M. tuberculosis was isolated from a patient with confirmed TB and pleural effusion at Montefiore Medical Center (Bronx, N.Y.). GC from liquid culture supernatant (Sauton's medium) of this strain was isolated by precipitation with 70% ethanol and was further purified by anion-exchange and gel filtration chromatography, using methods similar to those described by Lemassu and Daffe (21). The GC contained only glucose, as confirmed by gas-liquid chromatography and ¹³C magnetic resonance spectroscopy.

1-Cyano-4-dimethylaminopyridinium tetratfluoroborate (CDAP) chemistry was used to conjugate GC to recombinant Pseudomonas aeruginosa exoprotein A (rEPA) as described by Konadu et al. (20). Female 6- to 8-week-old BALB/cJ mice were immunized by intraperitoneal injection of 5 μg of GC-rEPA in 200 μl of 50% Freund's incomplete adjuvant (Sigma) in phosphate-buffered saline (PBS). Animals were given a booster dose with 5 μg of GC-rEPA in 200 μl of PBS at 2 and 4 weeks. The mice were bled from the retroorbital plexus 2, 4, and 6 weeks after the initial immunization. Binding of the sera to GC was measured by ELISA, as described below. The mouse with the highest titer was given a booster dose at 8 weeks with 5 μg of GC-rEPA in 200 μl of PBS, and the spleen was harvested 3 days later. Hybridomas were generated as described by de St. Groth and Scheidegger (10). Briefly, spleen cells were fused with NSO cells at a ratio of 4:1, suspended in medium containing hypoxanthine-aminopterin-thymidine (Sigma), plated in 20 96-well culture plates, and incubated at 37°C with 10% CO₂. Supernatants from wells containing hybridomas were screened for production of antibody to GC using ELISA. The hybridoma designated 24c5 was recovered and cloned twice on soft agar. The isotype of the antibody was determined by ELISA using isotype-specific secondary antibodies (Southern Biotechnology Associates, Birmingham, Ala.) in the GC ELISA. Supernatant from the 24c5 hybridoma was concentrated by using an Amicon 8400 concentrator and a YM100 membrane (Amicon/Millipore, Bedford, Mass.) under compressed N₂. The antibody concentration was determined by ELISA by comparison to a standardized amount of isotype-matched control antibody (ICN Biomedicals, Aurora, Ohio).

An ELISA of M. tuberculosis and other mycobacteria was performed as described previously (26). After washing of bovine serum albumin (BSA)-blocked microtiter plates containing whole-cell mycobacteria, MAb 24c5 was added in TBS, and the plates were incubated for 1 h at 37°C. After the plates were washed, 1 μg of goat anti-mouse alkaline phosphatase-conjugated (GAM-AP) immunoglobulin G1 (IgG1) antibody (Southern Biotechnology Associates) per ml in TBS was added to each plate, and the plates were incubated for 1 h at 37°C and then developed using p-nitrophenylphosphate (Sigma). The M. tuberculosis ELISA plates were processed simultaneously. ELISA measurements were obtained in triplicate for each sample and averaged. Experiments were performed twice (including culture of M. tuberculosis), with similar results.

Polystyrene microtiter plate wells were coated with 50 μl of GC (10 μg/ml) in carbonate buffer (0.015 M Na₂CO₃, 0.035 M NaHCO₃, 0.003 M NaN₃; pH 9.8) by incubating the plates overnight at 4°C. The wells were then blocked by adding 200 μl of 3% BSA in TBS and incubating the plates overnight at 4°C. MAb 24c5 or serum from M. tuberculosis-infected mice was added to the wells and incubated for 1 h at 37°C. For determination of the murine IgG titer to GC, serum was diluted in TBS with 0.075 M β-mercaptoethanol and heated for 1 h at 37°C to dissociate IgM pentamers, and the solution was immediately assayed for IgG reactivity to GC by ELISA. The plates were then washed, and 50 μl of a 1-μg/ml solution of GAM-AP antibody (GAM-AP IgG1 antibody recognizing MAb 24c5 and GAM-AP IgG or IgM antibody for murine serum) was diluted in TBS and added to each well for 1 h at 37°C. The ELISA plates were washed and developed by using p-nitrophenylphosphate substrate, as described by Schwebach et al. (26). Type VIII (slipper limpet) and type IX (bovine liver) glycogens (Sigma) were used in ELISA at concentrations of 1, 10, and 100 μg/ml; the conditions used were identical to those used for GC ELISA.

