Ideal melamine detection method

Commonly used in industry, melamine is often mixed with formaldehyde to make a plastic known for its flame retardant properties, melamine-formaldehyde resin. In addition to being used in countertops, writing boards, laminates, adhesives, glues, papers and fabrics, melamine has recently been found in some food products. Investigations have found that some raw material suppliers illegally add this nitrogen-rich chemical to food sources to create the appearance of increased protein content.

Some dairy farmers dilute milk to increase profits, especially in places where technology is slow and profit margins in milk production have not increased with efficiency. This dilution reduces the quality of the milk, significantly reducing the concentration of protein, fat and sugar. Some dairy farmers dilute their milk by as much as 30% and then use melamine to cover up the fraud. Quality control equipment detects normal levels of nitrogen (which is found in protein), and the high nitrogen content in melamine can fool the test, allowing the fraudulent milk to pass as a higher-quality product, significantly increasing profits.

Melamine, combined with cyanuric acid (an impurity often found in melamine waste), aggregates in the body to form insoluble crystals that are highly toxic. Melamine cyanurate is absorbed into the bloodstream, where it aggregates and acts in the urine in the microtubules of the kidneys. It then crystallizes to form large, spherical yellow crystals that clog and damage kidney cells in the microtubules, causing severe kidney dysfunction. Prolonged exposure can lead to other health problems, such as reproductive damage, bladder or kidney stones, and cancer. The standard methods for determining protein content in food are the Kjeldahl method and the Dumas combustion method, both of which measure nitrogen content. These methods cannot distinguish melamine nitrogen from naturally occurring nitrogen in amino acids. Highly sensitive methods have been developed to detect melamine, rather than nitrogen. Liquid chromatography (LC) can be used alone or in combination with tandem mass spectrometry (LC/MS/MS), or gas chromatography can be combined with tandem mass spectrometry (GC/MS/MS).

These chromatographic methods are very precise, but are very expensive to prepare and run, both for equipment and operation. There is an urgent need for a simple high-throughput screening method that can accurately detect melamine residues in milk at a reasonable cost.

Classical ELISA (enzyme-linked immunosorbent assay) measurements could be an ideal solution. This method can be performed in microplates, so a large number of samples can be run simultaneously, and the instrument costs are not high. Immunoassay kits have been developed that allow efficient, simple and sensitive detection of melamine contaminants in raw materials for dairy products, animal feed and pet food.

This article is about how to use simple ELISAs to screen milk samples for melamine with high sensitivity. These tests need to be sensitive enough to detect less than 10 micrograms of melamine, which is the limit set by regulatory agencies worldwide for non-infant foods.

The classic ELISA (enzyme-linked immunosorbent assay) measurement can be
an ideal solution for the determination of melamine content.

Principle of the assay

Two commercial melamine ELISA kits from different manufacturers, but based on the same basic principle, were used in this study. Unknown samples and horseradish peroxidase (HRP)-labeled melamine are placed in the wells of a microplate coated with a melamine antibody. HRP-labeled melamine competes with melamine in the unknown sample for binding to the antibody. The binding ratio corresponds to the ratio of the concentration of HRP label to free melamine. Therefore, the amount of HRP label bound to the antibody is inversely related to the amount of free melamine in the sample.

After the binding phase, unbound material is removed using a microplate washer and an HRP chromogenic substrate is added. The measured HRP enzyme activity is proportional to the amount of HRP-labeled melamine and is related to the concentration of melamine in the unknown sample. After the specified incubation time, the reaction is terminated and the amount of colored dye formed is measured. If melamine is not present in the sample, a higher level of enzyme activity is indicated by the amount of colored dye formed and the higher absorbance. As the amount of unlabeled free melamine increases, the activity level and absorbance decrease. Therefore, the concentration of melamine can be directly determined from the calibration curve (discussed later). [page]

Sample Preparation

Three different milk samples - natural whole milk, skim milk, and artificial milk made from natural milk powder - were spiked with pure melamine. The three different dairy products were mixed with a melamine stock solution (2 mg/mL in distilled water) to prepare the spiked samples. All three milks were spiked with 20 and 100 μg/L of melamine, and the whole and skim milk samples were additionally spiked with 550 and 1,000 μg/L of melamine.

