Introduction

Tylosin (MW = 916.14), is a macrolide antibiotic that is active against gram-positive and gram-negative bacteria. It is also effective in treating respiratory diseases caused by mycoplasms. It is widely used as a feed additive for growth promotion as well as therapeutically in food producing animals, including cattle, chickens and swine. Tylosin is also used for the reduction of the incidence of liver abscesses caused by sphaerophorous necrophorous. Food and Drug Administration of the United States has set the guidelines for the allowable level of tylosin in animal feeds and the specific tolerance for tylosin in edible products of animals. The tylosin EIA kit is a simple detection system for tylosin in feed extract.

Principle of Tylosin Determination

The enzyme immunoassay for tylosin is based on the competition between the tylosin to be assayed and the Tylosin-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against tylosin, coated onto microwells. The sample containing the tylosin, and the Tylosin-Alkaline Phosphatase conjugate, when added to the microtiter wells, compete for binding to a limiting number of antibody sites. After incubation, each well is rinsed in order to remove non-bound components. The bound enzymatic activity is then measured by the addition of a chromogenic substrate. The intensity of the color developed is inversely proportional to the concentration of tylosin in the sample. The concentration is calculated on the basis of a standard curve.

Reagents

• 96-well microtiter plate. Twelve strips of 8 detachable wells, coated with Anti-Tylosin antibody.
• One vial of 0.9 ml Negative Calibrator containing 0 ng/ml of tylosin.
• Three vials of Positive Calibrator (0.75 ml) containing 0.75, 3.5 and 20 ng/ml of tylosin.
• One bottle containing 10.5 ml of Tylosin-Alkaline Phosphatase (TLS-ALP) conjugate.
• One bottle containing 10.5 ml of p-Nitrophenyl Phosphate (pNPP) substrate.
• One bottle containing 15 ml of 10xWash Buffer (PBS-Tween).
• One bottle containing 5.5 ml of Stop Solution, 3N NaOH.

Assay Procedure

• Add 25 μl of standard or sample to the bottom of each well.
• Add 100 μl of TLS-ALP conjugate to each well. Incubate at room temperature for 40 minutes.
• Wash microtiter wells 3 times with wash buffer.
• Add 100 μl of pNPP substrate to each well. Incubate at room temperature for 20 min.
• Add 50 μl of stop solution to each well.
• Read absorbance at 405 nm using ELISA reader.

Cross Reactivity

Do not cross react with Gentamicin, Neomycin, Monensin, Sulfamethazine, Sulfadimethoxine, Zearalenone and Narasin.

Tylosin Inhibition Curve