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Nitrofuran (AOZ) ELISA Kit

Reactivity: Chemical Colorimetric Competition ELISA
Catalog No. ABIN400586
  • Target
    Nitrofuran (AOZ)
    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 AOZ in the samples. The coupling antigens are pre-coated on the micro-well stripes. The AOZ in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-AOZ 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 AOZ concentration in the sample. This value is compared to the standard curve and the content of the corresponding AOZ is subsequently obtained.
    Analytical Method
    Qualitative and Quantitative
    Purification
    The antibody is affinity-purified from rabbit antiserum by affinity chromatography using an epitope-specific phosphopeptide. The antibody against non-phosphopeptide is removed by chromatography using a non-phosphopeptide corresponding to the phosphorylation site.
    Components
    Micro-well strips: 12 strips with 8 removable wells each 6 standard solution (1 mL each): 0 ppb, 0.05 ppb, 0.15 ppb, 0.45 ppb, 1.35 ppb and 4.05 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, 2 concentrated redissolving solution (50 mL) transparent cap, 2-Nitrobenzaldehyde(C7H5NO3)( 10 mL) white cap
    Material not included
    Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex, 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: NaOH, ethyl acetate, N-hexane, HCI(approx 36.5%), K2HPO43H2O, (for all samples) K2Fe(CN)5NO3H2O and ZnSO47H2O (for milk sample only)
  • Plate
    Pre-coated
    Protocol
    Sample pre-treatment: Instructions The following points must be dealt with before the pre-treatment of any kind of sample: 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 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: The concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL 2 concentrated redissolving solution + 1 mL deionized water), used for sample redissolving. C solution (for milk sample): dissolve 12.5 g K2Fe(CN)5 NO3H2O in deionized water to 100 mL. D solution(for milk sample): dissolve 29.8 g ZnSO47H2O in deionized water to 100 mL. 0.1 M K2HPO4 :dissolve 22.8 g K2HPO43H2O in deionized water to 1 L. 1 M HCl: dissolve 8.6 mL HCI(approx 36.5%) in water to 100 mL. 1 M NaOH: dissolve 4 g NaOH in water to 100 mL. 5.1 Samples preparation a) Tissue, intestine, liver -Homogenize the sample, continue as described in (1 to 7, d). b) milk Take 5 mL milk into centrifuge tube, add C and D solution, 250 L each. Mix thoroughly with vortex, centrifuge at above 4000 r/min at 4-12oC for 10 min with centrifuge of constant temperatures, if centrifuge of constant temperature is not avaible, chill sample temperature to approx 8oC, then centrifuge. Continue as described in (1 to 7, d). C) honey Weigh 10.05 g into centrifuge tube. Dissolve in 4 mL of the deionized water, then add 0.5mL 1M HCI and 100L 2-Nitrobenzaldehyde solution, mix thoroughly. Continue as described in (2 to 7, d). d) continue above steps 1 Weigh 1 0.05 g of the homogenized sample (tissue, intestine or liver), or put 1.1 mL the supernatant of centrifugal milk (equivalent to 1 mL of milk sample) in a plastic tube, add 4 mL of the deionized water, 0.5 mL 1 M HCI and 100 L 2-Nitrobenzaldehyde solution to each tube, shake properly. 2 Incubate at 37oC over night ( approx 16 h) or incubate at 56oC in water bath(2 hours). 3 Add 5 mL 0.1 M K2HPO4, 0.4 mL 1 M NaOH, 5 mL ethyl acetate to each tube, shake vigorously for 5 min. 4 Centrifuge at above 4000 r/min at room temperature(20-25oC) for 10 min. 5 Transfer 2.5 mL ethyl acetate layer (upper layer) into a new vessel and evaporate to dryness by nitrogen or air at 50oC. 6 Dissolve the dry residue in 1 mL N-hexane ,add 1 mL of the diluted redissolving solution, and mix properly, centrifuge at above 4000 r/min at room temperature(20-25oC) for 10 min. 7 Take 50 L of the lower solution for analysis. Fold of dilution of the sample:2 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 at 1:19 to 800 mL (or just to the required volume) for use. Numbering: number the micro-wells according to samples and standard solution, each testing sample and standard preparation should be performed in duplicate, record their positions. Add 50 L of the sample or standard solution to separate duplicate wells, and add 50 L of the antibody working solution into each well, seal the microplate with the cover membrane, Mix gently by shaking the plate manually, incubate at 25oC for 1 h. 5 Pour the liquid out of micowell, flap to dry on absorbent paper, add 250 L/well of washing buffer to wash microplate for 15-30 s, repeat 4-5 times, then take out and flap to dry with absorbent paper. Add 100 L of the enzyme conjugate into each well, and incubate at 25oC for 30 min.Take out microplate, continue as described in step 5. 7 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, and incubate at 25oC for 30 min at dark for coloration. 8 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 the AOZ in the sample. 7.1 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 0.268, and that of the sample 2 is 1.230, the OD value of standard solution is: 1.671 for 0ppb, 1.425 for 0.05ppb, 1.103 for 0.15ppb, 0.567 for 0.45 ppb, 0.205 for 1.35 ppb ,0.104 for 4.05 ppb, accordingly the concentration range of the sample 2 is 0.45 to 1.35, and that of the sample 2 is 0.05 to 0.15 ppb. Quantitative determination: The mean values of the absorbance values is 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 B the average 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 the standard solution and the semilogarithm values of the AOZ standard solution (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 actual concentration of AOZ 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 balance at 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 2M 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 standard solution 1 (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
    Nitrofuran (AOZ)
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