Nitrofuran (AMOZ) ELISA Test Kit

Nitrofuran (AMOZ) ELISA Test Kit

Nitrofuran (AMOZ) ELISA Test Kit

Brand: Green Spring
1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of AMOZ in the
sample. The coupling antigens are pre-coated on the micro-well stripes. The AMOZ in the sample
and the coupling antigens pre-coated on the micro-well stripes compete for the anti-AMOZ
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 AMOZ in it. This
value is compared to the standard curve and the AMOZ concentration is subsequently obtained.
2. Technical specifications
Sensitivity: 0.1 ppb
Detection limit
Tissue(Chicken, duck, porcine meat, beef and mutton).......................0.2 ppb
Liver(Chicken, duck, porcine meat, beef and mutton)..........................0.2 ppb
Honey, milk, intestine............................................................................0.2 ppb
Fish, shrimp(some interference)...........................................................0.3 ppb
Recovery rate
Tissue, liver......................................................................................... 90±15%
Honey, milk, intestine.......................................................................... 80±15%
Cross-reaction rate
AMOZ...................................................................................................... 100%
AHD....................................................................................................... <0.1%
AOZ........................................................................................................<0.1%
SEM....................................................................................................... <0.1%
3. Components
1? Micro-well strips: 12 strips with 8 removable wells each
2? 6× standard solution (1 mL each): 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb and 8.1 ppb
3? Enzyme conjugate (7 mL)................................................................... red cap
4? Antibody working solution (7 mL).......................................................blue cap
5? Substrate A solution (7 mL).............................................................. white cap
6? Substrate B solution (7 mL)..............................................................black cap
7? Stop solution (7 mL)....................................................................... yellow cap
8? 20× concentrated washing buffer (40 mL)....................................... white cap
9? 2× concentrated redissolving solution (50 mL)...................... transparent cap
10? 2-Nitrobenzaldehyde (C7H5NO3)(10 mL)......................................... white 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);
2? Micropipettors: single-channel 20-200 μL, 100-1000 μL, and multi-channel 250 μL;

3? Reagents: NaOH, ethyl acetate, n-Hexane, HCI (approx 36.5%), K2HPO4·3H2O(for all
samples), K2Fe(CN)5NO·3H2O and ZnSO4·7H2O (for milk sample)
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) The 2×concentrated redissolving solution is diluted with deionized water at 1:1(1mL
concentrated redissolving solution + 1 mL deionized water), used for sample redissolving.
2) C solution (for milk sample): dissolve 12.5 g K2Fe(CN)5NO·3H2O in deionized water to 100 mL.
3) D solution (for milk sample): dissolve 29.8 g ZnSO4·7H2O in deionized water to 100 mL.
4) 0.1 M K2HPO4 :dissolve 22.8 g K2HPO4·3H2O in deionized water to 1 L.
5) 1 M HCl: dissolve 8.6 mL HCI (approx 36.5%) in deionized water to 100 mL.
6) 1 M NaOH: dissolve 4 g NaOH in deionized water to 100 mL.
5.1 Samples preparation
a) Tissue, intestine, liver
1.Homogenize the sample, continue as described in (1-7, d).
b) milk
1. Put 5 mL sample into centrifuge tube; add C and D solution, 250 μL each.
2. Mix thoroughly, use vortex, centrifuge at above 4000 r/min at 4-12 ? for 10 min with centrifuge
of constant temperatures, if centrifuge of constant temperature is not available, chill sample
temperature to approx 8 ?, then centrifuge.
3. Continue as described in (1-7,d).
C) honey
1. Weigh 1± 0.05 g into centrifuge tube.
2. Dissolve in 4 mL of the deionized water, then add 0.5 mL 1 M HCI and 100 μL 2-
Nitrobenzaldehyde solution, mix thoroughly.
3. Continue as described in (2-7, d).
d) continue above steps
1. Weigh 1± 0.05 g of the homogenized sample (tissue, intestine or liver), or put 1.1 mL of the
centrifuged supernatant (equivalent to 1mL milk sample) in a plastic tube, add 4 mL of the
deionized water, 0.5 mL 1 M HCI and 100 μL 2-Nitrobenzaldehyde (C7H5NO3) to each tube,
shake properly.
2. Incubate at 37 ? over night ( approx 16 hours) or incubate at 56? by water bath(2 hours).
3. Add 5 mL 0.1 M K2HPO4, 0.4 mL 1 M NaOH and 5 mL ethyl acetate to each tube, shake
strongly for 5 min.
4. Centrifuge at above 4000 r/min at room temperature (20-25 ?) for 10 min.

5. Transfer 2.5 mL of the ethyl acetate layer (upper layer) into a new centrifugal tube and
evaporate to dryness by nitrogen or air at 50 ?.
6. Dissolve the dry residues in 1 mL 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.
7. Take 50 μL of the lower for analysis.
Fold of dilution of the sample: 2
6. ELISA 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, store at 2-8 ?, not frozen.
2. Solution preparation: dilute 40 mL of the 20 × concentrated washing buffer with the distilled or
deionized water at 1:19 to 800 mL (or just to the required volume) for use.
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 50 μL of the sample or standard solution into separate duplicate wells; add 50 ul enzyme
conjugate and 50 μL of the antibody working solution into each well, mix gently by shaking the
plate manully. Seal the microplate with the cover membrane, and incubate at 25 ? for 1 h.
5. Pour liquid out of microwell, flap to dry on absorbent paper, add 250 μL/well of washing buffer to
wash microplate for 15-30 s, then take out and flap to dry with absorbent paper, repeat 5 times
6. Coloration: add 50 μL of the substrate A solution and then 50 μL of the B solution into each well.
Mix gently by shaking the plate manully, then incubate at 25 ? for 15 min at dark for coloration.
7. Determination: add 50 μL of the stop solution into each well. Mix gently by shaking the plate
manully. 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).
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 AMOZ concentration.
7.1 Qualitative determination
The concentration range (ng/mL) of AMOZ can be obtained from comparing the average OD value
of the sample with that of the standard solution. Assuming that the OD value of the sample? is
0.238, and that of the sample? is 1.130, the OD value of standard solutions is: 1.721 for 0 ppb,
1.454 for 0.1 ppb, 1.153 for 0.3 ppb, 0.667 for 0.9 ppb, 0.295 for 2.7 ppb, 0.106 for 8.1 ppb,
accordingly the concentration range of the sample? is 2.7 to 8.1 ppb, and that of the sample? is
0.3 to 0.9 ppb.
7.2 Quantitative determination

The mean values of the absorbance values is obtained 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 AMOZ 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 AMOZ 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. Bring all reagents and micro-well strips to balance at 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.
5. 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.
6. 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.
7. Mix evenly, otherwise there will be the undesirable reproducibility.
8. The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.
9. 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.
10. Put the unused microplate into an auto-sealing bag to re-seal it. The standard solution and the
colourless color former is light sensitive, and thus they cannot be directly exposed to the light.
11. 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.
12. The optimum reaction temperature is 25 ?, 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.

 Shenzhen Lvshiyuan Biotechnology Co.,Ltd

www.lsybt.com

Green Spring Brand

Product Origin: China
Model Number: LSY-10001
Brand Name: GREEN SPRING
   
   

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Company Name: Shenzhen Lvshiyuan Biotechnology Co.,Ltd
Contact Person: Bella Zou
Address:
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Zip: 518120
Telephone:
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E-mail: info@lsybt.com
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