IELisa Lackadine Detection Kit

This reagent kit is for applicationELISADrug residue detection products developed through technological research and development,Compared with instrument analysis technology, it can economically and quickly detect musclesorganization、In samples such as feed and urineofLackadamine content.

3、 Cross reactivity rate

Cross reactivity rate with similar substances:

Ractopamine…………………………………100%

Shatianaol……………………………………﹤1%

Domperidone hydrochloride………………………………﹤1%

Clenbuterol ………………………………﹤1%

4、 Composition of reagent kit

microplate1The block,96Kong/board

standard solution5Bottle（1mL/Bottle)：

0 ppbThe0. 1ppbThe0.3ppbThe0.9ppbThe2.7ppb

Enzyme marker ………………………………………7mL

Antibody working fluid ………………………………………10mL

Substrate solutionA ………………………………………7mL

Substrate solutionB………………………………………7mL

Stop Solution……………………. .…………………7mL

10×Concentrated washing solution………………………………40mL

2×concentrateCompound solution………………………………50mL

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

5Equipment and reagents to be provided by the user unit

（1）instrument

Microplate ELISA reader(450 nm)Nitrogen drying device;oscillator；centrifugeVortex meter; Electronic balance (sensitivity)0.01 g）.

（2）equipment

Volumetric flask:100 mL，500 mL，1000 mL

single trackMicro pipette:20 ML～200 ML，100 ML~1000 ML

multiple coursesMicro pipette:30 ML～300 ML

（3）Reagent

Methanol, acetonitrile, n-hexane, *, concentrated hydrochloric acid, deionized water

6、 Preparation of solution

Sample pre-treatment requires preparation：

solution preparation1：1M NaOHsolution

Weigh out4.0 g NaOHplusdeionized waterdissolution,constant volume to100 mL.

solution preparation2：0.1M HCl

measure0.83 mLAdd concentrated hydrochloric acid to deionized waterconstant volume to100 mL.

solution preparation3：25%methanol

measure25mLMethanol, added to75mLMix thoroughly in deionized water.

solution preparation4：25%methanol-0.1MHCLsolution

measure20mL 0.1Mhydrochloric acidsolutionjoin in80mL 25%methanolMix thoroughly in the solution.

solution preparation5：Sample complex solution

2×concentrateCompound solutionPlease press before use1：1Dilution（1copyconcentrateSample complex solution+ 1Deionized water).

solution preparation6：Washing working fluid

10×Please press the button for concentrated detergent before use1：9Dilution（1serving of concentratewashliquid + 9Deionized water (can be configured as needed).

7、 Sample pre-processing methods

Notice before sample processing：

（1）Disposable tips must be used in the experiment, and the tips should be replaced when sucking different reagents.

（2）Before conducting the experiment, it is necessary to check whether all experimental equipment is clean. Clean experimental equipment must be used to avoid contamination and interference with the experimental results.

samplefrontProcessing Steps：

（A）group Weaving treatment (chicken, duck, pig muscles)/Liver, eggs, fish, shrimp, etc

Solution 1:

1AccurateWeigh out2.0±0.02 ghomogenateSample in50 mLIn centrifuge tubes;

2Join0.5mLDiluted sample complex solution5）Fully oscillating2minJoin in8mL acetonitrile, fullyVortex oscillation3~5 minAt room temperature4000 r/mincentrifugation10 min；

3、 取2.5 mLUpper liquid toIn another centrifuge tube，50~60Dry with nitrogen at ℃;

4Join1 mL Dissolve the residue in n-hexane and then add1mLSample complex solution (preparation)5）Fully mixed30 sAt room temperature4000 r/mincentrifugation10 min；

5Remove the upper layer of n-hexane and take20 MLLower layer liquid is used for analysis.

Solution 2:

1AccurateWeigh out2.0±0.02 ghomogenateSample in50 mLIn centrifuge tubes;

2Join6mL25%methanol-0.1MHCLSolution,oscillationmix well3~5min，room temperature4000 r/minabovecentrifugation10 min(Note: If the oil content in the tissue sample is high, it can be added after oscillation.)85Centrifuge after 10 minutes of water bath at ℃）；

3The取1 mLUpper liquid toanother2mLIn the centrifuge tube，join20ML 1M*Mix the solution thoroughly (measure after mixing)pHThe value is approximately7-8）；

4The4000 r/mincentrifugation3~5 minTake the supernatant20 MLUsed for analysis.

