**3.2. Analysis of individual serum proteins**

**3.1. Protein fractionation**

110 Ruminants - The Husbandry, Economic and Health Aspects

clarity of the electrophoretic bands [84].

for a precise tracing of the electrophoretic separation [88].

Electrophoresis is the current standard and most widely used fractionation technique of serum proteins in clinical biochemistry and molecular biology [71]. Several fractionation techniques have been developed to separate and consequently quantify the proteins in serum [72]. The most of them depend on the initial determination of total serum proteins, and then the concentrations of the main fractions can be calculated from the total protein values. Electrophoresis is based on the movement of charged particles through a buffered medium when subjected to an electrical field [73]. Serum proteins have a negative charge, so in the electrophoretic chamber, they migrate toward the positive pole in an electrical field and are separated from each other in different bands according to their sizes [74]. The speed of their movement depends on the characteristics of the protein undergoing separation, including its electrical charge, size and shape, as well as on the strength of the electrical field, type of medium used for the separation, and temperature [75]. After separation, the protein fractions are fixed in an acid solution to denature the proteins and immobilize them on the support medium [3]. The proteins are then stained and quantified by density measuring, providing also graphical data for computer analysis according to the used electrophoretic system [76]. The separation of proteins in an electric field was introduced by Tiselius in the 1930s [77]. The application of serum protein electrophoresis in clinical biochemistry laboratories started in 1950s using paper strips [78], were replaced a few years later by microporous acetate membranes [79]. In the 1970s, agarose gel as a support medium was introduced in the electrophoretic separation of proteins [80], and became a most commonly used supporting substance in veterinary medicine. There are great differences between the electrophoretic methods, which are usually caused by the material of support medium used for the fractionation of proteins [81]. Luraschi et al. [82] stated that the electrophoretic patterns of proteins and the numbers of identified peaks are dependent on the used support material: cellulose acetate vs. agarose gel electrophoresis. The standard agarose gel electrophoresis is a labor-intensive method, but the introduction of prepackaged gels and the development of new equipments allowed the automatization of this procedure [81, 83]. However, the correction of electrophoretograms by visual inspection of an experienced interpreter is very important. Furthermore, agarose gel electrophoresis has several advantages compared to cellulose acetate. Indeed, agarose gel as a support medium provides better resolution, higher reproducibility of results, and greater

In past few years, capillary zone electrophoresis (CZE) is being used also in veterinary laboratories [85]. In CZE, the separation of protein fractions occurs in a free liquid medium created by the low viscosity buffer, in which the application of high voltage generates an electroosmotic flow causing rapid movement of proteins toward the cathode [86]. This allows better separation of proteins with similar physicochemical characteristics, thus generating multiple subpeaks or narrower peaks [87]. The higher resolution of CZE can often result in abnormal electrophoretic profiles caused by the aforementioned multiple subpeaks of unknown significance, which is the disadvantage of this method. Recently, laser densitometry was introduced Recently, several techniques, including high resolution electrophoresis, two-dimensional electrophoresis, and proteomic assays were developed for the separation of proteins. These methods allow simultaneous identification of many individual proteins and localize specific proteins within several subfractions. On the other hand, the agarose gel electrophoresis is able to separate serum proteins only into five or six fractions [7]. Changes in proteins with very low concentrations may not be detected by electrophoresis [22]. Immunoassays are another type of methods that may be used for the determination of specific serum proteins. These procedures require a specific antibody against the analyzed serum protein. In biomedical research, enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) belong to the most common analytical methods, which may be used for the identification and quantification of specific proteins, antigens or antibodies [94]. ELISA is based on the concept of an antigen/protein binding to its specific antibody, which allows to detect very small concentrations of antigen/protein [95]. Various types of ELISAs have been developed, while the basic step is the direct or indirect detection of antigen by adhering or immobilizing the antigen or antigen-specific capture antibody onto the well surface [96]. Direct ELISA is considered to be the simplest format of immunoenzymatic assays that determine an antigen immobilized to the plate using an antibody directly conjugated to an enzyme [97]. The indirect ELISA technique requires a secondary antibody to detect the presence of antigen, which is "sandwiched" between the capture antibody coated on the plate and an enzyme-labeled conjugate. Furthermore, the determination of some serum proteins is possible based on their biological activities. For example, the high affinity of haptoglobin for hemoglobin may be used to assess its concentrations. Subsequently, the peroxidase activity of the bound hemoglobin is maintained at low pH [98], the intensity of which is directly proportional to the concentration of Hp in the sample. This colorimetric reaction is not species specific and may be used in several animal species, including ruminants. On the other hand, ceruloplasmin has endogenous oxidase activities, which can be applied to measure its concentrations [99]. However, for the quantitative determination of the most of serum proteins in animals, species-specific assays should still be developed.
