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Clenbuterol ELISA Kit

Reactivity: Chemical Colorimetric Competition ELISA
Catalog No. ABIN400590
  • Target
    Clenbuterol
    Reactivity
    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 Clenbuterol in the pork and liver. The coupling antigen is pre-coated on the micro-well stripes. The Clenbuterol in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Clenbuterol antibodies. 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 Clenbuterol in the sample. This value is compared to the standard curve and the Clenbuterol residues 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 (7 mL) red cap, Antibody working solution (10 mL) blue cap, Substrate A solution (7 mL) white cap, Substrate B solution (7 mL) black cap, Stop solution (7 mL) yellow cap
    Material not included
    Equipments: microplate reader, printer, homogenizer, nitrogen-drying device, vortex, oscillator, centrifuge, measuring pipets, balance (a sensibility reciprocal of 0.01 g) Micropipettors: single-channel 20-200 L, 100-1000 L, and multi-channel 250 L, Reagents: Acetonitrile (CH3CN), NaOH, ethyl acetate, HCI (approx 36.5%)
  • 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: 0.1 M HCI: dissolve 0.86 mL HCI (approx 36.5%) in deionized water to 100 mL. 1 M HCI: dissolve 8.6 mL HCI (approx 36.5%) in deionized water to 100 mL. 1 M NaOH(for feed sample): dissolve 4 g NaOH in deionized water to 100 mL. 0.1 M NaOH(for tissue samples): dissolve 0.4 g NaOH in deionized water to 100 mL. CH3CN-0.1 M HCI solution(for tissue samples): ?CH3CN :?HCI =84:16 5.1 Urine and serum Take 20 L clear urine or serum, directly detect it (If urine and serum are muddy, must filter or centrifuge at 4000 r/min at 15 ? for 10 min, then take clear urine and serum). Store at frozen enviroment if don'tuse. 5.2 Tissue (liver, pork, etc.) Weigh 20.05 g sample, add 6 mL CH3CN-0.1 M HCI solution, vortex for 10 min, centrifuge at 4000 r/min at room temperature (20-25oC) for 10 min. Take 3 mL of clear supernatant, add 2 mL 0.1 M NaOH and 6 mL ethyl acetate, shake for 10 min, centrifuge at 4000 r/min at room temperature (20-25oC) for 10 min. Take supernatant (almost is clear), blow to dry with nitrogen or air at 50oC. Add 1 mL tri-deionized water(tri-deionized water should be distilled 3 times for use), redissolve residues properly. Take 20 L for analysis. Fold of dilution of sample: 1 5.3 Feed Grind sample, weigh 2.00.05 g, add 2 mL 1 M HCI and 16 mL deionized water. then homogenize it . Vortex for 3 min, then shake with oscillator for 15 min. Centrifuge at above 4000 r/min for 15 min, take supernatant (must be clear), add 2 mL 1 M NaOH, mix evenly, adjust pH to 6-8. Centrifuge at above 4000 r/min for 15 min, take supernatant (must be clear). Diluted with tri-deionized water at 1:9 (100 L clear upper liquid + 900 L tri-deionized water). Take 20 L for analysis. Fold of dilution of sample: 100. Sample storageUntreated samples are stored at frozen environment. HCI - acidified homogenates are stable at 2-8oC for up to 3 days 3. Prepared sample can be stable at 2-8oC for 1 week. 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. 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 20 L of the sample or the standard solution into separate duplicate wells, then add enzyme conjugate, 50 L/well, and antibody working solution, 80 L/well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, incubate at 25oC for 30 min. Pour liquid out of microwell, flap to dry on absorbent paper, add 250 L/well of tri-deionized water, wash for 4-5 times, 15-30 s each time, then take out and flap to dry with absorbent paper. Coloration: add 50 L of substrate A solution and 50 L B solution into each well. Mix gently by shaking the plate manually, and incubate at 25oC 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 450/630 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 Clenbuterol concentration in the sample. 8.1 Qualitative determination The concentration range (ng/mL) obtained from comparing the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sample 1 is 0.313, and that of the sample 2 is 1.032, the OD value of standard solutions is: 1.892 for 0 ppb, 1.501 for 0.1 ppb, 1.175 for 0.30 ppb, 0.751 for 0.90 ppb, 0.421 for 2.7 ppb ,0.198 for 8.1ppb, accordingly the concentration range of the sample 1 is 2.7 to 8.1 ppb, and that of the sample 2 is 0.30 to 0.90 ppb. Quantitative determination The mean values of the absorbance values obtained for 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 solution B0the average OD value of the 0 ng/mL standard solution Draw the standard curve with the absorption percentages of standard solutions and the semilogarithm values of Clenbuterol standard solutions (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 dilution fold, finally obtaining clenbuterol concentration in the sample. Using the professional software of this kit will be more convenient for accurate and rapid analysis of a large amount of samples. PrecautionsBring 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. 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 solution (0 ppb) of less than 0.5 indicates its degeneration. The optimum reaction temperature is 25oC, and too high or too low temperatures will result in the changes in the detecting sensitivity and OD values.
    Restrictions
    For Research Use only
  • Storage
    4 °C
  • Target
    Clenbuterol
    Target Type
    Chemical
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