Detect Melamine With ELISA Assays

With common industrial uses, melamine is frequently mixed with formaldehyde to produce melamine-formaldehyde resin, a type of plastic known for its flame retardant properties. Used in the manufacture of countertops, dry-erase boards, laminates, glues, adhesives, paper and textiles, melamine has more recently been identified in some food products. Investigations have identified raw materials suppliers who have been illegally adding this nitrogen-rich chemical to food sources in order to increase its apparent protein content.

Some dairy farmers have been diluting milk to boost their profits, particularly in parts of the world where technological advancement has been slow and where improved efficiency has not increased profit margins on milk output. This dilution has resulted in decreased quality, reducing the measurable concentrations of milk proteins, fats, and sugars. Some farmers dilute their milk by up to 30%, then use melamine to conceal this fraudulence. The high nitrogen content in melamine effectively raises profits by deceiving the quality control equipment, which detects the nitrogen (from protein) at normal levels, enabling the milk to be passed as high quality.

When combined with cyanuric acid (an impurity often found naturally in scrap melamine), it accumulates in the body, where it forms insoluble crystals that can result in major toxicity problems. When melamine cyanurate is absorbed into the bloodstream, it concentrates and can interact in the urine-filled renal microtubules. It then crystallizes, forming large numbers of round yellow crystals that, in turn, can block and damage the renal cells that line the microtubules, resulting in severe kidney malfunction. Prolonged exposure can also lead to other health problems, such as reproductive damage, bladder or kidney stones, and cancer.

The standard methods used to estimate protein content in food are the Kjeldahl and Dumas techniques, both of which measure nitrogen content. These methods fail to distinguish the differences between the nitrogen found in melamine and the nitrogen naturally occurring in amino acids. Highly sensitive techniques have been developed that can detect melamine, rather than nitrogen. Liquid chromatography (LC) can be used on its own or in combination with tandem mass spectrometry (LC/MS/MS), and gas chromatography combined with tandem mass spectrometry (GC/MS/MS) can also be used.

Such chromatographic methods are accurate but have significant setup and running costs, for both equipment and labor. A simple high-throughput screening assay is urgently needed to accurately measure melamine residuals in milk at a reasonable cost.

Classic ELISA (enzyme-linked immunosorbent assay) measurements provide an ideal solution. They can be performed in microplates, where large sample volumes can be simultaneously analyzed and instrumentation costs remain low. Immunoassay kits that detect melamine contamination in the raw materials used in milk products, animal feed, and pet food supplies have been developed with speed, simplicity, and sensitivity in mind.

This article describes how the screening of milk samples for melamine can be performed with high sensitivity using simple ELISAs. These assays are designed to be sensitive enough to detect melamine to levels below 10 µg, the threshold value established by regulatory agencies worldwide for non-infant food products.

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Table 1. Limits of Detection of Both Assay Kits With Different Thermo Scientific Instrumentation

Assay Principles

For this investigation, melamine assay studies were performed using two commercially available melamine ELISA kits from different manufacturers, both applying the same basic principle. Unknown samples and horseradish peroxidase (HRP) conjugated melamine were placed into a microplate well coated with an antibody raised against melamine. The HRP conjugated melamine competitively binds to the antibody against any melamine present in the unknown samples. This binding occurs in the same ratio as the concentration of HRP conjugate to free melamine. As a result, the amount of antibody-bound HRP conjugate is conversely dependent upon the amount of free melamine present in the sample.