In this regard, the Enzyme linked Immunosorbent Assay (ELISA) method is not a feasible option. While intensive efforts have been devoted to the standardization of this method 1, 4, 5, 6, 7, there are also ongoing efforts to develop alternative methods for absolute quantitation of biomarker protein levels objectively and consistently for routine clinical practice.Ĭonsidering that the majority of clinical samples are preserved as Formalin Fixed Paraffin Embedded (FFPE) block in pathological practice, one pre-requisite for any method suitable for routine clinical practice is that this method must be compatible with FFPE samples. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries.Īlthough Immunohistochemistry (IHC) is the prevailing method in protein biomarker assessment for solid tumors, the inherent problems, including the lack of objectivity and consistency, are also well recognized in the field 1, 2, 3. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC and 96.9% with combined results from both IHC and FISH analyses. We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study.
Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice.
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