Nitrofuran AHD ELISA Food Safety Test Kit for Milk Sensitivity 0.04ppb 96Wells/Kit

1. Nitrofuran AHD ELISA Food Safety Test Kit 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. Nitrofuran AHD ELISA Food Safety Test Kit​ Technical specifications

Sensitivity: 0.04ppb

Incubation Temperature: 25℃

Incubation Time: 30min～15min

Detection limit Tissue, egg, honey: 0.1ppb

Cross-reaction rate

AHD 100%

AMOZ < 0.1%

AOZ < 0.1%

SEM < 0.1%

Recovery rate

Tissue, egg 95±25%

Honey 75±25%

3. Components

4. Materials required but not provided

1) Equipments:microplate reader, printer, homogenizer, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (a reciprocal sensibility of 0.01 g), incubator, water bath;

2) Micropipettors: single-channel 20-200µL, 100-1000µL, and multi-channel 30～300µl;

3) Reagents: NaOH, ethyl acetate, n-Hexane, HCI (approx 36.5%), K2HPO4·3H2O

5. Sample pre-treatment

Instructions

The following points must be dealt with before the pre-treatment of any kind of sample:

1) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

2) 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:

a)0.1 M K2HPO4: dissolve 11.4g K2HPO4·3H2O in deionized water to 500mL.

b)1 M HCl: dissolve 8.6mL HCI (approx 36.5%) in deionized water to 100mL.

c)1 M NaOH: dissolve 4g NaOH in deionized water to 100mL.

d)the 2×concentrated redissolving solution is diluted with deionized water at 1:1(1mL concentrated redissolving solution + 1 mL deionized water), used for sample redissolving.

5.1 Samples preparation

a) Tissue, egg

1) Weigh 1± 0.05g of the homogenized sample, add 4mL of the distilled water, 0.5mL 1 M HCI and 100µL 2-Nitrobenzaldehyde (C7H5NO3) to each tube, shake properly for 2min;.

2) Incubate at37 ℃ over night ( approx16 hours) or incubate at56℃ by water bath(2 hours).

3) Add 5mL 0.1M K2HPO4, 0.4mL 1M NaOH and 6mL ethyl acetate to each tube, shake for 30s.

4) Centrifuge at above 4000r/min at room temperature (20-25 ℃) for 10min ( if there is Emulsification or ethyl acetate layer is not enough for 3ml, incubate sample at 80℃ water bath for 10min and centrifuge repeatedly; or increase speed and extend time of centrifuge).

5) Transfer 3mL of the ethyl acetate layer into a new centrifugal tube and evaporate to dryness by nitrogen or air at 50℃.

6) Dissolve the dry residues in 2mL N-hexane, 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; Remove up-layer N-hexane phase (if there is Emulsification, after removing up-layer N-hexane phase, incubate sample at 70℃ water bath for 10-20min, centrifuge repeatedly ).

7) Take 50 µL of the lower for analysis.

Fold of dilution of the sample: 2

b) Honey

1) Weigh 2± 0.05g of the homogenized sample (honey), add 4mL of the distilled water, 0.5mL 1 M HCI and 100 µL 2-Nitrobenzaldehyde (C7H5NO3) to each tube, shake properly for 2min;.

2) Incubate at37 ℃ over night ( approx16 hours) or incubate at56℃ by water bath(2 hours).

3) Add 5mL 0.1M K2HPO4, 0.4mL 1M NaOH and 6mL ethyl acetate to each tube, shake for 30s.

4) Centrifuge at above 4000r/min at room temperature (20-25℃) for 10min ( if there is Emulsification or ethyl acetate layer is not enough for 3ml, incubate sample at 80℃ water bath for 10min and centrifuge repeatedly; or increase speed and extend time of centrifuge).

5) Transfer 3mL of the ethyl acetate layer into a new centrifugal tube and evaporate to dryness by nitrogen or air at 50 ℃.

6) Dissolve the dry residues in 2mL N-hexane, add 1mL of the diluted redissolving solution, mix properly for 30 seconds, centrifuge at above 4000r/min at room temperature (20-25 ℃) for 10 min; Remove up-layer N-hexane phase (if there is Emulsification, after removing up-layer N-hexane phase, incubate sample at 70℃ water bath for 10-20min, centrifuge repeatedly ).

7) Take 50 µL of the lower for analysis.

