Dicyandiamide (DICY) ELISA kit instructions

Dicyandiamide (DICY) ELISA Kit Instructions This kit is for research use only. 1 Purpose: This kit is used for quantitative detection of dicyandiamide (DICY) residues in milk. 2 Experimental principle The kit adopts a competitive ELISA method, coated with dicyandiamide (DICY) conjugated antigen in a microplate, added dicyandiamide (DICY) standard or sample, free dicyandiamide (DICY) and microwell The pre-coated dicyandiamide (DICY) conjugated antigens compete with each other for anti-dicyandiamide (DICY) antibody enzyme markers, developed with TMB substrate, and the color changes from blue to yellow after addition of the stop solution. The standard instrument is tested at a wavelength of 450 nm. The absorbance value is inversely proportional to the content of dicyandiamide (DICY) in the sample. The content of dicyandiamide (DICY) in the sample is calculated by the standard curve. 3 Kit composition 3.1 Pre-coated dicyandiamide (DICY) conjugated antigen-removable enzyme labeling plate: 1 piece (12 wells × 8 strips). 3.2 Dicyandiamide (DICY) standard products: 6 bottles (1ml / bottle), the contents are: 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7ppb, 8.1 ppb. 3.3 Anti-dicyandiamide (DICY) antibody conjugate: 1 bottle (6ml). 3.4 Color developing solution A: 1 bottle (6ml). 3.5 Color developing solution B: 1 bottle (6ml). 3.6 Stop solution: 1 bottle (6ml), 2M sulfuric acid. 3.7 Sample diluent: 1 bottle (10 ×, 6ml), used for sample dilution. 3.8 Concentrated washing solution: 1 bottle (20 ×, 20ml) for washing plates. 3.9 A manual. 4 Materials needed but not provided 4.1 Equipment 4.1.1 Microplate reader with wavelength 450nm. 4.1.2 Crusher. 4.1.3 Measuring cylinder. 4.1.4 Oscillator. 4.1.5 Funnel. 4.1.6 Whatman No 1 or equivalent filter paper. 4.1.7 Micro pipette. 4.2 Reagents 4.2.1 Deionized water or distilled water. 4.2.2 Methanol. 5 Storage 5.1 The kit is stored at 2 ~ 8 ° C, do not freeze 5.2 Unused microplates should be sealed and stored dry 6 Cautions 6.1 Please read the instructions carefully before using the kit. 6.2 Do not use expired kits. 26.3 Before using the kit, return the reagents to room temperature (25 ± 2 ° C). It is recommended to return to temperature for at least 2 hours. 6.4 The standard product contains dicyandiamide (DICY). Special care should be taken when using it. Gloves should be worn during operation. 6.5 The stop solution contains sulfuric acid, which prevents burns to the skin and corrosion of clothing when used. 6.6 The tips used for different standards and samples can not be mixed, otherwise it will affect the test results. 6.7 The reagents in different batch kits should not be mixed; the tips used for different standards and samples should not be mixed, otherwise it will affect the experimental results. 6.8 The sample dilution in this kit must be used when diluting the sample, otherwise it will affect the experimental results. 6.9 Avoid blistering when mixing reagents. 7 Preparation of working solution 7.1 Dicyandiamide (DICY) standard solution: 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb 7.2 Concentrated washing solution: diluted with distilled water at 1:20 (1 + 19) for use 7.3 Sample diluent: 1:10 (1 + 9) diluted with distilled water and ready for use 7.3 Developer: ready for use, avoid direct light 7.4 Reaction stop solution: ready for use 8 Sample processing procedures (samples must be strict during the extraction process) Operate according to the instructions, the dilution should be accurate during the extraction process, otherwise the results will be inaccurate, and the sample should be stored in a cool and dark place and refrigerated) 8.1 Take 10g of crushed sample, add 20ml 70% methanol solution 8.2 Vibrate vigorously for 3 minutes Filter with Whatman No 1 filter paper 8.4 Take 25μl of the processed sample, add 25μl of sample diluent to the reaction well (sample dilution factor is 2) 9 Enzyme-free analysis step 9.1 Experimental instructions 9.1.1 Please put all reagents in the box before the experiment Fully return to room temperature (25 ± 2 ℃) for about 2 hours. After warming to room temperature (25 ± 2 ℃), take out the microporous strips again. Re-seal the microporous strips and dry immediately at 2 ~ 8 ℃. Note: Make sure that the temperature is sufficient, otherwise the accuracy and accuracy of the test will be affected. 