The acceptable recoveries from spiked samples in beef and pork suggested that this ELISA is a reasonable quantitative/screening method in animal tissues

The acceptable recoveries from spiked samples in beef and pork suggested that this ELISA is a reasonable quantitative/screening method in animal tissues. of the carrier protein (Fig. ?(Fig.1).1). In this method, the furthest group of ENR from the linking point is the piperazinyl moiety. The high similarity of structures of substitutents (Positions 1 and 7) between ENR and CIP may explain their high reactivity (87%). Therefore, this ELISA could potentially be applied to the determination of both ENR and CIP. Although norfloxacin has the same piperazinyl moiety at Position 7, the extra ethyl group located in Position 1 may be responsible for the different electronic structures. Thus, medium cross-reactivity (25.8%) is not surprising. The second group consists of marbofloxacin, ofloxacin, and sarafloxacin, which lack the cyclopropyl group at Position 1 of ENR, but have some heteroatom structures. A comparison of the IC50 values demonstrates that the loss of this group and substitution with other groups has a diverse degree of effects on binding. The combined effect PMSF of changing the group at Position 1 and at the piperazinyl ring can be evaluated by comparing the IC50 values for lomefloxacin, danofloxacin, and PMSF flumequine. These results reveal the importance of spatial volume of the group at Positions 1 and 7 for antibody recognition, and the aromatic ring as well as the electronegative fluorine would be expected to PMSF involve a number of binding interactions. It is believed that the hapten- or antigen-antibody interaction is dependent on molecular shape and low-energy interactions such as hydrogen bonding and hydrophobic interactions (Wang et al., 2007). 3.5. Matrix effect determination One of the most common challenges of immunosorbent assays for food analysis is matrix interference. Immunosorbent assays often have a high potential for nonspecific binding to nontarget analysis. Chemical substances present in samples or sample extracts, such as solvents, fat, salt, and other compounds, might affect the binding between the antibody and analyte, reduce the sensitivity, and lower the extent of color development. So removing the matrix effects is important in the ELISA assays. In the present study, the beef and pork muscle samples were simply extracted and no cleanup step was employed prior to analysis. Fig. ?Fig.55 shows the comparison of the standard curves in dilutions of extracts and in PBS buffer alone. If the two curves are superposable, the matrix effect is not significant and the samples can then be analyzed according to the calibration curve. Open in a separate window Fig. 5 ENR inhibition curves in the diluted muscle samples Each point represents the average absorbance at 450 nm of three separate assays in triplicate. Insets indicate the icELISA standard curves in PBS As the dilution of beef extracts increased from 1:2 to 1 1:20, the absorbance gradually increased to Comp approach the PBS buffer values. The average PMSF em B /em 0 (antibody binding with no competitor present) values for dilutions at 1:2, 1:10, and 1:20 had absorbencies of 1 1.515, 1.864, and 1.902, respectively, as compared to 1.898 in PBS. It can be seen that matrix effects actually can be ignored at a dilution of 1 1:10 in beef. When compared the inhibition curves in pork, similar analytical sensitivity was observed between 1:20 dilution and PBS, indicating that matrix interference was sufficiently low and 1:20 dilution allowed a significant gain in the detectability of the analyte. Although dilution of the sample is an effective means of reducing the matrix effect, it leads to a reduction of assay sensitivity. Considering the dilution schedule, the LODs for ENR in blank samples of beef and pork were 2.1 and 4.6 g/kg, respectively, which were much lower than MRLs for ENR in these matrices. Based on the LODs of ENR in diluted muscle extracts, the same values for CIP (CR, 87%) in blank samples of beef and pork should be 2.4 and 5.3 g/kg, respectively. Thus, the loss in assay sensitivity was acceptable. 3.6. Analysis of spiked samples The analytical performance of ELISA is commonly assessed by spiking matrix samples with the target analyte. Each spiked sample was evaluated three times in duplicate to verify the repeatability of the developed icELISA, and the results are summarized in Table ?Table2.2. In the analysis of beef, the average recoveries for ENR and CIP ranged from 89% to.