**3. Evaluation of protein fractions and individual proteins**

Another method for the determination of protein concentrations is the Bradford assay, which is based on the binding of the Coomassie brilliant blue dye to the proteins in an acidic solution to form a complex with increased molar absorbance [31]. This assay is rapid, practical, and suitable for simple quantification of proteins in cell lysates, cellular fractions, and recombinant protein samples [32]. It may be performed also in microtiter plates using micro volumes, but its application area is mainly restricted to research laboratories [33]. Unfortunately, the Bradford assay is linear over a short range (to 2000 μg/ml) and shows a curvature over this range of protein concentration, which necessitates the dilution of sam-

The concentrations of serum or plasma proteins may be measured also by physical methods. Refractometers are used by many veterinary practitioners, because of their ability to measure the protein concentrations in various biological fluids rapidly. Generally, the refractometric technique is based on the determination of the extent, how light is refracted when it passes from one medium to another of different densities (usually from air into the sample) [36]. The angle of refraction is proportional to the concentration of solute in solution. Seeing that proteins are the most important solute dissolved in serum, the refractive index indicates the concentration of proteins in the sample [37]. A good correlation between refractometry and the biuret method was found in human serum samples [38], but the results for veterinary samples are less consistent. Indeed, whether some authors have reported a good correlation of results for domestic mammals (biuret methods vs. refractometry), others showed either higher or lower values for refractometry compared to the biuret method [39, 40]. The differences between the methods were of 6 g/l and 2 g/l in dogs and cats, respectively [36]. However, the most marked differences between the biuret and refractometric methods were observed in avian samples due to the interference by high concentrations of other light-refractive non-protein components of the blood, such as glucose, cholesterol, or lipids [37, 41]. These variations might be caused by differences in the design of various refractometers assigned by the manufacturers, variation in the biuret reagent mixture, as well as assay [42]. Vandeputte et al. [43] evaluated four different refractometers for measuring serum total protein concentrations in beef calves in comparison with the results obtained by the biuret method. In this study, the refractometric measurements were highly correlated with those obtained by the biuret method indicating similar accuracy for measuring serum total protein values. Calloway et al. [44] and Wallace et al. [45] identified a similar ability to detect failure of passive transfer in calves with refractometers. As the index of refraction is influenced by the temperature of the solute, Automatic Temperature Compensation (ATC) refractometers were commercialized to avoid the impact of potential temperature variations on the results [43]. Recently, digital refractometers have been introduced also into the veterinary medicine, where they demonstrated excellent precision with good sensitivity and specificity [43, 45]. However, according to Hunsaker et al. [46], they did not introduce benefits in accuracy over manual refractometry in regards to potential

ples before further analysis [34, 35].

108 Ruminants - The Husbandry, Economic and Health Aspects

interference due to non-protein solutes.

**2.2. Physical methods**

The identification and quantification of individual serum proteins or groups of proteins are possible only if they are separated. In the protein analyses, the most important method available to measure independent proteins or protein groups is the fractionation technique. Blood serum consists of a large number of proteins; thus, the whole protein complex is not possible to analyze in a simple step by currently available separation technologies [47].

The two major types of proteins in the blood are albumin and globulins. Currently, the bromocresol green (BCG) and bromocresol purple (BCP) methods are the basis for the determination of serum albumin [48]. The BCG method is a dye-binding technique characterized by an ionic interaction between positively charged albumin and negatively charged dye molecules at acidic pH [49]. The bromocresol green binds quantitatively with albumin forming an intense bluegreen complex, and the intensity of the color produced is directly proportional to the albumin concentration in the sample [50]. This method is easy to perform, rapid, and cheap, but less sensitive and selective compared to immunoassays [51]. Factors such as optimal pH, ionic strength of buffer, sample preparation, dilution rate, incubation time, and interfering proteins may affect the accuracy of this technique [52, 53]. The reaction between serum and BCG is not specific for albumin; therefore; the BCG method often overestimates the concentrations of serum albumin, but its specificity can be improved by minimizing the contact time with the serum sample [54]. The BCG method is often used to determine the serum albumin concentrations also in animals, including ruminant species [55]. However, albumin methodologies in chemistry analyzers are optimized and designed to measure human albumin. Furthermore, bromocresol green can bind animal globulins with extended reaction times [56, 57]. Therefore, protein electrophoresis may be a better method to provide more accurate albumin quantification [58].

