Chloramphenicol

ELISA Kit
Catalog Number KA1415
96 assays
Version: 04

Intended for research use only

Introduction ................................................................................................... 3

Background ................................................................................................................... 3

Principle of the Assay .................................................................................................... 3

General Information ...................................................................................... 4

Materials Supplied ......................................................................................................... 4

Storage Instruction ........................................................................................................ 4

Materials Required but Not Supplied ............................................................................. 4

Precautions for Use ....................................................................................................... 4

Assay Protocol .............................................................................................. 6

Assay Procedure ........................................................................................................... 6

Data Analysis ................................................................................................. 7

Calculation of Results .................................................................................................... 7

Resources ...................................................................................................... 8

Plate Layout .................................................................................................................. 8

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Introduction

Background

Chloramphenical (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 the Assay

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 is then 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 is calculated on the basis of a standard curve.

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General Information

Materials Supplied

List of component
Component Amount
96-wells microtiter plate (#S). Eight strips of 12 detachable wells,
1
coated with rabbit Anti-Chloramphenicol antibody
Positive Calibrator containing 0.9 ml each of 0 ng/ml, 0.25 ng/ml, 2.0
4 vials
ng/ml and 10 ng/ml of chloramphenicol
(#3) containing 10.5 ml of chloramphenicol-alkaline phosphatase
1 bottle
conjugate (CPC-ALP)
(#5) containing 10.5 ml of p-Nitrophenyl phosphate (pNPP)
1 bottle
substrate. Ready to use
(#6) containing 15 ml of Wash Buffer (10xPBS-Tween). Dilute 10
1 bottle
fold with distilled or deionized water to 150 ml prior to use
(#7) containing 5.5 ml Stop Solution, 3N NaOH 1 bottle

Storage Instruction

o
Reagents are for in vitro research use only. All reagents of the kit are stable, if stored at 2-8 C, until the
expiration date stated on the kit

Materials Required but Not Supplied

Pipetters capable of delivering 50 µl and 100 µl.

Precautions for Use

Do not mix reagents from different lots.

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middle range of the standard curve.
Do not return unused reagents back into their original bottles.
Samples tested should have a pH of 7.0 (± 1.0). Excessive alkaline or acidic conditions may affect the
test results.
The stop solution contains NaOH. Do not allow to contact skin or eyes. If exposed, flush with water.
Dispose of all materials, containers and devices in the appropriate receptacle after use.

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Assay Protocol

Assay Procedure

Let the components of the kit equilibrate to room temperature before use.
1. Carefully add 50 µl of standard or sample to the bottom of each well. Slightly tap the side of the strip
holder to evenly distribute the sample.

2. Avoid touching the well with pipette tip and add 100 µl of CPC-ALP conjugate (#3) to each well. Slightly
tap the side of the strip holder to properly mix the sample and enzyme conjugate.

3. Incubate at room temperature for 40 minutes.

4. After incubation, dispose the solution in the wells by inverting and shaking. Wash microtiter wells 3 times
with wash buffer to remove the non-bound conjugate. Washing may be done manually as follows: use
squeeze bottle to fill wells gently with wash buffer, dumping the wells between each wash by inverting
and shaking. After the third wash, tamp holder with washed strips onto a piece of absorbent paper.

5. Add 100 µl of pNPP substrate (#5) to each well and incubate at room temperature for 20 min. To avoid
contamination, place the needed amount of substrate into a test tube and dispense only from the tube
itself.

6. Add 50 µl of Stop Solution (#7) to each well and tap the strip holder for proper mixing.

7. Read absorbance at 405 nm using an ELISA reader.

• Simplified Assay Procedure

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Data Analysis

Calculation of Results

1. Calculation
(a) Average the absorbance (ODs) for each standard concentration of chloramphenicol including 0
ng/ml (OD0).
(b) % of Inhibition = 100 -( ODs / OD0 ) x100
2. Plot values of % of Inhibition, step 1 (b), against their corresponding concentrations on Log10 paper.
Calculate chloramphenicol concentration in the sample by interpolation and multiply by sample’s dilution factor
to obtain the actual quantity of chloramphenicol.

Chloramphenicol Inhibition curve

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Resources
Plate Layout
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1

G
C

H
A

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