plotted using STATA SE.11.0 (StataCorp LP, Texas, USA) software. ROC curve describes the probability of correct and incorrect results at different cutoff values. Differences between different groups were considered statistically significant at P < 0.05.

Expression, purification, and immunoactivity of recombinant proteins

All three recombinant proteins yield high-level expression after induction with 1 mM IPTG for 4 h at 37°C. All the recombinant proteins were successfully purified using Ni²⁺-NTA agarose affinity column chromatography under denaturing conditions except SS-4 protein. The purified recombinant proteins were obtained as a clear band corresponding to the expected molecular weight 28 kDa (SS-1), 17 kDa (SS-4), and 41 kDa (SS-5) with a purity level >96% by SDS-PAGE. The yield was 3.0g (SS-1), 0.3g (SS-4), and 0.3g (SS-5) of purified proteins per liter culture [Figure 1].

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Figure 1:

SDS-PAGE and Western blot of purified recombinant proteins. (a) SS-1 (28kDa), (b) SS-4 (17kDa), and (c) SS-5 (41kDa). M: Protein marker, WCL: Whole cell lysate, FT: Flow through, P: Purified proteins.

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Isolation of hybridomas producing anti-M. tuberculosis antigen-specific antibodies

The fusion was performed using PEG-1500 between myeloma cells and mice splenocytes that showed a higher antibody titer against each targeted recombinant antigen. The antibody titer was approximately 3271 folds in SS-1, 902 folds in SS-4, and 1198 folds higher than control in SS-5 mice sera. After clone screening, 34 wells of SS-1, 41 wells of SS-4, and 58 wells of SS-5 antigen group were selected (>1.0 O.D. as cutoff) for subcloning. Post three rounds of subcloning procedures, stable hybridoma clones producing anti-SS-1mAbs, SS-4mAbs, SS-5mAbs named as SS-1C14, SS-1D40, SS-1A76, and SS-1C82 (04 clone); SS-4B14, SS-4A30, SS-4D4, and SS-4A76 (04 clone); and SS-54D, SS-5B14, SS-5A30, SS-5B48, SS-5A76, SS-5D88, and SS-5B98 (07 clone) were identified and selected for clone scale-up procedure [Figure 2].

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Figure 2:

A representative diagram of hybridomas clones. (a) Presence of hybridoma clones after HAT selection. The photographs were taken at ×400 magnification using an inverted microscope. (b) Progression of hybridomas in cRPMI medium. The image was taken at ×200 using an inverted microscope. Black arrow indicates the survived hybridoma clones.

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Purification and immunoactivity of monoclonal antibodies

The mAbs named as SS-1mAbs, SS-4mAbs, and SS-5mAbs were successfully purified by protein-A-based affinity chromatography with a >95% purity level achieved. The reactivity of mAbs with respective recombinant antigens was analyzed using western blotting, which indicates a strong reaction with the corresponding recombinant antigen [Figure 3].

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Figure 3:

SDS-PAGE analysis of monoclonal antibodies mAbs_SS-1, mAbs_SS-4, and mAbs_SS-5. (a) SDS PAGE analysis of purified monoclonal antibodies using protein-A column chromatography (Lane 1: Protein marker; lane 2: mAbs_SS-1; lane 3: mAb_SS-4; and lane 4: mAb_SS-5). (b) Western blot of purified mAbs with respective recombinant antigens.

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Sandwich ELISA for detection of M. tuberculosis antigen

A modified sandwich ELISA was developed to detect M. tuberculosis antigens by purified mAbs. The mAbs SS-1A76, SS-4D4, and SS-5D54 showed maximum absorbance with SS-1, SS-4, and SS-5 antigens, respectively. Hence, these three mAbs were selected for validation and evaluation study [Figure 4].

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Figure 4:

Activity of purified monoclonal antibodies with recombinant antigen (a) SS-1, (b) SS-4, and (c) SS-5, OD: Optical Density.

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Study subjects

After the standardization of ELISA, a total of n = 384 participants were tested for the assessment of the diagnostic potential of mAbs, as summarized in Table 2. Therefore, of the 384 participants, 209 were confirmed TB participants (141 PTB and 68 EPTB) and 175 control participants. Moreover, of the 209 TB participants, 132 (63.16%) were male and 77 (36.84%) female, with a mean age of 33.02 ± 15.42 years, whereas between the control groups, 115 (65.71%) were male and 60 (34.29%) were female with a mean age of 33.30 ± 19.09 years. Furthermore, of the 141 PTB participants, all were confirmed culture-positive, and of these 141 participants, 30 (21.28%) were HIV-positive, and 111 (78.72%) were HIV-negative. In addition, of the 68 EPTB patients, 18 (26.47%) were HIV-positive, and 50 (73.53%) HIV-negative. Besides this, 37 (26.24%) PTB and 11 (16.18%) EPTB patients were MDR. The 76 (36.36%) of the TB participants group were Mantoux positive, while 133 (63.63%) were Mantoux negative. In addition, all participants were examined for BCG vaccination (scar mark). Thus, out of 384 participants, 148 (38.54%) had BCG vaccination with a discernible scar, and 236 (61.46%) had no vaccination/no scar or unknown.

