Abstract 534: Development and validation of sandwich ELISA to quantify circulating GRP78 as a cancer biomarker

Glucose regulated protein 78 kDa (GRP78), also referred to as immunoglobulin heavy chain binding protein (BiP), is a chaperone protein belonging to HSP70 protein family. GRP78 resides primarily in the lumen of the endoplasmic reticulum (ER) and plays an important role in protein folding, by targeting misfolded proteins, controlling the activation of transmembrane ER stress sensor and Ca2+ homeostasis. It contains the KDEL sequence (Lys-Asp-Glu-Leu) on its C-terminus, which is responsible for its retention in the ER through binding onto KDEL receptor. Some ER proteins escape the ER retention when they associate with other proteins and thus block the KDEL domains. Several in vitro studies have shown that GRP78 is found on the cancer cell surface. In clinical studies, increased expression of GRP78 has been correlated with chemoresistance in breast, colon, lung, prostate and endometrial cancers. However, quantification of GRP78 in the circulation has not been described as a diagnostic marker in cancer patients. Although circulating autoantibodies against GRP78 have been detected at high levels in the sera from cancer patients, these assays are difficult to perform by ELISA due to presence of immune complex bounded GRP78 in sera. We have developed and validated an ELISA-based assay able to quantify the free levels of GRP78 with a lower level quantification (LLQ) of 25 ng/mL. Since GRP78 is highly protein bound, acid dissociation using glycine demonstrated a linear range from 12.5 to 1600 ng/mL. The actual amounts of GRP 78 in plasma were measured using sandwich ELISA using TMB substrate, where the absorbance was measured at 450 nm. Acid dissociated plasma at pH 3.0 was able to liberated GRP78 where with no interfering protein was detected. Standard curve with a range 25-1600 ng/mL of GRP78 was linear (R2>0.98). To validate this ELISA method, interday and intraday variability testing were performed and demonstrated that the assays were linear, robust, and reproducible, over their respective concentration ranges and R2≥0.96. To determine the utility of this assay, circulating GRP78 in plasma was determined in breast cancer and compared to non-cancer volunteers. Breast cancer patients had significantly higher circulating GRP78 when compared to healthy volunteers, where the circulating levels correlated with disease staging. To further confirm the utility of this assay, circulating levels of GRP78 was also evaluated in patients with recurrent cervical cancer underg