Introduction of detection methods for feed and tissue olaquindox residues

Introduction of feed and tissue olaquinone residue detection methods:

1 principle and use

This kit uses indirect competitive ELISA to detect olaquindox (Olaquindox, OQX ) in tissues, feeds, etc. The kit consists of pre-coated conjugated antigen-labeled plates, enzyme markers, antibodies, standards, and other supporting reagents. composition. At the time of testing, a standard or sample solution is added, and the olaquindox and the plate on the plate are pre-coated with the antigen to compete for the anti-olaline ethanol antibody, and after adding the enzyme label, the TMB substrate is used for color development, and the sample absorbance value is The olaquindox content is negatively correlated, and the residual amount of olaquindox in the sample can be obtained by comparison with a standard curve.

2 technical indicators

2.1 Kit sensitivity: 0.5ppb (ng / ml)

2.2 Reaction mode: 37 °C, 30min ~ 30min ~ 1 5min

2.3 Detection limit:

Organization.......................................1ppb

Feed..........................................50ppb

2.4 Cross-reaction rate:

Olaquindox..........................................100%

Kabado..........................................<0.1%

2.5 Sample recovery rate:

Organization..........................................80±15%

Feed..........................................85±15%

3 kit composition

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

Standard (black cover): 1ml each

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

High standard (black cover): 1ppm............1ml

Enzyme label (red cover)........................11ml

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: Olaquindox 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: Anhydrous acetonitrile, methanol

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: sample extract

Take methanol as needed and dilute with deionized water: methanol = water = 0.5: 9.5, mix and set aside.

Solution 2: 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 Tissue (pork liver, pork, etc.) samples

1) Weigh 2±0.05g of fat-removed homogenate sample, add 2ml deionized water, 8ml acetonitrile, mix well, then place in 56°C water bath for 10min, shake for 5min, centrifuge at room temperature 4000r/min for 10min;

2) Take 5 ml of the supernatant and blow dry at 50-60 ° C under nitrogen or air.

3) Dissolve the dried product with 1 ml sample solution, add 2 ml of n-hexane, mix well, and centrifuge at room temperature 4000r/min for 5 min;

4) The upper organic phase was removed, and 50 ul of the lower layer was taken for analysis.

Sample dilution factor: 1 detection limit: 1ppb

5.3.2 Feed samples

1) Weigh 1 ± 0.05g of the ground feed sample, add 10ml sample extract, mix thoroughly, then set the water bath at 56 ° C for 10min, shake for 5min, centrifuge at room temperature 4000r / min for 10min;

2) Take 50ul of the supernatant, add 450ul of the sample solution to dilute, and mix;

3) Take 50 ul of the supernatant diluted in 2) for analysis.

Sample dilution factor: 100 detection limit: 50ppb

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 Loading reaction : Add standard or sample 50μl/well to each microwell, then add 50μl/well of antibody working solution, gently shake for 5 seconds, mix and shake at 37°C for 30 minutes.

6.3 Washing : Drain the liquid in the well and wash it thoroughly with 250 μl/well of working washing solution for 5 times at a time. Finally, pat dry with absorbent paper (bubbles that have not been removed after pat drying can be pierced with a clean tip) ).

6.4 Enzyme reaction : 100 μl/well of the enzyme label was added, and the reaction was carried out in the dark at 37 ° C for 30 minutes.

6.5 Washing : Same as above

6.6 Color : Add 50 μl/well of substrate A, add 50 μl/well of substrate B, gently shake for 5 seconds, mix and incubate for 15 minutes at 37 °C.

6.7 End: Add 50 μl/well of stop solution, gently shake and mix to stop the reaction.

6.8 Measurement of absorbance: The absorbance value of each well was measured at 450 nm using an ethanol residue detection instrument (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.

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