Fold of dilution of the sample: 1

6. ELISA procedures

6.1 Instructions

1) Bring all reagents and micro-well strips to the room temperature (20-25 ℃) before use;

2) Return all reagents to 2-8 ℃ 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 ELISA the procedures;

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.

6.2 Operation procedures

1) Bring test kit to the room temperature (20-25 ℃) for at least 30min, 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.

2) Solution preparation: dilute 40mL of the 20 × concentrated washing buffer with deionized water at 1:19 (1 part 20 × concentrated washing buffer + 19 parts deionized water). Or prepare as needed.

3) Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate, record their positions.

4) Add 50µL of the sample or standard solution into separate duplicate wells; add 50ul enzyme conjugate then 50µL of the antibody working solution into each well, mix gently by shaking the plate manually. Seal the microplate with the cover membrane, andincubate at 25 ℃ for 30min.

5) Pour liquid out of microwell, flap to dry on absorbent paper, add 250µL/well of washing buffer to wash microplate for 15-30s, then take out and flap to dry with absorbent paper, repeat 4-5 times. (If there are the bubbles after flapping, cut them with the clean tips).

6) Coloration: add 50µL of the substrate A and then 50µL of the substrate B into each well. Mix gently by shaking the plate manually, then incubate at 25 ℃ for 15 min at dark for coloration.

7) Determination: add 50µL of the stop solution into each well. Mix gently by shaking the plate manually. Set the wavelength of the microplate reader at 450nm to determine the OD value of every well. (Recommend to read the OD value at the dual-wavelength 450/630nm within 5 minutes).

7. Result judgment

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 AHD.

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 sampleⅠ is 0.3, and that of the sampleⅡ is 1.0ppb, the OD value of standard solutions is: 2.243 for 0ppb, 1.816 for 0.04ppb, 1.415 for 0.12ppb, 0.74 for 0.36ppb, 0.313 for 1.08ppb, 0.155 for 3.24ppb, accordingly the concentration range of the sampleⅠ is 1.08 to 3.24ppb, and that of the sampleⅡ is 0.12 to 0.36ppb.

7.2 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,

B—the average (double wells) OD value of the sample or the standard solution

B0—the average OD value of the 0ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solution and the semilogarithm values of the AHD standard solution (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 AHD concentration in the sample.

8. Precautions

1) The room temperature is lower than 25℃ or the reagent temperature and sample are not returned to room temperature (20-25℃), which will cause the standard OD value to decrease.

2) During the washing process, the drying of the microplate will be accompanied by the non-linearity of the standard curve and the unsatisfactory reproducibility; therefore, proceed to the next step immediately after washing.

3) Mix well, otherwise there will be poor reproducibility.

4) The stop solution is 2 M sulfuric acid solution, avoid contact with skin.

5) Do not use the kit beyond the expiration date. The use of diluted or adulterated reagents from the kit can result in changes in sensitivity and detection OD values. Do not replace reagents in different batches of kits.

6) Reseal unused microplates in self-sealing bags. Standard solutions and colorless couplers are light-sensitive and cannot be directly exposed to light.

7) Discard any tinting solution whose color indicates that the solution has degraded. A detection value of less than 0.5 for standard solution 1 (0 ppb) indicates degradation.

8) The optimal reaction temperature is 25℃, and the detection sensitivity and OD value will change if the temperature is too high or too low.

9. Storage and expiry date

Storage: store at 2 to 8 ℃, not frozen.

Expiry date: 12 months; date of production is on box.

Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.

Q1: When will it be shipped?
A1: We will ship the goods for you as soon as possible within 7 working days after receiving the payment. (In the event of the epidemic and other external factors, there may be delays in shipping)

Q2: Does it support OEM/ODM?
A2: It can be supported, but the specific quantity needs to be more than 100,000 pieces to facilitate customized products.

Q3: How is your factory doing in terms of quality control?
A3: We have ISO9001 and ISO13485 certified by the state. Our production process is in accordance with the standard process, which can ensure that the quality of the products is optimal.

Q4: How is after-sales service provided?
A4: We provide professional online technical after-sales service. We can provide you with one-on-one guidance in the form of video, phone calls, etc.

Q5: What is the payment method?
A5: We receive payment by T/T.

Q6: How to ship?
A6: By obtaining quotations from our many cooperative carriers, choose the best way to ship for you, or you can ship according to your requirements.