Amantadine Diagnostic ELISA Kit For Chicken Pork Duck 96 Wells/Kit Sensitivity 0.2 Ppb

Amantadine ELISA Test Kit for Chicken Pork Duck 96 Wells/Kit Sensitivity 0.2 ppb

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of Amantadine. The coupling antigen is pre-coated on the micro-well stripes. The Amantadine in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti- Amantadine 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 Amantadine in the sample. This value is compared to the standard curve and the Amantadine residues is subsequently obtained.

2. Technical specifications

Sensitivity: 0.2ppb
Incubator temperature: 25℃
Incubator time: 30min～15min

Detection limit:
Chicken, duck 0.2 ppb

Cross-reaction rate:
Amantadine 100%

Recovery rate:
Chicken, duck 90±25%

3. Components

4. Materials required but not provided

1) Equipment: ELISA Reader (450 nm/630nm), homogenizer, shaker, centrifuge, balance: 0.01g quantity sensitive, nitrogen-drying device, incubator, graduated pipettes, printer

2) Micropipettes:single-channel 20ml ~ 200ml, 100ml ~ 1000ml, multi-channel 30～300 μl

3) Reagents: Acetonitrile, n-hexane.

5. Sample pre-treatment

Instructions(The following points must be dealt with before the pre-treatment)

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 utensil 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) Sample extract solution

6 parts Acetonitrile + 1 parts Extractant to obtain the ready to use sample extract solution.

2) Sampleredissolving solution

Use 1 part of concentrated redissolving solution (5X) and dissolve with 4 parts of deionized water to obtain the ready to use sample redissolving solution.

Samples Preparation

5.1 Preparation ofChicken, duck sample

1) Take 3.0±0.05g homogenized tissue sample into 50ml centrifuge tube; add 6ml sample extract solution, shake for 3min, centrifuge at above 4000r/min at room temperature (20 - 25 ℃) for 5 min;

2) Take 2ml clear organic phase into a dry container, blow to dry with nitrogen or air at 50~60 ℃;

3) Firstly add 1ml n-hexane, then add 0.5ml Sample redissolving solution, mix for 30s, centrifuge at above 4000r/min at room temperature (20 - 25℃) for 5 min, discard up-layer n-hexane;

4) Take down-layer liquid 50μl to test

Dilution factor:0.5

6. ELISA procedures

6.1Instructions

1. Bring ELISA reagents to room temperature (20 - 25 °C) before use.

2. Put ELISA reagents back to 2-8 ℃immediately after use

3. The ELISA reproducibility in the analysis process is largely depends on the consistency of the washing plate, the correct washing plate operation is the point of determination ELISA program

4. In all process of constant temperature incubation, avoid light exposure, seal the microplate with the cover membrane

6. 2 Operation procedures

1. Bring test kit to the room temperature (20-25 ℃) for at least 30 min, note that each reagent must be shaken evenly before use;

2. Put the required micro-well strips into plate frames. Re-sealed the unused microplate, stored at 2-8 ℃, not frozen.

3. Solution preparation: dilute the 15ml 20× concentrated washing buffer with deionized water to 300ml.

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

5. Add standard/sample: Add 50 µL of the sample or the standard solution into separate duplicate wells, then add enzyme conjugate, 50 µL/well; then antibody working solution, 50 µL/well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane,incubate at25 °C for 30 min in the dark.

6. Wash microplate: Carefully open the cover membrance, pour liquid out of microwell; add 250 µL/well of diluted washing buffer, wash fully for 4-5 times, 15-30 s each time, then take out and flap to dry with absorbent paper.(Use unused spear to pierce bubble after dry)

7. Coloration: add 50 µL of substrate A solution then 50 µL B solution into each well. Mix gently by shaking the plate manually, andincubate at25 °C for 15minin the dark for coloration.

8. 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 450nm to determine the OD value of every well. (Recommend to read the OD value at the dual-wavelength 450/630nm within 5 min).

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 Amantadine in the sample

7.1 Qualitative determination
The concentration range (ng/ml) can be obtained by compared the average absorbance value with standards. Suppose absorbance value of Sample One is 0.3, Sample Two is 1.0, and the standards are: 0ppb of 2.243; 0.2ppb of 2.054; 0.8ppb of 1.715; 3.2ppb of 1.074; 12.8ppb of 0.451; 51.2ppb of 0.155. Then the concentration of the sample one is in the range of 12.8ppb ~ 51.2ppb; Sample Two is 3.2ppb ~ 12.8ppb. The concentration range of Amantadine in the samples can be obtained by multiplied by the corresponding dilution of the sample.
 

7. 2 Quantitative Analysis
In order to calculate the concentration of samples, a standard curve should be made. Before standard curve is made, the concept of % absorbance should be know.
Calculation of % absorbance:

B—the average OD value of the sample or the standard solution
B0—the average OD value of the 0 ng/mL standard solution
The zero standard is thus made equal to 100 % and the absorbance values are quoted in percentages. The values calculated for the standards are entered in a system of coordinates on semilogarithmic graph paper against the the Amantadine concentration [ng/mL]. The Amantadine concentration in ng/mL (ppb) corresponding to the absorbance of each sample can be read from the calibration curve.
A special software for result analysis of ELISA will facilitate double or multiple determinations. If you need, please call to request.

8. Precautions

1. The standard OD value will be low if the room temperature is lower than 25℃ or the reagent temperature and the sample has not returned to room temperature (20-25℃).
2. The microplate is dried during the washing process, which will be accompanied by non-linearity of the standard curve and poor reproducibility; therefore, proceed to the next step immediately after washing.
3. Mix well before adding any reagents.
4. The stop solution is 2M 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. Storage: Store at 2-8°C, do not freeze. Reseal unused microplates in self-sealing bags. Standard materials and colorless couplers are sensitive to light and cannot be directly exposed to light.
7. Discard any tinting solution whose color indicates that the solution has degraded. The detection value (450/630nm) of 0 standard solution (0 ppb) is less than 0.5 ((A450nm<0.5)) indicating its denaturation.
8. The optimal reaction temperature is 25℃. If the temperature is too high or too low, the detection sensitivity and OD value will change.

9. Storage and expiry date

Storage: store at 2-8 ℃, 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 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.