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Nitrofuran (AHD) ELISA Test Kit

Shenzhen Biokau Sci-tech Co.,ltd

Nitrofuran (AHD) ELISA Test Kit

Place of Origin : Shenzhen

Brand Name : Biokau

Model Number : BKU-10003

Certification : ISO9001

Price : 350USD

Packaging Details : 96T/kit

Delivery Time : 3 days

Payment Terms : TT% in advance

MOQ : 1

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Aminohydantion (AHD) ELISA Test Kit

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Aminohydantion (AHD) in the sample. The coupling antigens are pre-coated on the micro-well stripes. The Aminohydantion (AHD) in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-AHD 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 AHD in it. This value is compared to the standard curve and the AHD concentration is subsequently obtained.


2. Technical specifications

Sensitivity: 0.05 ppb

Detection limit

Tissue(Chicken, duck, porcine meat, beef and mutton) 0.1 ppb

Liver(Chicken, duck, porcine meat, beef and mutton) 0.1 ppb

Milk, intestine, honey 0.1 ppb

Fish, shrimp (some interference) 0.15ppb

Recovery rate

Tissue, liver 90±15%

Honey, milk, intestine 80±15%

Cross-reaction rate

AHD 100%

AMOZ ﹤0.1%

AOZ ﹤0.1%

SEM ﹤0.1%


3. Components

1) Micro-well strips: 12 strips with 8 removable wells each

2) 6× standard solution (1 mL each): 0 ppb, 0.05 ppb, 0.15ppb, 0.45 ppb, 1.35 ppb and 4.05 ppb

3) Enzyme conjugate (12 mL) red cap

4) Antibody working solution (7 mL) blue cap

5) Substrate A solution (7 mL) white cap

6) Substrate B solution (7 mL) black cap

7) Stop solution (7 mL) yellow cap

8) 20× concentrated washing buffer (40 mL) white cap

9) 2× concentrated redissolving solution (50 mL) transparent cap

10) 2-Nitrobenzaldehyde (10 mL) white cap


4. Materials required but not provided

  • Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex, centrifuge, measuring pipets, and balance (a sensibility reciprocal of 0.01 g);
  • Micropipettors: single-channel 20-200 µL, 100-1000 µL, and multi-channel 250 µL;
  • Reagents: NaOH, ethyl acetate, n-Hexane, HCI (approx36.5%), K2HPO4 (for all samples), K2Fe(CN)5NO·3H2O and ZnSO4·7H2O(for milk sample)

5. 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 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

  • the 2×concentrated redissolving solution is mixed with deionized water at 1:1 (1 mL concentrated redissolving solution + 1 mL deionized water), used for sample redissolving.
  • C solution (for milk sample): dissolve 12.5 g K2Fe(CN)5NO·3H2O in deionized water to 100 mL.
  • D solution (for milk sample): dissolve 29.8 g ZnSO4·7H2O in deionized water to 100 mL.
  • 0.1 M K2HPO4: dissolve 22.8 g K2HPO4·3H2O in deionized water to 1 L.
  • 1 M HCl: dissolve 8.6 mL HCI (approx 36.5%) in water to 100 mL.
  • 1 M NaOH: dissolve 4 g NaOH in water to 100 mL.

5.1 Samples preparation

a) milk

  • Put 5 mL milk into centrifuge tube, add C and D solution, 250 μL each.
  • Mix thoroughly, use vortex, centrifuge at above 4000 r/min at 4-12 ℃ for 10 min, if centrifuge of constant temperature is not avaible, chill sample to approx 8 ℃, then centrifuge.
  • Continue as described in step (1-7,b).

b) Tissue, intestine, liver, honey

  • Weigh 1± 0.05 g of the homogenized sample (Tissue, intestine, liver or honey), or put 1.1 mL supernatant of centrifuged milk (equivalent to 1 mL milk sample) into a plastic tube, add 4 mL of the deionized water, 0.5 mL 1 M HCI and 100 μL 2-Nitrobenzaldehyde to the tube, shake properly.
  • Incubate at 37 ℃ over night ( approx 16 h) or incubate at 56℃ in water bath(2 hours).
  • Add 5 mL 0.1 M K2HPO4, 0.4 mL 1 M NaOH and 5mL ethyl acetate, shake vigorously for 5 min.
  • Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
  • Transfer 2.5 mL ethyl acetate (upper layer) into a new centrifugal tube and evaporate to dry by nitrogen or air at 50 ℃.
  • Dissolve the dry residue in 1 mL N-hexane, then add 1mL of the diluted redissolving solution. Mix properly for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
  • Take 50 µL of the lower for analysis.

Fold of dilution of the sample:2

c) Special samples(Fried cooked foods, Non-fried cooked food)

  • weigh 1± 0.05 g of the homogenized sample in 50 mL tube.

Fried cooked foods: Add 4.5 mL Methanol and 0.5 mL distilled water, shake strongly for 3 min. Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min, Discard all solutions. then add 5 mL Acetonitrile and 5 mL N-hexane, shake strongly for 3 min. Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min, Discard all solutions.

Non-fried cooked foods: Add 5 mL distilled water and 5 mL N-hexane, shake strongly for 3 min, Discard all solutions.

  • Add 4 mL distilled water, 0.5 mL 1 M HCl and 100µL 2-Nitrobenzaldehyde(C7H5NO3) solution to the tube, shake properly.
  • Incubate at 37 ℃ over night ( approx 16 hours) or incubate at 56℃ by water bath(2 hours) .
  • Add 5 mL 0.1 M K2HPO4, 0.4 mL 1 M NaOH and 10 mL ethyl acetate to each tube, shake vigorously for 5 min.
  • Centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min.
  • Transfer 5 mL ethyl acetate into a clean centrifuge tube and blow to dryness by nitrogen or air at 50 ℃.
  • Dissolve the dry residues in 1 mL N-hexane, add 1 mL of the diluted redissolving solution, mix properly, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 5 min.
  • Take 50 µL of the lower layer for the analysis.

Fold of dilution of the sample: 2


6. ELISA procedures

  • Bring test kits to room temperature (20-25 ℃) for at least 30 min. Note that each reagent must be shaken to mix evenly before use; put the required micro-well strips into plate frames. Re-sealed the unused microplate, store at 2-8 ℃, not frozen.
  • Solution preparation: dilute 40 mL of the 20×concentrated washing buffer with the distilled or deionized water at 1:19 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 50 µL of the sample or standard solution to separate duplicate wells, and add 50 µL of the antibody working solution into each well, then seal the microplate with the cover membrane, and incubate at 37 ℃ for 30 min.
  • Pour liquid out of the wells , flap to dry on absorbent paper, add 250 µL/well of washing buffer to wash microplate for 15-30 sec, then take out and flap to dry with absorbent paper, repeat 5 times.
  • Add 100 µL enzyme conjugate into each well; and incubate at 37 ℃ for 30 min.Take out microplate, continue as described in step 5.
  • Coloration: add 50 µL of the substrate A solution and then 50 µL of the B solution into each well. Mix gently by shaking the plate manually, and incubate at 37 ℃ for 15 min at dark for coloration;
  • Determination: add 50 µL 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 of every well(Recommend to read the OD value at the dual-wavelength 450/630 nm).

Shenzhen Biokau Biotechnology Co.,Ltd makes no warranty of any kind, either expressed or implied, except that the materials from which its products made are of standard quality. If any materials are defective, Biokau Biotechnology will provide a replacement product. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. Biokau Biotechnology shall not be liable for any damages, including special or consequential damage, or expense arising directly ot indirectly from the use of the this product.



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