Shrimp Nitrofuran AMOZ 0.03ppb Diagnostic ELISA Kit For Milk Food Safety Sensitivity 96Wells/Kit

Nitrofuran AMOZ Diagnostic ELISA Kit for Milk Sensitivity 0.03ppb 96Wells/Kit

1. Diagnostic ELISA Kit Principle

This detection kit is based on a competitive enzyme immunoassay for the detection of AMOZ in samples. Conjugated antigens are pre-coated on microwell strips. The AMOZ in the sample competes with the conjugated antigen pre-coated on the microwell strip for anti-AMOZ antibody. After the enzyme conjugate is added, TMB substrate is added for staining. The optical density (OD) value of a sample is negatively correlated with the AMOZ in it. This value is compared to a standard curve and the AMOZ concentration is subsequently obtained.

2. Technical specifications

Sensitivity: 0.03ppb

Incubation Temperature: 25℃

Incubation Time: 30min～15min

Detection limit Tissue, egg, honey: 0.1ppb

Cross-reaction rate

AMOZ 100%

AHD < 0.1%

AOZ < 0.1%

SEM < 0.1%

Recovery rate

Tissue 80 ± 25%

Honey 75 ± 25%

Egg 95 ± 25%

3. Nitrofuran AMOZ ELISA Food Safety Test Kit​ Components

4. Materials required but not provided
1) Equipment: microplate reader, printer, homogenizer, nitrogen dryer, vortexer, centrifuge, measuring tube, balance (reciprocal 0.01g), incubator, water bath;
2) Micropipettes: single channel 20-200µL, 100-1000µL, multi-channel 30-300µl;
3) Reagents: NaOH, ethyl acetate, n-hexane, HCl (about 36.5%), K2HPO4 3H2O

5. Sample Preparation

instruct
The following points must be addressed before any kind of sample pretreatment:
1) The experiment can only use disposable tips, and the tips must be replaced when aspirating different reagents;
2) Before the experiment, each experimental equipment must be cleaned and re-cleaned if necessary to avoid contamination interfering with the experimental results.

Solution preparation before sample pretreatment:
1) 0.1 M K2HPO4: Dissolve 11.4 g of K2HPO4 3H2O in deionized water to 500 mL.
2) 1 M HCl: Dissolve 8.6 mL of HCl (about 36.5%) in deionized water to 100 mL.
3) 1 M NaOH: Dissolve 4 g of NaOH in deionized water to 100 mL.
4) Dilute 2× concentrated redissolving solution with deionized water at 1:1 (1 mL concentrated redissolving solution + 1 mL deionized water) for sample redissolving.

5.1 Sample preparation

a) Tissue, eggs
1) Weigh 1±0.05g of homogeneous sample, add 4mL of distilled water, 0.5mL of 1M HCl and 100µL of 2-nitrobenzaldehyde (C7H5NO3) to each tube, and shake appropriately for 2 minutes;
2) Incubate overnight at 37°C (about 16 hours) or in a water bath at 56°C (2 hours).
3) Add 5mL of 0.1M K2HPO4, 0.4mL of 1M NaOH and 6mL of ethyl acetate to each tube and shake for 30 seconds.
4) Centrifuge at room temperature (20-25°C) above 4000r/min for 10min (if there is emulsification or the ethyl acetate layer is less than 3ml, incubate the sample in a water bath at 80°C for 10min, and centrifuge repeatedly; or increase the speed and prolong the centrifugation time).
5) Transfer the 3 mL ethyl acetate layer to a new centrifuge tube and evaporate to dryness with nitrogen or air at 50°C.
6) Dissolve the dried residue in 2 mL of n-hexane, add 1 mL of the diluted reconstituted solution, mix well for 30 seconds, centrifuge at room temperature (20-25°C) at a speed of more than 4000 rpm for 10 minutes; remove the upper n-hexane phase. (If emulsification occurs, remove the upper n-hexane phase, incubate the sample in a 70°C water bath for 10-20min, and centrifuge repeatedly).
7) Take 50 µL of the lower layer for analysis.
Sample dilution factor: 2

