氯霉素ELISA试剂盒

Chloramphenicol (CAP) is a broad spectrum antibiotic which was widely used in veterinary medicine. ABSbioTM Chloramphenicol ELISA Kit provide a rapid, sensitive and reliable assay for the determination of chloramphenicol in food. The High Sensitivity plate is coated with the antibody and pre-blocked to provide timesaving for high-throughput users. Chloramphenicol containing samples or standards and an antibody directed against chloramphenicol are given into the wells of the microtiter plate. The chloramphenicol contained in samples or standards will bind to the antibody which reacts with the binding protein coated onto the microtiter plate. After 60 minutes incubation at room temperature a chloramphenicol-peroxidase conjugate is added into the wells without a preceding washing step to saturate free antibody binding sites. After additional 30 minutes incubation at room temperature the wells are washed with diluted washing solution to remove unbound material. Finally, TMB substrates are added, incubated for detection, and a blue color is developed. Reaction is stopped and color turns to yellow when Stopping Solution (acidic) is added. The yellow color intensity proportionally correlates to the concentration of the chloramphenicol in samples.

试剂盒组分

应用:Measurement of Chloramphenicol in food samples.

样品类型:Fish, shrimp, honey, and milk samples

检测方法:Colorimetric Sandwish ELISA assay (OD450 nm)

检测时间:2 hrs

储存:2-8°C

保质期:6 months after receipt

运输:Icepacks

所需其他材料:
Multi-channel pipette for washing

Cell culture incubator

Orbital shaker

micro plate reader

试剂准备

1. Wash Solution: 10x dilute Wash Solution with dH2O to prepare 1x Wash Solution.

2. Detection Antibody: Immediately before use, add 60 μl of the anti-CAP ab into 6 mL of Assay Diluent for one plate (for partial plate assay, adjust the volumes accordingly).

3. HRP-chloramphenicol conjugate: Immediately before use, add 60 μl of the conjugate into 6 mL of Assay Diluent for one plate (for partial plate assay, adjust the volumes accordingly).

检测程序

1. It is recommended that all standards and samples should be run in duplicate. Set standard wells, testing sample and blank wells on the assay plate/strip. Transfer diluted standard 50 μl to standard wells, diluted sample 50 μl to sample wells, assay diluent 50 μl only to blank wells.

2. Immediately add 50 μl diluted anti-CAP ab into each well, and cover the plate with plate sealer, incubate the plate at room temperature for 1 hr on an oscillating shaker.

3.Add 50 µl of the prepared HRP‐Chloramphenicol conjugate into each wells without preceding washing.

4. Cover the plate with plate sealer and incubate at room temperature for 0.5 hour.

5. Decant as much liquid as possible, fill the wells with 250 μl wash solution, oscillate the plate on an oscillating shaker if available for 1 min, decant the wash solution and remove residual liquid with absorbent paper. Repeat wash five times.

6. Add 150 µl of TMB Solution to each well and incubate at room temperature for 10~30 minutes protect from light, or keep close monitoring on the developing process until desired developing blue color observed. Note: please be aware that the color may develop more quickly or more slowly that the recommended incubation time depending on the localized room temperature.

7. Add 50 µl of Stop Solution to each well to stop the reaction (the blue color change to yellow), gently tap the plate frame for a few seconds to ensure thorough mixing.

8. Read absorbance of the plate on a microplate reader at 450 nm within 15 min.

9. Average the duplicate readings for each standard and samples, subtract the average zero (blank) standard optical density. Construct standard curve (plotting the mean OD450 for each standard on the Y‐axis against concentration on the X‐axis, draw a best‐fit curve through the points) and calculate linear regression equation, then use corrected sample OD values and regression equation to calculate the corresponding sample concentration. It should be remembered that the sample has been diluted and its actual concentration should be justified by dilution factor (the measurement and calculation can be accomplished by software like SoftMax).

检测特点

Sensitivity The lowest Chloramphenicol level that can be measured by this assay is 0.03ng/mL. Standard range:0-5 ng/mL

Precision Intra-assay Precision (Precision within an assay) C.V. < 10%. Inter-assay Precision (Precision between assays) C.V. <15%.

Specificity Cross reactivity with Chloramphenicol 100%; Chloramphenicol glucuronide 25%; Thiamphenicol <1%