For the first test kit, the spiked milk sample was prepared according to the kit instructions:

◆ Add approximately 1 mL of spiked milk sample to a clean test tube;
◆ Centrifuge the sample at 1,500 g, 10 °C for 10 minutes;
◆ Transfer 200 μl of the lower fat layer to a clean test tube; and 800 μl of test diluent was added to the milk whey and the sample was carefully mixed and diluted.

For the second test kit, the sample preparation method was slightly modified to improve the test sensitivity based on information directly provided by the manufacturer. If the milk sample is prepared according to the original instructions, it will be found that the sensitivity of melamine detection is lower than that of the first method because the test sample is over-diluted according to the original instructions:

◆ Add approximately 1 mL of spiked milk to a clean test tube;
◆ Centrifuge the sample at 1,500 g, 10 °C for 10 minutes to separate into 3 layers;
◆ Transfer 250 μl of the middle layer to a clean test tube; and
◆ Remove the liquid and add 10% methanol/PBS solution (20 mM) in a ratio of 1:3.

"Classical ELISA (enzyme-linked immunosorbent assay) measurements could be an ideal solution. This method can be performed in microplates, so a large number of samples can be run simultaneously, and the instrument costs are low."

Both ELISA kits were used according to the manufacturer's instructions. Melamine standards or spikes were added to the antibody-coated wells in an amount of 100 or 150 μl (depending on the kit). Subsequently, 50 μl of HRP-melamine marker was added to each well, the plate was mixed and incubated at room temperature for 30 minutes. Unbound samples were removed by rinsing with distilled water. The rinse was repeated four times with 300 μl of distilled water. 100 μl of HRP-substrate was dispensed into each well and the plate was incubated again at room temperature for 20 or 30 minutes (depending on the kit). The reaction was stopped by adding 100 μl of stop solution and the absorbance was measured at 450 nm using six different microplate readers. A calibration curve was prepared for the determination of unknown concentrations. (The curve can be calculated based on absorbance or exact values, with the blank absorbance set to 100%.)

Calibration curves and assay sensitivity

The calibration curve for melamine prepared with the first kit was determined using six different microplate readers. The calibration curve for the second kit was calculated using only two microplate readers. Previous data showed that the detector had no effect on the results. The limit of detection (LOD) for the method was calculated using the standard IUPAC 3*SD method based on the calibration data and the data from the blank sample.

The measurement range required for these methods is consistent with the wavelength range where all microplate readers have good accuracy. The limit of detection for these methods is largely dependent on the accuracy and precision of the photometric reader. Therefore, there are only small, non-significant differences between the LOD values. For both kits, melamine concentrations below 10 μg/L can be easily detected. Therefore, the accuracy of the ELISA method for melamine is comparable to that of LC/MS/MS.

Determination of Melamine

Three different milks spiked with pure melamine were analyzed using both kits. The measurements were performed using a range of different photometers.

The data showed that the enzyme activities measured by both kits were very similar, with recoveries differing by less than 10%. In addition, both kits showed a common trend of slightly lower concentrations in samples with high melamine concentrations; however, this did not affect the use of these methods, as all samples were also measured as high-concentration positive samples for screening tests. Therefore, the results clearly show that ELISA tests are suitable for screening, including the detection of melamine in unknown milk samples.

Based on the above results, both tests can be used to accurately determine the amount of melamine residue in dairy products. The sensitivity of the tests is comparable to that of mass spectrometry. However, ELISAs have a cost advantage over chromatographic methods. This simple, high-throughput screening method can be used to screen thousands of samples at a low instrument cost and a moderate running price.

Although these methods have slightly lower values ​​for high-concentration melamine in milk samples, the sensitivity achieved is still suitable for screening and making a presence/absence judgment for melamine. When these methods are used for screening, positive samples still need to be subsequently tested using LC/MS/MS or GC/MS/MS methods to determine the exact amount of melamine present.

This rapid (total test time: approximately 1 hour) and cost-effective method for testing milk for melamine residues allows for efficient screening of dairy products, ensuring that any contaminants are quickly and easily detected.