Editing 293F Protein Production (section)

==== Analysis of Antibody Production<br><br>  ====

1. To measure recombinant antibody accumulation over time, 100 uL aliquots are taken daily during the production phase. After centrifugation to remove cells, the antibody concentration in each sample is measured by sandwich ELISA.<br>2. Coat a 96-well ELISA plate overnight at 4°C with 100 uL of goat anti-human kappa light chain IgG diluted in PBS (coating solution).<br>3. Remove the coating solution with a multichannel pipettor and wash each well three times with 200 uL of washing buffer. The final wash is performed just<br>before the samples are loaded into the wells. After the final wash, tap the plate on a paper towel to remove any remaining wash solution.<br>4. Samples from the culture are diluted 1:10 in blocking buffer, and then 200 uL of each is loaded in triplicate onto the plate. Two serial two-fold dilutions in blocking buffer (100 uL sample + 100 uL blocking buffer) are done directly on the plate.<br>5. Load the antibody standard as serial 1:2 dilutions in triplicate on the plate.<br>6. Incubate the plate for 1 h at 37°C and then remove the samples using a multichannel pipetter.<br>7. Wash each well three times with 200- uL washing buffer as in step 3.<br>8. Add AP-conjugated goat anti-human gamma chain IgG diluted 1,000-fold in PBS to each well. For each plate, dilute 11 uL of antibody in 11 mL of PBS and add 100 uL to each well.<br>9. Incubate the plate for 1 h at 37°C.<br>10. Remove solutions and wash each well three times in 200 uL washing buffer as in step 3.<br>11. Add 100 L substrate solution to each well and cover the plate with aluminum foil.<br>12. Incubate the plate for 15 min at room temperature with gentle agitation.<br>13. Stop the reaction by addition of 100 L of 3 M NaOH.<br>14. Measure the absorbance at 405 nm using a microplate reader.<br>15. Determine the antibody concentration in each sample after generation of the standard curve from the absorbance of the standard antibody samples.<br><br>