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Chloramphenicol ELISA Kit

Reactivity: Chemical Colorimetric Competition ELISA
Catalog No. ABIN400589
  • Target See all Chloramphenicol products
    Chloramphenicol
    Reactivity
    Chemical
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Application
    ELISA
    Purpose
    This test kit is based on the competitive enzyme immunoassay for the detection of Chloramphenicol in the sample. The coupling antigen is pre-coated on the micro-well stripes. The Chloramphenicol in the sample and the coupling antigen pre-coated on the micro-well stripes compete for the anti-Chloramphenicol 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 Chloramphenicol in it. This value is compared to the standard curve and the Chloramphenicol 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, 0.05 ppb, 0.15 ppb, 0.45 ppb, 1.35 ppb and 4.05 ppb, Enzyme conjugate (12 mL) red cap Concentrated antibody working solution (1 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, 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: Ethyl acetate, Acetonitrile (CH3CN), N-hexane, Na2Fe(CN)5NO2H2O, ZnSO4.7H2O, Beta-Glucuronidase (Merck, Art. No. 4114), CH3COONa, CH3COOH
  • 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 which interferes with the experimental results. Solution preparation before sample pre-treatment C solution (for milk sample): dissolve 10.7 g Na2Fe(CN)5NO2H2O in the deionized water to 100 mL . D solution (for milk sample): dissolve 28.8 g ZnSO4.7H2O in deionized water to 100 mL. pH 4.8 100 mM CH3COONa buffer(for urine sample): add 2.4 g CH3COONa and 1.2 mL acetic acid in deionized water to 500 mL, and adjust pH to 4.8. Acetonitrile-H2O solution: Vacetonitrile:V H2O = 84:16 The concentrated redissolving solution is diluted with deionized water at 1:1 (1 mL concentrated redissolbing solution + 1 mL deionized water). 5.1 Tissue(Shrimp, fish, meat/liver ) Homogenize the sample . Take 3 0.05 g of the homogenized sample into tube. Firstly add 3 mL deionized water and mix evenly, then add 6mL ethyl acetate, shake properly for 5 min, centrifuge at above 4000 r/min at room temperature (20-25?) for 10 min. Take 2mL of the supernatant (equivalent to 1 g sample), and evaporate to dryness by nitrogen in 50-60? waterbath. Dissolve the dry residues in 1 mL N-hexane, add 1 mL of the diluted redissolving solution, shake vigorously for 30 seconds, centrifuge at above 4000 r/min at room temperature for 5 min. Take 50 L of the lower for analysis. Fold of dilution of the sample: 1 5.2 Serum, plasma Transfer 1 mL sample into tube, add 2 mL ethyl acetate, shake properly for 1 min. Centrifuge at 4000 r/min at room temperature for 5 min. Transfer ethyl acetate layer (upper layer) into a new vessel and evaporate to dryness by nitrogen in 50oC waterbath. Dissolve the dry residue with 1 mL diluted redissolving solution for 30 seconds. Take 50 L for further analysis. Fold of dilution of the sample: 1 5.3 Urine Transfer 2 mL urine into centrifuge tube, mix with 0.5 mL100 mM CH3COONa buffer(pH 4.8). Add 40 L of the Beta-Glucuronidase into the diluted urine, hydrolyze at 37oC for 2 h (or over night). Return to room temperature (20-25oC), add 8 mL ethyl acetate, mix properly for 1 min. Centrifuge at 4000 r/min at room temperature (20-25oC) for 10 min, take out 4 mL of the upper layer and evaporate to dryness by nitrogen at 50-60oC. Dissolve the dry residue in 1 mL of the diluted redissolving solution for 30 seconds. Take 50 L for further analysis. Fold of dilution of the sample: 1 5.4 Honey Put 2.0 0.05 g honey into centrifuge tube, diluted in 4 mL of the deionized water. Add 4 mL ethyl acetate, shake upside and down for 5 min. Centrifuge at 4000 r/min at room temperature (20-25oC) for 10 min. Transfer 1 mL of the ethyl acetate layer (upper layer) into a new vessel, evaporate to dryness by nitrogen at 50-60oC. Dissolve the dry residue in 0.5 mL of the diluted redissolving solution for 30 seconds. Take 50 L for further analysis. Fold of dilution of the sample: 1 Detection limit: 0.05 ppb Quantitative limit: 0.15 ppb Note: Recommend 0.15 ppb as value of cut off for positive sample. 