Melamine ELISA test kit

Melamine ELISA test kit

Melamine ELISA Test Kit   

Brand: Green Spring
1. Principle
This test kit is based on the 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
Sensitivity : 15 ppb
Detection limit:
Milk.......................................................................................................300 ppb
Tissue(chicken, pork, duck, fish, shrimp and liver).............................. 150 ppb
Feed...................................................................................................1500 ppb
Egg.......................................................................................................300 ppb
Recovery rate:
Milk...................................................................................................... 95±15%
Tissue.................................................................................................. 85±10%
Feed.....................................................................................................85±10%
Cross-reaction rate:
Melamine................................................................................................. 100%
Cyanuric acid............................................................................................. 60%
Triazine...................................................................................................... <1%
Diamino atrazine........................................................................................<1%
3. Components
1? Micro-well strips: 12 strips with 8 removable wells each
2? 6× standard solution (1 mL each): 0 ppb,15 ppb, 45 ppb, 135 ppb, 405 ppb, 1215 ppb
3? Enzyme conjugate (12 mL)...................................................................red cap
4? Antibody working solution (7 mL)........................................................ blue cap
5? Substrate A solution (7 mL)............................................................... white cap
6? Substrate B solution (7 mL)...............................................................white cap
7? Stop solution (7 mL).........................................................................yellow cap
8? 20× concentrated washing buffer (40 mL).........................................white cap
9? 5× concentrated redissolving solution (50 mL)....................... transparent cap
4. Materials required but not provided
1) Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex,
centrifuge, measuring pipets, balance( a reciprocal sensibility of 0.01 g).

2) Micropipettors: single-channel 20~200 μL, 100~1000 μL; and multi-channel 250 μL;
3) Reagents: HCl, NaOH, Acetonitrile(CH3CN), N-hexane
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;
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) The 5×concentrated redissolving solution is diluted with deionized water at 1:4 (1 mL
concentrated redissolving solution + 4 mL deionized water)
2) 1 M HCl: dissolve 8.6 mL HCl(approx 36.5%) in the deionized water to 100 mL.
3) 0.1 M NaOH solution: dissolve 0.4 g NaOH in the deionized water to 100 mL.
4) 1 M NaOH solution: dissolve 4 g NaOH in the deionized water to 100 mL.
5) Acetonitrile-0.1 M NaOH solution: Mix 84 mL Acetonitrile with 16 mL 0.1 M NaOH solution.
5.1 milk
1 Take 2 mL milk sample(without fat) to tube;
2 Add 8 mL Acetonitrile-0.1 M NaOH solution, mix throughly for 10 min. Centrifuge at above 4000
r/min at 15 ? for 10 min.
Take 50 μL for analysis
Fold of dilution of sample :40
5.2 meat, liver( chicken)
1 Take 2±0.05 g homogenized sample(remove fat), add 6 mL 0.5% Trichloroacetic acid and 2 mL
Acetonitrile. Mix for 5 min.
2 Centrifuge at above 4000 r/min at room temperature for 10 min.
3 Transfer 2 mL supernatant into a new vessel, add 2 mL N-hexane, mix properly. Static for 3 min.
Take 0.5 mL clear solution(lower layer). Centrifuge at above 4000 r/min for 5 min at room
temperature.
3 Take 50 μL clear solution into a new vessel(If have two layers, remove upper layer liquid), add
450 μL of the diluted redissolving solution, mix evenly.
4 Take 50 μL for analysis.
Fold of dilution of sample :40
5.3 honey, Royal jelly
1 Weigh 2±0.05 g honey sample, add 4 mL of 0.1 M H3PO4, shake properly for 10 min.
2 Centrifuge at above 4000 r/min at room temperature (20-25 ?) for 5 min, until liquid is clear.
3 Add 900 μL 1 M NaOH, adjust PH to 7-9(For Royal jelly, Transfer the Supernatant to a new
vessel).
4..Centrifuge at above 4000 r/min at room temperature (20-25 ?) for 5 min, until liquid is clear.

5 Take 50 μL suppernatant, add 350 μL of the diluted redissolving solution, mix evenly.
6 Take 50 μL for further analysis.
Fold of dilution of sample :20
6. ELISA procedures
1 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. Resealed
the unused microplate, stored at 2-8 ?, not frozen.
2 Solution preparation: dilute 40 mL of the concentrated washing buffer (20×concentrated) with
the distilled or deionized water to 800 mL (or just to the required volume) for use;
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 and standard solution to separate duplicate wells, then add 50 μL of
antibody working solution to each well, shake properly, seal the microplate with the cover
membrane, and incubate at 37 ? for 30 min;
5 Pour liquid out of microwell, flap to dry on absorbent paper; add 250 μL/well of washing buffer
for 15-30 seconds, then take out and flap to dry with absorbent paper, repeat 5 times.
6 Add 100 μL of enzyme conjugate to each well, shake properly, seal the microplate with the
cover membrane, and incubate at 37 ? for 30 min; continue as step 5.
7 Coloration: add 50 μL of the substrate A solution, 50 μL of the B solution into each well. Mix
gently by shaking the plate manually, and incubate at 37 ? for 15 min in the dark for coloration;
8 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 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 sample? is
0.3, and that of the sample? is 1.0, the OD value of standard solutions is: 2.243 for 0 ppb, 1.816
for 0.1 ppb, 1.415 for 0.4 ppb, 0.74 for 1.6 ppb, 0.313 for 6.4 ppb, 0.155 for 25.6 ppb, accordingly
the concentration range of the sample? is 6.4 to 25.6 ppb, and that of the sample ? is 0.4 to
1.6 ppb.
7.2 Quantitative determinationThe 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
B—the average (double wells) OD value of the testing sample or the standard solution
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.
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 Bring all reagents and micro-well strips to the room temperature (20-25?).
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.
5 The room temperature below 20? 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.
6 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.
7 Mix evenly, otherwise there will be the undesirable reproducibility.
8 The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.
9 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.
10 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.
11 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.

12 Colouration time is about 20 min, if the color is light, prolong the time of colouration but don’t
exceed 30 min.
13 The optimum reaction temperature is 37 ?, 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

 Shenzhen Lvshiyuan Biotechnology Co.,ltd   www.lsybt.com

Green Spring Brand

Product Origin: China
Model Number: LSY-10026
Brand Name: GREEN SPRING
   
   

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Company Name: Shenzhen Lvshiyuan Biotechnology Co.,Ltd
Contact Person: Bella Zou
Address:
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Zip: 518120
Telephone:
+86-755-28438788
Fax: +86-755-28938800
E-mail: info@lsybt.com
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