It requires minimum time and effort for assay optimization, as the time-proven buffers used in Western blot analysis can be adopted directly in a QDB-based assay. To develop a QDB-based assay is also very straightforward. The consistency, sensitivity and accuracy of the results are also increased by counting the luminescence signal directly in a microplate reader. By introducing a nitrocellulose membrane-based 96-unit QDB plate in the assay, this method significantly increases the protein-binding capacity of individual unit (100 to 200 µg/cm 2) to meet the analytical need of immunoblot analysis. Recently, Quantitative Dot Blot (QDB) method has been developed in our company as an improvement over the traditional antibody-based, dot blot method to allow high throughput, absolute quantitation of a specific protein at tissue level 13, 14, 15, 16. For the same reason, although Selected Reaction Monitoring Mass Spectrometry (SRM-MS) has become the only method to measure several protein biomarkers successfully in FFPE samples absolutely and quantitatively 10, 11, 12, it is not an ideal option in daily clinical practice, especially in the local clinical laboratories and clinical labs in developing countries. In addition, its complicated analytical processes and high costs limit its usage in routine clinical practice. However, its results are relative, not suitable for comparison among experiments. Reverse Phase Protein Microarray (RPPA) analysis has been reported to measure the expression levels of several protein biomarkers in FFPE samples 8, 9. The low binding capacity of ELISA plate (400–600 ng/cm 2) limits the amount of bound antigen for further analysis due to heavy protein crosslinking in FFPE samples, and the concentrating effect of capture antibody in Sandwich ELISA is also nullified for the same reason. 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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