Introduction

Chloramphenicol (CPC) is an effective antibiotic against a wide range of bacteria and is especially useful in the treatment of gram-negative infections in cattle. Bone marrow depression, a potential precursor to aplastic anemia and other serious blood disorders, has been associated with chloramphenicol treatment of people already sensitized to the drug. The Food and Drug Administration have established a zero tolerance level for chloramphenicol residues in both domestic and imported animal products. The broad-spectrum activity of CPC and its low cost attracts its use by some food producers. Chloramphenicol may also be used in both the larval and grow out stages of cultured shrimp farm. Therefore, chloramphenicol could be present in shrimp tissue if its withdrawal periods prior to harvest are not appropriate monitored. The sensitivity of the chloramphenicol EIA kit is excellent, and is well suited for the determination of chloramphenicol in sample extracts.

Principle of Chloramphenicol Determination

The enzyme immunoassay for chloramphenicol is based on the competition between the chloramphenicol to be assayed and the Chloramphenicol-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against chloramphenicol, coated onto microwells. The sample containing the chloramphenicol, and the Chloramphenicol-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 then is measured by the addition of a chromogenic substrate. The intensity of the color developed is inversely proportional to the concentration of chloramphenicol in the sample. The concentration of chloramphenicol is calculated on the basis of a standard curve.

Reagents

• 96-well microtiter plate. Twelve strips of 8 detachable wells, coated with anti-Chloramphenicol antibody.
• One vial of 1.2 ml Negative Calibrator containing 0 µg/ml of chloramphenicol.
• Three vials of Positive Calibrator (0.9 ml) containing 0.25, 2.0 and 10 ng/ml of chloramphenicol.
• One bottle containing 10.5 ml of Chloramphenicol-Alkaline Phosphatase (CPC-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 50 μl of standard or sample to the bottom of each well.
• Add 100 μl of CPC-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 other unrelated antibiotics.

Chloramphenicol Inhibition Curve