Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae; Kang, Hwan-Goo

The presence and specificity of antibodies were tested by indirect, competitive ELISA as described below. The microplates were coated with ENR-BSA dissolved in 0.5 M carbonate buffer (100 ng per well, 100 L). Following overnight incubation at 4 o C, plates were washed three times with washing buffer and blocked with 1% casein in PBS (w/v, 250 L/well), after which they were incubated for 1 h at 37 o C. The blocking solution was then removed, after which PBS buffer or competitor in PBS buffer (80 L/well) was added to each well, followed by the addition of serum or culture supernatants(80 L/well). Samples were then incubated at room temperature for 1 h, after which they were washed three times, and 100 L of anti-mouse IgG-HRP conjugate (dilution 1 : 1000 in 1% skim milk) was added and incubated for 1 h. Each well was washed with washing buffer and 100 L of TMB solution for 5 min, after which color development was halted by adding 100 L of 2N H2SO4, and the absorbance at 450 nm was measured using a microtiter plate reader.