This protocol for which all of the experimental condition have been carefully optimized can be used to facilitate a successful and efficient chemical conjugation of the DEC-205 antibody to OVA antigen. The main advantages of this technique are that it permits a flexible selection of the protein antigen and antibody, and that it does not rely on genetic fusion of these components. We have also used this protocol to generate conjugates of hepatitis C virus proteins and anti-DEC-205 for in vivo DC targeting.
This could likewise be valuable for anti-tumor vaccination approaches. To start anti-DEC-205 production, resuspend a one milliliter volume of one to five million thawed cryo-preserved NLDC 145 cells in nine milliliters of 37 degrees Celsius ISF-1 medium supplemented with 1%penicillin and streptomycin, and seed the cells into a 25 square centimeter cell culture flask for incubation at 37 degrees Celsius and 5%carbon dioxide for 24 to 48 hours. When the cells reach 70%confluency, transfer the entire cell suspension into a 15 milliliter conical tube and collect the cells by centrifugation.
Resuspend the pellet in 12 milliliters of fresh complete 37 degree ISF-1 medium and re-plate the cells in a 75 square centimeter flask. After 48 to 72 hours of culture, split the 70%confluent culture between each of two new 75 square centimeter flasks and add six milliliters of fresh complete ISF-1 medium to each flask. When the cultures reach 70%confluency, use a 10 milliliter pipette to flush the cell culture flask bottoms and culture surfaces with the cell suspension to collect all of the cells and transfer 10 milliliters of each expanded NLDC 145 cell suspension into individual PETG roller bottles.
Then add 140 milliliters of complete ISF-1 medium to each roller bottle culture and culture the roller bottles at 37 degrees Celsius, 5%carbon dioxide and 25 rounds per minute for three days. For antibody purification from the NLDC 145 supernatant, place the end of a silicon tube into the supernatant-filled beaker and allow 800 milliliters of supernatant to run dropwise through a Protein G Sepharose column. For elution, add 100 microliters of 1.5 molar Tris-HCL into each of twenty 1.5 milliliter tubes and remove the rubber plug from the column.
Transfer one milliliter of 0.1 molar glycine to the upper chamber of the column and collect the antibody containing eluent into one of the prepared 1.5 milliliter tubes and repeat for each tube. When all of the antibody has been collected and the antibody-containing elutions have been pooled, close the bottom of a 20 centimeter length dialysis tubing with an appropriate dialysis tubing closure, and carefully pipette the antibody elution into the tubing. Close the top of the dialysis tubing with a second clamp and fix the upper clamp of the dialysis tubing to a floating stand.
Add a magnetic stir bar to a beaker of PBS and place the beaker onto a magnetic stirrer. After overnight dialysis at four degrees Celsius, open one clamp to allow loading of the complete dialysate to a centrifugal concentrator with a 10 kiloDalton molecular weight cutoff, and centrifuge the concentrator for 30 minutes at 693 times G and four degrees Celsius. At the end of the spin, load the centrifugal concentrator with 10 milliliters of PBS for a second centrifugation until a final one to 1.5 milliliter volume of antibody solution is obtained.
To conjugate OVA to the anti-DEC-205 antibody, add 200 microliters of PBS supplemented with 500 micrograms of OVA protein, 240 microliters of a freshly prepared TCEP-HCL solution, and 560 microliters of sterile ultrapure water to a 1.5 milliliter tube for a 1.5 hour incubation at room temperature. At the same time, add 2.5 milligrams of anti-DEC-205 in 900 microliters of PBS and 100 microliters of freshly prepared sulfo-SMCC to a second 1.5 milliliter tube for a 30-minute incubation at 37 degrees Celsius at 550 revolutions per minute in a heating block. At the end of the incubations, place desalting columns with loosened caps and twisted off bottoms into individual 15 milliliter conical tubes, and centrifuge the columns to remove the liquid.
After centrifugation, place the columns in new tubes with the caps removed, and slowly load the antibody sulfo-SMCC and OVA TCEP-HCL solutions to the center of the compact resin bed of one column per solution. After centrifuging, discard the columns and immediately mix the antibody and OVA solutions with gentle pipetting. Load the anti-DEC-205/OVA solution onto a centrifugal protein concentrator and fill the concentrator with PBS to a 15 milliliter volume.
Centrifuge the concentrator for five minutes at 2, 000 times G and room temperature and fill the concentrator with an additional 10 milliliters of PBS. After centrifuging the concentration for at least eight minutes under the same centrifuge conditions, gently collect the concentrated sample from the upper chamber. To verify the success of the conjugation by Western blot analysis, load an aliquot out of each sample on a protein standard onto an SDS gel and run the gel according to standard SDS-PAGE analysis protocols.
After gel blotting, stain the membranes with horseradish peroxidase conjugated antibodies to detect anti-DEC-205 or OVA according to standard protocols. The membranes can then be analyzed for the presence of the respective protein in each sample. To verify the success of the conjugation by ELISA, coat an appropriate 96-well ELISA plate with 100 microliters of the anti-OVA antibody per well and serially dilute anti-DEC-205/OVA at a one to two ratio and blocking buffer to obtain dilutions from six micrograms per milliliter to 93.8 nanograms per milliliter.
Add 100 microliters of each dilution to the appropriate wells of the antibody-coated plate for a one-hour incubation at room temperature. At the end of the incubation, add 100 microliters of horseradish peroxidase conjugated antibody against the anti-DEC-205/OVA conjugate to the plate for a one-hour incubation at room temperature, then add 50 microliters of horseradish peroxidase substrate to each well to allow analysis of the color reaction by spectrophotometry. Parallel Western blot analysis can be used to detect both conjugated OVA as well as conjugated anti-DEC-205.
Staining for OVA would allow the detection of excess free OVA if present, and staining for anti-DEC-205 verifies the success of the conjugation through an increase in the molecular weight between naked anti-DEC-205 and the conjugate. ELISA can also be used to assess the success of the conjugation with a positive association between the detected signal and the analyzed amount of protein indicating the successful generation of anti-DEC-205/OVA conjugant. Flow cytometry and immunofluorescence analysis clearly show that the anti-DEC-205 core efficiently binds bone marrow-derived CD11c-positive cells.
Vaccination with anti-DEC-205/OVA in conjunction with adjuvant induces an increase in OVA-specific IgG titers in mouse recipients compared to vaccination with OVA and adjuvant alone. Furthermore, anti-DEC-205/OVA efficiently induces over-specific CD4 and CD8-positive T-cell responses with the anti-DEC-205/OVA-induced CD8-positive T-cell responses significantly exceeding that induced by OVA alone. For a successful conjugation of OVA antigen to anti-DEC-205 antibody, it is important to prepare the materials as demonstrated and to perform the conjugation steps under consistent conditions.
Following successful conjugation of the antigen and the antibody, numerous subsequent approaches are possible, including binding analysis and the induction of pathogen or tumor-related immunity in vivo.
