This kit is intended for research use only. It is designed for the quantitative detection of Oxytetracycline residues in various samples, including feed, fish, shrimp, meat tissues (such as chicken, beef, and pork), eggs, honey, milk, serum, urine, soil, and other similar matrices. The method employed is a competitive ELISA, where Oxytetracycline antigen is pre-coated on microplate wells. When the sample or standard solution is added, free Oxytetracycline competes with the pre-coated Oxytetracycline-conjugated antigen for binding to the anti-Oxytetracycline antibody. After adding the TMB substrate, a color change occurs, which is stopped by sulfuric acid, turning the color from blue to yellow. The absorbance is measured at 450 nm, and the results are inversely proportional to the concentration of Oxytetracycline in the sample. A standard curve is used to calculate the exact concentration.
The kit includes a pre-coated Oxytetracycline conjugate plate (12×8), six Oxytetracycline standards (0 ppb, 1 ppb, 3 ppb, 9 ppb, 27 ppb, 81 ppb), anti-Oxytetracycline antibody conjugate, TMB color reagent A and B, stop solution, sample diluent, washing solution, and an instruction manual. All reagents should be stored at 2–8°C, and unused plates must be kept dry and sealed. Before use, all reagents should be brought to room temperature (25±2°C) for at least two hours.
Special precautions include wearing gloves when handling Oxytetracycline standards due to its potential toxicity. The stop solution contains sulfuric acid, so care must be taken to avoid skin contact or damage to clothing. Reagents from different batches should not be mixed, and tips used for different samples or standards must remain separate to prevent cross-contamination. Sample preparation involves precise dilution and extraction steps depending on the matrix type, such as using methanol for liquid samples or acetonitrile for solid samples.
For analysis, the microplate is incubated at 37°C for 30 minutes, followed by five washes with diluted washing solution. TMB reagent is then added, and the reaction is stopped after 10 minutes. Absorbance is read at 450 nm within 5 minutes. Results are calculated using a standard curve, with percent inhibition values plotted against log concentrations. For semi-quantitative results, visual comparison or instrumental assessment can be used. Cross-reactivity with Oxytetracycline is 100%, ensuring high specificity.
The detection limit of the kit is 1 ppb, with a B0 value above 1.0 and inter-assay variation less than 15%. The recovery rate for tissue samples is over 80%. Positive results should be confirmed using alternative methods like HPLC or GC/MS. Proper storage, accurate pipetting, and strict adherence to the protocol are essential for reliable and reproducible results.
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