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Detect Melamine With ELISA Assays
This simple, high-throughput screening method permits large-scale screening at low cost
by Jorma Lampinen, PhD
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.
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.
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.
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.
After the binding phase, unbound material is removed using a microplate washer, and chromogenic HRP substrate is added. The detected HRP enzyme activity is directly proportional to the amount of bound HRP conjugated melamine and subsequently dependent upon the mela-mine concentration within the unknown sample. The reaction is stopped after a set incubation time, and the amount of formed colored dye is measured. When melamine is not present in the sample, high levels of enzyme activity occur, indicated by the amount of colored dye formed and subsequent high absorbance. These levels of activity and absorbance decrease as levels of unlabeled, free melamine increase. Melamine concentration can then be directly determined using calibration curves (as discussed below).
Although these assays have a slight inclination to underestimate the high melamine concentrations in milk samples, the level of sensitivity obtained is still well within acceptable levels for melamine screening when a qualitative yes/no answer is required.
Three different milk samples—natural full-fat milk, fat-free milk, and artificial milk produced from natural milk powder—were spiked with pure melamine. The spiked samples were prepared by mixing the melamine stock solution (2 mg/mL dissolved in distilled water) with the different milk products. All three milk types were spiked with 20 and 100 µg/L, and the full-fat and fat-free samples were also spiked with 550 and 1,000 µg/L melamine.
When using the first of the two assay kits, the spiked milk samples were prepared as described in the kit instructions:
- Approximately 1 mL of spiked milk sample was added to a clean test tube;
- Samples were centrifuged at 1,500 g at 10 °C for 10 minutes;
- Aliquots of 200 µL were taken from below the fat layer and transferred to a clean test tube; and
- 800 µL of assay diluent was added to the milk serum and the diluted samples were carefully mixed.
When using the second kit, however, sample preparation was adapted slightly, based upon information provided directly from the manufacturer, to increase the assay sensitivity. If the milk samples had been prepared in accordance with the original instructions, lower melamine sensitivity would have been observed in comparison to the first assay, because the samples were—according to original instructions—diluted much more for the assay:
- Approximately 1 mL of spiked milk was added to a clean test tube;
- Samples were centrifuged at 10,000 g at 10 °C for 5 minutes, which produced three defined layers;
- A 250 µL aliquot was taken from the middle layer and moved to a clean test tube; and
- This aliquot was diluted at a ratio of 1:3 by adding 500 µL of 10% MeOH/ 20mM PBS solution.
Both ELISA kits were used according to the manufacturer instructions. Either 100 or 150 µL (kit dependent) of melamine standard or spiked sample was added to the antibody-coated well. Fifty µL of HRP-melamine conjugate was then added to each well, and the plate was mixed and incubated for 30 minutes at room temperature. All unbound samples were removed by washing with distilled water. Washing was repeated four times with 300 µL of water. One hundred µL of HRP-substrate was aliquoted into each well, and the plate was incubated at room temperature for a further 20 or 30 minutes (kit dependent). The reaction was stopped via the addition of 100 µL of stop solution, and the absorbance at 450 nm was measured using one of six different microplate photometers. Calibration curves were generated and used to identify the unknown concentrations. (This curve can be calculated based on either measured absorbances or normalized values, where absorbance of the zero control has been set to 100%.)
Calibration Curves and Assay Sensitivity
Melamine calibration curves generated using the first assay kit were measured with all six microplate photometer models. When calibration curves from the second kit were calculated, only two of the microplate photometers were used. Previous data showed that the reader had no influence over the obtained results. As shown in Table 1 (see p. 46), the limit of detection (LOD) for this assay was calculated based on the calibration data, as well as the data from the zero control samples, using the standard IUPAC 3*SD method.
The measurement range required for these assays is ideally suited to the wavelength in which all photometers have an excellent degree of accuracy. The detection limits of the assays are highly dependent upon the accuracy and precision of these photometric readouts. As such, only marginal differences, too small to be significant, were observed between the LOD values. With both ELISA kits, concentrations of melamine below 10 µg/L are easily detected. Therefore, the ELISA assays are able to detect melamine levels to the same degree of accuracy as LC/MS/MS tests.
Therefore, the results clearly show that ELISA assays are well suited for screening purposes, including the detection of melamine in an unknown milk sample.
Determination of Melamine
The three different types of milk were spiked with pure melamine and analyzed using both ELISA kits. These measurements were taken using a range of different photometer types. The results from one of the photometers are shown in Table 2 (see p. 47). Results obtained from all other photometers were fully compliant with the results shown in Table 2.
The data shown illustrate how both assay kits show very similar activity, with <10% difference in recovery efficiencies. Additionally, both kits have a common tendency to slightly underestimate melamine concentrations in those samples with a high melamine concentration; however, this does not affect the usability of these assays, because all samples are still detected as strong, positive ones in the screening assay. Therefore, the results clearly show that ELISA assays are well suited for screening purposes, including the detection of melamine in an unknown milk sample.
Based on the above results, both of these tests can be used to detect melamine residuals accurately in dairy products. The assay sensitivity obtained is on par with that of mass spectroscopy analyses. ELISAs present a cost advantage when compared to chromatographic methods, however. This simple, high-throughput screening method enables large-scale screening of thousands of samples with low instrumentation costs and affordable running prices.
Although these assays have a slight inclination to underestimate the high melamine concentrations in milk samples, the level of sensitivity obtained is still well within acceptable levels for melamine screening when a qualitative yes/no answer is required. When these assays are used for screening, positive samples should still be confirmed subsequently using LC/MS/MS or GC/MS/MS methods to verify the exact quantity of melamine present.
Melamine residuals in milk can be detected in a fast (total assay time: approximately one hour) and cost-effective way to efficiently screen dairy products, ensuring that any contamination is quickly and easily identified. ■