Chloramphenicol ELISA Kit

Catalog No. ABIN997087

Product Details Chloramphenicol ELISA Kit

Target: Chloramphenicol

Reactivity: Chemical

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: Enzyme Immunoassay for the Quantitative Determination of Chloramphenicol in Food

Analytical Method: Quantitative

Specificity: 90-115%

Sensitivity: 0.03 ng/mL

Application Details

Assay Time: 1 h

Plate: Pre-coated

Assay Procedure:

1. Prepare samples as described above.
   2. Pipet 100 μL standards or prepared samples in duplicate into the appropriate wells of the microtiter plate. Immediately add 50 μL anti-chloramphenicol antibody into each well.
   3. Cover the microtiter plate with a plastic foil and incubate for 30 minutes at room temperature.
   4. Without preceding washing add 50 μL chloramphenicol-peroxidase conjugate into each well.
   5. Cover the microtiter plate with a plastic foil and incubate additional 15 minutes at room temperature.
   6. Wash the plate three times as follows: Discard the contents of the wells (dump or aspirate). Pipet 300 μL of diluted washing solution into each well. After the third repetition empty the wells again and remove residual liquid by striking the plate against a paper towel. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbencies.
   7. Pipet 100 μL of substrate solution into each well.
   8. Allow the reaction to develop in the dark (e.g. cupboard or drawer, the chromogen is light-sensitive) for 15 minutes at room temperature.
   9. Stop enzyme reaction by adding 100 μL of stop solution (0.5 M H2SO4) into each well. The blue color will turn yellow upon addition.
   10. After thorough mixing, measure absorbance at 450 nm (reference wavelength 620 nm), using an ELISA reader. The colour is stable for 30 minutes.

Calculation of Results:

1. Calculate the average optical density (OD 450 nm) for each set of reference standards or samples.
   
   2. Construct a standard curve by plotting the mean optical density obtained for each reference standard against its concentration in ng/mL on semi-log graph paper with the optical density on the vertical (y) axis and the concentration on the horizontal (x) axis.
   
   3. Using the mean optical density value for each sample, determine the corresponding concentration of chloramphenicol in ng/mL from the standard curve. Depending on experience and/or the avail ability of computer capability, other methods of data reduction may be employed.
   4. The diluted samples must be further converted by the appropriate sample dilution factor. The factors are listed for each sample matrix in the sample preparation section. Note: Due to the extraction with ethyl acetate negative samples may show a certain blank value. In repetitive performed experiments with negative samples for each matrix the following blank values were identified. Milk (direct assay) < 0.1 ng/g Milk (ethyl acetate extraction) < 0.1 ng/g Honey < 0.2 ng/g Shrimps < 0.2 ng/g Meat < 0.2 ng/g Fish meal < 0.2 ng/g Whole egg < 0.05 ng/g These values are defined as the cut-off of the method for the respective matrices. Lower concentrations have to be considered as negative. TYPICAL STANDARD VALUES The following table contains an example for a typical standard curve. The binding is calculated as percent of the absorption of the 0 ng/mL standard. These values are only an example and should not be used instead of the standard curve which has to be measured in each new test. Chloramphenicol (ng/mL) % binding of 0 ng/mL 0 100 0.05 84 0.1 70 0.5 28 1 17 5 8 PERFORMANCE
   

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store at 2-8 °C

Target Details for Chloramphenicol

Target: Chloramphenicol

Abstract: Chloramphenicol Products

Target Type: Chemical