（B）Urine, serum

取20 MLDirect measurement of clear urine samples or serum (such as cloudy urine samples or serum)

Must be filtered or4000 r/minYu15℃centrifugation10 minUntil the Qing Dynasty

bright）Samples that are not currently in use should be frozen and stored.

8、 Enzyme linked immunosorbent assay steps

Notice before measurement:

1、 Before use, bring all reagents and microplates back to room temperature.

2、 Immediately put all reagents back after use2~8 ℃.

3、 Do not let the micropores dry during use.

4、 inELISAThe repeatability in the analysis largely depends on the consistency of the washing process, and the correct washing operation isELISAKey points in the testing program.

5、 During all constant temperature incubation processes, avoid exposure to light and usecoverThe membrane covers the microporous plate.

Measurement steps:

1Prepare the required reagentsAnd microporous plateRemove from refrigerated environment and equilibrate at room temperature30 minEach liquid must be shaken well before use. Please note that all standard solutions must be prepared2A parallel experiment.

2Add standard products/Sample:plusenterreference standard/sample20 ML/Kong，joinenzymemarker50 ML/Hole, add moreAntibody working fluid80 ML/Kong，Gently shake and mix well，usecover plateCover the membrane,room temperature25℃Lower light avoidance reaction30 min.

3Washing board:Carefully remove the cover film and shake off the liquid inside the hole,Add detergent 250 ML/Hole, every timesoak15~30 sWash thoroughly4~5Next, take a photo with absorbent paperDo it.

4Color rendering:Add each hole firstSubstrate solutionA 50 MLAnd then each one join inSubstrate solutionB 50 MLGentlyoscillationMix well and thenroom temperature25℃downlight avoidance reaction10~15 min.

5determination:Add each hole separately50 MLTermination liquid,Gently shake and mix wellIn the enzyme-linked immunosorbent assay (ELISA) analyzer450 nmLocation determinationODValue (recommended to use)450/630 nmDual wavelength detection, in5 minRead the data internally.

9Result Analysis

The average absorbance of the standard solution or sample measured（B）Divide by

A standard solution（0The absorbance of standard solution（B₀）Multiply the value again100%，

Obtain the percentage absorbance value.

Percentage absorbance value（%）＝

Based on the concentration of standard samples10The logarithm of the base isXAxis, with a percentage absorbance value ofYAxis, draw standard curve. Substitute the percentage absorbance value of the sample into the standard curve, and read the corresponding value of the sample from the standard curve as10The power of, multiplied by the dilution factor, is the amount contained in the sampleRactopamineThe quantity.

Using professional analysis software such as reagent kits for calculations is more convenient for accurate and rapid analysis of large quantities of samples.

10Sample dilution factor

animal tissue：2Double (Method 1)4Double (Method 2)

Urine and serum samples：1times

tenoneSensitivity, accuracy, and precision of the reagent kit

Sensitivity of reagent kit：0. 1ppb

Sample detection limit:

Organize samples (Method 1)…………………0.2ppb

Organize samples (Method 2)…………………0.4 ppb

Urine and serum samples………………………0.1ppb

rate of recovery:

organizationsample …………………………75±20%

Urine and serum samples………………………95±20%

precisionThe coefficient of variation of the reagent kit is less than10%

tentwoPrecautions

1Room temperature below20 ℃Or the reagents and samples have not returned to room temperature（20~25 ℃）Will lead to all standardsODThe value is low.

2If the plate holes dry during the washing process, the standard curve may not be linear and the repeatability may be poor，So after washing and drying the board, the next step should be carried out immediately.

3Do not use reagent kits that have exceeded their expiration date; Do not use any reagents from expired kits, as mixing expired kits can cause a decrease in sensitivity; Do not exchange reagents from different batch numbers of reagent kits.

4Save the reagent kit2~8 ℃Do not freeze, put unused microplates in self sealing bags and reseal；Standard substances and colorless colorants are sensitive to light, so direct exposure to light should be avoided.

5Color reagentsAny color indicating that the coloring agent has deteriorated should be discardedIt's.

6、Upon joiningSubstrate solutionAfterwards, the color usually appears15 minJust enough. If the color is light, the reaction time can be extended to20 min(or longer), but not exceeding30 min；On the contrary, reduce the reaction time.

tenthreeStorage conditions and shelf life

Storage conditions: in2~8 ℃Save the reagent kit.

Shelf life: The expiration date of this reagent kit is12A month.