Melamine ELISA Test Kit
Catalog No. LSY-10026
This test kit is based on the indirect competitive enzyme
immunoassay for the detection of Melamine in the sample. The
coupling antigen is pre-coated on the micro-well stripes. The
Melamine in the sample and the coupling antigens pre-coated on the
micro-well stripes compete for anti-Melamine antibodies. After the
addition of the enzyme conjugate, the TMB substrate is added for
coloration. The optical density (OD) value of the testing sample
has a negative correlation with the Melamine concentration in the
sample. This value is compared to the standard curve and the
Melamine concentration is subsequently obtained.
2. Technical specifications
Incubator temperature: 25℃
Incubator time: 30min～15min
Fresh milk, pure milk 5ppb
Yogurt, milk powder 10ppb
Fresh milk, pure milk 90±25%
Yogurt, milk powder 80±25%
12 strips with 8 removable
|2||6× standard solution (1 mL each)||0ppb||0.5ppb|
|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||20x sample extracting solution||50ml||transparent cap|
|10||10x sample dilution||50ml||blue cap|
4. Materials required but not provided
- Equipments: microplate reader, printer, vortex, centrifuge, measuring pipets,
balance( a reciprocal sensibility of 0.01 g).
- Micropipettors: single-channel 20~200 µL, 100~1000 µL; and multi-channel 30～300
5. Sample pre-treatment
Instructions (The following points must be dealt with before the pre-treatment
1) Only the disposable tips can be used for the experiments and the
tips must be changed when used for different reagents;
- 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.
A. Sample extracting solution
Dilute 20x sample extracting solution with deionized water at 1:19;
B. Sample dilution
Dilute 10x sample dilution with deionized water at 1:9.
5.1 Milk(pure milk, fresh milk)
1. Take 50ul fresh milk for analysis
Fold of dilution of sample: 1
5.2 Milk powder
1 Take 1 g fresh milk powder into 50 ml centrifuge tube, add 9ml
Sample extracting solution, vortex and shake for 3min, centrifuge
at above 4000 r/min at 15 ℃ for 10 min;
2 Leave aside the top layer of condensed protein fat, take 50ul of
the middle layer liquid for analysis
Fold of dilution of sample: 10
1 Take 1 g fresh yogurt sample into 10ml/15ml/50 ml centrifuge
tube, add 4.5ml Sample dilution, vortex and shake for 2min;
2 Take 50µL upper-layer liquid for analysis
Fold of dilution of sample: 5
6. ELISA procedures
- Bring all reagents and micro-well strips to the room temperature
- 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 the procedures of ELISA.
- 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.
- Solution preparation: dilute 15mL of the 20Xconcentrated washing
buffer with the deionized water to 300mL for use;
- 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/standard solution to separate duplicate
wells, then add 50µL enzyme conjugate, then add 50µL antibody
working solution to each well, shake properly, seal the microplate
with the cover membrane, and incubate at 25 ℃ for 30 min;
- Pour the liquid out of the wells, wash the microplate with the
diluted washing buffer at 250 µL/well for 5-6 times. Each time soak
the well with the diluted washing buffer for 15-30 seconds and then
flap to dry on absorbent paper (if there are the bubbles after
flapping, cut them with the clean tips).
- Coloration: add 50µL of the substrate A, then 50 µL of the
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 the content of Melamine.
7.1 Qualitative determination
The concentration range (ng/mL) can be obtained from comparing the
average OD value of the testing 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:
2.043 for 0ppb, 1.604 for 0.5ppb, 1.101 for 1.5ppb, 0.512 for
4.5ppb, 0.161 for 13.5ppb, 0.055 for 40.5ppb, accordingly the
concentration range of the sampleⅠ is 4.5 to 13.5ppb, and that of
the sample Ⅱ is 1.5 to 4.5ppb.
- Quantitative determination
The mean values of the absorbance values is equivalent to the
percentage of the average OD value (B) of the testing 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 testing sample or the
B0—the average OD value of the 0ng/mL standard solution
Draw the standard curve with the absorption percentages of the
standard solutions and the semilogarithmic values of the Melamine
standard solutions (ng/mL) as Y- and X-axis, respectively. Read the
corresponding concentration of the testing 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 Melamine
concentration in the sample.
- The room temperature below 25℃ or the temperature of the reagents
and the testing 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; So continue to next
step immediately after washing.
- Mix evenly, otherwise there will be the undesirable
- The stop solution is the 2 M sulfu ric 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 standard
solution 1(0 ppb)of less than 0.5 indicates its degeneration.
- The optimum reaction temperature is 25 ℃, and too high or low
temperatures will result in the changes in the detecting
sensitivity and OD values.
9. Storage and expiry date
Storage: stored 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.