For assessing the specificity of sandwich ELISA, a total of 175 participants were included in the study as DCs or HCs. These included HIV-positive non-TB cases (20), cases of visceral leishmaniasis (20), and acquired toxoplasmosis (10). The 125 healthy volunteers with no obvious disease at the time of inclusion in the study were considered as healthy control. Among HC participants, 75 (60%) were Mantoux negative and 50 (40%) were IGRA negative [Table 2].

Activity of monoclonal antibodies in clinical samples

The optimum concentration of mAbs and purified recombinant antigens yielding high sensitivity and specificity was determined at 50ng/well for mAb_SS-1 and mAb_SS-5 and 25 ng/well for mAb_SS-4 and 10ng/well of purified SS-1, SS-4, and SS-5 antigens, respectively. The optimum dilution for primary (serum) was 1:100 and 1:15,000 for detection antibody (IgG-HRP), respectively. Using these dilutions, n = 384 human sera were tested with sandwich ELISA and analyzed data using statistical tools. The cutoff value was determined by the mean+2SD of the control participants. The estimated cutoff values were 0.496 for mAb_SS-1, 0.523 for mAb_SS-4, and 0.436 for mAb_SS-5, respectively. The area under curve (AUC) of each mAb was calculated between control and diseased group. AUCs were ranged from 0.97 (0.94–1.0) to 0.99 (0.98–0.99) for mAb_SS-1; 0.89(0.84–0.94) to 0.99 (0.98–1.0) for mAb_SS-4; and 0.95 (0.92–0.99) to 0.98 (0.97–1.0) for mAb_SS-5, which indicate a strong discriminatory potential of developed test (P < 0.0001) between participants of TB-positive and healthy group [Figure 5]. The sensitivity and specificity of these mAbs among study groups/subgroups are shown in Tables 3-5.

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Figure 5:

ROC curves of the activity of three purified mAbs (mAb_SS-1, mAb_SS-4, and mAb_SS-5) generated against three Mycobacterium tuberculosis recombinant antigens (SS-1, SS-4 & SS-5) with various categories of TB (a) PTB, (b) EPTB, (c) HIV-TB, and (d) MDR-TB and control (HC and DC) group. ROC: Receiver operative characteristic, TB: Tuberculosis, HC: Healthy control, DC: Diseased control, EPTB: Extrapulmonary tuberculosis, HIV-TB: HIV-associated tuberculosis, PTB: Pulmonary tuberculosis, MDR-TB: Multidrug-resistant tuberculosis.

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The overall sensitivity and specificity of these mAbs were ranging 93.53% and 97.71% for mAb_SS-5, 93.32% and 98.29% for mAb_SS-1, and 82.96% and 96.57% for mAb_SS-4, respectively [Table 3]. A significant value was found between participants of the healthy and TB-positive group (PmAb_SS-1 <0.001, PmAb_SS-4<0.001, and PmAb_SS-5<0.001) that indicated the higher discriminatory power of the test [Figure 6]. The collective sensitivity of mAb_SS-1, mAb_SS-4, and mAb_SS-5 was between 86.49% and 95.95% in PTB; 89.74% and 97.44% in EPTB; 52.08% and 95.83% in HIV-TB; and 95.83% and 100% in MDR-TB cases, respectively [Table 3].

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Figure 6:

Scatter plots of sandwich ELISA results obtained by clinical samples (a) mAb_SS-1, (b) mAb_ SS-4, and (c) mAb_SS-5. The activity of mAbs in serum samples HC, DC, and TB patients (EPTB, HIV-TB, PTB, and MDR-TB). Scatter plot indicates the antibody level per subject analyzed. A dotted horizontal line (red color) is included to show the cutoff value for a positive response of antigens. Any sample exhibiting absorbance above the cutoff value was classified as positive. ELISA: Enzyme-linked Immunosorbent Assay, HC: Healthy control, DC: Diseased Control, EPTB: Extrapulmonary Tuberculosis, HIV-TB: HIV-associated tuberculosis, PTB: Pulmonary Tuberculosis, MDR: Multidrug-Resistant Tuberculosis; Dotted red line showed the cutoff value.

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The mAb_SS-5 displayed highest sensitivity in PTB (95.95%) and 100% with MDR-TB group while mAb_SS-4 in EPTB (97.44%) and mAb_SS-1 in HIV-TB (95.83%). The mAb_SS-1 shows the highest specificity (98.29%) than other antibodies [Figure 6; Table 3].