9.1.2 Please put the reagents back to 2 ~ 8 ℃ for storage immediately after use 9.1.3 Please do not change the analysis program 9.1.4 Please use accurate micro pipette 9.1.5 Once the operation starts, please do not interrupt any program 9.1.6 The reproducibility of ELISA results depends greatly on the operating procedures, please strictly follow the requirements 9.1.7 To avoid cross-contamination, each standard and sample should be loaded with a different tip 9.1.8 When loading Do not let the tip touch the solution or inner surface in the microwell. 9.2 Analysis step 9.2.1 Number in advance to mark the position of B0, standard and sample. It is recommended to perform double-well detection. 9.2.2 Take the required number of microwells (micro Hole strips are removable), re-seal the excess strips and immediately put them back at 2 ~ 8 ℃ to store 9.2.3 Sample dilution (10 ×) and concentrated washing solution (20 ×) are diluted into working solution (diluted with distilled or deionized water) ) 9.2.4 Add 50 μl of 0.0 ng / ml standard solution to well B0 9.2.5 Add 50 μl of standard solution to each well 9.2.6 Add 50 μl of sample solution to each well 9.2.7 Add to all wells Add 50μl of anti-dicyandiamide (DICY) abzyme conjugate 9.2.8 Gently shake the reaction Plate for a few seconds. 9.3 Warm bath at 37 ° C for 30min (Tap the reaction plate from time to time during the warm bath to reduce the error of the two wells) 9.3.1 Shake off the liquid in the well and wash the microplate 5 times with the washing solution. The last time should be tapped on the absorbent paper to complete Remove the liquid from the hole. 39.4 Reaction 9.4.1 After the washing procedure is completed, immediately add 50 μl of chromogenic solution A and then 50 μl of chromogenic solution B to each microwell with a micropipette; shake the reaction plate slightly to mix thoroughly 9.4.2 37 ° C Warm bath 10min 9.4.3 Add 50μl of stop solution to each well, mix evenly 9.4.4 Detect the absorbance at 450nm, the result is read within 5min. 10 Result calculation 10.1 Quantitative analysis 10.1.1 The average value (B) of the absorbance value of each concentration standard solution and sample obtained is divided by the absorbance value (B0) of the first standard (0 standard) and multiplied by 100%, ie Percent absorbance value. B—average absorbance value of standard solution or sample solution B0—0μg / L average absorbance value of standard solution 10.1.2 The logarithmic value of dicyandiamide (DICY) concentration is the X axis, and the percent absorbance value is the Y axis. Graph. According to the percent absorbance value of the sample, the abscissa of the corresponding point can be obtained from the curve, which is the logarithm value of the concentration of dicyandiamide (DICY), and the antilogarithm is the concentration C (ppb ) 10.1.3 Because the sample has been pre-diluted, the sample concentration obtained from the standard curve must be multiplied by its dilution factor. 10.2 Semi-quantitative determination 10.1.1 Visual semi-quantitative determination: First select an appropriate standard solution to run with the sample, and determine whether the sample concentration value is less than or greater than the standard value based on the comparison of the absorbance value of the sample and the standard. 10.1.2 Instrument semi-quantitative determination: first select an appropriate standard solution to run with the sample, and determine whether the concentration value of the sample is less than or greater than the standard value according to the color depth comparison of the sample and the standard product. 11 Specific substance cross-reaction dicyandiamide (DICY) 100% 12 Kit parameters The lower detection limit of this kit is 0.05ppbB0. The optimal value of absorbance should be greater than 1.0. The absorbance within the kit is less than 8%, and the inter-plate error is less than 15%. The recovery rate of the tissue sample extraction method provided in this specification is greater than 80%. 13 The standard curve range provided by the kit is 0.1ppb ~ 8.1ppb. 14 Limitations of analysis The samples tested positive by this kit should be confirmed by another method such as HPLC or GC / MS.

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