Development of a microwell-based lateral flow immunoassay for detection of tetracycline in cow milk

Abstract: In order to meet the increasing demand for dairy products in China big cattle farms have been formed throughout the country. To control the outbreak and spread of disease antibiotics are readily employed, and detection methods are needed to ensure that the produced milk does not contain levels of antibiotics exceeding the regulatory limits. The aim of this project was to develop and optimize a commercially utilizable microwell-based lateral flow immunoassay for the detection of the antibiotic tetracycline in cow milk. The final conditions for the developed test strip included a test line with an antigen (TC-BSA) concentration of 2μg/μl and a control line with a secondary antibody (goat anti-mouse IgG) concentration of 0.08μg/μl. Each microwell contained 5μl of detection antibody (anti-TC) with a concentration of 0.12μg/μl as well as 5μl of Tris-HCl buffer with pH 7.5 and 5μl of the surfactant Tetronic 1307. These conditions resulted in an assay detection limit of 30ppb tetracycline and an expected shelf life of at least 12 months, with a total assay time of around 6-7 minutes.