A recent increase in the incidence of urinary tract stones, renal disease (such as urolithiasis) and associated death in infants has been reported in China. Since urinary tract stones are such a rarity in children, an investigation into the cause was commissioned. As a result, it is now believed that the disorder is associated with the ingestion of infant formula contaminated with melamine, which was added to raw milk products to boost the apparent protein content. Regulatory authorities have consequently published tolerable daily intakes (TDI) in line with recommendations from the World Health Organisation (WHO). In order for compliance to occur, sensitive and specific analytical methods with rapid detection and quantification are required. This article investigates the use of ELISA as a simple, low-cost and high-throughput melamine screening assay in combination with different microplate photometers.
Melamine contamination
Melamine contaminated milk formula in China has been reported to have caused urinary tract stones in 290,000 children [1]. Many of these children presented asymptomatically, with detection via ultrasound the sole indicator of the condition. This demonstrates that without widespread screening, accurate diagnosis of the condition is difficult. The presence of urinary tract stones causes a rapid loss of renal function, resulting in a build-up of metabolites such as urea, and therefore presents a significant health risk. Smaller, softer stones are easily passed as sand or sediment after drinking copious amounts of water. More seriously affected patients suffer urinary obstruction with acute renal failure, which requires medical intervention to relieve the blockage. This can be carried out with either a nephrostomy (an artificial opening between the kidney and skin) or a urinary catheter. In addition to the discomfort, metabolic acidosis (acidification of the blood) and hyperkalaemia (raised potassium levels) are the two most serious biochemical manifestations of acute renal failure and may require more complex treatment with sodium bicarbonate or dialysis, for example. In the recent Chinese cases, the severity of the affliction has been shown to be dose-related, with the most severe cases associated with the consumption of milk with a very high melamine concentration, in excess of 2.5 g/kg [2].
Although this is the first reported incident of melamine contamination in humans, similar symptoms were previously observed following the contamination of pet food in 2007. Melamine and cyanuric acid were identified in kidney tissue of affected animals and were considered responsible for the formation of urinary obstructions that lead to acute renal failure and subsequent death. Kidney histology revealed the presence of striated crystals in the kidney along with fibrosis and inflammation [3]. Analysis of the pet food revealed the presence of a number of triazine compounds, including melamine and cyanuric acid. A clear association between the presence of these compounds and the observed symptoms indicates a severe health risk [4].
The chemistry of melamine
Melamine is an organic base and a trimer of cyanamide, with a 1,3,5-triazine skeleton, which contains 66% nitrogen by mass. It is commonly combined with formaldehyde to produce a melamine resin, which is used in the manufacture of countertops, dry erase boards, fabrics, glues and flame retardants. However, melamine has also been used illegally to artificially boost the apparent protein content of milk. Some farmers were diluting their milk in order to boost profits, particularly in areas of the world where profit margins have not been improved via technological advancements. The result was milk of a much poorer quality. In order to conceal this fraudulent dilution with water, melamine was added in order to pass the government protein content tests. The Association of Analytical Communities (AOAC) International, a scientific association that sets standards for analytical methods, lists the Kjedahl and Dumas techniques as standard, but these methods detect nitrogen levels, not protein itself. As such, the addition of nitrogen-rich melamine results in the protein content of diluted milk appearing much higher than its true value.
Unfortunately, ingested melamine can accumulate in the body. While melamine itself is of low acute toxicity, it was often combined with cyanuric acid, an impurity often found naturally in scrap melamine (an illegal form of melamine that was commonly used). Once absorbed into the blood stream, the melamine and cyanurate concentrate together and interact in the urine-filled renal microtubules, subsequently crystallising to form a large number of round, yellow crystals. These crystals will then damage the renal cells that line the tubes, resulting in progressive tubular blockage and cellular degradation.
Since melamine on its own is not metabolised and has a short half-life, it is thought to be its combination with cyanuric acid or the formation of analogues such as ammeline and ammelide that cause the crystallisation and subsequent stone formation [2]. Melamine cyanurate has a very low solubility [4] and its analogues act as inhibitors of hepatic uric oxidase, causing a build-up of uric acid, the final oxidation product of purine, which is normally excreted in the urine. Resulting high levels of circulating uric acid cause the urine to become highly acidic and the urate becomes insoluble. It therefore crystallises to form stones, which can obstruct any part of the urinary tract, causing abdominal pain and damage to the renal cells [2].
Control measures
There are often low levels of melamine found in food as a result of contact with various packaging materials. However, following the high volume of recent melamine-related hospitalisations and deaths in China, regulatory authorities have published safety assessments on melamine and its structural analogues. As a result, the WHO has recommended that different counties abide by their currently proposed tolerable daily intakes (TDIs) [3]. The TDI is defined as the estimated maximum amount of an agent to which individuals in a population may be exposed daily over their lifetimes without an appreciable health risk.
The US Food and Drug Administration (FDA) has published an interim safety/risk assessment on melamine and its structural analogues in response to the contamination of imported products from China. Margins of safety were explored and the resulting TDI was established at 0.63 mg/kg of body weight per day [5]. The European Food Safety Authority (EFSA) has published a statement which applies a TDI of 0.5 mg/kg of body weight per day. This value takes into consideration the possible health effects which might occur with repeated consumption over a relatively short time period [6]. However, the Canadian Food Inspection Agency has taken a number of actions including product sampling, testing, and food recalls related to products that may be contaminated. As such, Health Canada has adopted the TDI recommended by the WHO scientific experts on melamine and has adjusted its standard to 0.2 mg/kg body weight per day [7].
