Aquatic drug residue rapid detector operation steps
1 principle and use
The kit uses an indirect competitive ELISA method to detect furazolidone metabolites ( AOZ ) in honey, fish, shrimp, poultry, liver, etc. The kit consists of an enzyme-labeled plate, a horseradish enzyme label, and an antibody pre-coated with an antigen. Standard and other supporting reagents. During the test, the standard or sample solution is added, the furazolidone metabolite and the enzyme label on the sample are pre-coated with the antigen to compete against the anti-furazolidone metabolite 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 furazolidone metabolites contained in it, and the residual amount of furazolidone metabolite in the sample can be obtained by comparison with a standard curve.
2 technical indicators
2.1 Kit sensitivity: 0.02ppb (ng / ml)
2.2 Reaction mode: 25 °C, 45min ~ 1 5min
2.3 Detection limit:
Organization, liver..............................0.04ppb
Milk powder, egg powder, feed.....................0.04ppb
Honey, milk, casings........................0.04ppb
The fishery tissue such as fish/shrimp has a certain interference, and the lower limit of quantification is 0.1ppb.
2.4 Cross-reaction rate:
Furazolidone metabolites.....................100%
Furanone metabolites........................<0.1%
Nitrofurantoin metabolites........................<0.1%
Nitrofurazone metabolites........................<0.1%
2.5 Sample recovery rate:
Tissue, liver..............................80±25%
Honey, milk, casings........................75±15%
Milk powder, egg powder, feed.....................85±25%
3 kit composition
Enzyme plate.................................96 hole
Standard solution: 1ml each
0ppb, 0.02ppb, 0.06ppb, 0.18ppb, 0.54ppb, 1.62ppb
High standard solution (red cover): 100ppb............1ml
Derivatization reagent (black cover)..................10ml
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-E96S aquatic product drug residue rapid detector, homogenizer, nitrogen blow dryer, 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, sodium hydroxide, concentrated HCl, K 2 HPO 4 ·3H 2 O, potassium nitroferrocyanide (K 2 Fe(CN) 5 (NO) ▪ 2H 2 O), ZnSO 4 ·7H 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:
Solution 1: 0.36M potassium nitroferrocyanide solution (milk, milk powder sample)
11.9 g of potassium nitroferrocyanide plus deionized water was dissolved in 100 ml.
Solution 2: 1.04M zinc sulphate solution (milk, milk powder sample)
29.8 g of zinc sulfate plus deionized water was dissolved in 100 ml.
Solution 3: 0.1M K 2 HPO 4 solution
Weigh 11.4 g of K 2 HPO 4 â–ª 3H 2 O with deionized water to dissolve to 500 ml.
Dosing 4:1M HCL
8.6 mL of concentrated HCL plus deionized water to a volume of 100 mL.
Dosing 5:1M NaOH solution
Weigh 4g NaOH and dilute to 100ml with deionized water.
Solution 6: complex solution
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 Milk sample processing method
1) Take 5ml sample in a centrifuge tube, add 250ul potassium nitroferricyanide solution (dispensing solution 1) and mix for 30 seconds. Add 250ul zinc sulphate solution (liquid 2) and mix for 30 seconds, 15°C 4000 rpm. Centrifugation for 10 minutes;
2) Take the supernatant 1.1ml, add 4ml deionized water, 0.5 ml 1M hydrochloric acid solution and 100μl derivatization reagent, shake for 5 minutes;
3) Incubate at 37 ° C overnight (about 16 hours) or 50 ° C (more than 50 ° C will affect the stratification effect) incubation in water bath for 3 hours;
4) separately add 5 ml of 0.1 MK 2 HPO 4 solution, 0.4 ml of 1 M NaOH solution and 5 ml of ethyl acetate, and shake for 5 minutes;
5) Centrifugation at 4000 rpm for 10 minutes at room temperature;
6) Take out 2.5 ml of the supernatant liquid to another centrifuge tube, and blow dry at 50-60 ° C under nitrogen or air;
7) Dissolve the residue with 1 ml of n-hexane, add 1 ml of reconstituted working solution and mix thoroughly for 30 seconds; centrifuge at room temperature for 4,000 rpm for 10 minutes;
8) Remove the upper n-hexane and take 50 μl of the lower layer for analysis.
Sample dilution factor is 2 Detection limit: 0.04ppb
5.3.2 Milk powder and egg powder sample processing method
1) Weigh 1±0.05g homogenized sample in a centrifuge tube, add 4ml deionized water, 0.5ml 1M hydrochloric acid solution and 100μl derivatization reagent, shake for 5 minutes;
2) Incubate overnight at 37 ° C (about 16 hours) or 50 ° C (more than 50 ° C will affect the stratification effect) in a water bath for 3 hours;
3) Add 250ul of potassium nitroferrocyanide solution (dispensing solution 1) and shake for 30 seconds. Add 250ul of zinc sulfate solution (liquid 2) and mix for 30 seconds, and centrifuge at 1000°C for 10 minutes.
4) Take all the supernatant into another centrifuge tube, followed by “5.3.1 Milk Sample Processing Method, 4) â€
5.3.3 Honey, tissue, casing, liver, feed, egg sample processing methods
1) Weigh 1±0.05g homogenized sample in a centrifuge tube, add 4ml deionized water, 0.5ml 1M hydrochloric acid solution and 100μl derivatization reagent, shake for 5 minutes;
2) Follow-up “5.3.1 Milk Sample Processing Method, 3) â€
5.3.4 Method of processing cooked food samples
1) Weigh 1 ± 0.05g homogenate in a 50ml centrifuge tube, add 4.5ml methanol and 0.5ml deionized water, shake for 2min, room temperature 4000 rev / min, centrifuge for 5 minutes, remove all liquid;
2) Add 5 ml of acetonitrile and 5 ml of n-hexane, shake for 2 min, centrifuge at room temperature for 4000 rpm, and centrifuge for 5 minutes to remove all the liquid;
3) adding 4 ml of deionized water, 0.5 ml of 1 M hydrochloric acid solution and 100 μl of derivatization reagent to the precipitate, and shaking for 5 minutes;
Note: Please add high standards at this step for the addition and recovery test of cooked food.
4) Follow-up “5.3.1 Milk Sample Processing Method, 3) â€
6 enzyme-linked immunoassay steps
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 of each well was measured at 450 nm using a CSY-E96S aquatic product drug rapid 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
Percent absorbance value (%) = | A | ×100% |
A0 |
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.
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