5.5 Intestine Homogenize the sample(note that dry sample must be cut into less than 5 mm pieces, damp sample must be washed to eliminate salt for 20 min with the deionized water, reduce water and then homogenize). Weigh 1.00.05 g of the homogenized sample into centrifuge tube, add 10 mL ethyl acetate, shake properly for 5 min, centrifuge at above 4000 r/min at room temperature (20-25oC) for 10 min. Take out 5 mL of the upper layer (equivalent to 0.5 g sample), blow to dry with nitrogen at 50-60oC. Dissolve the dry residues in 1 mL N-hexane, add 0.5 mL of the diluted redissolving solution, shake strongly for 30 seconds, centrifuge at above 4000 r/min at room temperature for 5 min. Discard upper layer, take 50 L of the lower for further analysis. Fold of dilution of the sample: 1 5.6 Milk and milk powder First method for milk sample Centrifuge at above 4000 r/min at 10oC for 10 min, remove fat (upper layer). Put 5 mL sample without fat into centrifuge tube, add 150 L C solution and appear precipitation, shake properly, then add 150 L D solution, mix properly. Centrifuge at above 4000 r/min at 15 ? for 10 min, take upper layer. Dissolve the upper layer in the diluted redissolving solution at 1:1, mix for 30 seconds. Take 50 L for further analysis. Fold of dilution of the sample: 2 Detection limit: 0.1 ppb Quantitative limit: 0.15 ppb Note: if appear turbidity after centrifuge, repeat the step of precipitation. Second method for milk sample Put 5 mL milk removed fat into centrifuge tube. Add 250 L C solution and 250 L D solution, mix thoroughly, centrifuge at above 4000 r/min at 4-12oC for 10 min. if centrifuge of constant temperature is not avaible, chill sample temperature to approx 8oC, then centrifuge. Transfer 2.2 mL upper layer (equivalent to 2 mL sample) into a new vessel, add 4 mL ethyl acetate, shake upside down for 5 min. Centrifuge at above 4000 r/min at room temperature (20-25?) for 10 min. Transfer 2 mL of the ethyl acetate (upper layer, equivalent to 1 mL sample), blow to dry with nitrogen at 60oC competely. Dissolve dry residues in 0.5 mL of the diluted redissolving solution . Take 50 L for further analysis. Fold of dilution of the sample: 0.5 Detection limit: 0.025 ppb Quantitative limit: 0.05 ppb Note: Recommend standards 3 (0.15 ppb) as value of cut off for positive result, because negative sample causes interference, sometimes value of interference between standards 2 and standards 3. Milk powder sample Weigh 2.0 0.05 g milk powder into centrifuge tube, add 10 mL of the deionized water , shake thoroughly to dissolve. Add 1 mL C solution and 1 mL D solution, mix thoroughly, centrifuge at above 4000 r/min at 4-12oC for 10 min. if centrifuge of constant temperature is not avaible, chill sample temperature to approx 8oC, then centrifuge. Transfer 3.6 mL of the upper layer (equivalent to 0.6 g sample) into a new vessel, add 6 mL ethyl acetate, shake upside down for 5 min. Centrifuge at above 4000 r/min at room temperature (20-25oC) for 10 min. Transfer 4 mL of the ethyl acetate (upper layer, equivalent to 0.4 g sample), blow to dry with nitrogen at 60oC competely. Dissolve dry residues in 0.4 mL of the diluted redissolving solution for 30 seconds . Take 50 L for further analysis. Fold of dilution of the sample: 1 Detection limit: 0.05 ppb Quantitative limit: 0.15 ppb Note: Recommend standard 3 as value of cut off for positive result, because negative sample cause interference, sometimes value of interference between standards 2 and standards 3. 5.7 Egg Homogenize the sample (whole egg or yolk ). Weigh 3.0 0.05 g of the homogenized sample, add 9 mL of the acetonitrile-H2O solution (V acetonitrile:V H2O = 84:16), shake for 5 min, centrifuge at above 4000 r/min at 15 ? for 10 min. Transfer 3 mL of the upper layer into a centrifuge tube, add 3 mL of the deionized water, mix properly, then add 4.5 mL ethyl acetate, mix properly for 5 min, centrifuge at above 4000 r/min at 15 ? for 10 min. Transfer the organic phase (upper layer) into a new centrifuge tube, blow to dry with nitrogen at 50oC. Dissolve dry residues in 1 mL N-hexane, add 2 mL of the diluted redissolving solution, mix peoperly for 30 seconds, centrifuge to remove N-hexane. Take 50 L for further analysis. Fold of dilution of the sample: 2
    Restrictions
    For Research Use only
  • Storage
    4 °C
  • Target See all Chloramphenicol products
    Chloramphenicol
    Abstract
    Chloramphenicol Products
    Target Type
    Chemical
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