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


CONCLUSIONS
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.
Center for Translational Medicine in Women's Health, University of Washington, Seattle, WA 98109-8050, USA. vgoodell@u.washington.edu
Vivian
V
Goodell
Douglas
D
McNeel
Mary L
ML
Disis
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D000912:Antibodies, Neoplasm / Q000032:analysis*; D000917:Antibody Formation; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D016466:CHO Cells; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005260:Female; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity
D000912:Antibodies, Neoplasm; D029968:Escherichia coli Proteins; D018971:Insulin-Like Growth Factor Binding Protein 2; D033902:Periplasmic Binding Proteins; C018963:histidine-binding protein