This method is based on a competitive-type ELISA (Enzyme Linked Immuno SorbentAssay) for in vitro screening of chloramphenicol. The solid support of the reaction is a microtiter plate with divisible strips coated with sheep anti-rabbit IgG antibodies.
This method is suitable for the screening of chloramphenicol in poultry, beef, swine, and catfish muscle at levels ≥ 0.25 ppb.
EQUIPMENT
- Analytical balance – Cat. No. BL15005, Sartorius.
- Centrifuge – Model No. 5810, Eppendorf.
- Mechanical shaker – Eberbach.
- Polypropylene centrifuge tubes – 50 mL, Falcon.
- Glass centrifuge tubes – 15 mL, HS No. 45600-15, Kimble.
- Nitrogen Evaporator – Turbovap.
- Vortex mixer – Genie 2, Scientific Industries.
- Micropipettors – 2-250 µL, Rainin EDP.
- Repeater pipet (Distriman) with 1250 µL mini syringes, Gilson.
- Transfer pipettes – 10 mL in 1/10 serological, Kimble.
- Transia Plate Chloramphenicol Test Kit – BioControl AB0299.
Note: Store kit components in the dark at 2 – 8 °C when not in use. Reagents with different batch numbers should not be interchanged between kits. The reagents should come to room temperature before use. The microtiter plate
should be kept in the sealed pouch to avoid condensation. Each vial should be shaken well before use.
REAGENTS AND SOLUTIONS
Note: Equivalent reagents / solutions may be substituted. The stability time frame of the solution is dependant on the expiration date of the components used or the listed expiration date, whichever occurs first.
Reagents
a. Ethyl acetate – HPLC Grade, Cat. No. 4601-7, Caledon.
b. n-hexane – HPLC Grade, Cat. No. 5601-7, Caledon.
c. Distilled water
Solutions
a. Washing buffer 1X:
Dilute the washing buffer to 1:20 in distilled water (Ex. Pipet 2 mL of washing buffer and 38 mL of distilled water into bottle). Mix well. The washing buffer can be stored at 2 – 8 °C until the expiration date stated on the kit label.
b. Sample dilution buffer 1X:
Dilute the sample dilution buffer to 1:4 in distilled water by pipetting 20 mL of sample dilution buffer and 60 mL of distilled water into a bottle. Mix well. The sample dilution buffer must be stored at 2 – 8 °C until the expiration date stated on the kit label.
c. Reconstitution of conjugate solution:
Reconstitute the lyophilized conjugate with 4 mL of reconstitution buffer. Mix thoroughly and keep in the dark until use. This solution can be stored in the dark at 2 – 8 °C for a maximum of 2 months. Alternatively, the reconstituted conjugate solution may be stored in a freezer at -20 °C until the expiration date on the kit. Freeze aliquots immediately after reconstitution.
d. Reconstitution of antibody solution:
Reconstitute the lyophilized antibodies with 4 mL reconstitution buffer. Mix thoroughly and keep in dark until use. This solution can be stored in the dark at 2 – 8 °C for a maximum of 6 months. Alternatively, the reconstituted antibody solution may be stored in a freezer at -20 °C until the expiration date on the kit. Freeze aliquots immediately after reconstitution.
STANDARD(S)
Note: Equivalent standards / solutions may be substituted. Purity and counterions are to be taken into account when calculating standard concentrations. The stability time frame of the solution is dependant on the expiration date of the components used or the listed expiration date, whichever occurs first.
1. Standard Information
a. Standards are provided with the ELISA kit and come ready to use. All standards must be stored in the dark at 2 – 8 °C until the expiration date stated on the kit.
Block for 5ml centrifuge tubes | ||||
| H5000-5MT | Benchmark Scientific | 1 PC | 494.19 EUR | |
Block for 5mL centrifuge tubes - EACH | ||||
| MIX7218 | Scientific Laboratory Supplies | EACH | 714.15 EUR | |
Stand For Centrifuge Tubes For 1.5 ml, | ||||
| PW102-2NO | EWC Diagnostics | 1 unit | 8.01 EUR | |
Cool rack 9 wells for 15ml centrifuge tubes - EACH | ||||
| HOM2740 | Scientific Laboratory Supplies | EACH | 1120.69 EUR | |
Cool rack 4 wells for 50ml centrifuge tubes - EACH | ||||
| HOM2742 | Scientific Laboratory Supplies | EACH | 1120.69 EUR | |
50mL Rack for Conical Bottom Centrifuge Tubes - PK20 | ||||
| 4365 | Scientific Laboratory Supplies | PK20 | 58.05 EUR | |
15mL Rack for Conical Bottom Centrifuge Tubes - PK20 | ||||
| 431355 | Scientific Laboratory Supplies | PK20 | 45.9 EUR | |
Holder for up to eight 15ml centrifuge tubes, for Bench-Top Shaker | ||||
| 099ASR815 | Glascol | each | 648 EUR | |
Holder for up to eight 50ml centrifuge tubes, for Bench-Top Shaker | ||||
| 099ASR850 | Glascol | each | 648 EUR | |
Block, 4 x 15ml centrifuge tubes | ||||
| BCM1405-B150 | Bio Basic | 1 pcs, 1 UNIT | 227.29 EUR | |
Block, 2 x 50ml centrifuge tubes | ||||
| BCM1405-B500 | Bio Basic | 1 pcs, 1 UNIT | 227.29 EUR | |
Block, 12 x 15ml centrifuge tubes | ||||
| BCM1412-BSW15 | Bio Basic | 1 pcs, 1 UNIT | 233.21 EUR | |
Block, 5 x 50ml centrifuge tubes | ||||
| BCM1412-BSW50 | Bio Basic | 1 pcs, 1 UNIT | 233.21 EUR | |
Block, 4 x 15ml centrifuge tubes | ||||
| BSH100-150 | Benchmark Scientific | 1 PC | 142.76 EUR | |
Block, 2 x 50ml centrifuge tubes | ||||
| BSH100-500 | Benchmark Scientific | 1 PC | 142.76 EUR | |
Block, 12 x 15ml centrifuge tubes | ||||
| BSW15 | Benchmark Scientific | 1 PC | 149.04 EUR | |
Block, 5 x 50ml centrifuge tubes | ||||
| BSW50 | Benchmark Scientific | 1 PC | 149.04 EUR | |
2. Preparation of Standard Solution(s)
a. Fortification solution (10 ppb):
Add 100 µL of the 100 ng/mL standard and 900 µL of sample dilution buffer in a
small bottle. Mix well.
Homogenize samples prior to proceeding with extraction.