Vol 56, No 3 August 2003 International Journal of Dairy Technology 133 REVIEW *Author for correspondence. E-mail: mg@aua.gr © 2003 Society of Dairy Technology Blackwell Publishing Ltd. Recent developments in antibody-based analytical methods for the differentiation of milk from different species GOLFO MOATSOU and EMMANUEL ANIFANTAKIS Laboratory of Dairy Technology, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece The antibody-based analytical methods for the detection of milk from different species that have been developed in recent years are, for the most part, various enzyme-linked immunosorbent assay (ELISA) configurations. Polyclonal and, more recently, monoclonal antibodies against total or individual caseins, whey proteins and synthetic peptides corresponding to milk proteins sequences have been used. The assays have been successfully applied to the detection of substitution of ovine or caprine milk by bovine milk and of ovine milk by caprine milk in raw and heat-treated milks and cheeses. Keywords ELISA, Immunoassays, Milk adulteration, Milk species differentiation, Polyclonal–monoclonal antibodies. *Author for correspondence. E-mail: mg@aua.gr INTRODUCTION Traditional cheeses produced in the Mediterranean countries, e.g. feta, manchego and pecorino, are made from ovine milk, or from its mixtures with caprine milk, and are accepted by consumers worldwide. The composition of cheese milk affects the characteristics and the organoleptic properties of the final product. The seasonal production and the higher prices of caprine milk and especially of ovine milk in comparison to bovine milk are the main reasons for the admixture of cheese milk with bovine milk. In addition, the higher price of ovine milk and the existence of mixed flocks of goats and ewes result in the accidental or fraudulent sub- stitution of ovine milk by caprine. However, the increased demand for genuine and accurately labelled traditional products necessitates protec- tion against adulteration of milk species in dairy products. Several analytical techniques, including chromatographic, electrophoretic, immunological and, more recently, DNA-based techniques, have been presented for the detection of ovine and caprine milk adulteration and most of them are based on the analysis of milk protein fractions. The present work is focused on antibody-based analy- tical methods that have been reported mainly following the review of Ramos and Juárez, 1 and involve, for the most part, enzyme-linked immuno- sorbent assay (ELISA) configurations. Methods based on antigen–antibody precipita- tion reactions for differentiating the proteins in milks from different species have been applied since 1901. 1 At first, the most effective of them used rabbit antiserums to milk whey proteins, which produce more specific antiserums than caseins, or to blood serum proteins for the detection of bovine milk in ovine and caprine milk and cheese. The elimination of cross-reacting anti- bodies by immunoadsorption of the prepared anti- serum on the protein fraction from species of milk or blood that must not react during analysis has been an important step in this research field. These specific antiserums have been used to detect the addition of bovine milk to caprine and ovine milk and some of these methods have been commercialized. 2,3 Levieux 4,5 prepared an antiserum against chromato- graphically purified bovine immunoglobulin (Ig)G1 by immunizing goats or sheep to overcome the adsorption step. He used the methods of radial immunodiffusion or inhibition of haemagglutina- tion and the detection limits were 1% and < 1% addition of bovine milk, respectively. Both methods have been commercialized as a patent (CV test for detection of bovine IgG1 and BC test for caprine IgG1) and they have been applied to many cheese varieties. 6,7 García et al. 8 developed a cows’ milk identification test (COMIT) for the detection of 3 – 100% cows’ milk in ewes’ milk, which is a modifi- cation of the agar-gel immunodiffusion technique and uses polyclonal antibodies raised in goats against bovine whey proteins. Whey proteins are not affected by proteolysis during cheese ripening but they are affected by the heat treatment of milk. The immunodiffusion method was suitable for milks heated up to 74°C for 30 s. 9 Caseins are heat resistant and have been used for the elaboration of immunological methods, although preservation of their epitopes depends on protein hydrolysis during cheese ripening. An