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

Goodell V, McNeel D, Disis ML - BMC Immunol. (2008)

Bottom Line: The application of high throughput molecular techniques such as SEREX are resulting in the identification of a multitude of tumor associated antigens.Precision experiments resulted in CVs < 15%.Furthermore, significantly greater levels of IGFBP-2 antibody immunity were found in cancer patients compared to normal controls (p = 0.008).

View Article: PubMed Central - HTML - PubMed

Affiliation: 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.

Show MeSH

Related in: MedlinePlus

Correlation of patient sample results by capture ELISA and indirect ELISA is significant. (A) Results from samples positive by indirect ELISA (horizontal axis) and capture ELISA (vertical axis) were plotted and the strength of the association between results using both methods was assessed by Pearson's product-moment correlation. Results are in ug/ml, and the best-fit line is shown for reference. (B) The ROC curve was constructed using results from normal donors and patients with breast or colon cancer and plotted as diagnostic sensitivity vs. 1- diagnostic specificity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2414992&req=5

Figure 4: Correlation of patient sample results by capture ELISA and indirect ELISA is significant. (A) Results from samples positive by indirect ELISA (horizontal axis) and capture ELISA (vertical axis) were plotted and the strength of the association between results using both methods was assessed by Pearson's product-moment correlation. Results are in ug/ml, and the best-fit line is shown for reference. (B) The ROC curve was constructed using results from normal donors and patients with breast or colon cancer and plotted as diagnostic sensitivity vs. 1- diagnostic specificity.

Mentions: We developed an indirect ELISA using commercially available recombinant human IGFBP-2. This assay meets all clinical laboratory requirements and allows comparison of a standard indirect ELISA with the his-tag-based capture ELISA described here (Table 1). The relationship between level of antibody response to the his-tagged IGBFP-2 protein by capture ELISA and level of antibody response by indirect ELISA was measured using Pearson's product-moment correlation (Fig. 4A). Matched results for all patient samples measured by both assay methods (n = 80) revealed a strong (r = 0.537), significant (p = 0.003) positive correlation. Preliminary assessment of the discriminatory capability of the assay was represented by ROC curves, and indicated that the capture ELISA is a weak predictor of presence of cancer with an area under the curve of 0.643 +/- 0.034, significantly different from the area expected by chance (p < 0.001) (Fig. 4B).


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

Goodell V, McNeel D, Disis ML - BMC Immunol. (2008)

Correlation of patient sample results by capture ELISA and indirect ELISA is significant. (A) Results from samples positive by indirect ELISA (horizontal axis) and capture ELISA (vertical axis) were plotted and the strength of the association between results using both methods was assessed by Pearson's product-moment correlation. Results are in ug/ml, and the best-fit line is shown for reference. (B) The ROC curve was constructed using results from normal donors and patients with breast or colon cancer and plotted as diagnostic sensitivity vs. 1- diagnostic specificity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2414992&req=5

Figure 4: Correlation of patient sample results by capture ELISA and indirect ELISA is significant. (A) Results from samples positive by indirect ELISA (horizontal axis) and capture ELISA (vertical axis) were plotted and the strength of the association between results using both methods was assessed by Pearson's product-moment correlation. Results are in ug/ml, and the best-fit line is shown for reference. (B) The ROC curve was constructed using results from normal donors and patients with breast or colon cancer and plotted as diagnostic sensitivity vs. 1- diagnostic specificity.
Mentions: We developed an indirect ELISA using commercially available recombinant human IGFBP-2. This assay meets all clinical laboratory requirements and allows comparison of a standard indirect ELISA with the his-tag-based capture ELISA described here (Table 1). The relationship between level of antibody response to the his-tagged IGBFP-2 protein by capture ELISA and level of antibody response by indirect ELISA was measured using Pearson's product-moment correlation (Fig. 4A). Matched results for all patient samples measured by both assay methods (n = 80) revealed a strong (r = 0.537), significant (p = 0.003) positive correlation. Preliminary assessment of the discriminatory capability of the assay was represented by ROC curves, and indicated that the capture ELISA is a weak predictor of presence of cancer with an area under the curve of 0.643 +/- 0.034, significantly different from the area expected by chance (p < 0.001) (Fig. 4B).

Bottom Line: The application of high throughput molecular techniques such as SEREX are resulting in the identification of a multitude of tumor associated antigens.Precision experiments resulted in CVs < 15%.Furthermore, significantly greater levels of IGFBP-2 antibody immunity were found in cancer patients compared to normal controls (p = 0.008).

View Article: PubMed Central - HTML - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus