LSY-10066 Carbadox metabolites(QCA) ELISA Test Kit meat testing 96wells per kit

Carbadox metabolites(QCA) ELISA Test Kit

Catalog No. LSY-10066

1. Principle

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

2. Technical specifications

Sensitivity: 0.2 ppb

Incubator temperature: 25℃

Incubator time: 30min～15min

Precision

Coefficient of variation within and between plates..............................<10%

Detection limit:

Tissue........................................................................................... 0.5 ppb

Cross-reaction rate:

Carbadox metabolites(QCA).............................................................. 100%

Olaquindox metabolite (MQCA)......................................................... 120%

Olaquindox (OLA) ............................................................................. <1%

Carbadox(CAR)................................................................................. <1%

Sulfaquinoxaline (SQX)...................................................................... <1%

Recovery rate:

Tissue...................................................................................... 70%-120%

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, printer
2. Micropipettors: single-channel 20 to 200 µL and 100 to 1000 µL and multi-channel 30～300 µl;
3. Reagents: Ethyl acetate, n-hexane, H₂SO₄, deionized wate.

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) 2M H₂SO₄: Take 10ml H₂SO₄, add 80ml deionized water, mix it evenly.

5.1 Tissue:

1. Take 1± 0.01 g of the homogenized tissue sample into 50ml centrifuge tube, add 1ml deionized water, vortex for 1min;
2. Add 6ml Ethyl acetate, then add 0.5ml 2M H₂SO₄ 2ml CH₃CN, Vortex for 2min, Centrifuge at 4000 r/min at room temperature for 5 min;
3. Take 3 mL up-layer clear liquid into a 10ml centrifuge tube, below to dry with Nitrogen at

50-60℃ water bath;

4. Add 1ml N-hexane, vortex for 1min, then add 0.5ml Redissolving solution, vortex for 30s; centrifuge at 4000 r/min at room temperature for 5 min.
5. Remove the up-layer n-hexane phase and middle-layer impurities completely.
6. Take 50 µL down-layer 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 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 Operation procedures

1. Take out all the necessary reagents and place at the room temperature (20-25 ℃) for at least 30min. Note that each reagent must be shaken to mix evenly before use;
2. Take the required micro-well strips and plate frames. Re-sealed the unused microplate, stored at 2- 8 ℃;
3. Solution preparation: dilute 15 mL of the 20× concentrated washing buffer with deionized water at1 :19 (1 part 20× concentrated washing buffer + 19 parts deionized water), or prepare as quantity needed;
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 50 µL the sample or standard solution to separate duplicate wells, then add 50µL Enzyme conjugate, then add 50µL antibody working solution into each well. Mix by shaking gently, seal the microplate with the cover membrane, and incubate at 25℃ at dark for 30 min;
6. Wash the microplate with the washing buffer at 250 µL/well for four to five times; soak the well with the washing buffer for 15-30 sec, flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips);
7. Coloration: add 50 µL of the substrate A solution, then 50 µL of the substrate B solution into each well. Mix by shaking gently, and incubate at 25 ℃ for 15 min in the dark for coloration;
8. Determination: add 50 µL stop solution into each well. Mix by shaking gently. Set the wavelength of the microplate reader at 450nm to determine the OD value. (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 content of QCA.

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 sample Ⅰ is 0.236, and that of the sample Ⅱ is 0.666, while those of the standard solutions are as the followings: 1.949 for 0ppb, 1.434 for 0.2ppb, 0.939 for 0.6ppb, 0.487 for 1.8ppb, 0.157 for 5.4ppb and 0.06 for 16.2ppb, accordingly the concentration range of the sampleⅠis 1.8ppb-5.4ppb, and that of the sample Ⅱ is 0.6ppb-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 QCA 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 QCA 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 20 ℃ 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. After adding Substrate A and Substrate B, it is generally sufficient to develop color for 15 minutes. If the color is lighter, the reaction time can be extended to 20min (or longer), but not more than 30min. Otherwise, the reaction time is shortened.

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.

Shenzhen Lvshiyuan Biotechnology Co., Ltd

D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China

Tel. 86-755-28438788

Fax 86-755-28938800

Email: info@lsybt.com

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