title: Rapid mastitis detection assay on porous nitrocellulose membrane slides.
abstract: 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.
authors.affiliation: Biomolecular Sensing and Diagnostics, Food and Biobased Research, Wageningen University and Research Centre, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands. liyakat.mujawar@gmail.com
authors.forename: Liyakat Hamid
authors.initials: LH
authors.lastname: Mujawar
authors.forename: Antoine
authors.initials: A
authors.lastname: Moers
authors.forename: Willem
authors.initials: W
authors.lastname: Norde
authors.forename: Aart
authors.initials: A
authors.lastname: van Amerongen
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry*; D001681:Biological Assay; D002021:Buffers; D002244:Carbon / Q000737:chemistry; D002417:Cattle; D003352:Corynebacterium / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D016905:Gram-Negative Bacterial Infections / Q000150:complications / Q000175:diagnosis* / Q000382:microbiology; D016908:Gram-Positive Bacterial Infections / Q000150:complications / Q000175:diagnosis* / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D018796:Immunoconjugates / Q000737:chemistry; D007281:Ink; D056656:Lab-On-A-Chip Devices; D008414:Mastitis, Bovine / Q000150:complications / Q000175:diagnosis* / Q000382:microbiology; D045682:Mycoplasma bovis / Q000235:genetics / Q000302:isolation & purification; D053758:Nanoparticles / Q000737:chemistry; D011327:Printing; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification; D013291:Streptococcus / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000907:Antibodies, Bacterial; D002021:Buffers; D018796:Immunoconjugates; D002244:Carbon