Olaquindox 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 ofOlaquindox in the sample. The coupling antigens are pre-coated on the micro-well stripes. TheOlaquindox in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-Olaquindox 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 Olaquindox in it. This value is compared to the standard curve and the Olaquindox concentration is subsequently obtained.

2. Technical specifications

Sensitivity:0.2 ppb
Incubator temperature: 37℃
Incubator time: 30min~30min～15min

Detection limit:
Pork, chicken 0.2 ppb
Porcine liver 1 ppb
Feed 20ppb

Cross-reaction rate:
Olaquindox100%
Carbadox<7%

Recovery rate:
Tissue95%±25%
Feed70%±20%

3. Components

4. Materials required but not provided

1) Equipments:microplate reader (450nm/630nm), homogenizer, oscillator, centrifuge, measuring pipets,nitrogen-drying device and balance (a sensibility reciprocal of 0.01 g), Incubator.
2) Micropipettors: single-channel 20 to 200µL and 100 to 1000µL and multi-channel 30～300µl;
3) Reagents:Acetonitrile, N-hexane, Al2O3

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 utensil must be checked to be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results.

Sample preparation
1) Sampleredissolving solution: Dilute2× concentrated redissolving solution with deionized water at 1:1

5.1Tissues (Chicken, pork/liver)
1. Take 2± 0.05g of the homogenized sample into 50ml centrifuge tube, add10mLAcetonitrile (without water), shake properly for2min.Centrifuge at above4000r/min at room temperature (20 - 25℃) for 10min.
2. Take5mL supernatant, blow to dry with nitrogen or air at 56℃.
3. Dissolve the dry residuesin 2mlN-hexane, add1mL of the diluted redissolving solution, mixthoroughly for 30s, centrifuge at above4000r/min at room temperature (20-25℃) for5min. Remove the up-layerN-hexane phase.
4. Take 50 µL for analysis.
Fold of dilution of the sample:1

5.2Feed
1. Weigh 1± 0.05 g homogenized sample into centrifuge tube,add 2gAl2O3 , then 5mlAcetonitrile, shakeviolently for3min,centrifuge at above4000r/min at 25℃ for5min;
2. Take50ulsupernatant, mix with 950ul diluted redissolving solutionevenly,take 50µL up-layer liquid for analysis.
Fold of dilution of the sample: 100

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 the procedures of ELISA;
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 Operating Procedure
1) Take out all the required reagents and keep at room temperature (20-25℃) for at least 30min. Note that each reagent must be shaken and mixed well before use;
2) Take the desired microwell strip and plate frame. Unused microplates should be resealed and stored at 2-8°C;
3) Solution preparation: Dilute 40 mL of 20× concentrated washing buffer with deionized water at 1:19 (1 part of 20× concentrated washing buffer + 19 parts of deionized water), or prepare as needed;
4) Numbering: Number the microwells according to the samples and standard solutions; each sample and standard solution should be done in duplicate; record their positions;
5) Add 50 µL of sample or standard solution to separate wells, and then add 50 µL of antibody working solution to each well. Shake gently to mix well, cover the microplate with a cover film, and incubate at 37°C for 30 min;
6) Wash the microplate 4-5 times with 250 µL/well of washing buffer; soak the wells in the washing buffer for 15-30 seconds, and pat dry with absorbent paper (if any air bubbles are found after tapping, cut off with a clean pipette tip) );
7) Add 100 µL of Enzyme conjugate to each well, shake and mix gently, cover the microplate with a cover film, and incubate at 37°C for 30 min; pour out the liquid from the well, wash the microplate with washing buffer, and continue to step 6 ).
8) Color development: Add 50 µL of Substrate A solution and 50 µL of B solution to each well. Gently shake and mix, and incubate at 37°C for 15 minutes in the dark for color development;
9) Assay: Add 50 µL of stop solution to each well. Shake gently to mix. Set the wavelength of the microplate reader to 450nm to determine the OD value. (It is recommended to read the OD value at 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 ofOlaquindox.

7.1 Qualitative determination
The concentration range (ng/mL) can be obtained from the comparison 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, while those of the standard solutions are as the followings:2.243 for 0ppb, 1.816 for0.2ppb, 1.415 for0.6ppb,0.74 for1.8ppb, 0.313 for5.4ppb and 0.155 for16.2ppb, accordingly the concentration range of the sampleⅠis5.4 to16.2ppb, and that of the sampleⅡ is0.6 to 1.8ppb.

7.2 Quantitative determination
The mean values of the absorbance values is equivalent to the percentage of 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 0 ng/mL standard solution
Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the Olaquindox standard solutions (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, thus finally obtaining Olaquindox concentration in the sample.
Using the professional analyzing software of this kit will be more convenient for the accurate and rapid analysis of a large amount of samples. (Please contact us for this software)

8. Precautions

1. The room temperature below 25 ℃ or the temperature of the reagents and the samples being not returned to the room temperature (20-25 ℃) will lead to a lower standard OD value
2. Dryness of the microplate in the washing process will be accompanied by the situations including the non-linear standard curves and the undesirable reproducibility
3. Mix every reagent and reaction mixture evenly and wash the microplate thoroughly, otherwise there will be the undesirable reproducibility
4. The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin;
5. Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance and the colourless color former is light sensitive, and thus they cannot be directly exposed to the light
6. Do not use the kit exceeding its expiry date. The use of diluted or adulterated reagents from the kits will lead to the changes in the sensitivity and the detecting OD values. Do not exchange the reagents from the kits of different lot numbers to use
7. Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value of the 0 standard solution of less than 0.5 indicates its degeneration
8. The optimum reaction temperature is 37 ℃, and too high or too low temperatures will result in the changes in the detecting sensitivity and OD values.

9. Storage and expiry date

Storage: store at 2-8 ℃, not frozen.
Expiry date: 12 months; date of production is on the 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.

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