Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of ARID1A in Tissue Lysates

ARID1A is frequently mutated in cancers, especially in gynecologic ones, but very little attention has been paid to determine ARID1A levels in tumor specimen. Therefore, the main aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) to measure ARID1A protein levels in an accurate and precise manner. In addition, we performed western blot analysis to validate the ELISA results. As we can show the high specificity, accuracy, and precision of our assay, further research can now focus on whether ARID1A protein levels are of predictive value, as most current methods analyze its gene ARID1A only. In terms of functionality, ARID1A is a member of the SWI/SNF complex, but it is not clear how mutations in members of this chromatin remodeling complex lead to tumorigenesis. We hope to facilitate the quantitative analysis of proteins involved in tumorigenesis with this detailed ELISA protocol for the nuclear protein ARID1A. ARID1A is a subunit of the mammalian SWI/SNF complex, which is thought to regulate gene expression through restructuring chromatin structures. Its gene ARID1A is frequently mutated and ARID1A levels are lowered in several human cancers, especially gynecologic ones. A functional ARID1A loss may have prognostic or predictive value in terms of therapeutic strategies but has not been proposed based on a quantitative method. Hardly any literature is available on ARID1A levels in tumor samples. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for ARID1A based on the current EMA and FDA criteria. We demonstrated that our ELISA provides the objective, accurate, and precise quantification of ARID1A concentrations in recombinant protein solutions, cell culture standards, and tissue lysates of tumors. A standard curve analysis yielded a 'goodness of fit' of R-2 = 0.99. Standards measured on several plates and days achieved an inter-assay accuracy of 90.26% and an inter-assay precision with a coefficient of variation of 4.53%. When tumor lysates were prepared and measured multiple times, our method had an inter-assay precision with a coefficient of variation of 11.78%. We believe that our suggested method ensures a high reproducibility and can be used for a high sample throughput to determine the ARID1A concentration in different tumor entities. The application of our ELISA on various tumor and control tissues will allow us to explore whether quantitative ARID1A measurements in tumor samples are of predictive value.