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BMC Immunol. 2008 May 29;9:23. doi: 10.1186/1471-2172-9-23.

His-tag ELISA for the detection of humoral tumor-specific immunity.

Author information

1
Center for Translational Medicine in Women's Health, University of Washington, Seattle, WA 98109-8050, USA. vgoodell@u.washington.edu

Abstract

BACKGROUND:

The 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.

METHODS:

We 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.

RESULTS:

The 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).

CONCLUSION:

A 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.

PMID:
18510754
PMCID:
PMC2414992
DOI:
10.1186/1471-2172-9-23
[Indexed for MEDLINE]
Free PMC Article

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