Overview
This video demonstrates a technique for the detection of extrapulmonary tuberculosis. Superparamagnetic iron oxide (SPIO) nanoprobes, conjugated to Mycobacterium-specific antibodies, are injected into a mouse previously infected with Mycobacterium bovis BCG, labeling the granuloma and providing a better imaging contrast for its detection.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. SPIO-MtbsAb synthesis
- Synthesize SPIO-conjugated 2,2'-(ethylenedioxy)bisethylamine (EDBE) using previously reported methods.
- Synthesize SPIO-EDBE-succinic anhydride (SA).
- Stir an alkaline solution (5 M NaOH; 10 mL)) of SPIO-EDBE and SA (1 g; 10 µmol) at room temperature for 24 h.
- Dialyze the solution with 20 changes of 2 L of distilled water using molecular porous membrane tubing (12,000-14,000 MW cutoff). 6 h for each change.
- Finally, add 100 μL of SPIO-EDBE-SA (4 mg/mL of Fe) to 400 μL of 4.5 mg/mL Mycobacterium tuberculosis surface antibody (MtbsAb) to synthesize SPIO-MtbsAb by using 1-hydroxybenzotriazole and (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate as catalysts and stir the solution at room temperature for 24 h.
- Finally, separate the solutions from the unbound antibody through gel filtration chromatography.
- Load the reaction mixture (5 mL) on a 2.5 cm × 33 cm column and elute using a phosphate-buffered saline (PBS) buffer. Confirm Ab–nanoparticle complex (i.e., nanoprobe) using a bicinchoninic acid protein assay kit.
2. Particle morphology observation and relaxation tier measurement
- Examine average particle size, morphology, and size distribution using a transmission electron microscope at a voltage of 100 kV.
- Drop-cast the composite dispersion onto a 200-mesh copper grid and air dry at room temperature before loading it onto the microscope.
- Measure the relaxation time values (T1 and T2) of the nanoprobes using the NMR relaxometer at 20 MHz and 37.0 °C ± 0.1 °C.
- Calibrate the relaxometer before each measurement.
- Record the r1 and r2 values from the eight data points generated through inversion-recovery and the Carr-Purcell-Meiboom-Gill pulse sequence, respectively, to determine r1 and r2 relaxivities.
3. Cell imaging
- Cultivate human monocytes THP-1 in RPMI 1640 with 10% fetal bovine serum, 50 µg/mL gentamycin sulfate, 100 units/mL penicillin G sodium, 100 µg of streptomycin sulfate, and 0.25 µg/mL fungizone in a 5% CO2 incubator at 37 °C.
- Incubate SPIO-MtbsAb nanoprobes (2 mM) with 106 colony forming units (CFU) of Mycobacterium bovis BCG preincubated with 1 × 107 activated monocytes in microcentrifuge tubes (1 mL) in a 5% CO2 incubator at 37 °C for 1 h.
- Centrifuge tubes at 200 x g and discard the supernatant. Redissolve pellets in the medium (200 µL).
- Scan the samples using a fast gradient echo pulse sequence (Repetition time (TR) = 500; Echo time(TE) = 20; Flip angle = 10°) through 3.0-T MRI to determine the nanoprobe's specificity and sensitivity.
4. BCG (Bacillus Calmette–Guérin) inoculation
- Reconstitute the lyophilized vaccine or bacterial stock in Sauton's medium and then dilute the stock with saline until properly dispersed as previously described.
- Inoculate a live attenuated strain of M. bovis BCG, obtained from ADIMMUNE (Taipei, Taiwan) (Connaught strain; ImmuCyst Aventis, Pasteur Mérieux) at a volume of 0.1 mL/mouse (i.e., 107 CFU) intradermally into the left or right dorsal scapular skin of mice, as described previously. Inject saline into mice as a negative control. Monitor animals daily after BCG inoculation.
- Sacrifice animals 1 month after bacteria inoculation using carbon dioxide euthanasia. Harvest the tissue from the intradermal inoculation site. Fix the tissue in 10% formalin and embed it in paraffin for serial sections at 5-10 µm. Stain tissue sections with the hematoxylin/eosin and Ziehl-Neelsen stains for acid-fast bacteria and with Berlin blue for ferric iron.
5. In vivo MRI
- Inject ketamine (80 mg/kg of body weight) and xylazine (12 mg/kg body weight) subcutaneously into mice for animal anesthesia.
- Inject SPIO-TbsAb probes (2 nmol/200 µL) into the tail veins of mice. MR image mice before and immediately after probe injection and then every 5 min for 30 min to acquire T2-weighted fast spin-echo images (TR = 3000; TE = 90; field of view = 8).
- Quantitatively analyze all MR images using signal intensity (SI), a measurement of defined regions of interest in comparable locations of an Mtb granuloma center and the back muscle adjacent to a granulomatous area.
- Calculate relative signal enhancements using the SI measurement before (SIpre; control) and 0-3 h after (SIpost) injection of the contrast agents using the formula
[(SIpost - SIpre)/SIpre] × 100
where SIpre is the SI of the lesion on the pre-enhanced scan and SIpost is the SI of the lesion on the post-enhanced scan.
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Materials
Name | Company | Catalog Number | Comments |
(benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate | Sigma-Aldrich | ||
1-hydroxybenzotriazole | Sigma-Aldrich | ||
Human monocytic THP-1 | |||
M. bovis BCG | Pasteur Mérieux | Connaught strain; ImmuCyst Aventis | |
MRI | GE medical Systems | 3.0-T, Signa | |
NMR relaxometer | Bruker | NMS-120 Minispec | |
SPECTRUM molecular porous membrane tubing, 12,000 -14,000 MW cut off | Spectrum Laboratories Inc | ||
TB surface antibody- Polyclonal Antibody to Mtb | Acris Antibodies GmbH | BP2027 | |
transmission electron microscope | JEOL | JEM-2000 EX II |