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Xylazine (XYL) can be used for analgesia, anesthesia, is often used as a tranquilizer for horses, cattle, and sheep, and as a muscle relaxant in large animals. The mechanism of action of XYL is by activating presynaptic receptors of the central nervous system, thereby inhibiting the release of norepinephrine. Although xylazine can be rapidly metabolized in all animals, the excessive application of xylazine of some illegal farmers can cause xylazine to remine in the body. People who consume foods containing XYL residues over a long period of time may develop vomiting, nausea, weakness, and numbness of the limbs, and excessive consumption may also cause symptoms such as coma, and reduced bradycardia.
Currently, there are several analytical methods available for the detection of XYL. However, these methods are not perfect due to the complex sample processing required, high instrument costs and the need for professional technical staff, meaning that they cannot be widely applied. Compared to the above-mentioned detection methods, immunoassays have many advantages and have been widely used for the rapid detection of many illegal additives. Ic-ELISA based on antigen–antibody interaction is a quantitative detection method and has high throughput, and good efficiency and sensitivity. The lateral-flow immunochromatographic assay is another immunoassay method and is more convenient than ic-ELISA. The gold nanoparticle-based lateral-flow test strip assay does not need incubation and washing steps and is a rapid detection method, with rapid acquisition of the results in less than 10 minutes. The production of a highly sensitive and specific Anti-Xylazine antibody and the development of a versatile gold nanoparticle-based lateral-flow assay strip provide great convenience for the detection of XYL in milk samples.
Successful hapten design is crucial to generation monoclonal antibody, XYL has a molecular weight of less than 500 Da, so it is a low molecular weight compound that lacks immunogenicity. In order to stimulate a physiological immune response, XYL was coupled with a large molecular weight protein. Thus, from the derivatization of XYL to XYL-hapten was successful. The XYL-hapten was reacted with the carrier proteins (BSA and OVA) using the classic carbodiimide method. Immunogen (XYL-hapten-BSA) and coating antigen (XYL-hapten-OVA) were characterized used UV spectroscopy. The results showed that XYL-hapten was successfully conjugated to BSA. Similarly, XYL-hapten-OVA was successfully synthesized.
The sensitivity and specificity of the Anti-Xylazine antibody were determined by ic-ELISA. By optimizing ic-ELISA, the sensitivity and specificity of the mAb were evaluated more accurately. The result shows that the lowest IC50 value was obtained at pH 7.5 with 1% NaCl and the antibody titer was suitable. Under these optimum conditions, a standard curve was created based on the Anti-Xylazine antibody of subclass IgG2b. In addition, a standard curve was established by charting the optical density (OD) against XYL concentration and used to calculate the IC50 value and LOD, the equation of the standard curve of XYL was y = 0.061 + 1.7343/(1 + [x/0.399]1.012) and the correlation coefficient of the curve was 0.997. The CR values of XYL with other chemicals were less than 1% which showed the mAb against XYL was highly specific.
Fig 1. Characterization of the mAb against XYL.
(Source: New Journal of Chemistry, 2021)
The sensitivity of the gold nanoparticle-based lateral-flow test (GNT) strip was strongly influenced by the coating antigen and the antibody. The GNT-labeled mAb 4F12 was used for the establishment of the GNT strips due to its sensitivity and high affinity. When 200 ng mL-1 of XYL was added to the PBS, the color of the T-line disappeared for XYL-hapten-BSA, while a dark T-line was visible with XYL-hapten-OVA. Consequently, the higher sensitivity of XYL-hapten-BSA was chosen for the next optimization. The result showed that the color of the T-line was lighter when the coating antigen concentration was 0.3 mg mL-1 and the antibody concentration was 5 mg mL-1, while no T-line color was visible when the XYL content was 200 ng mL-1 in PBS. The conjugated GNP-labeled Anti-Xylazine antibody was the recognition element in the GNT strip, where the coating antigen competed with XYL in the sample bound on the T-line for the GNP-labeled mAb. The results showed that the GNP solution was stable when the pH was 9.0, the sensitivity and visibility of the GNT strip assay were optimal with 5 mg mL-1 mAb. Another factor affecting performance of a test strip is the conditions under which the coating antigen and goat anti-mouse IgG are immobilized on the NC membrane. The results showed that carbonate buffer provided the best sensitivity and signal. The suspension buffer also affects GNP-labeled mAbs. It was found that the optimal concentrations of Tween-20, BSA and sucrose were 0.3%, 0.3% and 3%.
Under optimum conditions, the results were evaluated with the naked eye within 10–15 min. When no XYL was added, the red color of both the C-line and the T-line appeared at the same time. With increasing XYL concentration, the red color of the T-line weakened, until it disappeared at 200 ng mL-1. Therefore, the cut-off value of XYL was 200 ng mL-1 in PBS. Next, sample analysis of the immunochromatographic strip was performed. When the concentration of XYL in the sample was less than 20 ng mL-1, the sample was considered negative; whereas when the concentration of XYL in the sample was between 20 and 200 ng mL-1, the sample was considered weakly positive; when the concentration of XYL was greater than 200 ng mL-1, the sample was considered positive.
In milk samples, recovery rates ranged from 95.1% to 105.1% for XYL using an ic-ELISA. These findings indicated that results using the GNT strip were highly consistent with the ic-ELISA. Moreover, using the GNT strip it was possible to test samples more rapidly and more conveniently than using the ic-ELISA. Overall, a sensitive and specific Anti-Xylazine antibody was produced for the detection of XYL in milk samples by developing an ic-ELISA and GNT strip. The developed GNT strip was able to specifically test XYL with no cross-reactivity to other chemicals tested(<1%). The GNT strip enabled the detection of XYL in milk while maintaining high analytical parameters.
| Target | Cat. No. | Product Name | Host | Isotype | Application | |
| Xylazine | CABT-Z547M | Mouse Anti-Xylazine Monoclonal Antibody, clone XLZ | Mouse | IgG | ELISA, LFIA | Inquiry |
| Target | Cat. No. | Product Name | Expression System | Tag/Conjugate | Application | |
| Xylazine | DAG-WT055 | Xylazine [OVA] | N/A | OVA | N/A | Inquiry |
| DAG-WT056 | Xylazine [BSA] | N/A | BSA | N/A | Inquiry |
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