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

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

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Animals
  • Antibodies, Neoplasm / analysis*
  • Antibody Formation
  • Breast Neoplasms / diagnosis*
  • Breast Neoplasms / immunology
  • Breast Neoplasms / pathology
  • CHO Cells
  • Cell Line, Tumor
  • Colorectal Neoplasms / diagnosis*
  • Colorectal Neoplasms / immunology
  • Colorectal Neoplasms / pathology
  • Cricetinae
  • Cricetulus
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / immunology
  • Escherichia coli Proteins / metabolism
  • Female
  • Humans
  • Insulin-Like Growth Factor Binding Protein 2 / genetics
  • Insulin-Like Growth Factor Binding Protein 2 / immunology*
  • Insulin-Like Growth Factor Binding Protein 2 / metabolism
  • Male
  • Middle Aged
  • Periplasmic Binding Proteins / genetics
  • Periplasmic Binding Proteins / immunology
  • Periplasmic Binding Proteins / metabolism
  • Predictive Value of Tests
  • Sensitivity and Specificity

Substances

  • Antibodies, Neoplasm
  • Escherichia coli Proteins
  • Insulin-Like Growth Factor Binding Protein 2
  • Periplasmic Binding Proteins
  • histidine-binding protein