Enrofloxacin ELISA Kit

Produktnummer ABIN400593

Produktdetails

Target: Enrofloxacin

Reaktivität: Chemical

Nachweismethode: Colorimetric

Methodentyp: Competition ELISA

Applikation: ELISA

Verwendungszweck: This test kit is based on the competitive enzyme immunoassay. The coupling antigens is pre-coated on the micro-well stripes. The enrofloxacin residue in the sample and coupling antigens pre-coated on the micro-well stripes compete for the anti-enrofloxacin 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 content of enrofloxacin residue in it. This value is compared to the standard curve and the content of the corresponding enrofloxacin residue is subsequently obtained.

Analytische Methode: Qualitative and Quantitative

Aufreinigung: Prepared from rabbit serum by affinity purification using a column to which the fusion protein immunogen was coupled.

Bestandteile: Micro-well strips: 12 strips with 8 removable wells each 6 standard solution (1 mL each): 0 ppb, 0.25 ppb, 0.75 ppb, 2.25 ppb, 6.75 ppb and 20.25 ppb, Enzyme conjugate (7 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

Benötigtes Material: Equipments: microplate reader, printer, homogenizer, nitrogen-drying device, oscillator, centrifuge, measuring pipets, and balance with a reciprocal sensibility of 0.01 g, Micropipettors: single-channel 20 to 200 L and 100 to 1000 L, and multi-channel 250 L, Reagents: NaOH, dichloromethane, N-hexane, acetonitrile, Na2HPO412H2O, NaH2PO42H2O and heparin sodium ( for blood samples).

Anwendungsinformationen

Plattentyp: Pre-coated

Protokoll: 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 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: Solution? (0.1 M NaOH): dissolve 0.4 g NaOH in the deionized water to 100 mL. Solution? (acetonitrile-0.1 M NaOH solution): Vacetonitrile: VNaOH = 84:16. Solution? (0.02 M PB buffer, pH 7.2): dissolve 5.16 g Na2HPO412H2O and 0.87 g of NaH2PO42H2O in the deionized water to 1 L. Solution ?: V N-hexane: V dichloromethane = 2:8 Solution ?: dilute the 2 concentrated redissolving solution with deionized water at 1:1 (1 mL concentrated redissolving solution+1 mL deionized water), used for sample redissolving. 5.1 High-detection-limit samples 5.1.1 Animal tissues (meat, liver of pork and chicken, shrimp, fish, etc) Weigh 2.0 0.05 g of the homogenized tissue into 50 mL centrifugal tube. Add 8 mL of the acetonitrile-0.1M NaOH solution, shake upside and down for 10 min, centrifuge at above 3000 r/min at 15 ? for 10 min. Take 2 mL of the supernatant, add 2 mL 0.02 M PB buffer and 6 mL Solution ?, mix for 2 min, and centrifuge at above 3000 r/min at 15 ? for 5 min. Discard the N-hexane phase(upper layer) and take the organic phase (lower layer ) into a dry container (clear and bright without impurity). Blow to dry with nitrogen at 50oC. Dissolve the dry residues in 0.5 mL of the diluted redissolving solution, add 1 mL N-hexane, mix for 2 min, centrifuge at above 3000 r/min at 15? for 5min, Discard the whole upper layers, take 50 L of the lower layer for further analysis. Fold of dilution of the sample: 2 5.1.2 Chicken blood Collect the chicken blood into a centrifugal tube with the addition of the heparin sodium at (20 to 30 u/mL blood) (we recommend to rinse the blood-collecting syringe with the heparin sodium). Place the blood sample for at least 1 h at room temperature. After the separation of plasma, centrifuge at above 4000 r/min at 15 ? for 10 min, and take 1 mL of the plasma. Add 4 mL acetonitrile(100%), shake upside down for 10 min, and centrifuge at above 4000 r/min at 15? for 10 min. Transfer the supernatant into another centrifugal tube, add 2 mL of 0.02 M PB buffer, and mix thoroughly, Add 5 mL dichloromethane, mix thoroughly for 10 min, and centrifuge at 4000 r/min at 15? for 10 min. Discard the upper layer , and transfer organic phase (the lower) into a drying bottle (clear and bright without impurity). Then blow to dry completely with the nitrogen at 50oC. Dissolve the dry residues in 1 mL of the diluted redissolving solution, add 1 mL N-hexane, mix for 2 min and centrifuge at above 4000 r/min at 15 ? for 5 min. Discard impurity of the upper and middle layers, take 100 L of the lower, add 100 L of the diluted redissolving solution. Take 50 L for further analysis. Fold of dilution of the sample:2 5.2 Low-detection-limit samples 5.2.1 Animal tissues (chicken, pork, shrimp, fish, etc.) 1) Weight 2.0 0.05 g of the homogenized tissue, add 8 mL of 0.02 M PB buffer, shake upside down thoroughly for 10 min, centrifuge at above 4000 r/min at 15? for 10 min (if the supernatant is too turbid, take 2 to 4 mL of it and then centrifuge it), 2) Take 50 L of the supernatant, add 200 L of the diluted redissolving solution, and mix evenly, 3) Take 50 L for further analysis. Fold of dilution of the sample: 20 5.3 Honey Take 1 g honey, add 2 mL 0.02 M PB buffer, dissolve it completely. Add 8 mL dichlormethane, vortex for 5 min and centrifuge at 4000 r/min for 5 min. Remove the upper layer, and take the organic phase (lower layer) into a dry container. Blow to dry with nitrogen or air at 50oC. Dissolve the dry residues in 1mL of the diluted redissolving solution, and dilute it at 1:3 (1 mL sample solution+3 mL of the diluted redissolving solution). Take 50 L for further analysis. Fold of dilution of the sample: 4 ELISA procedures Instructions: 1 Bring all reagents and micro-well strips to the room temperature (20-25oC). 2 Return all reagents to 2-8oC 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 temperature, all the samples and reagents must avoid light exposure, and each microplate should be sealed by the cover membrane. Operating procedures: 1 Take out all the necessary reagents 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. Solution: dilute the 20concentrated washing buffer (40 mL) with the distilled or deionized water to 800 mL (or just to the required volume) for use. Take the required micro-well strips and plate frames. Re-sealed the unused microplate, stored at 2-8oC, not frozen. 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 to separate duplicate wells, and add 50 L of the enzyme conjugate and then 50 L of the antibody working solution into each well. Mix by shaking gently, seal the microplate with the cover membrane, and incubate at 25oC for 1 h. Wash the microplate with the washing buffer at 250 L/well for four to five times. Each time soak the well with the washing buffer for 10 s and 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 by shaking gently, and incubate at 25oC for 15 min at dark for coloration . Determination: add 50 L of stop solution into each well. Mix by shaking gently. Set the wavelength of the microplate reader at 450 nm to determine the OD value (we 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 enrofloxacin. 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 1is 0.238, and that of the sample 2 is 0.946, while those of the standard solutions are as the followings: 1.845 for 0 ppb, 1.542 for 0.25 ppb, 1.130 for 0.75 ppb, 0.635 for 2.25 ppb, 0.326 for 6.75 ppb and 0.156 for 20.25 ppb, accordingly the concentration range of the sample I is 6.75 to 20.25 ppb, and that of the sample II is 0.75 to 2.25 ppb. 7.1 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 enrofloxacin 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 enrofloxacin 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 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. 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 reagent 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 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. 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Antigendetails

Target: Enrofloxacin

Substanzklasse: Chemical