Tetracycline enzyme-linked immunosorbent assay (ELISA) instructions

This kit is intended for research use only. It is designed for the quantitative detection of tetracycline residues in various biological and environmental samples, including feed, fish, shrimp, meat tissues (such as chicken, beef, and pork), eggs, honey, milk, serum, urine, soil, and more. The method employed is a competitive ELISA technique. Tetracycline-conjugated antigen is pre-coated on microplate wells. When the sample or standard solution is added, free tetracycline competes with the conjugated antigen for binding to the anti-tetracycline antibody. After incubation, the enzyme-labeled antibody binds to the antigen-antibody complex. A TMB substrate is then used to develop the color, which changes from blue to yellow upon addition of the stop solution. The absorbance is measured at 450 nm using a microplate reader, with results inversely proportional to the tetracycline concentration in the sample. A standard curve is used to calculate the exact tetracycline content. The kit includes the following components: a pre-coated tetracycline-conjugated antigen plate (12x8), six tetracycline standards (0ppb, 3ppb, 9ppb, 27ppb, 81ppb, 243ppb), anti-tetracycline antibody enzyme conjugate, TMB color reagent A and B, stop solution (2M sulfuric acid), sample diluent (10x), concentrated washing solution (20x), and an instruction manual. Essential equipment such as a microplate reader, centrifuge, pipettes, and filters are required but not included. Reagents like deionized water and methanol must be provided separately. Storage conditions require the kit to be kept at 2–8°C, avoiding freezing. Unused plates should be sealed and stored in a dry environment. Before use, all reagents should be brought to room temperature (25±2°C) for at least two hours. Proper personal protective equipment, such as gloves, should be worn when handling tetracycline standards. The stop solution contains sulfuric acid, so care must be taken to avoid contact with skin or clothing. Tips should never be mixed between standards or samples to prevent cross-contamination. Reagents from different batches should not be combined. Sample preparation involves extraction and dilution according to specific protocols for different matrices, such as animal tissue, feed, milk, and milk powder. The analytical procedure includes incubation, washing, color development, and measurement. Results are calculated by plotting a standard curve and determining the concentration based on absorbance values. Semi-quantitative assessments can also be made visually or instrumentally. The kit has a detection limit of 3ppb, with high specificity for tetracycline and acceptable inter-assay variability. Positive results should be confirmed using alternative methods like HPLC or GC/MS.

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