Angray KangASEEL ATEF SAEED ISMAIL2022-05-292022-05-29https://drepo.sdl.edu.sa/handle/20.500.14154/49565Aim: Develop a novel assay that could detect the presence of anti-drug antibodies against ustekinumab. Materials and methods: A single-chain variable fragment of synthetic antiustekinumab was developed using NEB assembly tool. Gibson assembly was used to join the DNA ustekinumab insert into a pk10 vector. The ustekinumab and vector were then transformed to E. coli, and plasmid isolation was done using a GenElute Plasmid Miniprep Kit. Dnluc was inserted between the variable heavy and light chains using BamH1 restriction enzyme. Protein expression was performed by transforming the Uste dnluc into NEB Express Iq Competent E. coli, and Globody was directed to the periplasm of NEB Iq E. coli for protein purification. Results: Successful novel assay was developed that in theory, is capable of detecting antibodies against ustekinumab, and subsequent luciferase activity levels were determined. Conclusion: Detection of anti-drug antibody is essential, and it may shed some light on ustekinumab efficacy. Our novel assay to detect anti-drug antibody showed great potential. However, utilizing it on standards with different concentrations should be done prior to its application on human sera.en