Source: Shivange, G., et al. Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody. J. Vis. Exp. (2020).
This video demonstrates a technique to study the in vivo localization of tumor-specific antibodies in a mouse tumor xenograft model. Upon subcutaneously injecting cancer cells into mice for tumor formation, fluorescently-labeled antibodies are injected that bind to tumor-specific receptors, enabling in vivo assessment of antibody localization via imaging.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Expression and purification of antibodies
2. Fluorescent labeling
NOTE: Antibodies are labeled with the infrared dye that contains an NHS ester reactive group, which couples to proteins and forms a stable conjugate. This reaction is pH-sensitive and works best at pH 8.5. Fluorescent conjugates labeled with the dye display an absorption maximum of 774 nm, and an emission maximum of 789 nm. pH 8.5 is key for effective conjugation.
3. Mouse xenograft studies
4. Antibody localization using an in vivo imaging system
NOTE: In vivo imaging equipment (see Table of Materials) used in this experiment uses a set of high-efficiency filters and spectral un-mixing algorithms for noninvasive visualization and tracking of cellular and genetic activity within a living organism in real time. The system provides both fluorescence and bioluminescence monitoring capability.
The authors have nothing to disclose.
FreeStyle CHO media | Gibco Life Technologies | Cat # 12651-014 | |
Anti-Anti (100X) | Gibco Life Technologies | Cat # 15240-062 | |
Anti-Clumping Agent | Gibco Life Technologies | Cat # 01-0057DG | |
BD Insulin Syringe | BD BioSciences | Cat #329420 | |
Caliper IVIS Spectrum | PerkinElmer | Cat #124262 | |
CHO CD EfficientFeed B | Gibco Life Technologies | Cat #A10240-01 | |
Corning 500 mL DMEM (Dulbecco's Modified Eagle's Medium) | Corning | Cat # 10-13-CV | |
Corning 500 mL RPMI 1640 | Corning | Cat # 10-040-CV | |
Cy5 conjugated Anti-Human IgG (H+L) | Jackson ImmunoResearch | Cat # 709-175-149 | |
GlutaMax-I (100X) | Gibco Life Technologies | Cat # 35050-061 | |
HiPure Plasmid Maxiprep kit | Invitrogen | Cat # K21007 | |
HiTrap MabSelect SuRe Column | GE Healthcare | Cat # 11-0034-93 | |
Infusion | Takara BioScience | STO344 | |
IRDye 800CW NHS Ester | LI-COR | Cat # 929-70020 | |
Isoflurane, USP | Covetrus | Cat # 11695-6777-2 | |
Lubricant Eye Ointment | Refresh Lacri-Lube | Cat #4089 | |
Matrigel | Corning | Cat # 354234 | |
PEI transfection reagent | Thermo Fisher | Cat # BMS1003A | |
Slide-A-Lyzer Dialysis Cassettes | Thermo Scientific | Cat # 66333 | |
Steritop Vacuum Filters | Millipore Express | Cat #S2GPT02RE | |
Trypsin-EDTA | Gibco Life Technologies | Cat # 15400-054 | |
Experimental Models: Cell lines | |||
Human: OVCAR-3 | American Type Culture Collection | ATCC HTB-161 | |
Human: CHO-K cells | Stable transformed in our lab | ATCC CCL-61 | |
Mouse: 4T1 | Kind gift from Dr. Chip Landen, UVA | ||
Mouse: MC38 | Kind gift from Dr. Suzanne Ostrand-Rosenberg, UMBC | Authenticated by STR profiling | |
Mouse: MC38 hFOLR1 | Generated in our laboratory (This paper) | ||
Experimental Models: Animal | |||
Mice: athymic Nude Foxn1nu/Foxn1+ | Envigo | Multiple Orders | |
Mice: NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ | Jackson Laboratory | Multiple Orders |