Phone:
+1 877 302 8632
Fax:
+1 888 205 9894 (Toll-free)
E-Mail:
orders@antibodies-online.com

Tetracycline (TC) ELISA Kit

Reactivity: Streptomyces Colorimetric Competition ELISA
Catalog No. ABIN400612
  • Target
    Tetracycline (TC)
    Reactivity
    Streptomyces
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Application
    ELISA
    Purpose
    This test kit is based on the competitive enzyme immunoassay for the detection of Tetracyclines in the sample. The coupling antigens are pre-coated on the micro-well stripes. The Tetracyclines in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Tetracyclines 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 Tetracyclines in it. This value is compared to the standard curve and the Tetracyclines concentration is subsequently obtained.
    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, 20 concentrated washing buffer (40 mL) white cap, 5 concentrated redissolving solution (50 mL) transparent cap
    Material not included
    Equipments: microplate reader (450 nm / 630 nm), homogenizer, oscillator, centrifuge, balance (a sensibility reciprocal of 0.01 g), measuring pipets Micropipettors: single-channel 20 to 200 L and 100 to 1000 L, and multi-channel 250 L, Reagents: NaOH, Trichloroacetic acid, Na2HPO412H2O, deionized water, C6H8O7H2O, EDTANa22H2O, H2C2O4, CH3OH(=99.5%), RIDAC18 column
  • Plate
    Pre-coated
    Protocol
    Sample pre-treatment: Instructions The following points must be dealt with before the pre-treatment of any kind of sample: 1) This product can detect the following tissue samples:animal tissue,poultry,and aquatic tissue, eg:chicken,duck, beef,rabbit,fish,shrimp,etc. 2) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents, 3) 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: Solution A: dilute 5 concentrated redissolving solution with deionized water at 1:4. Solution B(3% Trichloroacetic acid, for tissue sample): dissolve 3 g Trichloroacetic acid in deionized water to 100 mL. Solution C(1% Trichloroacetic acid, for honey sample): dissolve 1 g Trichloroacetic acid in deionized water to 100 mL. Solution D(0.1M NaoH,for tissue sample): dissolve 0.4 g NaOH in deionized water to 100 mL Solution E: dissolve 27.6 g Na2HPO412H2O, 12.9 g C6H8O7H2O and 37.2 g EDTANa22H2O in deionized water to 1000 mL(adjust pH 4.0) Solution F: dissolve 1.8 g H2C2O4 in CH3OH to 1L. 5.1 Tissue(Chicken, pork, porcine liver, shrimp or fish) Take 2 0.05 g of the homogenized sample into 50ml centrifuge tube, add 4 mL of Solution B, shake properly with oscillator for 5 minutes. Centrifuge at above 4000 r/min at room temperature (20 - 25oC) for 10 minutes. Take 100 L supernatant into another tube, add 100 L of 0.1 M NaOH and then diluted with 800 L Solution A,mix for 30s(If its not clear, Centrifuge at above 4000 r/min for 10 minutes). Take 50 L for analysis. Fold of dilution of the sample: 20 5.2 Honey Weigh 1 0.05 g homogenized sample into centrifuge tube, add 2 mL Solution C, shake properly with oscillator for 5 minutes. Centrifuge at above 4000 r/min for 10 minutes. Take 100 L supernate into another tube and add 1900 L Solution A to be diluted,mix for 30s Take 50 L for analysis. Fold of dilution of the sample: 40 5.3 Milk 1.Take 1ml milk into centrifuge tube, add 3 mL of Solution B, shake properly with oscillator for 5 minutes. 2.Centrifuge at above 4000 r/min at room temperature (20 - 25oC) for 10 minutes. 3.Take 100 L supernate into another tube and add 400 L Solution A to be diluted, mix for 30s 4.Take 50 L for analysis. Fold of dilution of the sample: 20 5.4 Tissue(Chicken, pork, porcine liver, shrimp or fish) by RIDAC18 column Take 5 0.05 g of the homogenized sample into 50ml centrifuge tube, add 25 mL of Solution E, shake properly with oscillator for 30 minutes. Centrifuge at above 4000 r/min at 15 ? for 10 minutes. Take all the supernatant to a new tube, extract the precipitate with 25 mL of solution E again. Put the two supernatant together, and filtered by filter paper. Take 5 mL filter solution for purification. Purification procedure Take 3 mL CH3OH(100%) to wash the C18 column. Take 2 mL deionized water to wash the C18 column. Add the 5 mL filter solution to the C18 column, 15 drop/min. Wash the column by 3 mL deionized water. Empty the water in the column. Washed by 1 mL Solution F, 15 drop/min. gather the wash sulution. Diluted it with Solution A at 1:9(50 L sample + 450 L Solution A). Take 50 L for analysis. Fold of dilution of the sample: 20 6.ELISA procedures Instructions 1. Bring all reagents and micro-well strips to the room temperature (20-25oC). 2. Return all reagents to 2-8oC immediatelyafteruse. 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. Operation procedures Take out all the necessary reagents from the kit and place at the room temperature (20 to 25oC) for at least 30 minutes. Note that each liquid reagent must be shaken to mix evenly before use. Dilute the six standard solution separately(use glass bottle): Take 50 L Standard solution 1 (2, 3, 4, 5, 6) to 450L the diluted redissolving solution, shaken to mix evenly. Take the required micro-well strips and plate frames. Re-sealed the unused microplate, stored at 2-8oC, not frozen. Solution preparation: dilute 40 mL of the 20 concentrated washing buffer 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 50L of the sample and standard solution to separate duplicate wells, and add 50 L of the antibody solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, and incubate at 37oC for 30 minutes. Pour liquid out of microwell, add 250 L/well of washing buffer for 10 sec, repeat four to five times, then flap to dry (if there are the bubbles after flapping, cut them with the clean tips). Enzyme conjugate: Add enzyme conjugate, 100 L each well. seal the microplate with the cover membrane, and incubate at 37oC for 30 minutes. Pour liquid out of microwell, add 250 L/well of washing buffer for 10 sec, repeat five times, then flap to dry (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 37oC for 15 minutes 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 the 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 minutes). 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 Tetracyclines in the sample. 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 0.310, and that of the sample 2 is 0.820, while those of the standard solutions are as the followings: 1.610 for 0 ppb, 1.350 for 0.05 ppb, 1.030 for 0.15 ppb, 0.660 for 0.45 ppb, 0.389 for 1.35 ppb and 0.198 for 4.05 ppb, accordingly the concentration range of the sample 2is 1.35 to 4.05ppb, and that of the sample 2 is 0.15 to 0.45 ppb. Quantitative determination: The mean values of the absorbance values is equivalent to the percentage of 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 0 ng/mL standard solution Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the Tetracyclines 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, thus finally obtaining Tetracyclines 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 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. 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 0 standard solution of less than 0.5 indicates its degeneration. 15 minutes for coloration after addition of subtrate A and B, if the coloration is light, prolong time and dont exceed 30 minutes. The optimum reaction temperature is 37oC, 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
    Tetracycline (TC)
You are here:
Support