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Malachite Green ELISA Kit

Reactivity: Chemical Colorimetric Competition ELISA
Catalog No. ABIN400597
  • Target
    Malachite Green
    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 Malachite green in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Malachite green 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 Malachite green in it. This value is compared to the standard curve and the content of Malachite green residues is subsequently obtained.
    Analytical Method
    Qualitative and Quantitative
    Components
    Micro-well strips: 12 strips with 8 removable wells each Standard solution(1 mL) 81 g/mL Standard solution (0.4 mL) 100 ppb, Enzyme conjugate (25) (0.8 mL) white cap Antibody working solution (9 mL) white cap, Substrate solution (2 bottle, 16 mL) black cap, Stop solution (12 mL) transparent cap, 5 concentrated washing buffer (2 bottle, 100 mL) white cap, Enzyme conjugate diluent (15 mL) transparent cap Sample diluent (50 mL) transparent cap
    Material not included
    (1) Equipments: microplate reader, homogenizer, nitrogen-drying device, vortex, oscillator, centrifuge, measuring pipets, balance (a sensibility reciprocal of 0.01 g). (2) Micropipettors: single-channel 20-200 L and 100-1000 L, and multi-channel 250 L. (3) Reagents: Acetonitrile (CH3CN), N-hexane, Distilled water.
  • Plate
    Pre-coated
    Protocol
    Sample pre-treatment: Instructions (The following points must be dealt with before the pre-treatment) (1) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents, (2) 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. Samples preparation (1) Homogenize the sample.Take 4 g of the homogenized sample into tube. (2) Add 5 mL 0.2 M HCl, and Shake properly for 5 min. (3) Centrifuge at 2000 g at room temperature (20-25oC) for 5 min. (4) Transfer 2.5 mL of the supernatant into clean tube, add 5 mL CH2Cl2, and shake properly for 5 min. (5) Centrifuge at 2000 g at room temperature (20-25oC) for 5 min. Discard the supernatant. (6) Take 2.5 mL of lower layer into clean tube, and evaporate to dryness by nitrogen at 50oC. (7) Dissolve the dry residues in 2 mL N-hexane. (8) Dilute at 1:10 (50 L extract of acetonitrile solution + 450 L sample diluent) with sample diluent. (9) Take 75 L for analysis. 6. Enzyme conjugate pre-treatment Enzyme conjugate in the test kit is 25 fold concentrated.The concentrated enzyme conjugate is diluted with enzyme conjugate diluent before use. For example, working solution of enzyme conjugate is obtained by mixing 40 L of 25 concentrated enzyme conjugate and 1 mL enzyme conjugate diluent 7. Sample diluent pre-treatment Mix 1 mL Acetonitrile (CH3CN) (100%) and 9 mL sample diluent properly. Standard sample preparation The standard sample is diluted with 10% Acetonitrile/sample diluent(V/V),abtain 0?0.01?0.05?0.1?0.5 and 1 g/L(ppb) Standard sample of Malachite green. (1) 1 ppb standard sample: 40 L 100 ppb deposited soluting and 4 mL standard sample diluent. (2) 0.5 ppb standard sample: 1 mL 1 ppb standard sample and 1 mL standard sample diluent. (3) 0.1 ppb standard sample: 200 L 1 ppb standard sample and 1.8 mL standard sample diluent. (4) 0.05 ppb standard sample: 1 mL 0.1 ppb standard sample and 1 mL standard sample diluent. (5) 0.01 ppb standard sample: 200 L 0.05 ppb standard sample and 800 L standard sample diluent. 8. Enzyme linked immunosorbent assay Instructions (1) Bring all reagents and micro-well strips to balance at the room temperature (20-25oC) before use. (2) Return all reagents to 2- 8 ? immediately after use. (3) 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. (4) 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. ELISA procedures (1) Bring test kit to the room temperature (20-25oC) for at least 30 min, note that each reagent must be shaken evenly before use. (2) Take the required micro-well strips and plate frames. Re-sealed the unused microplate, stored at 2- 8 ?. (3) Numbering: number the micro-wells according to samples and standard solution, each sample and standard solution should be performed in duplicate, record their positions. (4) Add 75 L of the sample and 75 L of the standard solution into each well, then add antibody working solution, 75 L/well, seal the microplate with the cover membrane, and incubate at 25oC for 30 min. (5) Pour the liquid out of microwell, wash the microplate with the washing buffer at 250 L/well for 4-5 times, each time for 10 sec, flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips). (6) Add 150 L enzyme conjugate into each well, seal the microplate with the cover membrane, and incubate at 25oC for 30 min, continue as described in step 5. (7) Coloration: add 150 L of substrate into each well. Mix gently by shaking, and incubate at 25oC for 30 min at dark. (8) Determination: add 100 L stop solution into each well. Mix gently by shaking. then, Set the wavelength of the microplate reader at 450 nm to determine the OD value of every well (in 5 min). 9. Result judgment 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 Malachite green concentration in the sample. (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 0.250, and that of the sample 2 is 0.720, the OD value of standard solutions is: 1.610 for 0 ppb, 1.380 for 0.01 ppb, 1.100 for 0.05 ppb, 0.620 for 0.1 ppb, 0.289 for 0.5 ppb ,0.108 for1 ppb, accordingly the concentration range of the sample 1 is 0.5 to 1 ppb, and that of the sample 2 is 0.05 to 0.1 ppb. (2) 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 Malachite green 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) 10. Precautions (1) 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. (2) 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. (3) Mix evenly, otherwise there will be the undesirable reproducibility. (4) The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin. (5) 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. (6) 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. (7) 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. (8) 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. 11. Storage and expiry date Storage: store at 2-8oC, not frozen. Expiry date: 12 months, date of production is on box.
    Restrictions
    For Research Use only
  • Target
    Malachite Green
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