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Neomycin ELISA Kit

Reactivity: Chemical Colorimetric Competition ELISA
Catalog No. ABIN400599
  • Target
    Neomycin
    Reactivity
    Chemical
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Application
    ELISA
    Purpose
    This test kit is based on the competitive enzyme immunoassay. The coupling antigen is pre-coated on the micro-well stripes. The Neomycin residues in the sample and the coupling antigen pre-coated on the micro-well stripes compete for the anti-Neomycin antibody. 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 Neomycin in it. This value is compared to the standard curve and the Neomycin concentration is subsequently obtained.
    Analytical Method
    Qualitative and Quantitative
    Purification
    Rabbit Anti-Pyk2 (Ab-402) Polyclonal Antibody is affinity-purified from rabbit antiserum by affinity chromatography using epitope-specific immunogen.
    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 and 40.5 ppb, Enzyme conjugate (7 mL) red cap, Antibody concentrated 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
    Material not included
    Equipments: microplate reader, printer, homogenizer, nitrogen-drying device, vortex, centrifuge, measuring pipets, and balance( a sensibility reciprocal of 0.01 g). Micropipettors: single-channel 20-200 L, 100-1000 L, and multi-channel 250 L. Reagents: Trichloroacetic acid(TCA), NaOH, deionized water,
  • 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 checked to 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 concentrated redissolbing solution + 1 mL deionized water). 3% Trichloroacetic acid(for tissue sample): dissolve 3 g Trichloroacetic acid in the deionized water to 100 mL. 2% Trichloroacetic acid(for honey sample): dissolve 2 g Trichloroacetic acid in the deionized water to 100 mL 1 M NaOH: dissolve 4 g NaOH in the deionized water to 100 mL 5.1 Tissue(Shrimp, fish, meat, liver) sample Take 2 0.05 g of the homogenized sample into 50 mL tube, add 8 mL 3% Trichloroacetic acid, shake properly for 10 min, centrifuge at above 4000 r/min at room temperature (20-25?) for 10 min. Take the supernatant to another tube, adjust PH to 7.0-7.5 with NaOH, Dilute it with diluted redissolving solution at 1:4(100 L sample+400L diluted redissolving solution) . Take 50 L for analysis. Fold of dilution of the sample: 20 5.2 Honey sample Take 2 0.05 g sample into 50 mL tube, add 8 mL 2% Trichloroacetic acid, shake properly for 10 min, centrifuge at above 4000 r/min at room temperature (25?) for 10 min. Take the supernatant to another tube, adjust PH to 7.0-7.5 with NaOH, Dilute it with diluted redissolving solution at 1:4(100 L sample+400L diluted redissolving solution) . Take 50 L for analysis. Fold of dilution of the sample: 20 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 ELISA the procedures, 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 (20-25oC) for at least 30 min. Note that each reagent must be shaken to mix evenly before use. Take the required micro-well strips and plate frames. Re-sealed the unused microplate, store at 2-8oC, not frozen. Solution preparation: dilute 40 mL of the 20 concentrated washing buffer with the deionized water at 1:19 (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 and standard solution to separate duplicate wells, Then 50 L enzyme conjugate and 50 L of the antibody working solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, and incubate at 25oC for 1 h. 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 sec, flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips). 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 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 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 Neomycin. 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 solution. Assuming that the OD value of the sample 1 is 0.120, and that of the sample 2 is 1.331, while those of the standard solutions are as the followings: 2.178 for 0 ppb, 1.628 for 0.5 ppb, 1.135 for 1.5 ppb, 0.518 for 4.5 ppb, 0.161 for 13.5 ppb and 0.067 for 40.5 ppb, accordingly the concentration range of the sample 2is 13.5 to 40.5 ppb, and that of the sample 2 is 0.5 to 1.5 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 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 Neomycin 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, thus finally obtaining the Neomycin 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 25oC 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. Mix every reagent and reaction mixture evenly and wash the microplate thoroughly, otherwise there will be the undesirable reproducibility. The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin, 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. 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. 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, If 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
    Neomycin
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