Abstract
BackgroundThe application of high throughput molecular techniques such as SEREX are resulting in the identification of a multitude of tumor associated antigens. As newly identified antigens are incorporated into a variety of clinical trials, standardization of immunologic monitoring methods becomes increasingly important. We questioned whether mammalian cell expression of a histadine-linked human protein could be used to produce antigen suitable for detecting tumor-specific humoral immunity and whether such an assay could be amenable to standardization for clinical use.MethodsWe designed a his-tagged capture ELISA based on lysate from genetically engineered CHO cells for detection of antibodies to insulin-like growth factor binding protein 2, a novel tumor antigen. We performed technical and preliminary clinical validation studies, including comparison to a standard indirect ELISA based on commercially prepared recombinant antigen.ResultsThe his-tagged capture ELISA could be standardized. Precision experiments resulted in CVs < 15%. Linearity and calibration experiments demonstrated r2 values of 0.99. In comparison to Western blot analysis, his-tag and indirect ELISA accurately identified 88% and 93% of samples, respectively. Sample concordance between capture and indirect assays was highly significant (p = 0.003). Furthermore, significantly greater levels of IGFBP-2 antibody immunity were found in cancer patients compared to normal controls (p = 0.008).ConclusionA genetically engineered cell lysate based ELISA can be amenable to standardization and can detect increased levels of antibody immunity to tumor-associated antigen in cancer patients compared to non tumor-bearing healthy controls.
Highlights
The application of high throughput molecular techniques such as serological analysis of recombinant cDNA expression libraries (SEREX) are resulting in the identification of a multitude of tumor associated antigens
Serological analysis of recombinant cDNA expression libraries has led to the identification of hundreds of cancer-specific antigens using sera from cancer patients to probe proteins encoded by tumor cDNA libraries
The quality of assays used for this purpose must meet standards mandated by the Clinical Laboratory Improvement Act (CLIA) in order to make assessment of clinical trials meaningful and to allow for comparison between trials conducted by different groups
Summary
The application of high throughput molecular techniques such as SEREX are resulting in the identification of a multitude of tumor associated antigens. Applications of high-throughput molecular techniques are resulting in the identification of a multitude of tumor associated antigens. Genomic and proteomic technologies have allowed immunogenic proteins to be determined for a wide variety of cancers Both approaches utilize humoral immune responses to screen for tumorassociated antigens. Given the need for both high quality and flexibility in assay methods, we questioned whether his-tagged capture ELISA could be utilized for evaluating antibody immunity against novel antigens. If such an assay could be optimized to meet clinical laboratory standards, it could serve as a template method for use with potentially any cDNA of interest
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