1. Principle

The BIOKAU^® Malachite green ELISA test kit is based on the competitive enzyme immunoassay. The coupling antigen is pre-coated on the micro-well stripes. The Malachite green in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Malachite green antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with the Malachite green in it. This value is compared to the standard curve and the content of Malachite green residues is subsequently obtained.

2. Technical specifications

Sensitivity:  0.025 ppb

Detection limit

Shrimp, Fish..................................................................................... 0.1 ppb

Water............................................................................................... 0.1 ppb

Recovery rate

Shrimp, Fish.................................................................................... 75±10%

Cross-reaction rate

Malachite green................................................................................... 100%

Leucomalachite green........................................................................... 0.1%

Gentian violet ....................................................................................... 95%

Leucocrystal violet................................................................................ 0.1%

3. Components

1）   Micro-well strips: 12 strips with 8 removable wells each

2）   Standard solution (1 mL)............................................................... 100 ppb

3）   Enzyme conjugate (12 mL)............................................................. red cap

4）   Antibody working solution (7 mL).................................................... red cap

5）   Substrate solution A (7 mL).......................................................... white cap

6）   Substrate solution B (7 mL)......................................................... black cap

7）   Stop solution (7 mL).................................................................. yellow cap

8）   20× concentrated washing buffer (40 mL)..................................... white cap

9）   Reagent A (50 mL).............................................................. transparent cap

10) Reagent B (50 mL)........................................................................ blue cap

4. Materials required but not provided

(1) Equipments: microplate reader, homogenizer, nitrogen-drying device, vortex, oscillator, centrifuge, measuring pipets, balance (a sensibility reciprocal of 0.01 g).

(2)  Micropipettors: single-channel 20-200 µL and 100-1000 µL, and multi-channel 250 µL.

(3)  Reagents: Acetonitrile (CH₃CN), HCl, NaCl, CH₂Cl₂, methanol, Distilled water.

5. Sample pre-treatment

Instructions (The following points must be dealt with before the pre-treatment)

(1) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

(2) Before the experiment, each experimental utensil must be clean and should be re-cleaned if  necessary, in order to avoid the contamination that interferes with the experimental results.

Solution preparation before sample pre-treatment

1)      A solution: dissolve 0.8 mL HCl(approx 36.5%) in 1 L Acetonitrile(CH₃CN).

2)      B solution(20% NaCl soulution): dissolve 25 g NaCl in water to 100 mL.

3)      C solution: Dilute the reagent A with deinoized water at 1:1.

4)      D solution: Mix 3 mL Methanol with 28 mL C solution(3:28), used for sample redissolving and dilution of standard solution.