Detection methods
In order for countries to comply with these newly established TDIs, there is an immediate requirement for analytical methods that are extremely sensitive and specific, with rapid detection and quantitative accuracy. The most common techniques used to detect the presence of melamine are liquid chromatography (LC) on its own, or in combination with tandem mass spectroscopy (LC/MS/MS). Alternatively, gas chromatography combined with tandem mass spectroscopy (GC/MS/MS) can also be used. Although these methods produce accurate data, they can often be time consuming and a drain on laboratory resources. Therefore a simple, low-cost and high-throughput melamine screening assay, such as an ELISA (Enzyme-Linked ImmunoSorbent Assay), provides an ideal technique.
A competitive ELISA can easily be performed in microplates, where large numbers of samples can be analysed simultaneously and instrumentation costs remain low. Most commercially available assay kits include melamine coated antibody microplates, horseradish peroxidise (HRP) conjugated melamine and chromogenic HRP substrate. Unknown milk samples and HRP conjugated melamine are added to the melamine antibody-coated microplate well, where they will compete for the antibody binding sites. In accordance with the competition principle, the binding ratio will be the same as the concentration of free to conjugated melamine. As such, the amount of bound HRP conjugated melamine is conversely dependent on the amount of free melamine in the sample. After binding, unbound material is removed and chromogenic HRP substrate is added. HRP enzyme activity is then directly proportional to the amount of bound HRP conjugated melamine. After an incubation period, the HRP enzyme reaction is stopped and high levels of enzyme activity are represented by the amount of coloured dye formation and high absorbance. The enzyme activity and resulting absorbance values will decrease as levels of unlabelled free melamine rise. Concentrations can then be determined directly through the use of calibration curves.
Assay sensitivities
In order to investigate the sensitivity of ELISAs for the accurate detection of melamine in milk, two different ELISA kits were used to test full-fat milk, fat-free milk and milk powder. The spiked samples were prepared by mixing melamine stock solution (2 mg/mL dissolved in distilled water) with each milk product. 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 1000 µg/L melamine.
When using the first of the two assay kits, the spiked milk samples were prepared as described in the kit instructions. For one of the kits, sample preparation was adapted slightly, based on information provided directly from the manufacturer to increase the assay sensitivity. This was done in order to make the sensitivities accurately comparable between the two tests.
Either 100 or 150 µL (kit dependent) of standard or spiked melamine sample was added to the antibody coated well. 50 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 sample was removed by washing four times with 300 µL distilled water. 100 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 melamine concentration in each milk sample. This curve can be calculated based on either measured absorbances or normalised 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 of the Thermo Scientific microplate photometers included in the study. The second assay was performed using only two of these microplate photometers, since it was evident from the previous data that the reader had no influence over obtained results. As shown in Table 1, 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 demonstrate excellent accuracy. Assay detection limits are highly dependent upon the accuracy and precision of the photometric read-outs. As such, only marginal, insignificant differences were observed between the LOD values. With both ELISA kits, concentrations of melamine less than 10 µg/L are easily detected.
Determination of Melamine from spiked milk samples
The three different milk types were spiked with melamine and analysed using each of the ELISA kits. These measurements were taken using a range of different photometers. The results from one of the photometers are shown below in Table 2; the data obtained from all other instruments were fully compliant with the results shown.
These data illustrate that both assay kits show very similar activity, with <10% difference in recovery efficiencies. However, both also have a common tendency to slightly underestimate melamine concentrations in those samples that have high melamine content. This does not impact on the usability of the assays, since all samples are still detected as strong and positive. Therefore, these results clearly show that ELISA assays are well suited for screening and accurately detecting melamine in unknown milk samples.
Conclusion
The introduction of low-cost, high-throughput ELISA methods to accurately detect the presence of melamine in milk products enables quality control laboratories to ensure that TDIs are not exceeded and high-quality dairy products are produced. The assay sensitivity obtained is equal to that of mass spectroscopy analyses and ELISAs present a significant cost-effective advantage in comparison to these chromatographic methods. The levels of sensitivity obtained are well within the detectable limits required from various regulatory bodies, including the FDA. However, when these assays are used for screening, it is still recommended that positive samples are subsequently confirmed using LC/MS/MS or GC/MS/MS methods to verify the precise quantity of melamine present.
References
1. Chen, Jun-shi. A worldwide food safety concern in 2008 – melamine-contaminated infant formula in China caused urinary tract stones in 290 000 children in China. Chinese Medical Journal 2009;122(3):243-244.
2. Editorial. Melamine-tainted milk products (MTMP) renal stone outbreak in humans. Hong Kong Med J 2008;14(6):424-426.
3. Dobson RL, Motlagh S, Quijano M
4. World Health Organisation. Melamine and Cyanuric acid: Toxicity, Preliminary Risk Assessment and Guidance on Levels in Food. Updated 30 October 2008.
5. Interim Melamine and Analogues Safety/Risk: Assessment www.fda.gov/Food/FoodSafety/FoodContaminantsAdulteration/ChemicalContaminants/Melamine/ucm164658.htm
6. European Food Safety Authority. Statement of EFSA on risks for public health due to the presences of melamine in infant milk and other milk products in China. The EFSA Journal 2008;807:1-10.
7. The Government of Canada Responds to Reports of Melamine in Food Products: www.hc-sc.gc.ca/fn-an/securit/chem-chim/melamine/index-eng.php.
et al. Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs. Toxicol Sci 2008;106:251-262.
The author
Jorma Lampinen
Senior Application Scientist
Liquid Handling and Consumables
Thermo Fisher Scientific
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