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1. Product Description

Melamine is is an organic base and a trimer of cyanamide, with a 1,3,5-triazine skeleton. like cyanamide, it contains 66% nitrogen by mass. Melamine is sometimes illegally added to food products in order to increase the apparent protein content. Standard tests such as the Kjeldahl and Dumas tests estimate protein levels by measuring the nitrogen content, so they can be misled by adding nitrogen-rich compounds such as melamine. Ingestion of melamine may lead to reproductive damage, or bladder or kidney stones, which can lead to bladder cancer.This ELISA KIT is a competitive enzyme immunoassay for the quantitative analysis of Melamine in feed, milk and milk powder.

 2. Test principle

The basis of the test is the antigen-antibody reaction. The microtiter wells are coated with capture antibodies directed against anti-melamine antibodies. Anti-melamine antibodies are added which are bound by the immobilized capture antibodies. After incubation and washing step standards or sample solution and melamine enzyme conjugate are added. Free and enzyme conjugated melamine compete for the antibody binding sites (competitive enzyme immunoassay). Any unbound enzyme conjugate is then removed in a washing step. Substrate/chromogen solution is added to the wells and incubated. Bound enzyme conjugate converts the chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm. The absorption is inversely proportional to the melamine concentration in the sample.

3. Kit Contents

Each kit contains sufficient materials for 96 measurements (including standard analyses). Each test kit contains:

Microtiter plate with 96 wells coated with Melamine antigen

Melamine standard solutions：0 ppb (zero standard), 0.05 ppb, 0.1 ppb, 0.25 ppb, 0.75 ppb, 1.5 ppb

10× Melamine Antibody............................................................................. 1.6 ml

10× HRP-Conjugated Antigen.............................................................................0.8 ml

TMB Buffer A............................................................................................................. 7 ml

TMB Buffer B ............................................................................................................ 7 ml

Stop Buffer.................................................................................................................. 7 ml

10× Washing Buffer.................................................................................................50 ml

Sample Buffer.................................................................................................50 ml

4. Required Materials Not Provided With the Kit

(1)Equipment:

- Microtiter plate reader (450 nm)

- Balance

- Rotary evaporator or Nitrogen-blow concentrator

- Vortex mixer

- 10, 20, 100 and 1000 μl pipettes

- Multi-channel pipette: 20-300μl

- Incubator

- Centrifuge

- Centrifuge Tube: 15ml, 50ml

- Volumetric Flask: 100ml, 1000ml

- Centrifuge tube

(2)Reagents:

- 1M NaOH

- Methanol

5. Solution preparation

- Washing buffer: Mix 1 volume 10×Washing Buffer with 9 volume of distilled water.

- Antibody working buffer: Mix 1 volume 10×Melamine antibody with 9 volume of wash buffer.

- HRP-Conjugated Antigen working buffer: Mix 1 volume 10×HRP-Conjugated Antigen with 9 volume of wash buffer.

- 60% methanol solution：Mix 60ml methanol and water to 100ml.

- 90% methanol solution：Mix 90ml methanol and water to 100ml.

6. Sample Preparation

(1) Feed

1) Homogenize the sample

2) Use 1±0.05g of the homogenized sample and add 3 ml 60% methanol. Mix for 5min. Centrifuge: 5 min / 3000 g / room temperature.

3. Use 50μl supernatant, and add 450μl sample buffer. Mix. Use 50μl in the test

Note: Dilution factor: 40.

(2) Milk

1) Use 1ml milk sample. Centrifuge: 5 min / 8000 g / room temperature.

2) Discard the fat layer. Use 25μl supernatant and add 475μl distilled water. Mix. Use 50μl in the test

Note: Dilution factor: 20.

(3) Milk powder

1) Use 0.5±0.05g milk powder.

2) Add 5ml 90% methanol, Mix vigorously for 5min. Centrifuge: 5 min / 3000 g / room temperature.

3) Use 1ml supernatant and evaporate by rotary evaporator or nitrogen-blow concentrator (50℃).

4) Add hexane 1ml, dissolve the residue. Add 1ml sample buffer and mix vigorously for 1 min (with up-side-down shaker or vortex)

5) Centrifuge: 5 min / 3000 g / room temperature

6) Use 50μl lower layer liquid (water layer) in the test

Note: Dilution factor: 10.

7. ELISA Testing protocol

(1) Insert a sufficient number of wells into the microwell holder for all standards and samples to be run in duplicate. Record standard and sample positions.

(2) Add 100μl of Melamine antibody buffer and incubate for 30 min at 37℃.

(3)Pour the liquid out of the wells and tap the microwell holder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. Fill all the wells with 250 μl washing buffer and pour out the liquid again. Repeat four more times.

(4) Add 50μl standard solution or sample solution. Add 50 μl of HRP-Conjugated Antigen working buffer to the bottom of each well. Incubate for 30 min at 37℃.

(5) Pour the liquid out of the wells and tap the microwell holder upside down vigorously (three times in a row) against absorbent paper to ensure complete removal of liquid from the wells. Fill all the wells with 250 μl washing buffer and pour out the liquid again. Repeat four more times.

(6) Add 100 μl of TMB Buffer (mix TMB buffer A 1:1 with TMB buffer B) to the bottom of each well. Incubate for 15 min at 37℃ in the dark.

(7) Add 50 μl of the stop solution to each well. Mix gently by shaking the plate manually and measure the absorbance at 450 nm. Read within 30 minutes after addition of stop solution.

8. Results

A standard curve can be constructed by plotting the mean relative absorbance (%) obtained from each refernce standard against its concentration in ng/ml on a logarithmic curve.

Use the mean relative absorbance values for each sample to determine the corresponding concentration of the tested drug in ng/ml from the standard curve. A special program with Excel functionality, Triphil ELISA test results. Please contact your local distributor or [email protected]

for further information.

In order to obtain the melamine concentration in ppb actually contained in a sample, the concentration read from the calibration curve must be further multiplied by the corresponding dilution factor.

9. Detection limit and Recovery rate

(1) Detection limit:

Feed..............................................................................................2 ppb

Milk...............................................................................1 ppb

Milk powder...............................................................................0.6 ppb

(2) Recovery rate:

Feed, Milk .......................................................................................... 75±15%

Milk powder...........................................................................110±15%

10. Specificity:

The specificity of the ELISA Kit test was determined by analyzing the cross-reactivities to corresponding substances.

Melamine.............................................................................................. 100.00%

Cyanuric acid.......................................................................................... 3.5 %

11. Warnings and Precautions

--Make sure plates and reagents are brought up to room temperature. Keep the kit components out of the kit box for a least 1hour before starting the assay.

--Shake up the reagent before using it.

--Carefully label each reagent to make sure the reagents are not intermixed. Kits with different expiration dates might generate different range of OD readings.

--In general, a value of less than 0.6 in zero standard reading may indicate certain degrees of deterioration of reagents.

--Incubate at 37℃, too high or too low temperature may generate different range of OD readings. And avoid sunlight beaming directly.

--The stop buffer is 1M H₂SO₄, Please caution.

--The TMB buffer becoming blue before use is indicating the deterioration of reagent.

--Always refrigerate plates in sealed bags at drying condition without light.

--Store the kit at 2-8℃. The shelf life is 12 months when the kit is properly stored.