Proceeding of Veterinary and Animal Science Days 2018, 6th- 8th June, Milan, Italy 

HAF © 2013 

Vol. V, No. 1s  ISSN: 2283-3927 

   

 

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Feed additives are commonly used to improve pig performance and health, but they need to be 

tested so new biomarkers for intestinal health, non- or minimally invasive, are under 

investigations. 

The quantification of Myeloperoxidase (MPO) and Pancreatitis Associated Protein (PAP) in feces 

could prove useful to non-invasively monitor intestinal health (Niewold, 2015). MPO is an 

enzyme that permits to quantify the number of inflammatory cells present in tissues and feces 

(Prokopowicz et al., 2012) , while PAP is a protein mainly produced in the small intestine with 

anti-inflammatory and bactericidal activity (Cash et al., 2006; Mukherjee et al., 2014). Because of 

the lack of a commercial ELISA kit for porcine PAP detection, the main aim of this study was to 

develop and validate a new sandwich ELISA test for the quantification of PAP in pig fecal 

samples. Our study consisted of two phases: test development (Figure 1) and test validation. 

During the development phase we used polyclonal antibodies previously immunized from rabbit 

serum with a pure peptide containing the N-terminus of pig PAP (Soler et al., 2015). The 

validation of the test was then performed with fecal extraction samples derived from animals 

with known high or low growth performance. 

Moreover, the temperature stability of PAP in feces and the optimal extraction method was 

tested. Even if only preliminary, our results seem to show a fair relationship between fecal 

consistency, used as health indicator, and PAP fecal concentrations. Furthermore, no relevant 

differences in PAP concentration after 24h of incubation at 37 °C, 4°C or room temperature were 

detected. 

To date, the present results suggest that PAP seems to be exceptionally stable in feces and is a 

very promising candidate as a non-invasive (fecal) biomarker for intestinal health and growth. 

 

 

 

 

Keywords 

Non-invasive biomarkers, 

Pancreatitis Associated Protein, 

Myeloperoxidase ELISA, 

intestinal health.  

 
CORRESPONDING AUTHOR 

Elena Mariani 

elena.mariani1@unimi.it 

 
JOURNAL HOME PAGE 

riviste.unimi.it/index.php/haf 

Non-invasive intestinal biomarkers: a 

new ELISA test for Pancreatitis 

Associated Protein detection in pig.  

E. Mariani1,2,*, G. Savoini1, T.A. Niewold2 

 

1 Department of Health, Animal Science and Food Safety, Università degli 
Studi di Milano, Via Celoria 10, 20133 Milan, Italy. 

2 Nutrition and Health Unit, Department of Biosystems, Faculty of 
Bioscience Engineering, KU Leuven. Kasteelpark Arenberg 30, B-3001 
Heverlee, Belgium. 

 

 

http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en


Proceeding of Veterinary and Animal Science Days 2018, 6th- 8th June, Milan, Italy 52 

HAF © 2013 

Vol. V, No. 1s  ISSN: 2283-3927 

 

 

  

References 

Cash, H.L., Whitham, C.V., Behrendt C.L., Hooper L.V., 2006. Symbiotic bacteria direct expression of an intestinal 

bactericidal lectin. Science. 313, 1126-1130. 

Mukherjee, S., Zheng, H., Derebe, M.G., Callenberg, K.M., Partch, C.L., Rollins, D., Propheter, D.C., Rizo, J., Grabe, M., 

Jiang, Q.X., Hooper, L.V., 2014. Antibacteria membrane attack by pore-forming intestinal C-Type lectin. Nature. 

505, 103-107.  

Niewold, T.A., 2015. Intestinal health biomarkers in vivo. In: Intestinal health. T.A. Niewold (ed.). Wageningen 

Academic Publishers. Chapter 9.  

Prokopowicz, Z., Marcinkiewicz, J., Katz, D.R. and Chain, B.M., 2012. Neutrophil Myeloperoxidase: Soldier and 

statesman. Archivum Immunologiae et Therapiae Experimentalis. 60, 43–54.  

Soler, L., Miller, I., Nöbauer, K., Carpentier, S., Niewold, T., 2015. Identification of the major regenerative III proteon 

(RegIII) in the porcine intestinal mucosa as RegIIIγ, not RegIIIα. Veterinary Immunology and Immunopathology. 

167, 51-56.  

 

 

Figure 1:   Standard curves with absorbance at 405 nm versus log 2 RegIIIγ peptide concentration (µg/ml). Capture 

antibody concentrations used were 10 µg/ml (           ); 5 µg/ml ml (           ); 3.125 µg/ml (             ) and 0 µg/ml (           ). 

Detection antibody concentration used was 5 µg/ml. 

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