β-agonists ELISA Test Kit
Catalog No. LSY-10036
The GreenSpring®β-agonists test kit is based on the competitive enzyme immunoassay
for the detection of β-agonists in the sample. The coupling antigen
is pre-coated on the micro-well stripes. The β-agonists in the
sample and the coupling antigens pre-coated on the micro-well
stripes compete for the anti-β-agonists 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 β-agonists in the sample. This value
is compared to the standard curve and the β-agonists residues is
2. Technical specifications
Sensitivity: 0.1 ppb
Incubation Temperature: 25℃
Incubation Time: 30min～15min
Porcine urine 0.3ppb
Porcine urine, pork 90%±30%
12 strips with 8 removable
|2||6× standard solution (1 mL each)||0ppb||0.1ppb|
|3||Enzyme conjugate||7ml||red cap|
|4||Antibody working solution||10ml||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||40ml||white cap|
|9||5× sample extracting solution||50ml||transparent cap|
4. Materials required but not provided
- Equipments: microplate reader, printer, homogenizer, vortex, oscillator,
centrifuge, Incubator, measuring pipets, balance (a sensibility
reciprocal of 0.01 g)
- Micropipettors: single-channel 20-200µL, 100-1000µL, and multi-channel 30-300µL;
- Reagents: NaOH, HCI.
5. Sample pre-treatment
Instructions (The following points must be dealt with before the pre-treatment
- Only the disposable tips can be used for the experiments and the
tips must be changed when used for absorbing different reagents;
- Before the experiment, each experimental utensil 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:
- 0.2M HCI: dissolve 17.2mL HCI in deionized water to 1L.
- 1M NaOH: dissolve 4g NaOH in deionized water to 100 mL.
3) Sample extracting solution: 1 part 5X sample extracting solution
+ 4 parts deionized water, mix evenly.
5.1 Porcine urine
Take 20 µL clear urine, directly detect it (If urine are muddy,
must filter or centrifuge at 4000 r/min for 10 min, then take clear
urine). Store at frozen environment if don’t use.
Fold of dilution of sample: 1
- Weigh 2±0.05g homogenized tissue sample into a 50ml centrifuge
tube, add 3ml 0.2M HCl, shake for 3 min.
- Then add 600ul 1M NaOH solution and 2.4ml Sample extracting
solution, shake for 3min, centrifuge at 4000 r/min at room
temperature (20-25 ℃) for 10 min.
- Take 20µL up-layer liquid for analysis.(Note: if there is fat layer
after centrifuge, remove fat layer or separate fat layer, take
clear liquid for analysis)
Fold of dilution of sample: 4
6. ELISA procedures
- Bring all reagents and micro-well strips to the room temperature
(20-25 ℃) before use;
- Return all reagents to 2-8 ℃ immediately after use;
- 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;
- 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
- Bring test kit to the room temperature (20-25 ℃) for at least 30
min, note that each reagent must be shaken evenly before use;
- Put the required micro-well strips into plate frames. Re-sealed the
unused microplate, stored at 2-8 ℃, not frozen.
- Dilute the 40ml 20X concentrated washing buffer at 1:19 with
deionized water (1 part 20X concentrated washing buffer + 19 part
deionized water). Or prepare 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 20µL of the sample or the standard solution into separate
duplicate wells, then add enzyme conjugate, 50ul/well; then add
antibody working solution, 80µL/well. Mix gently by shaking the
plate manually, seal the microplate with the cover membrane, incubate at 25 ℃ for 30 min.
- Pour liquid out of microwell, add 250µL/well of washing buffer,
wash for 4-5 times, 15-30s each time, then take out and flap to dry
with absorbent paper(if there are the bubbles after flapping, cut
them with the clean tips).
- Coloration: add 50µL of substrate A, then add 50µL substrate B into
each well. Mix gently by shaking the plate manually, and incubate at 25 ℃ for 15 min in the dark for coloration.
- Determination: add 50µL 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 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 β-agonists concentration in the sample.
7.1 Qualitative determination
The concentration range (ng/mL) obtained from comparing 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, the OD value of standard solutions is: 2.243 for 0ppb,
1.816 for 0.1ppb, 1.415 for 0.3ppb, 0.74 for 0.9ppb, 0.313 for
2.7ppb, 0.155 for 8.1ppb, accordingly the concentration range of
the sample Ⅰ is 2.7 to 8.1ppb, and that of the sample Ⅱ is 0.3 to
7.2 Quantitative determination
The mean values of the absorbance values 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%|
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 standard
solutions and the semilogarithm values of β-agonists 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 dilution
fold, finally obtaining β-agonists concentration in the sample.
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
4.The stop solution is the 2 M sulfuric acid solution, avoid
contacting with the skin.
- 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.
- 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.
- Discard the colouration solution with any color that indicates the
degeneration of this solution. The detecting value of the 0
standard solution (0 ppb) of less than 0.5 indicates its
- 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.
Remarks: If the vacuum package of microtiter plates has leakage,
the microtiter plate is normal and effective, do not affect the
experimental result. Please feel free to use.