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Benzyl penicillin ELISA Kit

Reactivity: Penicillium sp. Colorimetric Competition ELISA
Catalog No. ABIN400588
  • Target
    Benzyl penicillin
    Reactivity
    Penicillium sp.
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Application
    ELISA
    Purpose
    This test kit is based on the competitive enzyme immunoassay for the detection of Benzyl penicillin in the chicken, pork, duck, honey, milk, fish, shrimp and egg, etc. The antigens conjugated Benzyl penicillin is pre-coated on the micro-well stripes, Benzyl penicillin in the sample and the conjugated antigens pre-coated on the micro-well stripes compete for the anti-Benzyl penicillin 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 Benzyl penicillin concentration in the sample. This value is compared to the standard curve and concentration of Benzyl penicillin in the sample 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, 0.05 ppb, 0.15 ppb, 0.45 ppb, 1.35 ppb and 4.05 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
    Material not included
    Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (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,Acetonitrile(CH3CN), N-Hexane, deionized water, Na2Fe(CN)5NO2H2O and ZnSO47H2O, HCI(36%), 2M H2SO4
  • 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 different reagents. 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: 1 concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL 2 concentrated redissolving solution+1 mL deionized water), used for sample redissolving 2 0.1 M NaOH(for milk sample) :dissolve 0.4 g NaOH in deionized water to 100 mL 3 CH3CN-0.1 M NaOH solution: ?CH3CN :?NaOH =84:16 (84 mL CH3CN + 16 mL 0.1 M NaOH). 4 C solution(for milk sample)/0.36 M Na2Fe(CN)5NO2H2O:dissolve 10.7 g Na2Fe(CN)5NO2H2O in deionized water to 100 mL . 5 D solution(for milk sample)/1 M ZnSO4 :dissolve 28.8 g ZnSO47H2O in deionized water to 100 mL. 6 Acidic Acetonitrile (CH3CN) (for honey sample): add 150 L 2 M H2SO4 into 100 mL Acetonitrile (CH3CN),mix properly. 7 1M HCI (for honey sample): dissolve 41.7 mL HCI(36%) in deionized water to 500 mL. 8 1M NaOH(for honey sample):dissolve 4 g NaOH in deionized water to 100 mL. 5.1 Tissue (Chicken, duck, pork or pork liver, fish, shrimp) 1 Homogenize the sample 2 Take 2 0.05 g of the homogenized sample into 50 mL tube, add 8 mL of CH3CN-0.1 M NaOH solution, rock it with vortex for 2 min, shake for 5 min , centrifuge at above 4000 r/min at room temperature(20-25oC) for 10 min. 3 Take 1 mL of the supernatant, blow to dry by nitrogen or air at 56oC. 4 Dissolve the dry residues in 1 mL N-Hexane, add 1 mL of the diluted redissolving solution, shake vigorously for 30s, centrifuge at above 4000 r/min at room temperature (20-25oC) for 5 min. 5 Remove N-Hexane phase (upper layer), take aqueous phase (the lower) to dilute at 1:4 ( 50 L sample + 200 L the diluted redissolving solution), blend for 30s Take 50 L for analysis. Fold of dilution of the sample: 20 5.2 Honey 1 Put 4.0 0.05 g honey into centrifuge tube , add 0.5 mL 1 M NaOH, mix properly, then place it for 20 min. 2 Add 0.5 mL 1 M HCI, mix properly(pH=3), add 7 mL of the Acidic Acetonitrile (CH3CN) (pH=4), shake vigorously for 5 min, centrifuge at above 4000 r/min at room temperature (20-25?) for 10 min. 3 Taker 3 mL of the clear liquid (upper layer) and evaporate to dryness by nitrogen at 56oC. 4 Dissolve the dry residue in 1 mL of the diluted redissolving solution. 5 Dilute with the diluted redissolving solution at 1:19 (20 L sample+380 L diluted redissolving solution),blend for 30s 6 Take 50 L for further analysis. Fold of dilution of the sample: 20 5.3 Milk a) First method 1 Take 2 mL fresh milk into 5 mL centrifuge tube, add 50 L C solution, mix properly. 2 Add 50 L D solution ,shake for 1 min, centrifuge at 4000 r/min at 10oC for 10 min. 3 Take clear liquid(upper layer), dilute with the diluted redissolving solution at 1:19(20 L sample+380 L diluted redissolving solution). 4 Take 50 L for further analysis. Fold of dilution of the sample: 20 Note: Repeat again if centrifuged sample is muddy b) Second method 1 Put 2 mL milk (removed fat) into centrifuge tube. 2 Add 8 mL of the CH3CN-0.1 M NaOH solution, shake vigorously for 5 min, centrifuge at above 4000 r/min at 15 ? for 10 min, take 1 mL of the clear liquid (upper layer) and evaporate to dryness by nitrogen at 56oC. 3 Dissolve the dry residues in 1 mL N-Hexane, add 1 mL of the diluted redissolving solution, mix properly for 30s, centrifuge and remove N-hexane phase. 4 Take the lower to dilute 1:3 (50 L sample + 150 L the diluted redissolving solution). 5 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). 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. Operating procedures: 1 Take out the kit from 4oC 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. 2 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 concentrated washing buffer (20concentrated) 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 preparation, each testing sample and standard solutiond 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 antibody working solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, and incubate at 37oC for 30 min. Pour the liquid out of the wells, wash the microplate with the washing buffer at 250 L/well for 4-5 times,the time gap between each time is 15s---30s. Each time soak the well with the washing buffer for 10 s and then flap to dry on absorbent paper (if there are the bubbles after flapping, cut them with the clean tips). Add 100 L of the enzyme conjugate into every well, seal the microplate with the cover membrane, and incubate at 37oC for 30 min, continue as subscribed in 6. 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 15min at dark for coloration. 9 Determination: add 50 L stop solution into each well. Vortex evenly. 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 Benzyl penicillin in the sample. 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 1is 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 4.05 ppb, and that of the sample 2 is 0.15 to 0.45 ppb. 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 0 ng/mL standard solution Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithmic values of the Benzyl penicillin 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 the actual concentration of Benzyl penicillin 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 standard solution 1(0 ppb) of less than 0.5 indicates its degeneration, Colouration time is 15 min after the addition of the substrate A and then the B solution, if the color is light, prolong the time, dont exceed 30min, 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
    Benzyl penicillin
    Target Type
    Chemical
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