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Nitrofuran (SEM) ELISA 试剂盒

适用: 化学剂 Colorimetric Competition ELISA
产品编号 ABIN400601
发货至: 中国
  • 抗原
    Nitrofuran (SEM)
    适用
    化学剂
    检测方法
    Colorimetric
    实验类型
    Competition ELISA
    应用范围
    ELISA
    原理
    This test kit is based on the competitive enzyme immunoassay for the detection of semicarbazide (SEM) in chicken, fish, shrimp, milk, honey, whole egg, etc. The coupling antigens are pre-coated on the micro-well stripes. The SEM in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-SEM 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 SEM in it. This value is compared to the standard curve and the SEM concentration is subsequently obtained.
    Analytical Method
    Qualitative and Quantitative
    纯化方法
    The antiserum is produced against synthetic phosphopeptide derived from human androgen receptor around the phosphorylation site of serine 650 (T-T-SP-P-T).
    组件
    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 and 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, 2 concentrated redissolving solution (50 mL) transparent cap, 2-Nitrobenzaldehyde(C7H5NO3) solution (10 mL) white cap
    试剂未包括
    Equipments: microplate reader, printer, honogenizer, 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, K2Fe(CN)5NO3H2O and ZnSO47H2O(only for milk sample).
  • 板类型
    Pre-coated
    实验流程
    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 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: The 2 concentrated redissolving solution is diluted with deionized water at 1:1 (1 mL concentrated redissolving solution + 1 mL deionized water). used for sample redissolving. C solution (for milk sample): dissolve 12.5 g K2Fe(CN)5NO3H2O 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 Put 5 mL milk into centrifuge tube, add C and D solution, 250 L each. Mix thoroughly by vortex, centrifuge at above 4000r/min at 4-12oC for 10 min with centrifuge of constant temperatures, if centrifuge of constant temperature is not available, chill sample temperature to approx 8oC, then centrifuge. Continue as described in (1 to 7, d). C) honey Weigh 1 0.05 g into centrifuge tube. Dissolve in 4 mL of the deionized water, then add 0.5 mL 1 M HCI and 100 L 2-Nitrobenzaldehyde solution, mix thoroughly. Continue as described in (2 to 7,d ). d) continue based on above steps Weigh 1 0.05 g of the homogenized sample (tissue, intestine, liver), or 1.1 mL the supernatant of centrifugal milk(equivalent to 1 mL of milk sample), add 4 mL of the deionized water, 0.5 mL 1 M HCI and 100 L 2-Nitrobenzaldehyde solution to each well, shake properly. Incubate at 37oC over night ( approx 16 hours,for milk,9 hours is ok) or incubate at 56oC by water bath(2 hours) . Add 5 mL 0.1 M K2HPO4, 0.4 mL 1 M NaOH, 10 mL ethyl acetate to each tube, shake vigorously for 5 min. Centrifuge at above 4000 r/min at room temperature (20-25oC) for 10 min. Transfer 5 mL ethyl acetate into a clean centrifuge tube and blow to dryness by nitrogen or air at 50oC. Dissolve the dry residues in 1 mL N-hexane, add 1 mL of the diluted redissolving solution, mix properly, centrifuge at above 4000 r/min at room temperature (20-25oC) for 5 min. Take 50 L of the lower layer for the analysis. Fold of dilution of the sample: 2 ELISA procedures Bring necessary kits 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, store 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 sample and standard solution should be performed in duplicate, record their positions. Add 50 L of the sample or standard solution into separate duplicate wells, then add 50 L antibody working solution to every well, mix gently by shaking the plate manually, seal the microplate with the cover membrane, and incubate at 37oC for 30 min. 5 Pour the liquid, wash the microplate with the washing buffer at 250 L/well for 4-5 times. Each time soak the well with the washing buffer for 15-30 s, flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips) 6 Add 100 L enzyme conjugate into each well, and incubate at 37oC 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 37oC for 15 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 content of SEM. 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 solutions is: 1.671 for 0 ppb, 1.425 for 0.1 ppb, 1.103 for 0.3 ppb, 0.567 for 0.9 ppb, 0.205 for 2.7 ppb, 0.104 for 8.1 ppb, accordingly the concentration range of the sample 2 is 0.9 to 2.7 ppb, and that of the sample 2 is 0.10 to 0.30 ppb. (multiplied by the corresponding dilution fold) . 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 Bthe average 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 solution and the semilogarithm values of the SEM 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 SEM 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 (20-25oC) 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 reagents and the samples should not return 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 of less than 0.5 indicates its degeneration.
    限制
    仅限研究用
  • 储存条件
    4 °C
  • 抗原
    Nitrofuran (SEM)
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