Ideal melamine test method

Melamine, which is commonly used in industry, is often mixed with formaldehyde to form a plastic known as flame retardant - melamine-formaldehyde resin. In addition to the use of countertops, writing boards, laminates, adhesives, adhesives, paper, and fabrics, melamine has recently been found in some foods. The survey found that some raw material suppliers illegally added this nitrogen-rich chemical to food sources to create the illusion of increased protein content.

Some dairy farmers dilute milk to increase profits, especially where technology is slow to develop and the profitability of milk production does not increase with efficiency. This dilution behavior results in a decrease in the quality of the milk and a significant decrease in the concentration of protein, fat and sugar. Some dairy farmers dilute milk to as much as 30%, and then use melamine to cover up the fraud. The quality control equipment detects the normal level of nitrogen (protein contained), and the high content of nitrogen in melamine can be mixed in the test, allowing the fake milk to pass the test as a quality product, thereby significantly increasing profits.

The combination of melamine and cyanuric acid (an impurity commonly found in melamine waste) accumulates in the body, forming insoluble crystals, causing severe toxicity. Melamine cyanurate is absorbed into the bloodstream and accumulates in the urine of the kidney microtubules. Subsequent crystallization, the formation of a large number of spherical yellow crystals, block and damage kidney cells in the microtubules, leading to severe renal dysfunction. Prolonged contact time can lead to other health problems such as reproductive damage, bladder or kidney stones, and cancer. The standard method for the determination of protein content in foods is the Kjeldahl method and the Dumas combustion method, both of which measure the nitrogen content. These methods do not distinguish the nitrogen of melamine from the nitrogen naturally occurring in amino acids. There has been a highly sensitive method for detecting melamine instead of measuring nitrogen. Liquid chromatography (LC) can be used alone or in combination with tandem mass spectrometry (LC/MS/MS) or in combination with tandem mass spectrometry (GC/MS/MS).

These chromatographic methods are very precise, but the cost of preparation and operation is very high for equipment and operation. Therefore, a simple high-throughput screening method is urgently needed to accurately detect the residual amount of melamine in milk at a reasonable cost.

Classical ELISA (enzyme-linked immunosorbent assay) measurements can be an ideal solution. This method can be carried out in a microplate, so that the number of samples can be simultaneously performed, and the cost of the instrument is not high. Immunoassay kits have been developed to efficiently, easily and sensitively detect melamine contaminants in dairy, animal feed, and pet food ingredients.

This article is about how to use a simple ELISA to highly sensitively screen melamine in milk samples. These tests require sufficient sensitivity to detect melamine below 10 ,, which is the limit set by regulators around the world for non-baby foods.

Classical ELISA (enzyme-linked immunosorbent assay) measurements can be used as an ideal solution to determine melamine content.

Detection principle

In this study, two melamine ELISA commercial kits from different manufacturers but with the same basic principles were used. Unknown samples and horseradish peroxidase (HRP)-labeled melamine were placed in the wells of melamine antibody-coated microplates. HRP-labeled melamine competes with melamine in an unknown sample for binding to antibodies. The binding rate corresponds to the concentration ratio of HRP marker to free melamine. Thus, the amount of HRP label bound to the antibody is inversely related to the amount of free melamine in the sample.

After the binding stage, the unbound material was removed using a microplate washer and HRP chromogenic substrate was added. The measured HRP enzyme activity is directly proportional to the amount of HRP-labeled melamine and is related to the concentration of melamine in the unknown sample. The reaction was stopped after the prescribed incubation time, and the amount of the colored dye formed was measured. If melamine is not present in the sample, it is known that the level of enzyme activity is higher by the amount of colored dye formed and the higher absorption value. As the amount of unlabeled free melamine increases, the level of activity and absorption decrease. Therefore, the concentration of melamine can be directly determined based on the calibration curve (discussed later).

Sample Preparation

Three different milk samples - natural whole milk, skim milk and artificial milk made from natural milk powder - were added to pure melamine. These three different dairy products were mixed with a melamine stock solution (concentrated in 2 mg/mL of distilled water) to make spiked samples. The three types of milk were added with 20 and 100 礸/L of melamine, and the whole fat and skim milk samples were additionally added with melamine at a concentration of 550 and 1,000 Å/L.