b) honey
1) Weigh 2±0.05g homogeneous sample (honey), add 4mL distilled water, 0.5mL 1 M HCl and 100 µL 2-nitrobenzaldehyde (C7H5NO3) to each tube, shake appropriately for 2 minutes;
2) Incubate overnight at 37°C (about 16 hours) or in a water bath at 56°C (2 hours).
3) Add 5mL of 0.1M K2HPO4, 0.4mL of 1M NaOH and 6mL of ethyl acetate to each tube and shake for 30 seconds.
4) Centrifuge at room temperature (20-25°C) above 4000r/min for 10min (if there is emulsification or the ethyl acetate layer is less than 3ml, centrifuge the sample repeatedly in a water bath at 80°C for 10min; or increase the speed and prolong the centrifugation time).
5) Transfer the 3 mL ethyl acetate layer to a new centrifuge tube and evaporate to dryness with nitrogen or air at 50°C.
6) Dissolve the dried residue in 2 mL of n-hexane, add 1 mL of diluted reconstituted solution, mix well for 30 seconds, centrifuge at room temperature (20-25°C) at a speed of 4000 r/min or more for 10 minutes; remove the upper n-hexane phase. (If emulsification occurs, remove the upper n-hexane phase, incubate the sample in a 70°C water bath for 10-20min, and centrifuge repeatedly).
7) Take 50 µL of the lower layer for analysis.
Sample dilution factor: 1

6. ELISA procedure

6.1 Description
1) Bring all reagents and microwell strips to room temperature (20-25°C) before use;
2) Put all reagents back to 2-8°C immediately after use;
3) The reproducibility of ELISA assays is largely dependent on the consistency of plate washing. Correct operation of plate washing is the key to the ELISA procedure;
4) During constant temperature incubation, all samples and reagents must be protected from light, and each microplate should be sealed with a cover film.

6.2 Operating Procedure
1) Put the kit at room temperature (20-25°C) for at least 30 minutes, note that each reagent must be shaken and mixed well before use, and put the required microwell strips into the plate frame. Unused microplates should be resealed and stored at 2-8°C, not frozen.
2) Solution preparation: 40 mL of 20 × concentrated wash buffer diluted 1:19 with deionized water (1 part 20 × concentrated wash buffer + 19 parts deionized water). Or prepare as needed.
3) Numbering: Number the microwells according to the samples and standard solutions; each sample and standard solution should be made in duplicate and their positions recorded.
4) Add 50µL of sample or standard solution to separate replicate wells; add 50ul of enzyme conjugate to each well, then add 50µL of antibody working solution, and shake the plate by hand to mix gently. Seal the microplate with a cover film and incubate at 25°C for 30 minutes.
5) Pour out the liquid in the microwell, pat dry on absorbent paper, add 250 µL/well of wash buffer to wash the microwell plate for 15-30 seconds, then remove and pat dry with absorbent paper, repeat 4-5 times. (If there are air bubbles after tapping, cut them off with a clean tip).
6) Color development: Add 50 µL of Substrate A to each well, followed by 50 µL of Substrate B. Mix gently by shaking the plate by hand, then incubate in the dark at 25 °C for 15 min to develop the color.
7) Assay: Add 50 μL of stop solution to each well. Mix gently by shaking the plate by hand. Set the wavelength of the microplate reader to 450 nm to determine the OD value of each well. (It is recommended to read OD values at dual wavelengths 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 AMOZ concentration.

7.1 Qualitative determination

The concentration range (ng/mL) of AMOZ 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 1.0, the OD value of standard solutions is: 2.243 for 0ppb, 1.816 for 0.03ppb, 1.415 for 0.09ppb, 0.74 for 0.27ppb, 0.313 for 0.81ppb, 0.155 for 2.43ppb, accordingly the concentration range of the sampleⅠ is 0.81 to 2.43ppb, and that of the sampleⅡ is 0.09 to 0.27ppb.

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 OD value of the sample or the standard solution

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

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

8. Matters needing attention
1) The room temperature is lower than 25℃ or the reagent temperature and the sample have 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 from 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 validity period
Storage: Store at 2-8°C, do not freeze.
Expiration date: 12 months; production date is on the box.
Note: If the microplate vacuum packaging leaks, it can still be used without affecting the test results, so you can use it with confidence.

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 case of external factors such as the epidemic, the delivery may be delayed)

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

Q3: How is your factory doing in terms of quality control?
A3: We have nationally certified ISO9001 and ISO13485. Our production process conforms to standard procedures to ensure optimum product quality.

Q4: How to provide after-sales service?
A4: We provide professional online technical after-sales service. We can provide you with one-on-one guidance via video, telephone, etc.

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

Q6: How to ship?
A6: Choose the best shipping method for you by getting quotes from our many cooperative carriers, and also ship according to your requirements.