This protocol demonstrates the expression and purification of antibodies and real time monitoring of antibody distribution in tumor and tissue. Screening antibodies with described protocol will help select antibodies that have limited non-specific tissue distribution, and enrich tumor localizations. The described technique is quick and cost-effective.
Other techniques, such as single-photon emission computed tomography and positron emission tomography, are very expensive and require radioactive and other sophisticated tracers. The technique is not limited to cancer targeting antibodies. It can be applied to any other disease, such as lung or heart diseases, for the analysis of antibody distribution in the body.
Demonstrating the procedure, will be Jeremy Gatesman, a veterinary technician from my university. Raw CHO cells in 200 milliliters of media, in Delong, Erlenmeyer baffled flasks. Combine five milliliters of CHO freestyle media with 50 micrograms of variable heavy clone DNA, and 75 micrograms a variable light clone DNA in a 15 milliliter tube.
Then, vortex the tube. Leave the mixture at room temperature for five minutes. Add 750 microliters of one milligram per milliliter polyethylenimine stock to the DNA solution, and aggressively vortex it for 30 seconds.
Leave the mixture at room temperature for an additional five minutes. Add the entire mixture to the CHO cells while manually shaking the flask. Immediately incubate the cells at 37 degrees Celsius while shaking at 130 RPM.
On the next day, add two milliliters of 100 X anti-clumping agent, and two milliliters of 100 X antibacterial antimycotic solution to the cells. Incubate the flasks at 32 to 34 degrees Celsius, with shaking at 130 RPM. Continue culturing the cells for 10 to 11 days, adding 10 milliliters of Tryptone N1 feed, and two milliliters of 100 X glutamine supplement on every fifth day.
Count the cells on every third day to ensure that cell viability remains above 80%On day 10 or 11, harvest the medium for antibody purification by centrifuging the culture at 3000 times G, and four degrees Celsius for 40 to 60 minutes. Then, filter the clear media with a 0.22 micrometer bottle filter. To purify the antibodies, equilibrate the protein A column with two column volumes of binding buffer.
Pass the filtered media through the column at the flow rate of one milliliter per minute. Then wash the column with two column volumes of binding buffer. Use five milliliters of elution buffer to elute the antibody into 500 microliter fractions.
And neutralize the pH of the alluded antibody by adding 10 microliters of neutralization buffer for fraction. Measure the concentration of the purified antibody with a spectrophotometer using the default protocol for IgG. Dialyze 0.5 milliliters of the antibody using a dialysis cassette in one liter of conjugation buffer.
After four hours, transfer the dialysis cassette to fresh buffer and dialyze overnight. To set up a conjugation reaction, add 0.03 micrograms of IRR dye 800 CWU per one milligram of antibody in a total volume of 500 microliters. Incubate the mixture for two hours at 20 degrees Celsius, then purify the labeled conjugates by extensive dialysis against PBS.
Estimate the degree of labeling by measuring the absorbance of the dye 780 nanometers, and absorbance of the protein at 280 nanometers. To develop mouse xenographs, gently lift the skin of the animal and separate it from the underlying muscle layer. Slowly inject 100 microliters of cell suspension under the skin with a 26 gauge needle.
Wait for a few seconds before taking the needle out so that basement matrix medium inform the semi-solid gel-like structure along with cells under the skin, which will prevent the mixture from coming out of the injection site. To perform in vivo imaging, inject the dye labeled antibody via the tail vein. After anesthetizing the mouse, check for the lack of response to pedal reflexes, and dilate the vein by applying warm water.
Use a 1cc insulin syringe with a 26 gauge needle to inject 25 micrograms of the labeled antibody in a volume of 100 microliters. As a negative control, label and inject the non-specific IgG isotype antibody which does not target cancer cells. Perform in vivo imaging eight, 24 and 48 hours after antibody injections.
In the imaging software, click initialize. Confirm that the stage temperature is 37 degrees Celsius. In the control panel, set up fluorescence imaging through the imaging wizard, and set the excitation to 773 nanometers, emission to 792 nanometers.
Transfer the anesthetized mouse into the imaging chamber. Position it on the imaging field using the nose cone. When ready, click acquire on the control panel for the image acquisition, and click auto expose.
The generated image is the overlay of the fluorescence on the photographic image with optical fluorescence and density displayed in units of counts or photons. Positive cDNA clones with variable heavy and variable light domains were confirmed with colony PCR. Representative results of purified farletuzumab run on non-reducing and reducing SDS page are shown here.
Heavy and light chains produced 50 and 25 kilodalton bands after reduction. Binding of antibodies to native proteins in the cell surface was also confirmed. Fluorescently labeled antibodies were tail vein injected, into mice grafted with full R1 expressing tumors.
Animals were lived image at multiple time points using IVIS. the data confirs selective enrichment of full R1 and BaCa antibodies into the tumors. When attempting this protocol, keep in mind that held maintenance of CHO cells is critical for antibody yield.
And it is important to have a similar degree of fluorescent dye conjugation for competitive tumor localization. Following this procedure, other methods such as tissue immunohistochemistry and tissue or tumor specific ELISA can be performed to support the live mice imaging data.
