Preparation of Monoclonal Antibodies Against Mycobacterium tuberculosis TB10.4 Antigen

Construction of recombinant plasmids

The esxH gene was amplified from the genome of M. bovis BCG strain Pasteur 1173P2, with a pair of gene-specific primers (forward: 5′-GCGGGATCCATGTCGCAAATCATGTA-3′, reverse: 5′-TATCTCGAGCTAGCCGCCCCATTT-3′) containing the BamHI and XhoI restriction sites (underlined), respectively. The cycling parameters of the PCR reaction were: initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 7 min. After digestion with BamHI and XhoI (TakaRa, Dalian, China), the amplified esxH product was inserted into the corresponding region of the pET30a(+) (Novagen, Darmstadt, Germany) and pGEX-6P-1 expression vectors (Amersham Bioscience, Uppsala, Sweden) with T4 ligase (TakaRa), and the recombinant plasmids were confirmed by restriction analysis. The gene was identified with a Basic Local Alignment Search Tool (BLAST) analysis. Host Escherichia coli BL21 and BL21(DE3) cells were transformed with the recombinant plasmids pGEX-6p-1–esxH and pET-30(a)–esxH, respectively.

Expression and purification of recombinant TB10.4 protein

BL21(pGEX-6p-1–esxH) and BL21(DE3)(pET-30(a)–esxH) cells were induced with 0.1 mM isoloropyl-β-D-thiogalactopyranoside (IPTG) at 20°C and 27°C, respectively, to produce recombinant proteins rGST–TB10.4 and rHis–TB10.4, respectively. The induced bacteria were centrifuged at 12,000 g for 10 min at 4°C and washed twice with phosphate-buffered saline (PBS). The bacteria were resuspended in PBS and disrupted by sonication. The rGST–TB10.4 and rHis–TB10.4 proteins were purified with a glutathione–Sepharose column (GE Healthcare, Piscataway, NJ) and a Ni²⁺ Sepharose column (Novagen), respectively. The purified proteins were collected and analyzed on a 12% SDS-PAGE gel stained with Coomassie Brilliant Blue.

Immunization of mice and establishment of hybridomas

BALB/c mice (female, 6–8 weeks old) were injected subcutaneously with 100 μg of purified rHis–TB10.4 protein mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO). A booster injection of the same antigen mixed with an equal volume of Freund's incomplete adjuvant (Sigma-Aldrich) was given after a 2-week interval. Serum titers were detected with an indirect ELISA and purified rGST–TB10.4 protein before we proceeded to the cell fusion stage. The mice were given a final injection exactly 3 days before fusion. The splenocytes were harvested and fused with SP2/0 myeloma cells using standard hybridoma methods.⁽¹⁴⁾

Screening hybridoma cells and MAb production

The titers of the hybridoma supernatants were determined with an indirect ELISA with purified rGST–TB10.4 protein. In this assay, a polyclonal antibody (serum from BALB/c mice immunized with rHis–TB10.4) was used as the positive control, and the supernatant of SP2/0 myeloma cells was used as the negative control. Hybridoma cells that secreted MAbs directed against TB10.4 protein were subcloned and cultured on a larger scale. After two to three rounds of subcloning, 5×10⁵ hybridoma cells were intraperitoneally injected into BALB/c mice to induce ascites containing MAbs directed against TB10.4 protein.

Isotype and titer analysis of MAbs

The isotypes of the MAbs directed against TB10.4 were determined with mouse monoclonal antibody isotyping reagents (Sigma-Aldrich). The titers were determined with an indirect ELISA with purified rGST–TB10.4 protein, performed as previously described.⁽¹⁵⁾ The ascites containing the MAbs were diluted in a range from 1:1000 to 1:1,024,000; the last dilution of the ascites at which the ratio OD₄₅₀ sample/OD₄₅₀ control was ≥2.1 was deemed to be the MAb titer in the ascites.

Immunoblotting analysis

An immunoblotting analysis was performed as previously described.⁽¹⁵⁾ In brief, purified rHis–TB10.4 and rGST–TB10.4 proteins were resolved with 12% SDS-PAGE, and the proteins were then transferred onto polyvinylidene difluoride membrane. The MAbs directed against TB10.4 from the hybridoma cell lines were used as the primary antibodies and horseradish-peroxidase-conjugated goat anti-mouse IgG antibody was used as the secondary antibody. The protein bands were visualized with 3,3′-diaminobenzidine.

Construction of recombinant plasmids and expression of recombinant TB10.4 proteins

The esxH gene was amplified from the genome of M. bovis BCG strain Pasteur 1173P2 and was inserted into the pET30a(+) and pGEX-6P-1 expression vectors. The recombinant plasmids pET30(a)–esxH and pGEX-6p-1–esxH were identified with restriction digestion and PCR analysis. The expected band size of the restriction digest products was 300 bp.

The expression of the fusion proteins was induced with IPTG and the predicted molecular weights of the recombinant proteins were 16 kDa from pET30(a)–esxH and ∼35 kDa from pGEX-6p-1–esxH. The recombinant proteins were purified with a Ni²⁺ Sepharose column and a glutathione–Sepharose column, respectively. SDS-PAGE analysis confirmed the successful production and purification of rGST–TB10.4 and rHis–TB10.4 proteins from pGEX-6p-1–esxH and pET30(a)–esxH, respectively (Fig. 1). Bands of the expected size were observed only from samples transfected with esxH that were induced with IPTG.

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FIG. 1.

Expression and purification of rGST–TB10.4 (A) and rHis–TB10.4 (B). M, unstained protein molecular weight marker; lane 1, BL21(pGEX-6p-1–esxH) in absence of IPTG; lane 2, BL21(pGEX-6p-1–esxH) induced with IPTG; lane 3, purified rGST–TB10.4 protein; lane 4, BL21(DE3)-pET-30(a) induced with IPTG; lane 5, BL21(DE3)-pET-30(a)–esxH in absence of IPTG; lane 6, BL21(DE3)-PET-30(a)–esxH induced with IPTG; lane 7, purified rHis–TB10.4 protein.

Generation of MAbs against TB10.4

The supernatants of growing colonies were screened for the presence of secreted antibodies specifically directed against TB10.4 using an indirect ELISA, and eight colonies were chosen and subcloned. After three rounds of subcloning and detection, eight hybridomas were established and mouse ascites containing MAbs were prepared as described in the Methods section.

Isotype and titer analyses of MAbs

Isotype analysis of the MAbs produced in mouse ascites and directed against TB10.4 indicated that all eight MAbs were of the IgG1 isotype (Table 1). The indirect ELISA assay indicated that the titers of these MAbs ranged from 10⁵ to 10⁶ (Table 1).

MAbs recognize recombinant TB10.4

To confirm that all the MAbs produced recognized both the rHis–TB10.4 and rGST–TB10.4 proteins, the purified MAbs were tested with immunoblotting. The results are shown in Figure 2, in which the expected 16 kDa and 35 kDa bands were detected. This confirms that all the MAbs bound the rHis–TB10.4 and rGST–TB10.4 proteins.

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FIG. 2.

Western blot analysis of His–TB10.4 and GST–TB10.4 with TB10.4 MAbs. Recombinant His–TB10.4 (A) and GST–TB10.4 (B) were detected with MAbs B1, C5, D2, F5, C12, G2, E11, and B3, as shown in lanes 1–8, respectively.