Introduction to the determination method of sulfonamide drug residue detector

Introduction to the determination method of sulfonamide drug residue detector:

1 principle and use

The kit uses an indirect competitive ELISA method to detect sulfonamides (Salfamides, SAs ) in tissues, serum, honey, milk, urine, eggs, etc. The kit consists of an enzyme-labeled plate pre-coated with antigen, and an enzyme label. Composition of antibodies, antibodies, standards and other supporting reagents. During the test, a standard or sample solution is added, and the sulfonamide and the microplate on the sample are pre-coated with the antigen to compete for the anti-sulfonamide antibody, and after adding the enzyme label, the TMB substrate is used for color development, and the sample absorbance value is obtained. It is inversely related to the content of sulfonamides contained in it, and the residual amount of sulfonamides in the sample can be obtained by comparison with the standard curve.

2 technical indicators

2.1 Kit sensitivity: 0.5ppb (ng / ml)

2.2 Reaction mode: 25 °C, 45min ~ 1 5min

2.3 Detection limit:

Organization (high detection limit method)..................0.5ppb

Organization (low detection limit method)..................2.5ppb

Serum, urine, eggs..................... 2ppb

Honey.......................................0.5ppb

Milk....................................10ppb

Water sample....................................1ppb

2.4 Cross-reaction rate:

2.5 Sample recovery rate:

Tissue, honey, water sample..................95±25%

Urine, milk, serum..................85±25%

Eggs....................................90±25%

3 kit composition

Enzyme plate.................................96 hole

Standard solution: 1ml each

0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb, 40.5ppb

High standard solution (red cover): 1ppm............1ml

Enzyme label (red cover)........................5.5ml

Antibody working solution (blue cap)..................5.5ml

Substrate A (white cover)........................6ml

Substrate B (black cover)........................6ml

Stop solution (yellow cover)..............................6ml

20X concentrated washing solution (white cover)..................40ml

2X complex solution (yellow cover)........................50ml

Instruction manual..............................1 copy

4 equipment and reagents needed

4.1 Instrument: CSY-E96Y drug residue detector, homogenizer, nitrogen blower, oscillator, centrifuge, graduated pipette, balance (sensitivity 0.01g)

4.2 Micropipette: single channel 20μl-200μl, 100μl-1000μl, multi-channel 300μl

4.3 Reagents: ethyl acetate, n-hexane, acetonitrile, Na ₂ HPO ₄ · 12H ₂ O, NaOH, concentrated HCL, NaH ₂ PO ₄ · 2H ₂ O

5 sample pretreatment

5.1 Notes before sample processing:

Laboratory equipment must be clean and use disposable tips to avoid contamination and interference with experimental results.

5.2 Dosing:

Dosing 1:0.2M NaOH solution

0.8g NaOH is dissolved in deionized water 100ml

Dosing 2: 0.02M PB buffer

2.58 g of Na ₂ HPO ₄ ·12H ₂ O and 0.44 g of NaH ₂ PO ₄ ·2H ₂ O were weighed and dissolved in 500 ml of deionized water.

Solution 3: 0.5M hydrochloric acid solution

4.3 ml of concentrated HCL was added to deionized water to make up to 100 ml.

Solution 4: complex solution

(Note: If the test sample is a water sample, do not equip this liquid)

The 2× complex solution was diluted 2-fold with deionized water for reconstitution of the sample, and the complex solution was stored for one month at 4 °C.

5.3 Sample pre-processing steps:

5.3.1 Tissue sample (high detection limit) processing method

1) Weigh 3±0.05g homogenized tissue sample into the centrifuge tube, add 3ml 0.02M PB buffer and mix well by shaking, then add 4ml ethyl acetate and 2ml acetonitrile, shake well for 10min, centrifuge at 4000r/min or higher. 10min;

2) Pipetting 2 ml of the upper liquid (about 1 g of the sample) to dry at 50-60 ° C under nitrogen or air;

3) Add 1 ml of n-hexane to dissolve the dried residue, add 1 ml of the complex solution, vigorously shake for 1 min, 4000 r / min, centrifuge for 5 min;

4) The upper layer of n-hexane was removed, and 50 μl of the lower aqueous phase was taken for analysis.