For binding an interphase extract of M. tuberculosis culture supernatant known to contain GC, 300 ml of a day-25 M. tuberculosis culture supernatant was filtered twice with a 0.2-μm-pore-size Millipore filter (Amicon/Millipore) and vacuum concentrated 10-fold at room temperature (RT). The concentrated supernatant was extracted with chloroform-methanol-water (3:4:3, vol/vol/vol). The resulting interphase was collected for ELISA analysis by extraction with 10 ml of distilled H₂O. One hundred microliters of the extracted interphase was mixed with 800 μl of TBS and incubated for 1 h at 37°C in 16 wells (50 μl per well) of a 96-well polystyrene microplate (Corning) before blocking with 3% BSA-TBS overnight. An ELISA using MAb 24c5 was then performed with this preparation and all preparations described below by diluting MAb 24c5 threefold from a starting concentration of 100 μg/ml. ELISA of M. tuberculosis and GC (1 μg/ml) were performed as described above; for the M. tuberculosis ELISA M. tuberculosis Erdman cells grown for 20 days in 7H9 medium were used. An AM (10 μg/ml) ELISA was performed as described by Schwebach et al. (26). MAb binding to purified M. tuberculosis Erdman lipoarabinomannan (10 μg/ml), phosphatidylinositol mannoside (10 μg/ml), mycolyl-arabinogalactan-peptidoglycan complex (1 mg/ml), total lipid fraction (1 mg/ml), and fast-growing mycobacterial lipomannan (10 μg/ml) was examined as described by Glatman-Freedman et al. using ELISA (16). These antigens were kindly supplied by P. J. Brennan and J. T. Belisle (Department of Microbiology, Colorado State University, Fort Collins). M. tuberculosis AM was isolated as described previously (26). GAM-AP IgG1 secondary antibody was used in all these ELISA.

Treatment of M. tuberculosis with proteinase K during ELISA was done as previously described (16). Briefly, cells of M. tuberculosis Erdman grown in 7H9 medium for 20 days were added to ELISA plates as described above. The M. tuberculosis was then treated with 1 mg of proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) per ml in PBS or with PBS alone (as a control) at RT for 20 h before continuation of the ELISA as described above. This enzyme preparation was active in similar experiments used to digest organ homogenate (26).

For immunofluorescence (IF) studies, MAb 24c5 and the irrelevant isotype-matched control MAb 2D10 (recognizing Cryptococcus neoformans glucuronoxylomannan) were purified from murine ascites fluid using a protein G column (Pierce, Rockford, Ill.). The purified antibodies were then conjugated to the ALEXA-488 fluorophore (Molecular Probes, Eugene, Oreg.) by following the manufacturer's directions. Cells of M. tuberculosis Erdman grown for 25 days in 7H9 medium were centrifuged (2,000 × g, 8 min), washed in PBS, and fixed in 5% PBS-buffered formalin at room temperature for several days (which is a safe method for M. tuberculosis inactivation [27]). The fixed cells were washed three times in PBS. The M. tuberculosis-PBS suspension was then placed on poly-l-lysine slides (Sigma) and air dried overnight. The slides were then blocked with 1% BSA-TBS for 1 h at RT. The blocking solution was then removed, and a 1:100 dilution (10 μg/ml) of conjugated MAb (or conjugated irrelevant control) in 1% BSA-TBS was added to the slides. The slides were then incubated for 1 h at 37°C and washed three times with TBS. Coverslips were placed on the slides before viewing with an AX-70 fluorescence microscope (Olympus, Melville, N.Y.).

