Nitrofuran
(AOZ) ELISA Test Kit
Catalog
No.: LSY-10002
1. Principle
This test kit is based on the competitive enzyme immunoassay for the
detection of AOZ in the sample. The coupling antigens are pre-coated on the micro-well
stripes. The AOZ in the sample and the coupling antigens pre-coated on the micro-well
stripes compete for the anti-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 AOZ concentration is
subsequently obtained.
2. Technical specifications
Sensitivity: 0.02ppb
Incubation
Temperature: 25℃
Incubation
Time: 30min～15min
Detection
limit Tissue, egg: 0.1ppb
Cross-reaction
rate
AOZ................................................................................................ 100%
AMOZ............................................................................................ <0.1%
AHD.............................................................................................. <0.1%
SEM.............................................................................................. <0.1%
Recovery
rate
Tissue, egg.................................................................................. 95±25%
3. Components
|
1
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Micro-well strips
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12 strips with 8 removable
wells each
|
|
2
|
7× standard solution (1mL each)
|
0ppb
|
0.02ppb
|
|
0.06ppb
|
0.18ppb
|
|
0.54ppb
|
1.62ppb
10ppb
|
|
3
|
Enzyme conjugate
|
7ml
|
red cap
|
|
4
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Antibody working solution
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7ml
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blue cap
|
|
5
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Substrate A
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7ml
|
white cap
|
|
6
|
Substrate B
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7ml
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black cap
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7
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Stop solution
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7ml
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yellow cap
|
|
8
|
20× concentrated washing buffer
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40ml
|
white cap
|
|
9
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2× concentrated
redissolving solution
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50ml
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transparent cap
|
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10
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2-Nitrobenzaldehyde
(C7H5NO3)
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10ml
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black cap
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4.
Materials required but not provided
1） Equipment: microplate reader, printer, homogenizer, nitrogen-drying device,
vortex, centrifuge, measuring pipets, balance (a reciprocal sensibility of 0.01
g), incubator, water bath;
2） Micropipette: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30～300 µl;
3） Reagents: NaOH, ethyl acetate, n-Hexane, HCI (approx 36.5%), K2HPO4·3H2O
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) 0.1 M K2HPO4 : dissolve 11.4 g K2HPO4·3H2O in
deionized water to 500mL.
2) 1 M HCl: dissolve
8.6mL HCI (approx 36.5%) in
deionized water to 100mL.
3) 1 M NaOH: dissolve 4g NaOH in
deionized water to 100mL.
4) the
2×concentrated redissolving solution is diluted with deionized water at 1:1(1mL
concentrated redissolving solution + 1mL deionized water), used for sample
redissolving.
5.1 Samples preparation
Tissue, egg
1) Weigh 1± 0.05g of the homogenized sample, add 4mL of the
distilled water, 0.5mL 1 M HCI and 100µL 2-Nitrobenzaldehyde (C7H5NO3) to each tube, shake properly for 2min;.
2) Incubate at 70℃ by
water bath for 20 minutes(Or incubate in 75℃ incubator for 25min).
3) Add 5mL 0.1M K2HPO4, 0.4mL 1M NaOH and 6mL ethyl acetate to each tube, shake
for 30s.
4) Centrifuge at above 4000r/min at
room temperature (20-25℃) for 5min (if there is Emulsification or ethyl acetate layer is not enough for 3ml, incubate sample
at 80℃ water bath for 10min and centrifuge repeatedly; or increase speed
and extend time of centrifuge).
5) Transfer 3mL of the ethyl acetate layer
(up-layer liquid) into a new centrifugal tube and evaporate to dryness by
nitrogen or air at 50℃.
6) Dissolve the dry residues in 2mL
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 5 min;
Remove up-layer N-hexane phase (if there is Emulsification, after removing up-layer N-hexane phase, incubate sample at 70℃ water bath
for 10-20min, centrifuge repeatedly).
7) Take 50 µL of the lower for
analysis.
Fold of dilution of the sample: 2
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 ELISA the procedures;
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. 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 40mL
of the 20 × concentrated washing buffer with deionized water at 1:19 (1 part 20
× concentrated washing buffer + 19 parts deionized 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 50 µL of
the sample or standard solution into separate duplicate wells; add 50 ul enzyme conjugate then 50 µL of the antibody working
solution into each well, mix gently by shaking the plate manually. Seal the
microplate with the cover membrane, and incubate
at 25 ℃ for 30min.
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 4-5 times. (If there are the bubbles
after flapping, cut them with the clean tips).
6. Coloration: add 50 µL of the
substrate A and then 50 µL of the substrate B into each well. Mix gently by
shaking the plate manually, 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 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 AOZ 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 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 for 0.54ppb, 0.155 for
1.62ppb, accordingly the concentration range of the sampleⅠ is 0.54ppb to
1.62ppb, and that of the sampleⅡ is 0.06ppb to 0.18ppb.
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,
|
Percentage
of absorbance value =
|
B
|
×100%
|
|
B0
|
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 standard solution 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 of the standard solution 1(0 ppb) of less than 0.5 indicates its
degeneration.
8. 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.
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
www.lsybt.com