NIRM (Near InfraRed Microscopy)

NIRM analysis, like light microscopy, is a particle-based method. Briefly explained a NIRM is a combination of a near infrared spectrometer associated to a conventional light microscope. The identification of PAPs relies on the analysis of the spectral absorbance of single animal particles exposed to a near infrared beam.  Effectively, particles of a given nature deliver a unique NIR spectrum which is a signature reflecting its molecular composition.  

The method does not imply any treatment of the sample.  Particles of raw feed are simply spread on a sample plate and inserted under the infrared beam of the microscope. Spectra are recorded and automatically compared by chemometric analysis with data from libraries of spectra that are known to occur in compound feeds.  NIRM, compared to light microscopy, can identify and segregate fish, mammals and poultry particles, but it cannot discriminate at species level.

Mass spectrometry (MS)-based proteomics

Mass spectrometry (MS) is an analytical technique that identifies ionized molecules according to their mass-to-charge ratio (m/z). On the other side, proteomics studies of an organism’s proteome, just as genomics does for its genome. 

In MS-based proteomics, proteins/peptides are identified based on the mass to charge ratio (m/z) of its primary structure, the amino acid sequence. 

Peptide sequences, as in the case of DNA, have specific polymorphisms that can be traced to their species/taxonomic origin. Although the genome remains broadly unchanged in different tissues of the same organism, peptides can be specific of a type of tissue/by-product. Accurate selection of peptide biomarkers will provides information about the tissue AND the species of origin of the detected by-product. Another important characteristic of proteins is the relatively good resistance of their primary structure, the amino acid sequence, to processing. That is why mass spectrometry-based proteomics is perfectly adapted to the feed ban regulation.

The sample preparation as it is currently developed (bottom-up workflow) involves the four following steps:

  • Protein extraction
  • Reduction and alkylation of cysteine residues
  • Enzymatic digestion with trypsin
  • Purification and concentration

Analyses are performed by ultra-high performance liquid chromatography coupled with a tandem mass spectrometer (UHPLC-MS/MS). Liquid chromatography (LC) is coupled to the mass spectrometer in order to separate compounds from a complex matrix before MS analysis.

EURL-AP is currently developing a multi-targeted MS method for the detection of animal proteins in feed.

Advantages and drawbacks of these methods are summarised in this document.


Immunoassays are based on the detection of proteins, more precisely on the specific interaction and binding of antibodies to antigens of animal origin present in the feed. Selected antibodies should target heat resistant protein as PAPs are heat treated in their production; furthermore they should reveal differences in molecular structure according to the taxonomic groups of interest (ruminant, porcine, etc). Several kits are available on the market for the purpose but their performances are still not satisfactory.

Two main applications are to be found:

  • Enzyme linked immunoabsorbant assay (ELISA) for laboratory use
  • Dipstick or lateral flow for field use

The use of immunoassays is limited to screening purposes and shall never be confirmatory for the presence of PAPs in feed. The EURL-AP team realised evaluations of several commercialized test kits.  These studies revealed some inconsistencies of sensitivity (e.g lack of detection of ruminant PAPs in fish meals) but also of specificity (e.g false detection of PAPs in presence of beet pulp). It seems that those tests, not developed inside the European Union, are incompatible with the requirement of severe heat treatments for PAPs production.

Advantages and drawbacks of these methods are summarised in this document.