LSY-10009 Sulfonamides residues (SAs) ELISA detection kit 0.4ppb Honey test kit

Green Spring Sulfonamides (SAs) ELISA Kit (0.4ppb)

Catalog No. LSY-10009

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:  30min～15min

Detection limit

Tissue (high-detection-limit method) 0.4 ppb

Honey………………………………….……………………………………0.4 ppb

Serum, urine 1.6 ppb

Milk 8 ppb

Cross-reaction rate

Sulfamerazine (SM₁) 100%

Sulfadiazine (SD or SDZ) 130.2%

Sulfamonomethoxine (SMM) 181.8%

Sulfamethoxazole (SMZ) …..   86.6%

Sulfafurazole(SIZ) 76.5%

Sulfathiazole (ST) 103.3%

Sulfaquinoxaline(SQX)………………………………………………………59.4%

Sulfamethoxypyridazine (SMP)……………………………………………68.6%

Sulfaclozine (Esb3) ………………………………………………………… 72.1%

Sulfachloropyridazine (SCPA)……………………………………………… 49.6%

Sulfamethoxydiazine(SMD) 194.8%

Sulfanethazine(SM2)………………………………………………………. 79.3%

Sulfisomidine (SM2′) …………………………………………………..… 100.6%

Sulfadimethoxine(SDM)…………………………………………………… 140.8%

Sulfadoxine (SDM ′) …………………………………………………….. 132.1%

Sulfadimoxine (SDM2) ……………………………………………………. 144.7%

Phthalylsulfathiazole (PST) 73.9%

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

Sulfamidin(SG) ……………………………........................................……. 0.1%

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.

Recovery rate

Tissue, urine, milk 85±25%

Honey, serum 80±23%

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	6× standard solution (1 mL each)	0ppb	0.4ppb
		0.8ppb	1.6ppb
		3.2ppb	6.4ppb
3	Enzyme conjugate	7ml	red cap
4	Antibody working solution	7ml	blue cap
5	Substrate A	7ml	white cap
6	Substrate B	7ml	black cap
7	Stop solution	7ml	yellow cap
8	20× concentrated washing buffer	40ml	white cap
9	20× concentrated redissolving solution	50ml	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, K₂HPO₄·3H₂O, 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) 0.1M K₂HPO₄ Solution: Weigh 11.4g K₂HPO₄·3H₂O, dissolve with 500ml deionized water.

2) 0.02 M PB buffer: dissolve 5.16g Na₂HPO₄·12H₂O and 0.87g NaH₂PO₄·2H₂O in the deionized water to 1L.

3) Acetonitrile (CH₃CN) -Methylene chloride mixing solution

V_acetonitrile-V_{methylene chloride}  =  1 : 4

4) The 20× concentrated redissolving solution is diluted with deionized water at 1:19 (1 part concentrated redissolving solution + 19 part deionized water).

 

5.1 Tissue

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 8 ml Acetonitrile (CH₃CN) -Methylene chloride mixing solution, 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 container, 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 50µl down 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 to dissolve the dry residues, then add 1 mL of the diluted redissolving solution, shake strongly for 1 min. Centrifuge at 4000 r/min at room temperature for 5 min, remove the upper layer

4) Take 50 µL down layer solution for further analysis.

Fold of dilution of the sample: 1

5.2 Serum

1)  Place the serum sample in the room temperature for 30 min, centrifuge at above 4000r/min at 10 ℃ for 10 min, separation of the serum or filter serum

2)  Take 1 mL serum and add 3mL 0.02 M PB buffer. Mix for 30 s.

3)  Take 50 µL for further analysis

Fold of dilution of the sample: 4

5.3 Honey

1. Weight 2.0 ± 0.05 g honey into 50 mL centrifugal tube, then add 2 mL 0.1 M K₂HPO₄ Solution, vortex and shake for 2min;

2. Add 8 mL Ethyl acetate, vortex and shake for 3min, centrifuge at above 4000 r/min at 15 ℃ for 10 min;

3. Take 4mL supernatant, blow to dry with nitrogen at 56 ℃, add 1 mL of the diluted redissolving solution to redissolve, vortex and shake for 1min.

