Abstract 323: Generation and application of a novel monoclonal antibody to UBQLN4 to characterize the dsDNA damage response
Authors: McWilliams Brian D. , Spencer Mike 
Published: June 2022

Ubiquilin-4 (UBQLN4) is a member of the ubiquilin protein family whose primary function is to maintain protein homeostasis in cells. Recent research indicates that UBQLN4 contributes to genomic stability through interactions with ubiquitylated MRE11. In a dysregulated state, UBQLN4 can shift double-strand break repair toward the homologous recombination repair (HRR) pathway or canonical non-homologous end joining (c-NHEJ) pathway. The latter pathway is associated with an overexpression of UBQLN4 and can be suggestive of a sensitivity to PARP1 inhibitors.

As with any protein with diagnostic or therapeutic implications, having trustworthy antibodies is required to generate actionable and repeatable data. To help standardize the validation of these antibodies, the International Working Group for Antibody Validation was created in correlation with a 2016 Nature publication by Uhlen et al. In this publication, a series of “validation pillars” were proposed to give guidance on validating antibodies for scientific use. In response to that publication, we have institutionally adapted that language and many of those principles to supplement our existing validation scheme of antibodies produced on site, including a novel monoclonal antibody against UBQLN4.

Four UBQLN4 peptides or full-length UBQLN4 were synthesized and purified internally and used for rabbit immunization, B-cell sorting, and screening for activity via ELISA. Variable regions were sequenced to avoid clonal redundancy, and a subset of these clones were selected to be characterized via a strip-style western blot. Clones were also assessed by immunohistochemistry (IHC) using tissue microarrays with multiple tissue and cell types, and specificity was determined via reciprocal IPs and western blot.

Over 200 clones were sequenced and screened by ELISA. These results allowed us to narrow the set down to 153 clones that were screened by strip-style western blot. 74 of these were assessed by IHC, 9 clones were screened by reciprocal IPs, and finally, 6 clones by western blot. Specificity was assessed throughout the process by the utilizing independent antibodies, complementary assays, a UBQLN4 knockout cell line, and UBQLN4-overexpressing cells.

Over the course of narrowing this clone set from 200 clones to a single antibody, we were able to meet the pillar requirements outlined in our validation scheme, empowering us to identify a superior antibody useable in several assays, including WB, IP, IHC, and ICC. Going forward, to further detail the use of this target's role in DNA repair, multiplex immunofluorescent studies will be performed in conjunction with Co-IP to identify additional protein binding partners unique to the DNA repair process.

Citation Format: Brian D. McWilliams, Mike Spencer. Generation and application of a novel monoclonal antibody to UBQLN4 to characterize the dsDNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 323.