Detection of clenbuterol (Ventipulmin) in the horse.

The research article presents the development of an immunoassay for detecting clenbuterol, a drug commonly used in horses, in equine blood and urine.

Introduction

• The researchers developed an enzyme-linked immunosorbent assay (ELISA), a popular lab technique used to measure the concentration of analytes like peptides, proteins, antibodies, and hormones.
• The focus of this study was clenbuterol, a β2 agonist used typically as a bronchodilator in horses. The drug stimulates the beta 2 adrenergic receptors leading to the relaxation of smooth muscle in the lungs and hence, improved breathing.

Methodology

• The antiserum used in this ELISA was created using clenbuterol-diazo-BSA as the antigen and clenbuterol-diazo-horseradish peroxidase as the enzyme conjugate. Antiserum is blood serum containing antibodies against the antigen.
• The concentration of clenbuterol that could reduce tracer binding by 50% was determined to be 27.50 +/- 4.20 picogram (pg)/well. This is referred to as the IC50 value indicating the concentration of a drug that is required for 50% inhibition in vitro.
• Antibodies generated in the study also had some degree of cross-reactivity with other beta agonists like salbutamol (30% cross-reactivity), terbutaline (14% cross-reactivity), and cimaterol (1% cross-reactivity).

Testing and Validation

• The ELISA was used to screen horse serum directly and diluted urine samples for clenbuterol presence.
• Positive results from the screenings were then confirmed using two independent high-performance liquid chromatography (HPLC) systems, combined with off-line detection using the developed clenbuterol-ELISA. HPLC is a method used in biochemistry and analytical chemistry to identify, quantify and purify individual components in a mixture.
• Additionally, salbutamol was used as an internal standard to verify the relative retention of the drug in the samples.

Results and Conclusion

• The detection limit of clenbuterol in serum and urine was found to be 0.04 nanogram (ng)/ml. Thus, the newly developed ELISA showed high sensitivity in detecting clenbuterol.
• Moreover, when clenbuterol was administered intravenously to horses, using a marketed product called Ventipulmin, it could be detected in the serum for up to 24 hours and in urine for up to 96 hours.
• When orally administered, the drug could be detected in the serum for up to 48 hours and urine for up to 75 hours.
• This study, therefore, suggests that the developed ELISA could serve as an efficient tool in equine drug testing especially for residues of clenbuterol.