Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates

1. Production of αDEC-205 from the hybridoma cell line NLDC-145

1. For antibody production, thaw cryopreserved NLDC-145 cells producing αDEC-205 at 37 °C in a water bath. Expand the cells at 37 °C and 5% CO₂. Two 75 cm² bottles will be needed to proceed to antibody production. Cell culture procedures should be performed in a safety cabinet to ensure safe working conditions and prevent contamination of the cultures.
   1. Resuspend 1 mL of thawed cells (1 x 10⁶ – 5 x 10⁶ cells/mL) in 9 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin (pre-warmed to 37 °C) into a cell culture flask (25 cm²). Place the flask horizontally in a cell culture incubator at 37 °C, 5% CO₂.
   2. Culture the cells at 37 °C and 5% CO₂, until 70% confluence. This should normally be achieved after 24 – 48 h.
   3. Once the cells are 70% confluent, transfer the complete NLDC-145 cell suspension (10 mL) into a 15 mL conical centrifuge tube using a pipette controller with a pipette in a volume range from 1-10 mL. Pellet the cells by centrifugation at 250 x g for 10 min at room temperature.
   4. Pre-warm ISF-1 medium to 37 °C in a water bath and resuspend the pellet in 12 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin. Transfer the resuspended cell suspension into a fresh cell culture flask (75 cm²).
   5. Culture and expand the cells in ISF-1 medium supplemented with 1% penicillin/streptomycin at 37 °C and 5% CO₂, until 70% confluent and 99% viable. This should normally be achieved after 48 – 72 h.
   6. Split the cells into two 75 cm² flasks. To do this first flush the cell culture flask bottom/culture surface with the cell suspension to remove all NLDC-145 cells from the surface. Transfer 6 mL of the NLDC-145 cell suspension each into one of two fresh 75 cm² bottles and add pre-warmed ISF-1 medium supplemented with 1% penicillin/streptomycin up to 12 mL.
      NOTE: Do not renew the cell culture medium as the NLDC-145 cells will have conditioned the medium. Transfer the cells together with their medium and fill the culture up to the desired volume with fresh medium. This is crucial for viability and maximum antibody production by the NLDC-145 cells.
   7. Expand the cell cultures at 37 °C and 5% CO₂, until about 70% confluent which is generally achieved after 48 – 72 h.
   8. Once the cells are 70% confluent, transfer 10 mL of the expanded NLDC-145 cell suspension from each of the 75 cm² bottles into one PETG (polyethylene terephthalate glycol) roller bottle (1,050 cm²). For this, flush the cell culture flask bottom/culture surface with the cell suspension to remove all cells from the surface using a pipette controller and 10 mL pipette.
   9. Add 140 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin (pre-warmed to 37 °C) directly out of the medium bottle to each of the NLDC-145 containing roller bottles filling them up to the 150 mL mark.
      NOTE: See note in step 1.1.6.
   10. Culture the roller bottles at 37 °C, 5% CO₂, and 25 rounds/min for three days.
   11. Add 150 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin (pre-warmed to 37 °C) to each of the NLDC-145 containing roller bottles filling them up to the 300 mL mark.
   12. Culture the roller bottles now containing 300 mL culture each at 37 °C, 5% CO₂, and 25 rounds/min for another three days.
   13. Add another 100 mL of ISF-1 medium supplemented with 1% penicillin/streptomycin (pre-warmed to 37 °C) to each of the NLDC-145 containing roller bottles, thereby filling them up to the 400 mL mark.
   14. Culture the roller bottles now containing 400 mL culture each at 37 °C, 5% CO₂, and 25 rounds/min for another seven days.
       NOTE: During this week, the culture gets very dense (up to 95% density) and viability decreases (down to as little as 50%), enabling maximum antibody release.
2. For the purification of αDEC-205 from the culture supernatant pour the NLDC-145 cell suspension (from both roller bottles) directly into 500 mL autoclaved centrifugation bottles.
   NOTE: A total volume of 800 mL of culture should be collected.
   1. Centrifuge the culture for 30 min at 8,600 x g and 4 °C to remove cells and debris.
   2. Collect the supernatants, discarding the pellets, and pooling the supernatants in a sterile reagent bottle.
      NOTE: Purification of αDEC-205 can be commenced immediately (step 2.) or the supernatant can be stored short-term at 4 °C.

