Melamine ELISA Test Kit for Detection Milk 96Wells/Kit ISO9001

1. Principle

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. Milk Test Kit Components

3. Technical specifications

Sensitivity: 0.5ppb

Incubator temperature: 25℃

Incubator time: 30min～15min

Detection limit:

Butter, candy, cheese 10ppb

Cross-reaction rate:

Melamine 100%

Recovery rate:

Butter 90±25%

Candy, cheese 95±25%

4. Materials required but not provided

1) Equipments:microplate reader, printer, vortex, centrifuge, measuring pipets, balance( a reciprocal sensibility of 0.01 g), water bath of 70℃;

2) Micropipettors: single-channel 20~200 µL, 100~1000 µL; and multi-channel 30～300 μl;

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

1) Sample extracting A solution

Dilute 10x sample extracting A solution with deionized water at 1:9;

2) Sample extracting B solution

Dilute 20x sample extracting B solution with deionized water at 1:19.

5.1 Butter

1) Take 1 g fresh butter into 50 ml centrifuge tube, add 5ml Sample extracting A solution, incubate at water bath of 70℃for 5min, vortex andshake for 5min, centrifuge at above 4000 r/min at room temperature for 5 min;

2) Take 50ul down-layer liquid for analysis

Fold of dilution of sample: 5

5.2 Candy, cheese

1) Take 1 g fresh sample into 50 ml centrifuge tube, add 5ml Sample extracting B solution, incubate at water bath of 70℃for 5min, vortex andshake for 5min, centrifuge at above 4000 r/min at room temperature for 5 min;

2) Take 50ul down-layer liquid for analysis

Fold of dilution of sample: 5

6. ELISA procedures

6.1 Instructions

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.

6.2 Operation 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. Re-sealed the unused microplate, stored at 2-8 ℃, not frozen.

2) Solution preparation: dilute 15mL of the 20Xconcentrated washing buffer with the deionized water to 300mL 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 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 at25℃ for30 min;

5) 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).

6) Coloration: add 50µL of the substrate A, then 50 µL of the substrate B into each well. Mix gently by shaking the plate manually, andincubate at25 ℃ for15min in the dark for coloration;

7) 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.

7.2 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

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.

8. Precautions

1) 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.

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 reproducibility.

4) The stop solution is the 2 M sulfu ric acid solution, avoid contacting with the skin.

5) 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.

6) 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.

7) 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.

8) 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.

Q1: How long will it take for you to ship?
A1: Usually ships within 7 working days.

Q2: Do you support OEM/ODM?
A2: can be supported. We can customize according to your specific needs and specific quantities.

Q3: How is your factory doing in terms of quality control?
A3: We have ISO9001 certification and ISO13485 certification, so far all the production process, we have standard rules, we comply with the relevant behavior and laws of the government.

Q4: Is after-sales service guaranteed?
A4: We provide professional online technical after-sales service. If the product fails during the experiment, we can provide
one-to-one guidance through telephone, video and other forms.

Q5: What is your minimum order quantity?
A5: 1Kit.

Q6: What is the shipping method?
A6: It can be shipped by express (FEDEX, UPS, DHL, EMS, etc.) or by air and ground.Please confirm with us before placing an order.