For the first test kit, the spiked milk sample is prepared according to the kit operation method:

â—† Add about 1mL of spiked milk sample to the clean test tube;
◆The sample is centrifuged at 1,500 g, 10 °C for 10 minutes;
◆ Take the lower part of the fat layer 200 礚 into a clean test tube; and add 800 礚 test dilution to the milk whey, carefully mix and dilute the sample.

For the second kit, the sample preparation method is slightly adjusted to improve the detection sensitivity according to the information directly provided by the manufacturer. If a milk sample is prepared according to the original instructions, the melamine detection sensitivity is found to be lower than the first method because the sample is diluted in excess according to the original instructions:

â—† Add about 1mL of spiked milk to the clean test tube;
◆The sample is centrifuged at 1,500g, 10 °C for 10 minutes, and divided into 3 layers;
◆ Take the middle layer 250 礚 and transfer it to a clean tube; and ◆ Remove the liquid and add 10% methanol/PBS solution (20 mM) in a ratio of 1:3.

“Classic ELISA (Enzyme Linked Immunosorbent Assay) measurement can be an ideal solution. This method can be performed in a microplate, so the number of samples can be performed simultaneously and the cost of the instrument is not high.”

Both ELISA kits are used with reference to the manufacturer's instructions. The melamine standard or standard addition is added to the antibody-coated microwells in an amount of 100 or 150 Å (depending on the kit). Then 50 礚HRP-melamine label was added to each well, and the wells were mixed and incubated for 30 minutes at room temperature. Rinse water rinse to remove unbound sample. Rinse 4 times with 300 礚 distilled water. 100 礚 HRP - Substrate is dispensed into each well and the plate is incubated again for 20 or 30 minutes at room temperature (depending on the kit). The reaction was stopped by the addition of 100 Torr stop solution, and the absorbance was measured at 450 nm using six different microplate readers. Make a calibration curve to determine the unknown concentration. (The curve can be calculated based on the absorption value or the exact value, and the absorption value of the blank control is set to 100%.)

Calibration curve and test sensitivity

The melamine calibration curve made using the first kit was measured using six different types of microplate readers. When calculating the calibration curve for the second kit, only two microplate readers were used. Previous data showed that the detector had no effect on the results. The detection limit (LOD) of this method was calculated using the standard IUPAC 3*SD method based on calibration data and data from blank control samples.

The measurement range required for these methods is consistent with the range of wavelengths with good accuracy for all microplate readers. The detection limits of these methods depend to a large extent on the accuracy and precision of the metering reader. Therefore, there is only a small difference between the LOD values ​​that is not significant. For both kits, a concentration of melamine below 10 礸/L can be easily detected. Therefore, the accuracy of ELISA for the detection of melamine is comparable to LC/MS/MS.

Determination of melamine

Three different milks were added to the pure melamine and analyzed using two kits. Measurements were made using a range of different photometers.

The data shows that the enzyme activities measured by the two kits are very close, and the difference in recovery is less than 10%. In addition, there is a similar trend in both kits, that is, the concentration measurement of melamine high concentration samples is slightly lower; however, this will have no effect on the use of these methods, because all samples will also be used as high concentrations for screening detection. Positive samples were assayed. Therefore, the results clearly indicate that ELISA assays are suitable for screening, including detection of melamine in unknown milk samples.

Based on the foregoing results, both tests can be used to accurately determine the residual amount of melamine in dairy products. The sensitivity of the test is comparable to the sensitivity of mass spectrometry. However, ELISAs have cost advantages over chromatographic methods. This simple, high-throughput screening method enables large-scale screening of thousands of samples at a lower instrument cost and moderate operating price.

Although these methods have slightly lower values ​​for high concentrations of melamine in milk samples, the level of sensitivity is still suitable for screening melamine and making a presence/absence. When these methods are used for screening, the positive sample then still needs to determine the exact amount of melamine using LC/MS/MS or GC/MS/MS methods.

This fast (total detection time: approx. 1 hour) and cost-effective method for detecting melamine residues in milk for efficient screening of dairy products ensures that any contaminants can be quickly and easily Find.

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