2. Purification of the αDEC-205 antibody from the NLDC-145 cell supernatant

NOTE: From the NLDC-145 cell supernatant, αDEC-205 is purified using a protein G Sepharose column (reusable). The column dimensions are 15 mm x 74 mm and 5 mL protein G Sepharose are packed per column.

1. For preparation and washing of the protein G Sepharose column, put an airtight rubber plug on the upper opening of the protein G Sepharose column. Puncture the rubber plug with two sterile cannulas (20 G x 1 1/2", 0.90 x 40 mm).
   1. Connect a 10 mL syringe to one of the two cannulas and a flexible silicon tube (approximately 100 cm long, 2.5 – 3 mm diameter) with a tubing connector to the second cannula.
      NOTE: The syringe/rubber plug construction is reusable and provides a vacuum resulting in a continuous flow of the large volume of culture supernatant to the protein G Sepharose column. To this end, slightly pull back the plunger of the syringe to ensure continuous fluid flow in the following steps.
   2. Wash the column with 50 mL of 0.1 M glacial acetic acid (pH 2) to remove potentially remaining antibody from any previous antibody purification. Put the end of the silicon tube in the 0.1 M glacial acetic acid (pH 2) filled reagent bottle. As a result of the induced vacuum, 50 mL of the 0.1 M glacial acetic acid (pH 2) dropwise run through the protein G Sepharose column.
      NOTE: 0.1 M glacial acetic acid (pH 2) should be stored in a reagent bottle or freshly filled in a beaker.
   3. Wash the column with 100-200 mL phosphate-buffered saline (PBS). Put the end of the silicon tube into a PBS-filled reagent bottle or beaker. Let 100-200 mL PBS run dropwise through the protein G Sepharose column.
2. For antibody purification from the NLDC-145 supernatant, load 800 mL of the NLDC-145 supernatant (obtained from step 1.2.2.) onto the column. Put the end of the silicon tube into the NLDC-145 supernatant-filled reagent bottle. Let 800 mL NLDC-145 supernatant run dropwise through the column.
   1. Wash the column with 500 mL of PBS. Put the end of the silicon tube in a PBS-filled reagent bottle or beaker. Let 500 mL PBS run dropwise through the column.
   2. For elution, use 20 1.5 mL tubes and pipette 100 µL of 1.5 M Tris-HCl (pH 8.8) into each 1.5 mL tube. Remove the rubber plug from the column and pipette 1 mL of 0.1 M glycine (pH 3) to the upper chamber of the protein G Sepharose column to elute the antibody from the column. Collect the flow-through directly as eluate in one of the prepared 1.5 mL tubes.
   3. Repeat the elution step (2.2.2.) for all 20 tubes (1.5 mL).
   4. Determine the optical density of all elution fractions at 280 nm (OD₂₈₀) using a spectrophotometer in order to identify the antibody-containing fractions.
      NOTE: Use the first elution fraction as blank.
   5. Pool all fractions with an OD₂₈₀ greater than 0.5 (approximately 10 fractions).
   6. Store the protein G Sepharose column filled with 20% ethanol at 4 °C.
3. Dialyze the pooled elutions against 1000 mL PBS (in a 2000 mL beaker) at 4 °C overnight using dialysis tubing with a molecular weight cut-off (MWCO) of 12 – 14 kDa.
   1. Cut the dialysis tubing into pieces of 20 cm. Boil the dialysis tubing in 500-800 mL of 10 mM EDTA (pH 7.5) for 30 min in a beaker using a hot plate to remove contamination. Discard the 10 mM EDTA (pH 7.5) solution and boil the dialysis tubing in deionized water for 10 min.
      NOTE: The dialysis tubing can be used directly or stored in 0.01% sodium azide (NaN₃)/H₂O solution at 4 °C until the next usage.
   2. Close the bottom of the dialysis tubing with an appropriate dialysis tubing closure/single-piece, hinged clamp and carefully pipette the antibody elution into the dialysis tubing. Close the top of the dialysis tubing with a second clamp.
   3. Fix the upper clamp of the dialysis tubing to a floating stand, put it together with a magnetic stir bar into the PBS-filled beaker, and place the beaker on a magnetic stirrer.
   4. Dialyze overnight stirring at 4 °C.
4. To increase the concentration of αDEC-205, load the complete dialysate to a centrifugal concentrator with 10 kDa MWCO. Open one clamp of the tubing and carefully pipette the complete dialysate out of the dialysis tubing into the centrifugal concentrator (10 kDa MWCO).
   NOTE: Do not touch the concentrator bottom with the pipette tip.
   1. Centrifuge for 30 min at 693 x g (2,000 rpm) and 4 °C.
   2. Load the centrifugal concentrator with 10 mL of PBS and centrifuge at 693 x g (2,000 rpm) and 4 °C until the final volume of antibody solution left is 1-1.5 mL.
      NOTE: If necessary, repeat the centrifugation step of 2.4.1. to adjust to the desired amount.
   3. Using a spectrophotometer, determine the optical density of the concentrated αDEC-205 solution at 280 nm (OD₂₈₀). Use PBS as blank.
   4. Calculate the concentration of αDEC-205 using the following formula:
      concentration [mg/mL] = OD₂₈₀/1.4.
   5. Filter the αDEC-205 solution using a 0.22 µm syringe filter unit.
      NOTE: Purification of αDEC-205 from the NLDC-145 hybridoma cell supernatant can also be achieved by FPLC (fast protein liquid chromatography). Purified αDEC-205 can be stored at 4 °C or at -18 °C for long-term storage.