For whole-mount transmission electron microscopy, M. tuberculosis Erdman was grown for 25 days in 7H9 medium, collected by centrifugation (2,000 × g, 8 min), washed twice with 0.1 M phosphate buffer (PB) (Fisher), and collected by centrifugation again. A solution containing 1 mg of poly-l-lysine (Sigma) per ml in PB was applied to Formvar/carbon-coated 300-mesh nickel grids (Polysciences, Inc., Warrington, Pa.) for 5 min. Bacteria were then suspended in PB and allowed to settle onto the grids for 15 min. Bacteria were fixed onto the grids using 2.5% glutaraldehyde (Polysciences) in PB. The grids were washed five times (3 min each) in PB and blocked (10 min) in 0.26 M NH₄Cl (Fisher) before further blocking for 15 min in 0.5% BSA-1% coldwater fish gelatin PB (BSA-gel PB) (Aurion, Wageningen, The Netherlands). Hybridoma supernatants containing either MAb 24c5 or the irrelevant isotype-matched MAb control (MAb C293, recognizing dipeptidyl peptidase V of Aspergillus fumigatus, kindly provided by M. Feldmesser) were added to BSA-gel PB at a final concentration of 100 μg/ml, and grids were incubated in these solutions for 2 h at RT. The grids were washed five times (3 min each) in BSA-gel PB and transferred to BSA-gel PB containing 0.05% goat anti-mouse IgG conjugated to 5-nm gold (Aurion) for 1 h at 37°C. The grids were washed in BSA-gel PB three times (3 min each) and then in PB three times (3 min each), and then they were fixed overnight by incubation in PB containing 2.5% glutaraldehyde and 2% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, Pa.) at 4°C. The fixed grids were moved to pathogen level 2 for washing in PB (five times, 5 min each) and in distilled H₂O (five times, 5 min each) and staining for 90 s with 1% phosphotungstic acid (Fisher). Excess phosphotungstic acid was removed from the grids using Whatman no. 5 filter paper, and the grids were viewed with a 1200EX transmission electron microscope (JOEL, Peabody, Mass.) at an accelerating voltage of 80 kV.

Capture ELISA were used to detect GC antigen in culture supernatants. These assays were modeled on capture assays developed for detection of C. neoformans glucuronoxylomannan (3), M. tuberculosis lipoarabinomannan (17), and M. tuberculosis AM (26). Serum from a female New Zealand White rabbit immunized intraperitoneally with 50 μg of GC-rEPA in Freund's incomplete adjuvant (Sigma) was used for detection of GC captured by MAb 24c5. ELISA plates were first incubated with 50 μl of TBS containing 2 μg of unlabeled goat anti-mouse IgG1 antibody per ml for 1 h at 37°C. The wells were blocked by adding 200 μl of 1% BSA in TBS and incubating the plates overnight at 4°C. The plates were then washed between steps with TBS containing 0.05% Tween 20 (Sigma) as described above. After the washing, 1 μg of MAb 24c5 per ml in TBS was added to each well of the 96-well plates, and the plates were incubated for 1 h at 37°C. Aliquots of concentrated M. tuberculosis culture supernatant were added to the plates after the washing. Purified GC, added to 7H9 medium, 7H9-T medium, or TBS and serially diluted in TBS, was used as a standard and a positive control. The same media without purified GC were also used as negative controls. Following incubation at 37°C for 1 h, day 7 rabbit serum (stored frozen at −20°C) diluted 1:100 in TBS was added to the 96-well plates. After 1 h of incubation at 37°C, the plates were washed, 1 μg of goat anti-rabbit alkaline phosphatase-conjugated IgM (Southern Biotechnology Associates) per ml in TBS was added, and the assay mixture was developed as described above. An absorbance signal that was ≥1.5 times the background signal was considered positive for detection of GC.

The specificity of MAb 24c5 was explored by testing the reactivity of this MAb with purified M. tuberculosis cell wall fractions (Fig. 2C). Binding of MAb 24c5 was limited to GC and the GC-rich interphase (21) recovered from a chloroform-methanol extract of M. tuberculosis Erdman supernatant (Fig. 2C). MAb 24c5 did not bind M. tuberculosis AM, lipoarabinomannan, phosphatidylinositol mannoside, mycolyl-arabinogalactan-peptidoglycan, total lipid fraction, or mycobacterial lipomannan, which are components of the remainder of the nonprotein M. tuberculosis cell surface (2, 4, 7). In addition, MAb 24c5 did not react with an M. tuberculosis protein antigen(s), as treatment of M. tuberculosis whole cells with proteinase K did not reduce its binding to the bacteria (Fig. 2D). Hence, MAb 24c5 binds M. tuberculosis GC.

