Nitrofuran (AOZ) ELISA Test Kit (Feed)

Catalog No. LSY-10002f

1. Principle

This test kit is based on the competitive enzyme immunoassay for the detection of AOZ in thefeedsample. The couplingantigens are pre-coated on the micro-well stripes. TheAOZ in the sample andthecoupling antigens pre-coated on the micro-well stripescompetefor theanti-AOZ 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 AOZ in it. This value is compared to the standard curve and the AOZconcentrationis subsequently obtained.

 

2. Technical specifications

Sensitivity: 0.02ppb

Incubation Temperature:  25℃

Incubation Time:  30min～15min

Detection limit    Feed: 4ppb

Cross-reaction rate

AOZ100%

AMOZ<0.1%

AHD<0.1%

SEM<0.1%

Recovery rate

Feed95±25%

 

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	6× standard solution (1mL each)	0ppb	0.02ppb
		0.06ppb	0.18ppb
		0.54ppb	1.62ppb
3	Enzyme conjugate	7ml	red cap
4	Antibody working solution	7ml	blue cap
5	Substrate A	7ml	white cap
6	SubstrateB	7ml	black cap
7	Stop solution	7ml	yellow cap
8	20× concentrated washing buffer	40ml	white cap
9	2× concentrated redissolving solution	50ml	transparent cap
10	2-Nitrobenzaldehyde (C7H5NO3)	10ml	black cap

 

4. Materials required but not provided

1） Equipments:microplate reader, printer, homogenizer, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (a reciprocal sensibility of 0.01 g), incubator, water bath;

2） Micropipettors: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30～300 µl;

3） Reagents: NaOH, ethyl acetate, n-Hexane, HCI (approx36.5%), K2HPO4·3H2O,Ethanol absolute 

 

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 equipment 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) 0.1M K2HPO4: dissolve11.4gK2HPO4·3H2O in deionized water to500mL.

2) 1M HCl: dissolve 8.6mL HCI (approx36.5%) in deionized water to 100mL.

3) 1M NaOH: dissolve 4g NaOH in deionized water to 100mL.

4) the 2×concentrated redissolving solution isdiluted with deionized water at 1:1(1mL concentrated redissolving solution + 1mL deionized water), used for sample redissolving.

5) 50% Ethanol absolute: 1 part of Ethanol absolute + 1 part ofdeionized water, mix it evenly.

5.1 Samples preparation

         Feed

1) Weigh 1± 0.05g of the homogenized sample, add9mL of the50% Ethanol absolute,then add5mL n-Hexane, fully shake for 3min;

2) Centrifuge at above 4000r/min at room temperature (20-25 ℃) for 10min;

3) Take5mL middle-layer water phase into another50mL clean centrifuge tube, add0.5mL 1 M HCI and 100µL 2-Nitrobenzaldehyde (C7H5NO3),fully shake for 2min;

4) Incubate at 70℃ by water bath for 20 minutes;

5) Add 5mL 0.1M K2HPO4, 0.4mL 1M NaOH and10mL ethyl acetate,fullyshake for 30s;

6) Centrifuge at above 4000r/min at room temperature (20-25 ℃) for 10min;

7) Transfer1mL oftheethyl acetate layer into a newcentrifugal tubeand evaporate to dryness by nitrogen or air at 50℃.

8) Dissolve the dry residues in2mL N-hexane, add 1mL of the diluted redissolving solution, mix properly for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25 ℃) for 10 min; Remove up-layerN-hexane phase;

9) Take 200µl down-layer clear liquid and 800µlof the diluted redissolving solution, mix it evenly;

10) Take 50 µLliquid for analysis.

Fold of dilution of the sample:100

 

6. ELISA procedures

6.1Instructions 

1) Bring all reagents and micro-wellstrips to the room temperature (20-25 ℃) before use;

2) Return all reagentsto2-8 ℃ immediately after use;

3)The reproducibility of theELISA analysis, to alargedegree, depends on the consistencyof platewashing.The correct operationof platewashing is the keypoint inELISA theprocedures;

4)For theincubationatconstant temperatures, all the samples and reagents mustavoid light exposure, andeach microplate should be sealed by the covermembrane.

6.2Operation procedures

1. Bring test kit to the room temperature (20-25 ℃) for at least 30 min, note that each reagent must be shaken to mix evenly before use, put the required micro-well strips into plate frames. Re-sealed the unused microplate, storeat 2-8 ℃, not frozen.

2. Solution preparation: dilute 40mL of the 20 × concentrated washing buffer with deionized waterat 1:19 (1 part20 × concentrated washing buffer + 19 partsdeionized water). Or prepare as needed..

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

4. Add 50µL of thesampleorstandardsolution intoseparate duplicate wells;add 50 ul enzyme conjugatethen50 µL of the antibody working solution into each well,mix gently by shaking the plate manually. Seal the microplate with the cover membrane, andincubate at 25 ℃ for30min.

5. Pourliquid out of microwell, flap to dry onabsorbent paper,add 250 µL/well of washing buffer to wash microplate for 15-30 s, then take out and flap to dry withabsorbent paper, repeat4-5 times. (If there are thebubbles after flapping, cut them with the clean tips).

6. Coloration: add 50 µL of the substrate A and then 50 µL of thesubstrateB into each well. Mix gently by shaking the plate manually,then incubate at 25 ℃ for15min at dark for coloration. 

7. 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 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 AOZ concentration.

7.1 Qualitative determination

The concentration range (ng/mL)of AOZcan 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 0.02ppb, 1.415 for 0.06ppb, 0.74 for 0.18ppb, 0.313 for0.54ppb, 0.155 for1.62ppb, accordingly the concentration range of the sampleⅠ is0.54ppb to1.62ppb, and that of the sampleⅡ is 0.06ppb to 0.18ppb.

7.2 Quantitative determination

The mean values of the absorbance valuesisobtained 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 AOZ 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 AOZ concentration in the sample.

 

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, otherwise there will be the undesirable reproducibility.

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 lots to use.

6. Put the unused microplate into an auto-sealing bag to re-seal it. The standardsolutionand 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 of the standard solution 1(0 ppb) of less than 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.