3. Chemical conjugation of OVA to αDEC-205

NOTE: A ratio of 0.5 mg OVA protein to 2.5 mg αDEC-205 (1:5) is required for optimal chemical conjugation. However, this ratio can vary for other proteins and antibodies and needs to be optimized for alternative conjugates. Reduction of the disulfide bonds of the OVA protein is performed through incubation with 30 mM TCEP-HCl, which exposes the sulfhydryl-groups for chemical conjugation to αDEC-205 and 240 µl of TCEP-HCl are needed in step 3.2. Both steps, TCEP-induced reduction of OVA (step 3.1.) and sulfo-SMCC activation of αDEC-205 (step 3.2.) should preferably be performed in parallel.

1. Freshly prepare a 125 mM TCEP-HCl solution (pH 7.0). Weigh out the desired amount of TCEP-HCl and dissolve the TCEP-HCl in 0.9 M Tris base (pH 8.8). Use pH indicator strips to test the pH of the 125 mM TCEP-HCl solution (which should be neutral) and adjust the pH with Tris base (pH 8.8).
   1. Pipette 200 µL OVA protein solution (containing 0.5 mg OVA) into a 1.5 mL sterile tube. Add 240 µl 125 mM TCEP-HCl and 560 µL of sterile ultrapure water to the OVA protein using a pipette to a final concentration of 0.5 mg/mL OVA protein and 30 mM TCEP-HCl (OVA/TCEP-HCl).
      NOTE: 2.5 mg EndoGrade OVA (lyophilized) is dissolved in 1 mL PBS resulting in a 2.5 mg/mL OVA solution.
   2. Incubate the resulting OVA/TCEP-HCl at room temperature for 1.5 h.
      NOTE: Do not extend this incubation step.
2. To activate αDEC-205 for conjugation, dissolve 2 mg sulfo-SMCC in 100 µL of ultrapure water.
   NOTE: Sulfo-SMCC is susceptible to hydrolysis. Therefore, larger amounts of undissolved sulfo-SMCC should be handled rapidly or available 2 mg aliquots should be used.
   1. Dilute αDEC-205 in PBS so that 2.5 mg is contained in 900 µL.
   2. Mix 2.5 mg of αDEC-205 (900 µL volume; obtained from step 3.2.1.) and 100 µL of sulfo-SMCC (obtained from step 3.2.) in a 1.5 mL tube, resulting in a total volume of 1 mL.
   3. Incubate the αDEC-205/sulfo-SMCC solution for 30 min at 37 °C and 550 rpm in a heating block.
3. Following these incubations, excess sulfo-SMCC and TCEP-HCl are immediately removed from the solutions using desalting columns (MWCO 7 kDa; 5 mL column volume).
   1. Twist off the columns (MWCO 7 kDa) bottom closure, loosen the cap, and place the column into a 15 mL conical tube.
   2. Centrifuge for 2 min at 1,000 x g at room temperature to remove the liquid.
   3. Place the column in a fresh tube and remove the cap. Slowly load the antibody/sulfo-SMCC and the OVA/TCEP-HCl, respectively, to the center of the compact resin bed of one column each.
   4. Centrifuge for 2 min at 1,000 x g at room temperature.
   5. Discard the columns after use. The solutions containing antibody and OVA primed for conjugation are in the tubes.
   6. Immediately mix both solutions by pipetting for conjugation of αDEC-205 and OVA.
   7. Incubate the resulting αDEC-205 and OVA mixture overnight at 4°C.
4. Following conjugation, excess unbound OVA is removed from the solution and the coupled αDEC-205/OVA is concentrated using a centrifugal protein concentrator (MWCO 150 kDa).
