Preparation and application of immunoaffinity in-tube solid phase microextraction column with oriented antibody-immobilized porous layer open tubular capillary for high sensitive quantification of serum extracellular domain of human epidermal growth factor receptor 2 levels.

Human epidermal growth factor receptor 2 (HER2) gene expresses a transmembrane glycoprotein that is over-expressed in 15-30% breast, 3% lung, and other several digestive cancers. So HER2 is a good biomarker for tumor diagnostic and treatment monitoring. Clinically, detection of HER2 often employs invasive approaches with tissue samples, which at large extent limit its universal application. Shedding of the extracellular domain (ECD) of the HER2 (HER2-ECD) into the circulation has led to the development of a serum test of HER2-ECD as an additional approach to probe the HER2 overexpression. However, few methods were developed due to the high sensitivity required by the serum HER2-ECD determination. In this work, we prepared a novel immunoaffinity in-tube solid phase microextraction (IT-SPME) sorbent for selective enrichment of HER2-ECD. Two clinical available monoclonal antibodies against to HER2, trastuzumab and pertuzumab, were selected as immunoaffinity ligands. Porous layer open tubular capillary with oriented antibody immobilization were fabricated and systematically optimized to afford a higher extraction capacity. The capacity was reached to 120.4 μg/m, which is more than 1000 times higher than that obtained by a common method (directly antibody immobilization on a naked capillary). After sample extraction and enrichment by the IT-SPME, the eluent were determined by a particle-enhanced turbidimetric immunoassay (PETIA). Sensitive quantification of HER2-ECD by the PETIA was thereby accomplished. HER2-ECD concentrations in 82 clinical serum samples were determined by the developed IT-SPME/PETIA method, and the results were well-correlated with that by the clinical used chemiluminescence immunoassay (CLIA). Besides, the IT-SPME/PETIA method was found providing 5 times higher sensitivity than the CLIA, and 500 times higher than the PETIA without IT-SPME. The results indicate that the developed method is suitable for high-sensitive quantification of HER2-ECD in clinical samples.