Sample dilution factor: 1 detection limit: 0.5ppb

5.3.2 Tissue sample (low detection limit) processing method

1) Weigh 2.0±0.05g homogenized tissue sample in a centrifuge tube; add 8ml 0.02M PB buffer, shake for 2min, centrifuge at 4000r/min for 10min;

2) Take 50 μl of liquid for analysis.

Sample dilution factor: 5 detection limit: 2.5ppb

5.3.3 Serum sample processing method

1) The blood sample is allowed to stand at room temperature for 30 min, centrifuged at 4000 r/min or more for 10 min, and serum or filtered serum is separated;

2) Take 1ml serum, add 3ml 0.02 M PB buffer and mix for 30s;

3) Take 50 μl for analysis.

Sample dilution factor: 4 detection limit: 2ppb

5.3.4 Honey sample processing method

1) Weigh 1 ± 0.05g honey sample in a 50ml centrifuge tube, add 1ml 0.5M hydrochloric acid in a 37 ° C environment for 30min;

2) Add 2.5ml of 0.2M sodium hydroxide (adjust the pH to about 5) and then add 4ml of ethyl acetate to shake for 5min, centrifuge at 4000r/min for 10min at room temperature;

3) Take 2ml of the upper liquid and blow dry at 50-60 ° C, add 0.5ml of the diluted solution to reconstitute, mix for 30s;

4) Take 50 μl for analysis.

Sample dilution factor: 1 detection limit: 0.5ppb

5.3.5 urine sample processing method

1) mixing with 3 ml of 0.02 M PB buffer and 1 ml of clear urine sample after centrifugation, mixing for 30 s;

2) Take 50 μl of liquid for analysis.

Sample dilution factor: 4 detection limit: 2ppb

5.3.6 Milk sample processing method

1) Dilute the milk sample 20 times with 0.02 M PB buffer (such as 20 μl milk + 380 μl 0.02 M PB buffer) and mix for 30 s;

2) Take 50 μl for analysis.

Sample dilution factor: 20 detection limit: 10ppb

5.3.7 Water sample processing method

1) Take 200ul of water to add 200ul 2X complex solution, mix for 30s;

2) Take 50 μl for analysis.

Sample dilution factor: 2 detection limit: 1ppb

5.3.8 Egg sample processing method

1) homogenize the egg sample with a homogenizer to mix the egg white and egg yolk thoroughly;

2) Weigh 2.0±0.05g of the homogenized egg sample (1g of egg powder and 3ml of deionized water and mix 2ml of 2g fresh eggs) into a 50ml centrifuge tube, add 8ml of acetonitrile, and immediately shake with the oscillator for 10min. , centrifuge at room temperature 4000r/min for 5min;

3) Pipette 1 ml of the supernatant into a 10 ml clean dry glass test tube, and blow dry at 50-60 ° C under nitrogen or air;

4) Add 1ml of n-hexane, vortex for 30s to dissolve the dried residue, add 1ml of reconstituted working solution, vortex for 1min with vortex, centrifuge at room temperature 4000r/min for 5min;

5) The upper organic phase was removed and 50 ul of the lower aqueous phase was taken for analysis.

Sample dilution factor: 4 detection limit: 2ppb

6 CSY-E96Y Sulfonamide Residue Detection Test Procedure

Remove the required reagent from the 4 ° C refrigerated environment, and equilibrate at room temperature for more than 30 min. When the washing solution is refrigerated, the crystal may be returned to room temperature to be fully dissolved. Each liquid reagent should be shaken before use. Remove the required number of microplates and frames, place the unused microplates in a ziplock bag and store at 2-8 °C.

Before the start of the experiment, the 20X concentrated washing solution was diluted with 20 times of deionized water into a working washing solution.

6.1 No.: The micropores corresponding to the sample and the standard are numbered sequentially, and each sample and the standard are made in parallel with 2 holes, and the position of the standard hole and the sample hole is recorded.

