**2.1. Chemical methods**

In biochemical laboratories, the most widely used analytical technique to assess the concentrations of total proteins is the biuret method. This method is based on colorimetric principle, in which the copper ions from the biuret reagent react with the amide groups from the proteins at strong alkaline pH, creating a violet color [24, 25]. However, this method is not sensitive enough to measure lower protein concentrations found, for example, in cerebrospinal fluid [26]. Despite of this disadvantage, the biuret assay is still frequently used because of simple analytical procedure, easy preparation of reagents, and when compared with other copper-based assays, this method is less susceptible to chemical interference [27]. Many of the total protein assay kits developed for the automated use in wet biochemical analyzers, as well as dry chemistry analyzers, are based on this principle. This technique is very cheap and this favored its wide application in veterinary medicine.

The biuret method was modified by using the Folin phenol reagent (Folin-Ciocalteu), which is more sensitive and thus more appropriate to measure low concentrations of proteins [28]. In this method, the phenolic groups of tyrosine and tryptophan in proteins react with the Folin-Ciocalteu reagent producing a blue-purple colored complex [29]. The disadvantages of the Lowry method are the sensitivity to the amino acid composition of the protein and the interference with a range of substances, including buffers, drugs, and nucleic acids [30]. 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 samples before further analysis [34, 35].

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

to analyze in a simple step by currently available separation technologies [47].

may be a better method to provide more accurate albumin quantification [58].

BCP reagent [63, 64].

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

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].

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

The Use of Serum Proteins in the Laboratory Diagnosis of Health Disorders in Ruminants

http://dx.doi.org/10.5772/intechopen.72154

109

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

#### **2.2. Physical methods**

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 interference due to non-protein solutes.
