1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Sulfonamides in the chicken, pork, milk, honey, egg, etc. The coupling antigens are pre-coated on the micro-well stripes. The Sulfonamides in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Sulfonamides 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 Sulfonamides in the sample. This value is compared to the standard curve and the Sulfonamides concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 1 ppb

Detection limit

Tissue............................................................................................. 1 ppb

Serum, urine.................................................................................... 4 ppb

Milk............................................................................................... 20 ppb

Recovery rate

Tissue....................................................................................... 70 ±10%

Egg.......................................................................................... 65 ±10%

Milk, honey, serum..................................................................... 70 ±10%

Cross-reaction rate

Sulfadiazine (SD or SDZ)................................................................. 100%

Sulfamonomethoxine (SMM)........................................................... 97.6%

Sulfamerazine (SM₁)........................................................................... 12%

Sulfamethoxazole (SMZ).................................................................. 100%

Sulfathiazole (ST).......................................................................... 195.0%

Phthalylsulfathiazole (PST)................................................................. 73%

Sulfamethyltiadiazolum..................................................................... 110%

Sulfapyridine..................................................................................... 48%

Sulfamethoxydiazine(SMD)............................................................. 26.4%

Sulfafurazole(SIZ)........................................................................... 95.0%

This test kit is used to detect 10 Sulfonamides based on cross-reaction rate, its detection limit as follows:

SD-1 ppb  SMM- 1 ppb  SMZ -2 ppb  ST - 0.5 ppb  SM1-10 ppb  PST-2 ppb

SMD-10 ppb  Sulfamethyltiadiazolum-1 ppb   Sulfapyridine-2 ppb   SIZ-1 ppb

3. Components

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

2）   6× standard solution (1 mL each): 0 ppb, 1 ppb, 3 ppb, 9 ppb, 27 ppb and 81 ppb

3）   Enzyme conjugate (7 mL)............................................................... red cap

4）   Antibody working solution (10 mL)................................................ blue cap

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

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

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

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

9）   2× concentrated redissolving solution (50 mL)..................... transparent cap

4. Materials required but not provided

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

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

3)      Reagents: Acetonitrile (CH₃CN), ethyl acetate, N-hexane, Na₂HPO₄·12H₂O, NaH₂PO₄·2H₂O, NaOH, HCl

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)      0.2 M NaOH: dissolve 0.8 g NaOH in deionized water to 100 mL.

2)      0.5 M HCl (for honey): dissolve 4.3 mL HCI (36%) in deionized water to 100 mL.

3)      0.02 M PB buffer: dissolve 2.58 g Na₂HPO₄·12H₂O and 0.44 g NaH₂PO₄·2H₂O in the deionized water to 500 mL. (for low-detection-limit samples)

4)      The 2× concentrated redissolving solution is diluted with deionized water at 1:1 (1 mL concentrated redissolbing solution + 1 mL deionized water), used for the treated sample redissolving.

5.1 Tissues(meat , liver, shrimp, fish, egg. etc)

1）  Homogenize the sample at 10000 r/min for 1 min

2）  Weigh 3 ± 0.05 g of the homogenized sample, put into centrifugal tube, add 3 mL 0.02 M PB buffer, shake properly. Then add 4 mL ethyl acetate and 2 mL Acetonitrile (CH₃CN), shake properly for 10 min, centrifuge at above 4000 r/min at 15 ℃ for 10 min;

3）  Transfer 2 mL supernatant(approx 1 g sample) into a new centrifugal tube, blow to dry with nitrogen or air completely by rotary evaporation at 50-60 ℃

4）  Add 1 mL N-hexane, then add 1 mL of the diluted redissolving solution, shake strongly for 30 s. Centrifuge at 4000 r/min at room temperature for 5 min, remove the upper layer

5）  Take 20 µL for further analysis.

Fold of dilution of the sample: 1

It needs five fold to dilute the sample(1mL sample+4 mL of the diluted redissolving solution) if the detection is stipulated in the most residue (100 ppb) of national regulation. 

5.2 Honey

1.      Weight 1.0 ± 0.05 g honey into 50 mL centrifugal tube, then add 1 mL 0.5 M HCI. Be static at 37 ℃ for 30 min.

2.      Add 2.5 mL 0.2 M NaOH (adjust pH to 5), then add 4 mL ethyl acetate, shake for 5 min, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.

3.      Take 2 mL supernatant, blow to dry with nitrogen at 50 ℃, add 0.5 mL of the diluted redissolving solution, redissolve it for 30 s.

4.      Take 20 µL for further analysis

Fold of dilution of the sample: 1

5.3 Urine

1.      Add 3 mL 0.02 M PB buffer and 1 mL of the centrifuged clear sample, mix properly.

2.      Take 20 µL for further analysis

Fold of dilution of the sample: 4     Detection limit: 4 ppb

5.4 Milk&