MAb 24c5 bound to different sources of glycogen in ELISA, indicating antigenic similarity between M. tuberculosis GC and glycogen (Fig. 2E and F). This is not suprising as GC has structural resemblance to glycogen (21). More binding was observed with higher concentrations of glycogen, and the apparent affinity of binding to type ix glycogen was greater than the apparent affinity of binding to type viii glycogen. Differences in the reactivity of MAb 24c5 with the two forms of glycogen may reflect differences in the branching of these molecules.

Previous studies described M. tuberculosis GC on the surface of mycobacteria (22) or M. tuberculosis (21) without determining the quantities of surface GC for different periods of culture growth. To measure the amounts of GC antigen present on the surface of M. tuberculosis after different periods of growth, M. tuberculosis was cultured with (7H9-T medium) and without (7H9 medium) Tween 80 to compare bacteria grown as a pellicle and bacteria grown as a diffuse suspension, respectively, and cells were tested for reactivity with MAb 24c5. For this measurement, the number of bacteria used in the ELISA was standardized on the basis of the amount of protein in sedimented bacterial samples of the same volume as described previously (23, 26). MAb 24c5 bound with similar intensities to M. tuberculosis Erdman and CDC 1551 grown in 7H9 or 7H9-T medium, regardless of the period of growth (Fig. 3). M. tuberculosis CDC 1551 grown in 7H9 medium exhibited only slightly greater binding during later periods of growth than when it was grown for shorter periods of time. Binding for M. tuberculosis Erdman grown in 7H9 medium for 11 days or for CDC 1551 grown in 7H9-T medium for 11 days was only slightly less than the binding observed after growth for longer periods of time. These results indicate that a stable amount of GC antigen is associated with the M. tuberculosis cell surface during in vitro culture. Thus, the relatively constant expression of the GC antigen is different than expression of the AM antigen previously studied (26), as the amount of detectable AM antigen on M. tuberculosis Erdman increased after longer culture periods. In addition, the AM antigen was more prevalent on M. tuberculosis strain Erdman whole cells than on CDC 1551 whole cells, while similar amounts of the GC antigen were detected on the two M. tuberculosis strains (Fig. 3). These differences in AM and GC may have immunological implications, such as implications in the phagocytosis of M. tuberculosis (13, 32).

MAb 24c5 was used to determine whether GC is found in nontuberculosis mycobacteria. The MAb 24c5 antigen was detected on the surfaces of M. kansasii, BCG Pasteur, M. smegmatis, M. phlei, M. fortuitum, and M. avium grown in vitro by ELISA (Fig. 6). This is in agreement with the work done by Lemassu et al. (22), which indicated that glucose is present in the extracellular carbohydrates of various mycobacteria. It should be noted that other growth conditions could influence the amount of MAb 24c5 binding to whole-cell mycobacteria. Our results, which are qualitative, could be very different if extended periods of growth were used, for example.

The increased amounts of polysaccharide (34) and polysaccharide antigen (26) in the culture media of M. tuberculosis grown for longer periods imply that M. tuberculosis cells shed polysaccharide antigen during prolonged culture. We therefore developed a capture ELISA to detect the presence of GC in M. tuberculosis culture supernatants (Fig. 7). The plot of absorbance versus GC concentration has a parabolic shape (revealing prozone-like effects for MAb binding to GC) with a limited slope and no conspicuous inflection point after dilution of GC in the assay mixture (Fig. 7A). As the assay tapered to background upon dilution of antigen, we were able to quantify the amounts of 24c5 antigen present in various culture supernatants relative to purified GC in the same media (Table 1). Day 11 M. tuberculosis culture supernatants did not have detectable concentrations of GC antigen (Table 1). Day 20 and day 25 M. tuberculosis culture supernatants were found to contain the GC antigen, irrespective of the presence of Tween 80 in the medium. Both M. tuberculosis Erdman and CDC 1551 accumulated this antigen (Fig. 7) at concentrations of ≥1 mg/ml after 25 days of growth (Table 1). The presence of Tween 80 diminished the amount of GC antigen found at day 25. We were unable to detect the 24c5 GC antigen in M. tuberculosis-infected BALB/c murine organ homogenates using this assay (data not shown). These results are in accord with the accumulation of AM antigen in the media of culture supernatants with or without the detergent Tween 80 (26).