Differential quantification of SCCA1 and SCCA2 cancer antigens using a hydrogel biochip
Methods employing hydrogel-based microarrays (biochips) allow the simultaneous monitoring of protein interactions with different antibodies immobilized in gel elements. The method was applied for the simultaneous differential quantification of two highly homologous antigens of squamous cell carcinomas (SCCs) SCCA1 and SCCA2 in a single analysis. Two panels of monoclonal antibodies against recombinant SCCA1 and SCCA2 were generated, and two antibodies, C5 (anti-SCCA1) and A11 (anti-SCCA2), were selected for further evaluation based on their ability to specifically interact with their cognate antigens. Using a sandwich analysis, these antibodies were further tested in combination with anti-SCCA antibodies (H31 and SCC107) recognizing both of the SCCA antigens, thus allowing a quantitative independent measurement of both antigens. The intra- and inter-assay coefficients of variation for all resultant tests did not exceed 10% for the range of SCCA concentrations tested and were independent of whether SCCA1 and SCCA2 concentrations were determined simultaneously. The lower limit of detection (LOD) was estimated as 0.006 ng ml−1 for SCCA1 and 0.011 ng ml−1 for SCCA2 using the SCC107-Cy5 developing antibody and 0.014 ng ml−1 and 0.01 ng ml−1 concentrations, respectively, of the H31-Cy5 developing antibody. This assay provides a simple and accurate procedure for the differential quantitation of SCCA1 and SCCA2 using a single analysis of human serum on a biochip.