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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.

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The IGFBP-2 capture ELISA can discriminate between cancer patients and controls. (A) Data is expressed as IGFBP-2 IgG in ug/ml. Lines represent mean of antibody responses to IGFBP-2 by capture ELISA. (B) Bars represent percentage of samples positive for antibodies to IGFBP-2 by capture ELISA.
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Figure 3: The IGFBP-2 capture ELISA can discriminate between cancer patients and controls. (A) Data is expressed as IGFBP-2 IgG in ug/ml. Lines represent mean of antibody responses to IGFBP-2 by capture ELISA. (B) Bars represent percentage of samples positive for antibodies to IGFBP-2 by capture ELISA.

Mentions: Two hundred samples from healthy volunteers, 80 samples from breast cancer patients, and 80 samples from colon cancer patients were assayed for antibodies to IGFBP-2 using the capture ELISA. Magnitude of antibody response was significantly increased in breast and colon cancer patients compared to control samples (Fig. 3A) (p < 0.001) when 3-way analysis of continuous data was performed by Kruskal-Wallis test. Levels of IGFBP-2 antibodies were increased in breast cancer patients compared to normal volunteers (p = 0.013), and in colon cancer patients compared to normal volunteers (p < 0.001) when analyzed by Mann-Whitney U test. Continuous results from all samples were dichotomized using the 0.18 ug/ml cut-point derived from the reference range and subjected to Chi2 test. The capture assay found that the presence of antibodies to IGFBP-2 was significantly increased in the population of cancer patient samples, where 23% of patients were positive, compared to control samples, with a positive rate of only 1% (p = 0.008). Furthermore, in comparisons between controls and each type of cancer, 5% of breast cancer patients (p = 0.032) and 40% of colorectal cancer patients (p < 0.001) had IGFBP-2 specific antibodies, compared to controls (Fig. 3B).


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

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

The IGFBP-2 capture ELISA can discriminate between cancer patients and controls. (A) Data is expressed as IGFBP-2 IgG in ug/ml. Lines represent mean of antibody responses to IGFBP-2 by capture ELISA. (B) Bars represent percentage of samples positive for antibodies to IGFBP-2 by capture ELISA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: The IGFBP-2 capture ELISA can discriminate between cancer patients and controls. (A) Data is expressed as IGFBP-2 IgG in ug/ml. Lines represent mean of antibody responses to IGFBP-2 by capture ELISA. (B) Bars represent percentage of samples positive for antibodies to IGFBP-2 by capture ELISA.
Mentions: Two hundred samples from healthy volunteers, 80 samples from breast cancer patients, and 80 samples from colon cancer patients were assayed for antibodies to IGFBP-2 using the capture ELISA. Magnitude of antibody response was significantly increased in breast and colon cancer patients compared to control samples (Fig. 3A) (p < 0.001) when 3-way analysis of continuous data was performed by Kruskal-Wallis test. Levels of IGFBP-2 antibodies were increased in breast cancer patients compared to normal volunteers (p = 0.013), and in colon cancer patients compared to normal volunteers (p < 0.001) when analyzed by Mann-Whitney U test. Continuous results from all samples were dichotomized using the 0.18 ug/ml cut-point derived from the reference range and subjected to Chi2 test. The capture assay found that the presence of antibodies to IGFBP-2 was significantly increased in the population of cancer patient samples, where 23% of patients were positive, compared to control samples, with a positive rate of only 1% (p = 0.008). Furthermore, in comparisons between controls and each type of cancer, 5% of breast cancer patients (p = 0.032) and 40% of colorectal cancer patients (p < 0.001) had IGFBP-2 specific antibodies, compared to controls (Fig. 3B).

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