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Salbutamol ELISA Kit

Reactivity: Chemical Colorimetric Competition ELISA Tissue Samples, Urine
Catalog No. ABIN400604
  • Target
    Salbutamol
    Reactivity
    Chemical
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Application
    ELISA
    Purpose
    The test kit is based on the competitive enzyme immunoassay for the detection of Salbutamol in the sample. The coupling antigen is pre-coated on the micro-well stripes. The Salbutamol in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Salbutamol 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 Salbutamol in it. This value is compared to the standard curve and the Salbutamol residues is subsequently obtained.
    Sample Type
    Urine, Tissue Samples
    Analytical Method
    Qualitative and Quantitative
    Components
    Micro-well strips: 12 strips with 8 removable wells each 6 standard solution (1 mL each): 0 ppb, 0.5 ppb, 1.5 ppb, 4.5 ppb, 13.5 ppb ,40.5 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, 20x concentrated washing buffer (40 mL) white cap, 2x concentrated redissolving solution (50 mL) transparent 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 and 100-1000 L, and multi-channel 250 L, Reagents: Acetonitrile (CH3CN), NaOH, ethyl acetate, N-hexane, HCI (approx 36.5%), isopropanol
  • 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: 1 M HCI: dissolve 8.6 mL HCI (approx 36.5%) in deionized water to 100 mL 1 M NaOH: dissolve 4 g NaOH in deionized water to 100 mL The 2 concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL concentrated redissolbing solution + 1 mL deionized water, for redissolving) and 1:3(for extracting sample). 5.1 Samples preparation a) Urine: Take 50 L clear urine, directly detect it (If urine is muddy, must filter or centrifuge at 4000 r/min at 15 ? for 10 min, then take clear urine). Store at frozen if don'tuse. Some interference in urine, we recommend 1 ppb as cut off value of positive sample. b) First method of recovery (liver, pork) Weigh 2 0.05 g sample, add 1 mL 0.5 concentrated redissolving solution(dilluted at 1:3), mix properly, then add 1 mL isopropanol, vortex for 10 min, centrifuge at 4000 r/min at room temperature (20-25oC) for 10 min. Take 3 mL supernatant, add 50 L 1 M NaOH, mix properly, then add 7 mL Ethyl acetate solution, shake for 10 min, centrifuge at 4000 r/min at room temperature (20-25oC) for 10 min, take all supernatant, blow to dry with nitrogen or air at 50oC. Dissolve residues in 1 mL 1diluted redissolving solution(dilluted at 1:1), shake properly, centrifuge at room temperature(20-25oC) for 10 min. Take 50 L for analysis. Fold of dilution of sample: 1 Second method of recovery (feed) Take 2 0.05 g grinded sample, add 2 mL 1 M HCI, 16 mL deionized water, homogenize. Vortex for 3 min, shake for 15 min with oscillator . Centrifuge at 4000 r/min for 15 min, take the supernatant (upper layer) , add 1 mL 1 M NaOH, adjust PH value to 6-8. Centrifuge at 4000 r/min for 15 min(If its not clear, should be centrifuged at higher speed). Dilute supernatant with diluted redissolving solution at 1:9(100 L supernatant + 900 L diluted redissolving solution). Take 50 L for further analysis. Fold of dilution of sample: 100 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 (20 concentrated) 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, and add antibody working solution, 50 L/well, seal the microplate with the cover membrane, and incubate at 37oC for 30 min, Pour the liquid out of microwell, wash the microplate with the washing buffer at 250 L/well for four to five times. Each time soak the well with the washing buffer for 15 sec, 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 each well, seal the microplate with the cover membrane, and incubate at 37oC for 30 min, continue as described in 5. 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 37oC for 15 min at dark for coloration, Determination: add 50 L 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 Salbutamol concentration in the sample. 8.1 Qualitative determination The concentration range (ng/mL) can be 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 0.5 ppb, 1.175 for 1.5 ppb, 0.751 for 4.5 ppb, 0.421 for 13.5 ppb ,0.198 for 40.5 ppb, accordingly the concentration range of the sample 1 is 13.5 to 40.5 ppb, and that of the sample 2 is 1.5 to 4.5 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 0ng/mL standard solution Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the 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 corresponding dilution fold, finally obtaining the Salbutamol 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). 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.
    Restrictions
    For Research Use only
  • Storage
    4 °C
  • Target
    Salbutamol
    Target Type
    Chemical
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