Bioluminescence Immunoassays For The Detecting Of 21 Sulfonamides And 20 Fluoroquinolones Residues In Milk Based On Single Chain Variable Fragment

Chemical hazard in animal origin food is an important part of food safety. Development of efficient, high sensitive, fast and multiresidues detection methods is an efficient way for the ensurement of food safety. The traditional polyclonal antibody and monoclonal antibody in immunoassay has little possibility for the modification after preparation. While, the recombinant antibody allows us to modify, transform, directed lable the antibody from the gene level, and in vitro direct expression. Furthermore, its single binding characteristics made the immunoassays more sensitivethe. The Bioluminescence has been applied in many areas because of its excellent sensitivity and broad spectrum. And that the luciferases used in the bioluminescence has high luminescence, rapid luminescence and can be expressed by the prokaryotic expressionsystem. The aim of this paper was to study the bioluminescence immunoassay in the sulfonamides (SAs) and fluoroquinolones (FQs) residues in the milk sample, and then provide rapid, sensitive and high thoughout analytical menthods for the monitoring of veterinary drug residues.We directed labeled the alkaline phosphatase (ALP) and Renilla luciferase (Rluc) to the anti-fluoroquinolones (FQs) single chain variable fragment (scFv), and prepared two fusion proteins FQs-scFv-ALP and FQs-scFv-Rluc. Based on the two fusion proteins, we established chemiluminescence direct competitive immunoassay (CL-cdELISAFQs-scFv-ALP) and bioluminescence direct competitive immunoassay (BL-cdELISAFQS-scFv-Rluc), all of which could detect 20 FQs. The standard curves were established with norfloxacin (NOR). In the CL-cdELISAFQS-scFv-ALP, the half-maximal inhibitory concentration (IC50) of NOR was 0.15 μg/L, the linear range was 0.04-1.08 μg/L, the limit of detection (LOD) was 0.021 μg/L and the cross-reactivities (CRs) with other 19 FQs were 0.43-133.89%. In the BL-cdELISAFQs-scFv-Rluc, the IC50 of NOR was 0.031 μg/L, the linear range was 0.006-0.16 μg/L, the LOD was 0.124 μg/L and the CRs with other 19 FQs were 0.45-129.7%. The LODs in CL-cdELISAFQs-scFv-ALP was lower than BL-cdELISAFQS-scFv-Rluc, but the milk was diluted by 40 times in BL-cdELISAFQs-scFv-Rluc, and the sample processing method in CL-cdELISAFQs-scFv-ALP was very complex. The two immunoassaies were used to determine ciprofloxacin (CIP), enrofloxacin (ENR), marbofloxacin (MAR), danofloxacin (DAN), and flumequine (FLU) in spiked milk samples to confirm its performance. The average intra-and inter-assay recoveries from spiked milk of were 78.4-117.2%å'Œ 77.8-117.9%, the coefficients of variations (CVs) were 6.9-13.4% and 7.4-12.9%. The results demonstrated that the IC5o values of the two methods established in this study were 2 and 10 times lower than the enzyme linked immunosorbent assay (ELISA). Meanwhile, the BL-cdELISAFQs-scFv-Riuc was more sensitive than the CL-cdELISAFQs-scFv-ALP, and costed littler time. Furthermore, the milk sample processing method of BL-cdELISAFQs-scFv-Riuc was simpler than the CL-cdELISAFQs-scFv-ALP· Though the CV of BL-cdELISAFQs-scFv-Rluc was higher than the CL-cdELISAFQs-scFv-ALP, all of them were less than 15% and the recovires were between 75-120%. Therefore, the two methods could meet the requirement of veterinary drug residues.In this study, we developed a dual-luciferases competitive direct bioluminescent immunoassay (DBL-cdELISA) for the simultaneous screening of 20 fluoroquinolones (FQs) and 21 sulfonamides (SAs) residues in milk below the maximum residue limits (MRLs) based on the SAs-scFv-Fluc and FQs-scFv-Rluc fusion proteins. The standard curves were established with NOR and sulfamethazine (SMZ). The IC50 values for NOR and SMZ were 0.051 μg/L and 0.211 μg/L, respectively. The CRs with other 19 FQs and 20 SAs were between 0.56-130.46% and 2.25-1406.67%. Milk matrix effect was eliminated by 80 times dilution. The LODs of NOR and SMZ in milk sample were 0.232 μg/L and 1.894 μg/L. The average recoveries for five fluoroquinolones (CIP, ENR, MAR, DAN and FLU)were 76.6-118.4% with CV ranging from 5.7%-9.7%, and for five sulfonamides (SMZ, SMD, SMM, SQX and SMX)were 79.2-118.6% with a CV ranging from 5.4%-9.6%, which could meet the requirement of detection. It is the first report that realized the simultaneous detection based on microtiter plates and enzymes with only one substrates addition step followed by simultaneous signal acquisition immediately.In this paper, we describe a general bioluminescence resonance energy transfer (BRET) homogeneous immunoassay based on quantum dots (QDs) as the acceptor and Rluc as the donor (QD-BRET) for the determination of FQs. Coelenterazine-h was the most appropriate substrate. The calculated Forster distance (Ro) was 7.86 nm. The distance between QDs and Rluc was 8.9 run. The standard curve was established with ENR. Under optimized conditions, the IC50 for ENR was 0.782 μg/L and the linear range covered four orders of magnitude (0.023 to 25.60 μg/L), the LOD was 2.54 ng/L. The CRs with 7 representative FQs were 2.55-111.6%, which was similar to the data obtained in the section 2 and 3. The average intra-and inter-assay recoveries from spiked milk of were 78.5-118.2% and 79.3-116.6%. The CVs were less than 10%. Although the sensitivity was not ideal with the DBL-cdELISA, but it could meet the requirement of detection and it was a homogeneous immunoassay that did not need coating and washing steps. Therefore, it cost littler time with simpler operations.In conclusion, the homogeneous and heterogeneous bioluminescence immunoassaies based on scFv and luciferase fusion proteins in this study could be applied for the detection of 21 SAs and 20 FQs in milk samples. Copmared with chemiluminescence imminoassaies, it was more sensitive and cost little time with simple milk processing method. Furthermore, it realized the real simultaneous detection based on microtiter plates and enzymes with different luciferases. Besides that, the results demonstrated that the QD-BRET could be applied for the analysis of small molecules and provide rapid, sensitive and high thoughout analytical menthods for the monitoring of veterinary drug residues.