4. Take 50 µL for further analysis

Fold of dilution of the sample: 1

5.4 Urine

1. Add 3 mL 0.02 M PB buffer and 1 mL of the centrifuged clear urine sample, mix properly for 30s.

2. Take 50 µL for further analysis

Fold of dilution of the sample: 4    

5.5 Milk

1. Take 1 mL milk, add 0.02 M PB buffer, dilute at 1:19(Ⅴ/Ⅴ) (20 µL milk + 380 µL 0.02 M PB buffer), mix for 30s.

2. Take 50 µL for further analysis

Fold of dilution of the sample: 20       

6. ELISA procedures

6.1 Instructions

1. Bring all reagents and micro-well strips to the room temperature (20-25 ℃) before use;

2. Return all reagents to 2-8 ℃ immediately after use;

3. The reproducibility of the ELISA analysis, to a large degree, depends on the consistency of plate washing. The correct operation of plate washing is the key point in the procedures of ELISA;

4. For the incubation at constant temperatures, all the samples and reagents must avoid light exposure, and each microplate should be sealed by the cover membrane.

6.2 Operation procedures

1. Take out all the necessary reagents and place at the room temperature (20-25 ℃) for at least 30min. Note that each reagent must be shaken to mix evenly before use;

2. Take the required micro-well strips and plate frames. Re-sealed the unused microplate, stored at 2- 8 ℃;

3. Solution preparation: dilute 40 mL of the 20× concentrated washing buffer with deionized water to 800 mL for use;

4. Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate; record their positions;

5. Add 50 µL of the sample or standard solution to separate duplicate wells, add 50 µL of enzyme conjugate, then add 50 µL of the antibody working solution into each well. Mix by shaking gently, seal the microplate with the cover membrane, and incubate at 25℃ for 30 min;

6. Wash the microplate with the washing buffer at 250 µL/well for four to five times.;soak the well with the washing buffer for 15-30 sec, flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips);

7. Coloration: add 50 µL of the substrate A solution and 50 µL of the B solution into each well. Mix by shaking gently, and incubate at 25 ℃ for 15 min in the dark for coloration;

8. Determination: add 50 µL of stop solution into each well. Mix by shaking gently. Set the wavelength of the microplate reader at 450 nm to determine the OD value. (recommend to read the OD value at the dual-wavelength 450/630 nm within 5 min) .

7. Result judgment

There are two methods to judge the results; the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with the content of Sulfonamides in the sample.

7.1 Qualitative determination

The concentration range (ng/mL) obtained from the comparison the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sampleⅠ is 0.3, and that of the sampleⅡ is 1.0, the OD value of standard solutions is: 2.243 for 0ppb, 1.816 for 0.4ppb, 1.415 for 0.8ppb, 0.74 for 1.6ppb, 0.313 for 3.2ppb, 0.155 for 6.4ppb, accordingly the concentration range of the sampleⅠ is 3.2ppb to 6.4ppb, and that of the sampleⅡ is 0.8ppb to 1.6ppb. (multiplied by the corresponding dilution fold)

7.2 Quantitative determination

The mean values of the absorbance values is equivalent to the percentage of the average OD value (B) of the sample and the standard solution divided by the OD value (B₀) of the first standard solution (0 standard) and subsequently multiplied by 100%, that is,

Percentage of absorbance value =	B	×100%
	B0

B—the average (double wells) OD value of the sample or the standard solution

B₀—the average OD value of the 0ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the Sulfonamides standard solutions (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the sample from the standard curve by incorporating its absorption percentage into the standard curve. The resulting value is subsequently multiplied by the corresponding dilution fold, finally obtaining the Sulfonamides concentration in the sample.

Using the professional analyzing software of this kit will be more convenient for the accurate and rapid analysis of a large amount of samples. (Please contact us for this software)

7.3 The individual Sulfonamides result calculation method

    Results multiply the cross-reactivity rate

8. Precautions

1. The room temperature below 25 ℃ or the temperature of the reagents and the samples being not returned to the room temperature (20-25 ℃) will lead to a lower standard OD value.

2. Dryness of the microplate in the washing process will be accompanied by the situations including the non-linear standard curves and the undesirable reproducibility; So continue to next step immediately after washing.

3. Mix evenly, otherwise there will be the undesirable reproducibility.

4. The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.

5. Do not use the kit exceeding its expiry date. The use of diluted or adulterated reagents from the kits will lead to the changes in the sensitivity and the detecting OD values. Do not exchange the reagents from the kits of different lot numbers to use.

6. Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance and the colourless color former is light sensitive, and thus they cannot be directly exposed to the light.

7. Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value of the 0 standard solution of less than 0.5 (A450 nm< 0.5 ) indicates its degeneration.

8.  The optimum reaction temperature is 25 ℃, and too high or too low temperatures will result in the changes in the detecting sensitivity and OD values.

9. Storage and expiry date

Storage: store at 2-8 ℃, not frozen.

Expiry date: 1 year; date of production is on box.

 Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.

 

Shenzhen Lvshiyuan Biotechnology Co., Ltd

D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China

Tel. 86-755-28438788

Fax 86-755-28938800

Email: info@lsybt.com   

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