   1. Pre-rinse the centrifugal protein concentrator (MWCO 150 kDa) by pipetting 12 mL of PBS on the column and centrifuging for 2 min at 2,000 x g at room temperature.
      NOTE: If necessary, repeat the centrifugation step (3.4.1.) until a volume of about 5 mL has passed through the column.
   2. Before loading the αDEC-205/OVA onto the centrifugal protein concentrator, save a 20 µL sample of the un-concentrated αDEC-205/OVA for western blot analysis. Store this aliquot at 4 °C until analysis.
   3. Load the αDEC-205/OVA onto the centrifugal protein concentrator by pipetting.
      NOTE: Avoid any contact with the bed of the upper chamber of the centrifugal concentrator.
   4. Fill the concentrator to 15 mL with PBS and centrifuge the concentrator for 5 min at 2,000 x g at room temperature.
   5. Save a sample of the flow-through (flow-through I) for western blot analysis and discard the remaining flow-through.
   6. Fill the concentrator to 10 mL with PBS and centrifuge the concentrator for at least 8 min at 2,000 x g at room temperature.
   7. Save a sample for the second flow-through (flow-through II) for western blot analysis and discard the remaining flow-through.
   8. Once the desired enrichment is achieved (around 1.5 mL of the αDEC-205/OVA solution should be left in the upper chamber) gently aspirate the concentrated sample.
      NOTE: If too much fluid is left in the upper chamber, centrifugation can be repeated but should be kept as short as possible.
5. Determine the protein concentration of the resulting αDEC-205/OVA using a microvolume spectrophotometer. Use PBS as blank.
6. Filter the αDEC-205/OVA using a 0.22 µm syringe filter unit.
   NOTE: For later analysis and in vivo experiments, αDEC-205/OVA can be stored at 4 °C or -18 °C.

4. Verification of the chemical conjugation by ELISA

1. Perform ELISA for further verification of successful chemical conjugation resulting in αDEC-205/OVA.
2. Coat an appropriate 96-well ELISA plate with 100 µL/well of 3 ng/µL rabbit αOVA antibody in coating buffer (0.1 M sodium bicarbonate (NaHCO₃) pH 9.6 diluted in H₂O).
   1. Incubate the plate overnight at 4 °C.
3. Following coating, wash the plate three times with PBS, e.g., using an ELISA washer.
4. Block the plate by pipetting 200 µL of blocking buffer (10% FCS in PBS) in each well of the plate and incubate the plate for 30 min at room temperature.
5. Serially dilute αDEC-205/OVA (obtained from step 3.6.) 1:2 in blocking buffer (10% FCS in PBS) to obtain dilutions ranging from 6 µg/mL down to 93.8 ng/mL αDEC-205/OVA and add 100 µL/well of these decreasing amounts of αDEC-205/OVA to the wells.
   1. Incubate the plate for 1 h at room temperature.
6. Wash the plate three times with PBS, e.g., using an ELISA washer.
7. Add 100 µL of the goat αrat-IgG+IgM(H+L)-HRPO antibody (diluted to 1:2,000 in blocking buffer (10% FCS in PBS)) to each well of the plate.
   1. Incubate for 1 h at room temperature.
8. Wash the plate three times using PBS, e.g. using an ELISA washer.
9. Add 50 µL of HRPO-substrate to the wells. When observing a clear color reaction, stop the reaction through addition of 150 µl stopping solution (1M H₂SO₄) per well.
10. After 5 min, read absorption at 450 nm using an ELISA reader.