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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

MIX7218 Scientific Laboratory Supplies EACH 634.8 EUR

Block for 5ml centrifuge tubes

H5000-5MT Benchmark Scientific 1 PC 479.8 EUR

Cool rack 9 wells for 15ml centrifuge tubes

HOM2740 Scientific Laboratory Supplies EACH 996.17 EUR

Cool rack 4 wells for 50ml centrifuge tubes

HOM2742 Scientific Laboratory Supplies EACH 996.17 EUR

50mL Rack for Conical Bottom Centrifuge Tubes

4365 Scientific Laboratory Supplies PK20 51.6 EUR

15mL Rack for Conical Bottom Centrifuge Tubes

431355 Scientific Laboratory Supplies PK20 40.8 EUR

Centrifuge Tubes S 2420 30ml Qfit

QBC24C24R0 Scientific Laboratory Supplies PK10 240 EUR

Oakridge Centrifuge Tubes 30ml

31390030 Scientific Laboratory Supplies PK10 273.6 EUR

CapLock 0.5mL Centrifuge Tubes

ARG1782 Scientific Laboratory Supplies PK500 65.8 EUR

CapLock 1.5mL Centrifuge Tubes

ARG1808 Scientific Laboratory Supplies PK1000 117.5 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 138.6 EUR

Block, 2 x 50ml centrifuge tubes

BSH100-500 Benchmark Scientific 1 PC 138.6 EUR

Block, 12 x 15ml centrifuge tubes

BSW15 Benchmark Scientific 1 PC 144.7 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.

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