Generation of an anti-NAGase single chain antibody and its application in a biosensor-based assay for the detection of NAGase in milk.
Bovine mastitis, an inflammation of the mammary gland in cows, is a major challenge for the dairy industry worldwide as it lowers milk yield, reduces milk quality and increases overall production costs. Early diagnosis is of the utmost importance. N-acetyl-β-D-glucosaminidase (NAGase) is an enzyme released into milk during inflammation and acts as an early indicator of mastitis. This paper describes the selection of anti-NAGase single chain fragment variable antibodies (scFv) from naïve human antibody libraries and their incorporation into an automated optical biosensor-based immunoassay to detect NAGase in milk. The scFv with the highest affinity for NAGase was first characterized by inhibition ELISA, followed by further evaluation using a surface plasmon resonance platform. Purified NAGase was immobilized on the surface of a CM5 chip and spiked NAGase milk samples were analyzed. The limit of detection for the assay for the assay was determined as 1μg/ml.
Welbeck
Katherine
K
Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.
Leonard
Paul
P
Gilmartin
Niamh
N
Byrne
Barry
B
Viguier
Caroline
C
Arora
Sushrut
S
O'Kennedy
Richard
R
D000118:Acetylglucosaminidase / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D057205:Automation, Laboratory; D015415:Biomarkers / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods; D002417:Cattle; D042241:Early Diagnosis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008414:Mastitis, Bovine / Q000175:diagnosis* / Q000276:immunology; D008892:Milk / Q000737:chemistry*; D012015:Reference Standards; D057127:Single-Chain Antibodies / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D015415:Biomarkers; D057127:Single-Chain Antibodies; D000118:Acetylglucosaminidase