Bromocresol purple is an another related dye that may be used for the determination of albumin concentrations, giving more accurate results and thus has better diagnostic utility [54, 59]. Bromocresol purple is an albumin selective dye, which minimizes globulin interference that occurs with bromocresol green by long incubation (more than 30 seconds) [60, 61]. Good correlation was observed between the serum albumin values obtained by the BCP method and immunoassay [61, 62]. Discrepancies may be observed between the serum and plasma albumin values determined by the BCP method. Plasma albumin concentrations may be falsely increased by turbidity due to the precipitation of fibrinogen when plasma is diluted into the BCP reagent [63, 64].

A number of methods have been developed to measure the concentration of globulins. One type of these techniques is based on the precipitation of globulins using solutions of metal salts, e.g., sodium sulfite or zinc sulfate [65, 66]. The addition of salts causes turbidity, which may be visually evaluated or measured by spectrophotometer as units of turbidity. This method may be used as a field test for the evaluation of suckling efficiency or failure of passive transfer of maternal immunity via colostrum in calves and foals [67–69]. However, protein electrophoresis is recommended to accurately determine globulin distribution, allowing to efficiently and precisely detect, as well as quantify several globulin fractions (α-, β-, and γ-globulins) [70].

#### **3.1. Protein fractionation**

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].

Electrophoresis in ruminant species is normally used in serum, but plasma or other body fluids (urine, cerebrospinal fluid) may also be processed. Serum is the best material for protein electrophoresis, as it does not contain fibrinogen. Electrophoretic technique may be used also for the analysis of urinary proteins, which is a fundamental step in the early diagnosis and subsequent monitoring of renal diseases [89]. It was found that polyacrylamide gel electrophoresis may localize the origin of urinary proteins based on their molecular weight, providing a diagnostic sensitivity comparable to results obtained by kidney biopsy [90]. It is considered a very sensitive method to discriminate between glomerular, tubular, or mixed proteinuria [91]. Agarose gel electrophoresis may be applicable also to separate the main protein fractions in cerebrospinal fluid (CSF) samples. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system, as well as possible disturbances in the blood-brain barrier [92]. Cerebrospinal fluid contains only a small amount of proteins when compared with serum. Therefore, the proteins in CSF should be concentrated to increase the sensitivity of CSF protein detection by electrophoresis [93].

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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.

**3.2. Analysis of individual serum proteins**

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 clarity of the electrophoretic bands [84].

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

Electrophoresis in ruminant species is normally used in serum, but plasma or other body fluids (urine, cerebrospinal fluid) may also be processed. Serum is the best material for protein electrophoresis, as it does not contain fibrinogen. Electrophoretic technique may be used also for the analysis of urinary proteins, which is a fundamental step in the early diagnosis and subsequent monitoring of renal diseases [89]. It was found that polyacrylamide gel electrophoresis may localize the origin of urinary proteins based on their molecular weight, providing a diagnostic sensitivity comparable to results obtained by kidney biopsy [90]. It is considered a very sensitive method to discriminate between glomerular, tubular, or mixed proteinuria [91]. Agarose gel electrophoresis may be applicable also to separate the main protein fractions in cerebrospinal fluid (CSF) samples. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system, as well as possible disturbances in the blood-brain barrier [92]. Cerebrospinal fluid contains only a small amount of proteins when compared with serum. Therefore, the proteins in CSF should be concentrated to increase the sensitivity of CSF protein detection by electrophoresis [93].
