Development of Dairy Cow Mastitis Test Kit Based on Immunochromatographic Strip Using Monoclonal Antibody Against <I>Staphylococcus aureus,/I>

Abstract

This study aimed to develop an immunochromatographic strip test based on immunological techniques using monoclonal antibodies against Staphylococcus aureus, an infection mastitis pathogen in raw milk. Specificity of monoclonal antibodies to S. aureus and cross-reactivity to other potential mastitis pathogens, such as Enterococcus faecalis, Streptococcus agalactiae, and Escherichia coli  were evaluated at the bacterial levels of 10⁶ - 10⁹ CFU / mL, using indirect enzyme-linked immunosorbent assay (indirect- ELISA). The results have shown that the optimal concentration of monoclonal antibodies in the indirect - ELISA test was 1: 2,500, which could bind S. aureus, E. faecalis, S. agalactiae and E. coli (10⁹ CFU / mL) with binding 100, 30, 13 and 10 %, respectively. To develop the strip, monoclonal antibodies were linked with colloidal gold (size 40 nm, pH 9.0), at the wavelength of 532 nm. After that, 1 mg / mL of monoclonal antibodies (0.5 µL), and 1.0 mg / mL of goat anti - mouse IgG (H + L) (0.5 µL), were coated on nitrocellulose membrane at the control line and test line, respectively. The strip was then incubated at 37 ^ºC for 14 - 16 hours and assembled with the other components to create a prototype of the immunochromatographic strip test. To test the specificity of the strip test device, suspension of bacterial cells prepared at 10⁹ CFU / mL in running buffer was dropped onto the sample pad. The samples containing S. aureus formed purple- pink bands at the control line and the test line, while the samples containing other bacteria created a purple- pink band only at the control line. In conclusion, the developed mastitis detection kit was highly specific to S. aureus and could be used as a laboratory- level of the prototype for rapid determination of S. aureus infection in investigation causing mastitis.