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

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.

• Authors: Vivian Goodell, Douglas McNeel, Mary L Disis
• Journal: BMC immunology (BMC Immunol) Vol. 9 Pg. 23 (May 29 2008) ISSN: 1471-2172 [Electronic] England
• PMID: 18510754 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural, Validation Study)

Chemical References

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

Topics

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