Norfloxacin is an antibiotic in the group of fluoroquinolone. This antibiotic is used for the prevention and treatment of various diseases from bacteria in livestock. Residue of norfloxacin may found in animal product and this might affect to consumers. It can cause mutation and drug resistance in bacteria. Various analytical methods can be used to detect norfloxacin, including chemical method, such as HPLC, and serological methods such as RIA and ELISA. ELISA method has become the most popular method for screening of norfloxacin due to its high sensitivity, high specificity, simplicity and appropriate format for screen large numbers of samples. In this research, ELISA test kits were developed. A direct competitive ELISA (Ag captured) has been validated to be the most suitable method with the average IC50 value, limit of detection (LOD) and detectable range of 8.28, 1.01, and 1-50 ng/ml respectively. The prototype test kit was specific to quinolones and fluoroquinolones with %cross-reactivities to other tested antibiotics of less than 0.1%. The developed test kit was used to test different samples including chicken muscle and egg, yielding recovery of 86.28-109.68% and 89.49-111.34%, respectively. Furthermore, comparative quantification of norfloxacin using the developed ELISA test kit, the commercially available test kit (EURO-DIAGNOSTICA) and HPLC technique showed that all three methods gave comparable values. In addition, the inter-assay and intra-assay variations of the developed test kit were also investigated and found to be 4.29-9.37% and 5.27–11.01%, respectively. These result indicated that the prototype test kit is suitable and efficient for sample screening for norfloxacin, quinolones and fluoroquinolones.
