Rapid mastitis detection assay on porous nitrocellulose membrane slides
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We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase at their surface. The antibodies were inkjet printed onto three different nitrocellulose membrane slides, Unisart (Sartorius), FAST (GE Whatman), and Oncyte-Avid (Grace-Biolabs), and the final assay signals on these slides were compared. The blackness of the spots was determined by flatbed scanning and assessment of the pixel gray volume using TotalLab image analysis software. The black spots could be easily read by the naked eye. We successfully demonstrated the detection of specific amplicons from mastitis-causing pathogens in less than 3 h. Using a similar protocol, we also showed that it was possible to detect specific amplicons from four different mastitis-causing pathogens (six strains) on the same pad. The influence of two different printing buffers, phosphate-buffered saline (pH 7.4) and carbonate buffer (pH 9.6), on the functionality of the primary antibodies was also compared.
KeywordsMastitis Biochip Nitrocellulose PCR Microarray immunoassay Carbon nanoparticles
This research was supported by the Dutch Technology Foundation STW, Applied-Science Division of NWO (Dutch Organisation for Scientific Research), and the Technology Program of the Ministry of Economic Affairs of The Netherlands. Dr. Gerard Wellenberg from the Animal Health Service, Deventer, The Netherlands, is acknowledged for providing pathogen primer sequences and DNA template samples.
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