1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Sulfonamides residue. 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:  0.4 ppb

Incubator temperature:  25℃

Incubator time:  60min～20min

Detection limit

Tissue (high-detection-limit method)............................................... 0.4 ppb

Tissue (lower-detection-limit method)……………………………………...2 ppb

Honey, egg …………………………………………………………………0.4 ppb

Serum, urine................................................................................. 1.6 ppb

Milk................................................................................................. 8 ppb

Recovery rate

Tissue, honey............................................................................. 85±25%

Urine, milk, serum........................................................................ 70±20%

Cross-reaction rate

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

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

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

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

Sulfamerazine (SM₁)........................................................................... 92%

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

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

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

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

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

Sulfanethazine(SM2)………………………………………………………. 140%

Sulfadimethoxine(SDM)…………………………………………………… 110%

Sulfadoxine (SDM2)…………………………………………………………..80%

Sulfaquinoxaline(SQX)………………………………………………………160%

Sulfamethoxypyridazine (SMP)………………………………………………80%

Sulfachlorinepyridazine (SPDZ)……………………………………………...75%

Sulfabenzoyl (SML)…………………………………………………………..104%

Conclusion drawn from the cross-reaction rate: This kit can be used for the test of Sulfonamides, totally meet the national testing demand of Sulfonamides.

3. Components

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

2）   6× standard solution (1 mL each): 0 ppb, 0.4 ppb, 1.2 ppb, 3.6 ppb, 10.8 ppb and 32.4 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 30-300 µL;

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

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 5.16g Na₂HPO₄·12H₂O and 0.87g NaH₂PO₄·2H₂O in the deionized water to 1L.

4)      Acetonitrile (CH₃CN) -Methylene chloride mixing solution

V_acetonitrile-V_{methylene chloride}  =  1 : 4

5)      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 (chicken, duck, pork/liver, egg, fish, shrimp etc.)

A. High-detection-limit method

Method one

1)  Weigh 2.0 ± 0.05 g of the homogenized tissue sample into 50 ml centrifuge tube

2)  Add Acetonitrile (CH₃CN) -Methylene chloride mixing solution 8 ml, shake for 2 min, centrifuge at above 4000 r/min at 15 ℃ for 10 min

3)      Take 4 ml the clear organic phase (upper layer) into a dry tube, blow to dry with nitrogen or air completely by rotary evaporation at 50-60 ℃

4)      Dissolve the dry residues in 1 mL of the diluted redissolving solution, add 1 mL N-hexane, shake vigorously for 30 seconds; centrifuge at above 4000 r/min at 15℃ for 5 min.

5)      Remove the upper layer, take 20µl lower layer solution for further analysis.

 Fold of dilution of the sample: 1

Method two

1)  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;

2)       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 ℃

3)       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

4)