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Diethylstilbestrol ELISA Kit

Reactivity: Chemical Colorimetric Competition ELISA
Catalog No. ABIN400592
  • Target
    Diethylstilbestrol
    Reactivity
    • 1
    • 1
    Chemical
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Application
    ELISA
    Purpose
    This test kit is based on the competitive enzyme immunoassay for the detection of Diethylstilbestrol(DES) in the feed, urine, liver, meat, shrimp and fish. The conjugate antigen is pre-coated on the micro-well stripes. The Diethylstilbestrol in the sample competes with the conjugate antigen pre-coated on the micro-well stripes, to interact with the antibodies against Diethylstilbestrol. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with the content of Diethylstilbestrol in the sample. This value is compared to the standard curve and the content of the corresponding Diethylstilbestrol is subsequently obtained.
    Analytical Method
    Qualitative and Quantitative
    Components
    Micro-well strips: 12 strips with 8 removable wells each 6 standard solution (1 mL each): 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 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, 5 concentrated redissolving solution (50 mL) transparent cap
    Material not included
    Equipments: microplate reader, printer, homogenizer, nitrogen-drying device, votex, centrifuge, measuring pipets, balance (a sensibility reciprocal of 0.01 g) Micropipettors: single-channel 20-100 L ,100-1000 L, and multi-channel 250 L, Reagents: NaOH, Acetonitrile (CH3CN), Acetone, deionized water, H3PO4 (85%), CHCl3
  • Plate
    Pre-coated
    Protocol
    Sample pre-treatment: Instructions (The following points must be dealt with before the pre-treatment) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents, Before the experiment, each experimental utensil 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 6 M H3PO4:dissolve 100 mL H3PO4 in 150 mL deionized water, mix properly 1 M NaOH:dissolve 4 g NaOH in deionized water to 100 mL 2 M NaOH:dissolve 8 g NaOH in deionized water to 100 mL Acetonitrile- Acetone: add 80 mL Acetonitrile and 20 mL Acetone, mix evenly The 5concentrated redissolving solution is mixed with deionized water at 1:5 (1 mL concentrated redissolving solution + 4 mL deionized water), used for the treated sample redissolving 5.1 Feed Weigh 2 0.05 g of the homogenized sample, add 8 mL Acetonitrile, shake properly for 10 min, centrifuge at above 3000 r/min at 15 ? for 10 min Take 2 mL supernatant into a new centrifuge tube, blow to dry withnitrogen or air at 60oC. Add 0.5 mL CHCl3, vortex for 20 sec, add 2 mL 1 M NaOH, vortex for 30 sec, centrifuge at above 3000 r/min for 5 min. Take 1 mL supernatant, add 100 L 6 M H3PO4, vortex for 5 sec Dulition: Compound feed-- take 50 L sample, add 950 L of the diluted redissolving solution, Concentrated /Premixed feed-- take 25 L sample, add 975 L of the diluted redissolving solution Take 50 L for analysis Fold of dilution of the sample: Compound feed ---------100 Concentrated / Premixed feed -----------200 5.2 Meat, liver, shrimp, fish Weigh 2 0.05 g of the homogenized sample, add 6 mL Acetonitrile- Acetone, shake for 10 min, and centrifuge at above 3000 r/min at 15 ? for 10 min. Transfer 3 mL supernatant into a new centriffuge tube, blow to dry with nitrogen or air at 60oC. Add 0.5 mL CHCl3, vortex for 20 sec, add 2 mL 1 M NaOH, vortex for 30 sec, centrifuge at above 3000 r /min for 5 min. Take 1 mL supernatant, add 200 L 6 M H3PO4, vortex for 5 sec. Add 3 mL Acetonitrile (CH3CN) for extraction, shake properly for 10 min, centrifuge at above 3000 r/min at room temperature (20-25oC) for 10 min, take the upper layer, blow to dry with nitrogen or air at 60oC. Dissolve dry residues in 1 mL of the diluted redissolving solution. Dilution: shrimp and fish-----directly take 50 L water phase for detection, Meat and liver-----take 50 L water phase, add 450 L of the diluted redissolving solution, shake properly. Take 50 L for analysis. Fold of dilution of the sample: shrimp and fish----2 meat and liver-----20 5.3 Urine Take 2 mL urine into centrifuge tube, centrifuge at above 3000 r/min at room temperature (20-25) for 10 min, stop when it is clear. Transfer 1 mL clear urine into centrifuge tube, add 1 mL 1 M NaOH, shake vigorously for 5 min Add 100 L 6 M H3PO4, vortex for 30 sec Add 8 mL CHCl3 for extraction, shake properly for 10 min, centrifuge at above 3000 r/min at 15? for 10 min. Remove the upper layer (water phase), take 4 mL of the lower layer, blow to dry with nitrogen or air at 60oC. Dissolve dry residues in 3 mL of the diluted redissolving solution? take 50 L for analysis. Fold of dilution of the sample:6 ELISA procedures Instructions: Bring all reagents and micro-well strips to the room temperature (20-25oC) before use. 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. Operating procedures: Take out the kit from the refrigerated environment. Take out all the necessary reagents from the kit and place at the room temperature (20-25?) for at least 30 min. Note that each liquid reagent must be shaken to mix evenly before use Take the required micro-well strips and plate frames. Re-sealed the unused microplate, stored at 2-8oC, not frozen Solution preparation: dilute the 20 concentrated washing buffer 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 or standard solution to separate duplicate wells, add 50 L of the antibody working solution into each well. Seal the microplate with the cover membrane, and incubate at 37? for 30 min Pour the liquid out of the microwells, add 250 L/well of washing buffer for 10 sec, repeat four to five times, then flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips). Add 100 L enzyme conjugate into every well, seal the microplate with the cover membrane, react at 37? for 30 min, continue as described in 6 Coloration: add 50 L of the substrate A solution and then 50 L of the B solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane and incubate at 37oC for 15 min at 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 (Recommend to read the OD value at the dual-wavelength 450/630 nm 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 Diethylstilbestrol (DES). 7.1 Qualitative determination The concentration range (ng/mL) can be obtained from the comparison the average OD value of the sample with that of the standard solutin. Assuming that the OD value of the sample 1is 0.310, and that of the sample 2 is 0.820, the OD value of standard solutins is: 1.510 for 0 ppb, 1.320 for 0.1 ppb, 1.03 for 0.3 ppb, 0.660 for 0.9 ppb, 0.389 for 2.7 ppb ,0.198 for 8.1 ppb, accordingly the concentration range of the sample 1 is 0.8-1.6 ppb, and that of the sample 2 is 0.2-0.4 ppb. (multiplied by the corresponding dilution fold) . Quantitative determination: The mean values of the absorbance values is equivalent to the percentage of the average OD value (B) of the 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 sample or the standard solutin B0the average OD value of the 0ng/mL standard solutin Draw the standard curve with the absorption percentages of the standard solutins and the semilogarithm values of the Diethylstilbestrol(DES) standard solutins (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the 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, thus finally obtaining the Diethylstilbestrol (DES) 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 The room temperature below 20oC or the temperature of the reagents and the samples being not returned to the room temperature (20-25oC) 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 the 0 standard solutin of less than 0.5 (A450 nm< 0.5 ) indicates its degeneration The optimum reaction temperature is 37oC, and too high or low temperatures will result in the changes in the detecting sensitivity and OD values.
    Restrictions
    For Research Use only
  • Storage
    4 °C
  • Target
    Diethylstilbestrol
    Target Type
    Chemical
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