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Protokoll
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Sample pre-treatment: Instructions (The following points must be dealt with before the pre-treatment ) 1) Only the disposable tips can be used for the experiments and the tips must be changed when used for different reagents, Before the experiment, each experimental equipment must be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results. Solution preparation before sample pre-treatment: 1) The 10concentrated redissolving solution is diluted with deionized water at 1:9 (1 mL concentrated redissolving solution + 9 mL deionized water) 2) 0.5% Trichloroacetic acid(for chicken and liver sample): dissolve 0.5 g Trichloroacetic acid in the deionized water to 100 mL. 3) 0.1 M H3PO4 (for honey sample): dissolve 680L H3PO4 in the deionized water to 100 mL. 4) 1 M NaOH(for honey sample): dissolve 4 g NaOH in the deionized water to 100 mL. 5.1 milk 1 Take 1 mL milk sample, diluted at 1:39 (1950 L diluted redissolving solution+ 50 L milk) 2 Take 50 L for analysis Fold of dilution of sample :40 5.2 meat, liver( chicken) 1 Take 20.05 g homogenized sample(remove fat), add 6 mL 0.5% Trichloroacetic acidand 2 mL Acetonitrile. Mix for 5 min. 2 Centrifuge at above 4000 r/min at room temperature for 10 min. 3 Transfer 2 mL supernatant into a new vessel, add 2 mL N-hexane, mix properly. Static for 3 min. Take 0.5 mL clear solution(lower layer). Centrifuge at above 4000 r/min for 5 min at room temperature. 3 Take 50 L clear solution into a new vessel(If have two layers, remove upper layer liquid), add 450 L of the diluted redissolving solution, mix evenly. 4 Take 50 L for analysis. Fold of dilution of sample :40 5.3 honey, Royal jelly 1 Weigh 20.05 g honey sample, add 4 mL of 0.1 M H3PO4, shake properly for 10 min. 2 Centrifuge at above 4000 r/min at room temperature (20-25oC) for 5 min, until liquid is clear. 3 Add 900 L 1 M NaOH, adjust PH to 7-9(For Royal jelly, Transfer the Supernatant to a new vessel). 4.Centrifuge at above 4000 r/min at room temperature (20-25oC) for 5 min, until liquid is clear. 5 Take 50 L suppernatant, add 350 L of the diluted redissolving solution, mix evenly. 6 Take 50 L for further analysis. Fold of dilution of sample :20 ELISA procedures Bring test kit to the room temperature (20-25oC) for at least 30 min, note that each reagent must be shaken evenly before use, put the required micro-well strips into plate frames. Re-sealed the unused microplate, stored at 2-8oC, not frozen. Solution preparation: dilute 40 mL of the concentrated washing buffer (20concentrated) with the distilled or deionized water to 800 mL (or just to the required volume) for use, Numbering: number the micro-wells according to samples and standard solution, each sample and standard solution should be performed in duplicate, record their positions. Add 50 L of the sample and standard solution to separate duplicate wells, then add 50 L of antibody working solution to each well, shake properly, seal the microplate with the cover membrane, and incubate at 37oC for 30 min, Pour liquid out of microwell, flap to dry on absorbent paper, add 250 L/well of washing buffer for 15-30 seconds, then take out and flap to dry with absorbent paper, repeat 5 times. Add 100 L of enzyme conjugate to each well, shake properly, seal the microplate with the cover membrane, and incubate at 37oC for 30 min, continue as step 5. Coloration: add 50 L of the substrate A solution, 50 L of the B solution into each well. Mix gently by shaking the plate manually, and incubate at 37oC for 15 min in the dark for coloration, Determination: add 50 L of the stop solution into each well. Mix gently by shaking the plate manually. Set the wavelength of the microplate reader at 450 nm to determine the OD value of every well. (Recommend to read the OD value at the dual-wavelength 450/630nm within 5 min). Interpretation of results There are two methods to judge the results, the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with the content of Streptomycin. 7.1 Qualitative determination The concentration range (ng/mL) can be obtained from comparing the average OD value of the testing sample with that of the standard solution. Assuming that the OD value of the sample 1 is 0.3, and that of the sample 2 is 1.0, the OD value of standard solutions is: 2.243 for 0 ppb, 1.816 for 0.1 ppb, 1.415 for 0.4 ppb, 0.74 for 1.6 ppb, 0.313 for 6.4 ppb, 0.155 for 25.6 ppb, accordingly the concentration range of the sample 2 is 6.4 to 25.6 ppb, and that of the sample 2 is 0.4 to 1.6 ppb. Quantitative determination The mean values of the absorbance values is equivalent to the percentage of the average OD value (B) of the testing sample and the standard solution divided by the OD value (B0) of the first standard solution (0 standard) and subsequently multiplied by 100%, that is Percentage of absorbance value = B 100% B0 Bthe average (double wells) OD value of the testing sample or the standard solution B0the average OD value of the 0ng/mL standard solution Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithmic values of the Streptomycin standard solutions (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the testing sample from the standard curve by incorporating its absorption percentage into the standard curve. The resulting value is subsequently multiplied by the corresponding dilution fold, finally obtaining the Streptomycin concentration in the sample. Using the professional analyzing software of this kit will be more convenient for the accurate and rapid analysis of a large amount of samples. (Please contact us for this software) Precautions Bring all reagents and micro-well strips to the room temperature (20-25?). Return all reagents to 2-8oC immediately after use. The reproducibility of the ELISA analysis, to a large degree, depends on the consistency of plate washing. The correct operation of plate washing is the key point in the procedures of ELISA. For the incubation at constant temperatures, all the samples and reagents must avoid light exposure, and each microplate should be sealed by the cover membrane. The room temperature below 20? or the temperature of the reagents and the testing samples being not returned to the room temperature (20-25?) will lead to a lower standard OD value. Dryness of the microplate in the washing process will be accompanied by the situations including the non-linear standard curves and the undesirable reproducibility, So continue to next step immediately after washing. Mix evenly, otherwise there will be the undesirable reproducibility. The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin. Do not use the kit exceeding its expiry date. The use of diluted or adulterated reagents from the kits will lead to the changes in the sensitivity and the detecting OD values. Do not exchange the reagents from the kits of different lot numbers to use. Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance and the colourless color former is light sensitive, and thus they cannot be directly exposed to the light. Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value of standard solution 1 (0 ppb)of less than 0.5 indicates its degeneration. Colouration time is about 20 min, if the color is light, prolong the time of colouration but don'texceed 30 min. The optimum reaction temperature is 37oC, and too high or low temperatures will result in the changes in the detecting sensitivity and OD values.
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Bestandteile
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Micro-well strips: 12 strips with 8 removable wells each 6 standard solution (1 mL each): 0 ppb, 0.1 ppb, 0.4 ppb, 1.6 ppb, 6.4 ppb, 25.6 ppb, Enzyme conjugate (12 mL) red cap, Antibody working solution (7 mL) blue cap, Substrate A solution (7 mL) white cap, Substrate B solution (7 mL) black cap, Stop solution (7 mL) yellow cap, 20 concentrated washing buffer (40 mL) white cap 10 concentrated redissolving solution (50 mL) transparent cap
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Benötigtes Material
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Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance( a reciprocal sensibility of 0.01 g). Micropipettors: single-channel 20~200 L, 200~1000 L, and multi-channel 250 L, 3) Reagents: H3PO4, NaOH(for honey sample), Acetonitrile(CH3CN), N-hexane
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