QTZ for In Vivo Immuno-Bioluminescence Imaging[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Reagent Preparation: Dissolve QTZ in an in vivo injection buffer to prepare a stock solution at a suitable concentration (can be dissolved up to ~2mM).
2. Paired Enzyme Preparation: Prepare the amber light-emitting mutant luciferase QLuc, which pairs with QTZ. This can be done by using an adeno-associated virus (AAV) system carrying a QLuc expression gene driven by a liver-specific TBG promoter, or by constructing and expressing/purifying a QLuc fusion protein with a targeting antibody fragment (e.g., anti-Her2 scFv).
3. Animal Model and Labeling: For immuno-bioluminescence imaging (ImmunoBLI), inoculate AkaLuc-labeled tumor cells in a xenograft mouse model. Inject AAV expressing QLuc (or the scFv-QLuc fusion protein) via the tail vein, or administer the purified scFv-QLuc fusion protein into tumor-bearing mice via tail vein injection.
4. Imaging Procedure: After QLuc expression is established in the target tissue (e.g., liver) or the scFv-QLuc fusion protein has circulated and accumulated in vivo (e.g., wait for 6 hours post-protein injection), administer QTZ to the mice via intraperitoneal (i.p.) or intravenous (i.v.) injection (e.g., 0.1μmol).
5. Signal Acquisition: Using a cooled CCD camera or bioluminescence imaging system equipped with appropriate filters (consider using a far-red filter of 600-700nm to reduce background interference), perform in vivo imaging on anesthetized mice to capture the amber light emitted by the QLuc-QTZ reaction (emission peak ~585nm).
Precautions:
(1) The QLuc-QTZ luciferase-luciferin pair emits red-shifted light (peak ~585nm), with over 52% of its total emitted light having wavelengths above 600nm, enhancing tissue penetration and signal-to-noise ratio for deep-tissue in vivo imaging.
(2) A The in vitro intrinsic brightness of the QLuc-QTZ pair is approximately 24% of the NLuc-FRZ pair but over 60 times brighter than the FLuc-D-luciferin pair. In a mouse liver model, its long-lasting luminescence signal (>30 minutes) outperforms other tested small single-domain NLuc mutants (teLuc, LumiLuc).
(3) The solubility of QTZ in water is approximately 62.5-125μM, and its solubility in the in vivo injection buffer can reach ~2mM.
(4) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Xiong Y, Zhang Y, Li Z, et al. Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo. J Am Chem Soc. 2022 Aug 10;144(31):14101-14111. |
