Selection Of Aptamers And The Development Of Chemiluminescent Enzyme Immunoassay For Enrofloxacin And Sulfamethazine

Enrofloxacin (ENR) and sulfamethazine (SM2) have been wildly used to treat infections in livestock and aquatic animals. Therefore, the intensive residue monitoring for these two drugs in animal products is essential. The immunoassay, which employs antibody as the recognition element, is the most common used method for the rapid detection of veterinary drugs. However, the preparation of antibody not only needs the laboratory animals, but also requires strict storage condition. Moreover, it would be difficult to be reproduced, therefore the application of the antibody is limited. Aptamers are single-strand deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) that can specifically bind to targets (proteins, drugs or other molecules). Compared with antibody, aptamers are selected by in vitro selection, which is an animal-friendly method. Besides, aptamers are more stable than antibodies, and they can be modified easily. In this study, ENR-specific and SM2-specific aptamers were selected by in vitro selection for the first time. And the direct competitive chemiluminescent enzyme immunoassays (dc-CLEIA) for detecting ENR and SM2were developed based on the newly developed aptamers.ENR and ofloxacin (OFL) were immobilized onto magnetic beads (MBs) by forming a covalent bond. The Mag-SELEX system was developed for selecting several aptamers that can specifically bind with ENR by introducing OFL-MBs as counter selection. After8rounds of selection, six aptamer were selected, which were No.1l, No.17, No.19, No.21, No.27and No.49. Three of them, No.17, No.19and No.49, showed the relatively high affinity towards ENR. The dissociation constants (K4) of the three aptamers were188nM,355nM and459nM, respectively. Then, the specific tests of individual aptamer were conducted among the three aptamers. The results showed that No.17and No.19were more specific to ENR than No.49did. Interestingly, No.17also exhibited a weak affinity towards cipfloxacin (CIP). But No.17was still capable to tell ENR from CEP and OFL according to the statistical analysis. Therefore, No.17will be chosen to develop the method for detection of ENR.Besides, SM2and sulfamerazine (SMR) were attached onto MBs by forming a covalent bond as well. As the tool for counter selection, SMR-MBs was introduced to develop the Mag-SELEX system for the selection of aptamers that can specifically bind to SM2. After the ninth round selection, four aptamers were selected, which were SA04, SA06, SA07and SA41. All of these aptamer showed a high affinity towards SM2, the Kd values of them were203nM,264nM,79nM and122nM separately. And the specific tests of SA07and SA41were conducted. The results showed that both aptamers were highly specific for SM2. So, SA07, the aptamer with the highest affinity, would be chosen to develop the method for the detection of SM2.The direct competitive chemiluminescent enzyme immunoassay for detecting ENR was developed by introducing the streptavidin-biotin system, the biotin-labeled No.17as the recognition element and the ENR-spacer-HRP as the enzyme-labeled antigen. The optimized method displayed a detection limit (LOD) of2.26ng/mL. And a low degree of cross-reactivity between ENR and CIP (2.5%) was observed. These results revealed that the developed method was specific for ENR. Besides, recovery of spiked milk samples with ENR was studied, the recoveries of ENR were90.6-104.7%. Assay reproducibility was satisfactory, with coefficient of variation (CV) ranging from9.8%to18.9%. These results indicated that ENR in bovine milk can be detected by this method efficiently.Meanwhile, the dc-CLEIA for the detection of SM2was also developed. The biotin-labeled SA07and SM2-GA-HRP were used in this assay as the recognition element and the enzyme-labeled antigen, respectively. The LOD values of this method was0.92ng/mL, and the working linear range was from1.85ng/mL to21.57ng/mL. The specificity test for the determination of SM2was performed by employing27SAs. And a low degree of cross-reactivity between SM2and27SAs was observed, suggesting a high specificity of the method. Besides, the analysis of SM2fortified milk samples by the developed method showed average recoveries from88.0-98.2%with CV ranged from10.0%to23.4%. Thus, these results suggested that the SA07-based dc-CLEIA was suitable for screening SM2in milk.