Tetracyclines(TCs) ELISA Test Kit
Catalog No. LSY-10006-2
This test kit is based on the competitive enzyme immunoassay for
the detection of Tetracyclines in the honey sample. The coupling
antigens are pre-coated on the micro-well stripes. The
Tetracyclines in the sample and the coupling antigens pre-coated on
the micro-well stripes compete for the anti-Tetracyclines 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 Tetracyclines in it. This value
is compared to the standard curve and the Tetracyclines
concentration is subsequently obtained.
2. Technical specifications
Sensitivity: 0.1 ppb
Incubation Temperature: 25℃
Incubation Time: 30min—15min
12 strips with 8 removable
|2||6× standard solution (1mL each)||0ppb||0.1ppb|
|3||Enzyme conjugate||7ml||red cap|
|4||Antibody working solution||7ml||blue cap|
|5||Substrate A||7ml||white cap|
|6||Substrate B||7ml||black cap|
|7||Stop solution||7ml||yellow cap|
|8||20× concentrated washing buffer||15ml||white cap|
|9||20× sample extract A||15ml||yellow cap|
|10||2× sample extract B||50ml*2||transparent cap|
|11||20× sample diluent||10ml||black cap|
4. Materials required but not provided
- Equipments: microplate reader, homogenizer, oscillator, centrifuge, balance
(a sensibility reciprocal of 0.01 g), graduated pipette, incubator.
- Micropipettors: single-channel 20~200 µL and 100~1000 µL, and multi-channel
- Reagents: NaOH.
5. Sample pre-treatment
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) Sample extract A
1 part of 20× sample extract A + 19 parts of deionized water
2) Sample extract B
1 part of 2× sample extract B + 1 part of deionized water
3)1M NaOH solution
Weigh 4g NaOH, add deionized water to 100ml
4) Sample diluent
1 part of 20× sample diluent + 19 parts of deionized water
1) Take 2± 0.05 g of the honey sample into 50 mL centrifuge tube,
add 3mL diluted Sample extract A, shake for 3min;
2) Then add 600ul 1M NaOH solution and 2.4ml diluted Sample extract
B, shake for 3min; centrifuge at above 4000 r/min at room
temperature (20 - 25 ℃) for 5 minutes.
3) Take 50ul up-layer clear liquid, add 450ul diluted Sample
diluent, mix it evenly;
4) Take 50ul above mixed solution for analysis.
Fold of dilution of the sample: 40
6. ELISA procedures
1. Bring all reagents and micro-well strips to the room temperature
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.
- Take out all the necessary reagents from the kit and place at the
room temperature (20 to 25 ℃) for at least 30 minutes. Note that
each liquid reagent must be shaken to mix evenly before use.
- Take the required micro-well strips and plate frames. Re-sealed the
unused microplate, stored at 2-8 ℃, not frozen.
- Washing buffer preparation: dilute 15 mL of the 20× concentrated
washing buffer with the deionized water at 1:19 (1 part 20×
concentrated washing buffer + 19 parts deionized water ). Or
prepare washing buffer as quantity needed.
- Numbering: number the micro-wells according to samples and standard
solution; each sample and standard solution should be performed in
duplicate; record their positions.
- Add 50µL of the sample or standard solution to separate duplicate
wells, and add 50ul Enzyme conjugate then 50 µL of the antibody
solution into each well. Mix gently by shaking the plate manually,
seal the microplate with the cover membrane, and incubate at 25℃ for 30 minutes.
- Pour liquid out of microwell, add 250 µL/well of washing buffer for
15-30 seconds, repeat four to five times, then flap to dry (if
there are the bubbles after flapping, cut them with the clean
- 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, and incubate at 25 ℃ for 15 minutes at dark for coloration.
- 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 (Recommend to
read the OD value at the dual-wavelength 450/630 nm within 5
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 Tetracyclines in the sample.
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: 2.243 for 0 ppb, 1.816 for 0.1 ppb, 1.415
for 0.3 ppb, 0.74 for 0.9 ppb, 0.313 for 2.7 ppb and 0.155 for 8.1
ppb, accordingly the concentration range of the sampleⅠis 2.7 to
8.1ppb, and that of the sample Ⅱ is 0.3 to 0.9ppb.
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%|
B—the average (double wells) OD value of the sample or the standard
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
Tetracyclines 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 Tetracyclines 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)
- 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.
- Dryness of the microplate in the washing process will be
accompanied by the situations including the non-linear standard
curves and the undesirable reproducibility.
- Mix every reagent and reaction mixture evenly and wash the
microplate thoroughly, otherwise there will be the undesirable
- The stop solution is the 2 M sulfuric acid solution, avoid
contacting with the skin.
- 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.
- 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.
- 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.
- 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.