Shenzhen Wensidun Technology Co., Ltd. |
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MQCA ELISA Test Kit for Chicken Pork Duck 96 Wells/Kit Sensitivity 0.5 ppb
1. Principle
This test kit is based on the competitive enzyme immunoassay for
the detection of MQCA in the sample. The coupling antigens are
pre-coated on the micro-well stripes. The MQCA in the sample and
the coupling antigens pre-coated on the micro-well stripes compete
for the anti-MQCA 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 MQCA in it. This value is compared to the standard curve and
the MQCA concentration is subsequently obtained.
2. Technical specifications
Sensitivity:0.5 ppb
Incubator temperature: 25℃
Incubator time: 30min~15min
Detection limit:
Tissue 1 ppb
Cross-reaction rate:
MQCA 100%
Olaquindox <0.1%
Recovery rate:
Tissue 85%±10%
3. Components
1 | Micro-well strips | 12 strips with 8 removable wells each | |
2 | 6× standard solution (1 mL each) | 0 ppb | 0.5 ppb |
1.5 ppb | 4.5 ppb | ||
13.5 ppb | 40.5 ppb | ||
3 | Concentrated Enzyme conjugate | 1 ml | red cap |
4 | Antibody working solution | 8 ml | blue cap |
5 | Substrate A | 7 ml | white cap |
6 | SubstrateB | 7 ml | black cap |
7 | Stop solution | 7 ml | yellow cap |
8 | Redissolving solution | 50 ml | transparent cap |
9 | 20× concentrated washing buffer | 40 ml | white cap |
4. Materials required but not provided
1) Equipments:microplate reader (450 nm / 630 nm), homogenizer,
oscillator, centrifuge, measuring pipets, nitrogen-drying device
and balance (a sensibility reciprocal of 0.01 g), Incubator.
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, HCl, NaCl, CH3CN
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) Sample extract (Store at 2~8℃for 1 month): Take 8.6ml
concentrated HCl, add deionized water to 500ml, then add 10g NaCl
to get the sample extract.
5.1Livestock and poultry tissue(Chicken, duck, pork):
1. Take 2± 0.05 g of the homogenized sample into 50ml centrifuge
tube;
2. Add 4 mL Sample extract, shake thoroughly for 1min;
3. Add 4ml Ethyl acetate and 2ml CH3CN, shake properly for 10s;
Centrifuge at 4000 r/min at room temperature (20 - 25 ℃) for 10 min
(Note: This step only need shake for 10s, shake too long will lead
toEmulsification, ifEmulsification appeared, take a certain
supernatant, then shake tube lightly for 5s, centrifuge again then
can get enough supernatant);
4. Take 3 mL up-layer organic phase into a clear container, below
to dry with Nitrogen or air at
56℃water bath;
5. Add 1ml N-hexane, shake for 30s, then add 0.5ml Redissolving
solution, shake and mix thoroughly for 30s; centrifuge at 4000
r/min at room temperature (20-25 ℃) for 5 min.
6. Remove the up-layer organic phase.Take 50 µL down-layer for
analysis.
Fold of dilution of the sample:0.5
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 40 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) Prepare mix solution of antibody working solution and
Concentrated Enzyme conjugate: Mix antibody working solution and
Concentrated Enzyme conjugate at 10:1 evenly (1000ul antibody
working solution + 100ul Concentrated Enzyme conjugate, this mix
solution should be used once prepared, it can not be stored, the
package quantity has extra amount, prepare as proportion is OK.)
6) Add 20 µL the sample or standard solution to separate duplicate
wells, then add 70 µL mix solution of antibody working solution and
Concentrated Enzyme conjugate into each well. Mix by shaking
gently, seal the microplate with the cover membrane,and incubate
at25℃ at darkfor30 min;
7) 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);
8) Coloration: add 50 µL of the substrate A solution and 50 µL of
the B solution into each well. Mix by shaking gently,and incubate
at25 ℃ for15 min in the dark for coloration;
9) Determination: add 50 µL stop solution into each well. Mix by
shaking gently. Set the wavelength of the microplate reader at 450
nm to determine the OD value. (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 the content of MQCA.
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 0.3, and
that of the sample Ⅱ is 1.0, while those of the standard solutions
are as the followings: 2.043 for 0 ppb, 1.716 for 0.5 ppb, 1.215
for 1.5 ppb, 0.74 for 4.5 ppb, 0.313 for 13.5 ppb and 0.155 for
40.5 ppb, accordingly the concentration range of the sampleⅠis 13.5
to 40.5ppb, and that of the sample Ⅱ is 1.5 to 4.5 ppb.
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,
Percentage of absorbance value = | B | ×100% |
B0 |
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 MQCA
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 MQCA 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 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
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. 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 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.
Q1: When will it be shipped?
A1: We will ship the goods for you as soon as possible within 7
working days after receiving the payment. (In case of external
factors such as the epidemic, the delivery may be delayed)
Q2: Does it support OEM/ODM?
A2: It can be supported, but the specific quantity needs to be more
than 100,000 pieces, which is convenient for customized products.
Q3: How is your factory doing in terms of quality control?
A3: We have nationally certified ISO9001 and ISO13485. Our
production process conforms to standard procedures to ensure
optimum product quality.
Q4: How to provide after-sales service?
A4: We provide professional online technical after-sales service.
We can provide you with one-on-one guidance via video, telephone,
etc.
Q5: What is the payment method?
A5: We receive payment by T/T.
Q6: How to ship?
A6: Choose the best shipping method for you by getting quotes from
our many cooperative carriers, and also ship according to your
requirements.