T2 toxin enzyme linked immunoassay (ELISA)

T2 toxin enzyme linked immunoassay (ELISA) kit instruction manual This kit is for research use only. 1 Purpose: This kit is used for quantitative detection of T2 toxin residues in feed, fish, shrimp and meat tissues (such as chicken, beef and pork), eggs, honey, milk, serum and urine samples. 2 Experimental principle The kit adopts a competitive ELISA method. The T2 toxin conjugate antigen is coated on the microplate, and the T2 toxin standard or sample is added. Compete with anti-T2 toxin antibody enzyme markers, develop color with TMB substrate, the color changes from blue to yellow after adding the stop solution, and detect with a microplate reader at a wavelength of 450nm. The content of T2 toxin in the sample was calculated by the standard curve. 3 Kit composition 3.1 Pre-coated T2 toxin conjugated antigen removable enzyme labeling plate: 1 piece (12 wells × 8 strips). 3.2 T2 toxin standard product: 6 bottles (1ml / bottle), the contents are: 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb. 3.3 Anti-T2 toxin antibody enzyme conjugate: 1 bottle (6ml). 3.4 Color Rendering Solution A: 1 bottle (6ml). 3.5 Color Rendering 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), used 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. 6.3 Before using the kit, return the reagent to room temperature (25 ± 2 ℃). It is recommended to return to temperature for at least 2 hours. 6.4 The standard contains T2 toxin. 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 T2 toxin standard solution: 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb 7.2 Concentrated washing solution: 1:20 (1 + 19) diluted with distilled water for use 7.3 Sample diluent : Dilute with distilled water at 1:10 (1 + 9) for use 7.3 Developer: Used, avoid direct light 7.4 Reaction Stop Solution: Used 8 Sample processing procedures (Samples must be operated in strict accordance with the instructions during the extraction process, It should be accurately diluted during the extraction process, otherwise the results will be inaccurate, and the sample should be kept in a cool and dark place and refrigerated) 8.1 Take 10g of the crushed sample, add 20ml 70% methanol solution 8.2 Vibrate vigorously for 3 minutes 8.3 Use Whatman No 1 Filter paper to filter 8.4 Take 100μl of the processed sample, add 400μl of sample diluent 8.5 Take 50μl of the diluted sample for analysis (dilution factor 5) 9 Enzyme-free analysis step 9.1 Experimental notes 9.1.1 Please keep all reagents outside 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 ° C to store 9.2.3 Sample diluent (10 ×) and concentrated washing solution (10 ×) are diluted into working fluid (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-T2 toxin abzyme conjugate 9.2.8 Gently shake the reaction plate 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. 9.4 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—The average absorbance value of the standard solution or the sample solution. The average absorbance value of the B0—0 μg / L standard solution is 10.1.2. Taking the logarithm of the T2 toxin concentration as the X axis and the percent absorbance value as the Y axis, draw the standard curve. 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 of the T2 toxin concentration, and the antilogarithm is the T2 toxin concentration C (ppb) in the measurement solution. 10.1.3 Because the sample has passed Pre-dilute, so 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 Cross-reaction of specific substances T-2 toxin: 100% Acetyl T-2 toxin: about 114% HT-2 toxin: about 7% Iso T-2 toxin: about 2% 12 Kit parameters The lower detection limit of this kit is 0.05 The optimal value of ppbB0 absorbance should be greater than 1.0. The error within the absorbance plate of the kit is less than 8%, and the error between the plates is less than 15%. The recovery rate of the tissue sample extraction method provided in this specification is greater than 80%. 13 Standard curve mode (for reference only) 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. Shanghai Yuping Biological Technology Co., Ltd. mainly deals with ELISA kits of various brands and grades, with quality assurance and perfect after-sales service. And provide free generation testing. Serving universities and immunology research units. Technicians serve you better.

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