Product For Feed

LSY-10063 Nicarbazine ELISA Test Kit for feed

Nicarbazine ELISA Test Kit

Catalog No. LSY-10063

1. Principle

This test kit is based on theindirectcompetitive enzyme immunoassay for the detection ofNicarbazine. The coupling antigen is pre-coated on the micro-well stripes. TheNicarbazine in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti- Nicarbazine 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 theNicarbazine inthe sample. This value is compared to the standard curve and theNicarbazine residues is subsequently obtained.


2.  Technical specifications

Sensitivity: 0.1 ppb

Incubator temperature:  25℃

Incubator time:  30min~15min

Detection limit:

Tissue  10ppb

Feed  20ppb

Cross-reaction rate:

Nicarbazine   100%

Recovery rate:

Tissue, Feed   70%-120%


3. Components


Micro-well strips

12 strips with 8 removable wells each


6× standard solution (1 mL each)








Enzyme conjugate


red cap


Antibody working solution


blue cap


Substrate A


white cap




black cap


Stop solution


yellow cap


20× concentrated washing buffer


white cap


Sample dilution


transparent cap


4. Materials required but not provided

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

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

3)  Reagents: Acetonitrile.


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.

Samples Preparation

5.1  Preparation ofTissue sample

1) Take1.0±0.05g tissue sample into10ml centrifuge tube, add2mlacetonitrile,vortex for5min;

2) Centrifuge at above 4000r/min atroom temperature(20℃-25℃) for5 min;

3) Take50ul supernatant, add950ulSampledilution, shakeviolently to mix for 30s; 

4) Take50μlto test.

Dilution factor: 40


5.2 Preparation ofFeed sample

1) Take5.0±0.05g feed sample into 50ml centrifuge tube; add20mlacetonitrile, thenvortex for5min;

2) Centrifuge at above 4000r/min at room temperature(20℃-25℃)for5min;

3) Take50ul supernatant, add950ulSampledilution, shakeviolently to mix for 30s;

4) Take50μlto test.

Dilution factor:80


6.  ELISA procedures


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

2.  Put ELISA reagents back to2-8℃ immediately after use

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

4. In all process of constant temperature incubation, avoid lightexposure, 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: take the15ml20× concentrated washing buffer, dissolve with deionized water at 1:19 (1 part20× 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 standard/sample:Add50 µL of the sampleor the standard solution into separate duplicate wells,then add enzyme conjugate, 50 µL/well; thenantibody 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; add250 µL/well ofwashing buffer, wash fullyfor4-5 times, 15-30 s each time,then take out and flap to dry with absorbent paper.(Use unusedspeartopierce bubble after dry)

7.  Coloration: add 50 µL of substrate A solutionthen 50 µL substrate 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 450 nm to determine the OD value of every well. (Recommend to read the OD value at the dual-wavelength 450/630 nm 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 theNicarbazine in the sample

7.1  Qualitative determination

The concentration range (ppb) can be obtained by compared the average absorbance value with standards. Suppose absorbance valueof Sample One is 0.3,SampleTwo is 1.0,and thestandards are: 0ppbof2.243; 0.1ppbof1.816; 0.3ppbof 1.415; 0.9ppb of 0.74;2.7ppbof 0.313;8.1ppb of 0.155. Then the concentration of the sample one is in therange of2.7ppb ~8.1ppb;SampleTwois0.3ppb ~0.9ppb. The concentration range ofNicarbazine in the samplescan be obtainedbymultipliedbythe corresponding dilution of the sample.

7. 2  Quantitative Analysis

In order tocalculate 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:

Percentage of absorbance value =




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 theNicarbazine concentration [ng/L]. TheNicarbazine concentration in ng/L (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 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; So continue to next step immediately after washing.

3. Mix evenly before adding any reagents.

4.  The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.

5.  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.

6.  Storage:store at 2-8 ℃, not frozen.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.

7.  Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value(450/630nm) of the 0 standard solution (0 ppb) of less than 0.5((A450nm<0.5)) indicates its degeneration.

8. The optimum reaction temperature is25 ℃, 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 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.



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Company: Shenzhen Lvshiyuan Biotechnology Co.,Ltd

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