False Biomarker Discovery due to Reactivity of a Commercial ELISA for CUZD1 with Cancer Antigen CA125

Abstract

BACKGROUND By using proteomics and bioinformatics, we have previously identified a group of highly pancreas-specific proteins as candidate pancreatic ductal adenocarcinoma (PDAC) biomarkers. With the use of commercially available ELISAs, the performance of some of these candidates was initially evaluated in a relatively small serum cohort (n = 100 samples). This phase revealed that CUB and zona pellucida-like domains protein 1 (CUZD1) may represent a new, promising PDAC biomarker. METHODS We performed detailed experiments to investigate the specificity of the commercial CUZD1 ELISA assay. CUZD1 was expressed in house in both bacteria and yeast expression systems. Recombinant CUZD1 and biological samples containing CUZD1, as well as commercial CUZD1 ELISA standards, were analyzed by Western blot, size exclusion HPLC, and mass spectrometry (LC-MS Orbitrap). RESULTS We confirmed that instead of CUZD1, the commercial assay is recognizing a nonhomologous, known cancer antigen [cancer antigen 125 (CA125)]. CONCLUSIONS We conclude that poor characterization of commercial ELISA assays is a factor that could lead to false biomarker discovery. To our knowledge, this is the first report documenting that a commercial ELISA marketed for one analyte (CUZD1) may, in fact, recognize a different, nonhomologous antigen (CA125).

Validation of New Cancer Biomarkers: A Position Statement from the European Group on Tumor Markers

“…The potential dangers of using unreliable and nonvalidated immunoassays in biomarker development have been addressed by Diamandis and coworkers (28,29 ). Among the suggestions made by these authors to minimize this possibility were to purchase such reagents from companies with a proven quality record, perform local validation, and report any identified problems.…”

Translational researchers beware! Unreliable commercial immunoassays (ELISAs) can jeopardize your research

“…For instance, Gutiérrez et al recently reported that an ELISA kit from USCN Life Sciences (Wuhan, China), which was designed to recognize human hemojuvelin, was not able to identify the analyte of interest but rather, an unknown protein, which was subsequently found to be the unrelated antigen, ferritin [7]. In our own recent report on a similar incident, we have shown that a kit purchased from the same company, designed to quantify CUB and Zona Pellucida-Like Domains Protein 1 (CUZD1), was unable to detect the analyte of interest but instead, the kit was quantifying the known ovarian cancer antigen, CA125 [8]. In the past, reports for unreliable ELISAs were mainly related to poor performance (e.g., precision) or possible cross-reactivities with other analyte(s).…”

“…We spent almost 2 years and approximately $500,000 to identify the antigen that the commercial assay for CUZD1 was measuring (CA125) [8]. Incidences like these also highlight additional possible harms, such as rejection of probably promising biomarkers, a situation that calls for a re-examination of such candidates by alternative technologies.…”

Aberrantly high expression of the CUB and zona pellucida-like domain-containing protein 1 (CUZD1) in mammary epithelium leads to breast tumorigenesis

“…Leong et al demonstrated the importance of CUZD1 in cell growth and proliferation of a human ovarian cancer cell line and proposed a potential role of CUZD1 in chemotherapeutic resistance (61,62). Efforts have also been made to develop serum-based assays using CUZD1 as a biomarker for ovarian cancer and pancreatic cancer, however controversial reports support the need for additional studies (61,(63)(64)(65)(66)(67)(68)(69). Our recent work fills a gap in the body of knowledge surrounding CUZD1 by detailing the molecular signaling pathway of CUZD1-induced proliferation in mammary epithelial cells (51).…”