6.2 Addition reaction: Add standard or sample 50μl/well to the respective microwells, then add 50μl/well of enzyme label, then add 50μl/well of antibody working solution, seal the plate with cover membrane, and gently shake 5 Mix in seconds and react at 25 ° C for 45 minutes.

6.3 Washing: Carefully uncover the cover film, dry the liquid in the well, and wash it thoroughly with the working washing liquid 250μl/well 5 times, each time interval 30 seconds, pat dry with absorbent paper (bubbles that have not been removed after pat drying) A clean gun puncture).

6.4 Color: 50 μl of substrate A was added to each well, and 50 μl of substrate B was added. The mixture was gently shaken for 5 seconds, and mixed at 25 ° C for 15 minutes.

6.5 termination: 50 μl of stop solution was added to each well, and the mixture was gently shaken to terminate the reaction.

6.6 Measured absorbance: The absorbance value per well was measured at 450 nm using a sulfa drug residue detector (double wavelength 450/630 nm is recommended). The assay should be completed within 10 minutes of terminating the reaction.

7 results analysis

7.1 Calculation of percent absorbance

The percent absorbance of the standard solution or sample is equal to the average value of the absorbance value of the standard solution or sample (double well) divided by the absorbance value of the first standard solution (0 ppb), multiplied by 100%, ie

A—the average absorbance value of the standard solution or sample solution

Average absorbance value of A0—0ppb standard solution

7.2 Drawing and calculation of standard curve

The semi-logarithmic plot of the standard solution is plotted with the percent absorbance of the standard solution as the ordinate and the logarithm of the corresponding standard solution concentration (ppb) as the abscissa. Substituting the percent absorbance of the sample into the standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the corresponding dilution factor is the actual concentration of the analyte in the sample.

If the kit professional analysis software is used for calculation, it is more convenient for accurate and rapid analysis of a large number of samples. (Welcome to call)

8 considerations

8.1 Room temperature below 25 ° C or reagents and samples not returned to room temperature (25 ° C) will result in low OD values ​​for all standards.

8.2 If the plate hole is dry during the washing process, the standard curve will not be linear and the repeatability is not good. Therefore, the next step should be taken immediately after the plate is patted dry.

8.3 The mixing should be uniform, the washing should be thorough, and the reproducibility in the ELISA analysis depends largely on the consistency of the washing.

8.4 Seal the microplate with a cover film during all incubations to avoid light exposure.

8.5 Do not use kits that have expired. Do not exchange reagents from different batches.

8.6 If any color of the coloring solution indicates deterioration, it should be discarded. A 0 standard absorbance value of less than 0.5 units (A450nm < 0.5) indicates that the reagent may deteriorate.

8.7 The reaction stop solution is corrosive and avoids contact with the skin.

9 storage and storage period

Storage conditions: The kit is stored at 2-8 ° C to avoid freezing.

Shelf life: The product is valid for 1 year, and the production date can be found in the box.

CSY-E96Y sulfonamide drug residue detector technical parameters:

☆ wavelength range: 300nm-1000nm

☆ Wavelength accuracy: ± 2nm

☆ Absorbance range: 0.000~4.000ABS

☆ Resolution: 0.001Abs

☆ Stability : ±0.001A/hr

☆ Transmittance repeatability: ≤0.5%T

☆Light source : Imported LED

☆ sample cell : microplate

ADL Adult Diaper

Adult diapers are disposable urinary incontinence products, one of the adult care products, and a disposable diaper mainly suitable for incontinent adults. Most products are flaky and shorts type. Use adhesive sheets to connect into a pair of shorts. The adhesive sheet also has the function of adjusting the waist size so as to fit different fat and thin body shapes. Generally, the structure of diapers is divided into three layers from the inside to the outside. The inner layer is close to the skin and made of non-woven fabric; the middle layer is water-absorbent fluff pulp, added with polymer water-absorbing agent; the outer layer is an impermeable breathable film.

Adult Briefs,Depends Adult Diapers,Adult Diapers For Sale,Adult Disposable Diapers

Shandong Kangshun Daily Products Co., Ltd , https://www.centurybenifit.com