Markus Hollas

Optimization of a Microarray Sandwich-ELISA against hINF-γ on a Modified Nitrocellulose Membrane

The highly specific and highly sensitive ELISA (enzyme linked immunosorbent assay) technique is the most commonly used method for immunological diagnostics in general. In combination with protein microarrays and their ability to allow performing thousands of experiments in parallel, a promising tool for global analytical approaches with reduced consumption of time, analytes, and reagents is given. In this study a protein microarray‐based sandwich‐ELISA for human interferon‐γ (hINF‐γ) is established. In consideration of the immense importance of the surface chemistry, a new black nitrocellulose matrix that generates very high signal‐to‐noise ratios (SNR) and a very low autofluorescence was tested and optimized as microarray substrate. A validation of the applicability of the system was performed with a comparison to different commercially available systems. Experimental results show that the microarray‐based ELISA is faster and easier to perform and shows a lower limit of detection (LOD) than a comparable system in a 96‐well plate. The spotted slides with the capture antibody can be stored up to 1 month with no significant loss of signal intensity. A second model system with immobilized His‐tagged restriction enzyme EcoRV and an anti‐His antibody shows in coincidence the good applicability of the black nitrocellulose membrane and no cross‐reactivity toward the ELISA.

Protein microarrays: Reduced autofluorescence and improved LOD

In protein microarray performance, the choice of an appropriate surface is a crucial factor. Three‐dimensional substrates like nitrocellulose are known to have higher binding capacities than planar surfaces. Furthermore, they can enable the immobilization of proteins in a functional manner. One disadvantage of todays nitrocellulose‐based microarrays is the high background fluorescence, which can interfere with the detection of low‐abundance proteins. We have developed an innovative black nitrocellulose membrane‐based protein microarray that exhibits low autofluorescence in combination with increased sensitivity and improved LOD (limit of detection). The applicability of the novel material was demonstrated with main focus on reversed‐phase microarray experiments. In comparison to various commercially available microarrays, a higher sensitivity in regard to the spotted protein was achieved. In contrast to other porous nitrocellulose‐based microarrays, the black nitrocellulose provides a significant lower autofluorescence and background intensity.