High Throughput Screening of Melamine in Infant Formula Milk Powder

Creative Diagnostics - Food & Feed Analysis

High Throughput Screening of Melamine in Infant Formula Milk Powder

Tsoi, T. H., & Wong, W. T. (2015).
A simple, highly sensitive, high throughput and organic solvent-free screening method for melamine by microsphere-based flow cytometry immunoassay.
Analytical Methods, 7(14), 5989-5995. DOI: 10.1039/c5ay00648a.


Melamine is also known as 2,4,6-Triamino-1,3,5-triazine. As an industrial chemical, it is commonly used in many manufacturing processes like melamine resin tableware, coatings, commercial filters, glues or adhesives, fertilizers and flame retardants. Although it is prohibited to add into foods or food products, melamine is still possible to contaminate foods by using melamine resin tableware. And as a metabolite product of cyromazine, it will contaminate foods due to the application of this insecticide on animals or crops too. In addition to being an environmental contaminant, its abuse as an adulterant in infant formula milk powder to mimic the presence of high protein levels is another serious problem. Adding melamine into infant formula milk powder may result the occurrence of kidney stones in infants, which may lead to death. The WHO has recommended that the level of melamine in any foods containing more than 15% milk should be below 2.5 mg/ml. And many countries stipulate that the melamine level in infant formula milk powder must be no more than 1 mg/kg.

Therefore, the development of rapid test of melamine in infant formula milk powder is very meaningful.


Tsoi, et al. use microsphere-based flow cytometry, a novel multiplexing immunochemical method for food-testing, to screen for melamine in various brands of infant formula powder (Figure 1). There is a competition between the free analyte in the sample and analyte-coupled microspheres, and by measuring the fluorescence emitted from labelled antibodies (Melamine, Creative Diagnostics), they had quantified the content of the analyte indirectly. The coating antigen (AETT) was synthesized by a two-step reaction and then immobilized onto carboxylated microspheres by NHS/EDC conjugation chemistry. Those chosen formula milk powders of different brands had been tested by a commercial ELISA to confirm the absence of melamine.

Figure 1. Schematic diagram of microsphere-based competitive inhibition type flow cytometry immunoassay.


To test for the detection ability of the method, six brands of formula powders designed for infants were used. The melamine (0.1 mg/g, low concentration level; 10 mg/g, high concentration level; 0.5 and 1.5 mg/g, close to the legislated acceptable level) was fortified into the milk powder. Not only did the developed method give satisfactory spike recoveries, but also the relative standard deviations of all spike levels were < 8%. To validate the specificity of the method, cross-reactivity values to other structurally similar compounds were determined. There is no significant cross-reactivity value to melamine (< 0.5%) between all compounds, except for cyromazine. Cyromazine was found to respond exactly in the same manner to the primary antibody as melamine (CR value = 100%).


This simple, high throughput, highly sensitive (7.58 ng/ml, limit of detection = 0.70 ng/ml) and organic solvent-free immunoassay towards melamine based on the flow cytometry principle has been developed and successfully applied to detect the melamine in various brands of infant formula powder, fulfilling the legislated tolerance level. This method is suitable to be a high-throughput screening, or even quantitative/semiquantitative tool for melamine in infant formula powder.

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