Abstract:

The study aims to evaluate the inhibitory effects on tumor cell migration using early screened hybridoma supernatant. Firstly， anti-αvβ3 hybridoma positive clones were screened by enzyme-linked immunosorbent assay （ELISA）. After accumulating the fermentation broth， Biacore was used to detect the affinity， and 9 mixed clone supernatants with different affinity gradients were randomly selected. Using genetically engineered H-αvβ3-A375 cells （stably highly expressing αvβ3 receptor） as the model， the inhibitory effect of 9 mixed clone supernatants on tumor cell migration was detected by Transwell method， and 5 clones with strong-medium-weak gradient inhibitory effects were screened out. These 5 clones were subcloned， and the migration inhibitory activity of the subclone supernatants was also detected by Transwell method. Chimeric antibodies were synthesized based on the sequencing results of the subclones， and 4 concentration gradients （160， 80， 40， 20 μg·mL^-1） and a negative control （no antibody treatment group） were set to verify the activity consistency between the chimeric antibodies and the supernatants. The results showed that there were significant differences in the inhibition rates of the 9 mixed clone supernatants on the migration of H-αvβ3-A375 cells （P0.05）， ranging from 26.77% to 99.89%. There was no statistical difference in the inhibitory effect between the 5 selected subclone supernatants and the corresponding mixed clone supernatants （P0.05）， while the difference in inhibition rates between groups was significant （P0.05）. The migration inhibition rates of the chimeric antibodies at each concentration gradient were consistent with those of the mixed clone and subclone supernatants （P0.05）. The "hybridoma supernatant-Transwell" combined evaluation method established in this study can directly realize the early screening of antibody migration inhibition function at the mixed clone stage without antibody purification steps， with an accuracy rate close to 100%. The method significantly shortens the antibody research and development cycle and reduces the early research cost， providing a reliable technical means for the efficient development of anti-metastatic antibodies.

Key words: early antibody evaluation, migration inhibition, hybridoma supernatant