Quantitation of Acute Phase Proteins and Protein Electrophoresis in Monitoring the Acute Inflammatory Process in Experimentally and Naturally Infected Mice

Serologic screening for infectious disease in sentinel mice from rodent colonies is expensive and labor-intensive, often involving multiple assays for several different infectious agents. Previously, we established normal reference ranges for the protein fractions of several laboratory strains of mice by using a commercially available agarose system of protein electrophoresis. In the current study, we address protein fractionation and quantitation of acute phase proteins (APP) in mice experimentally infected with Sendai virus or mouse parvovirus. We further investigate this methodology by using samples from sentinel mice from colonies with endemic infection. All study groups showed significant increases in γ globulins. Various other protein fractions showed mild variable changes; significant differences were not detected for individual APP. These results contrast the significant changes observed in APP and protein electrophoresis by using the standard methods of inducing inflammatory responses through injection of complete Freund adjuvant or LPS. These present data suggest that although quantitation of individual APP may not be helpful, γ globulin levels may reflect infection in laboratory mice and provide a possible adjunct to traditional screening methods.Abbreviations: APP, acute phase protein; CFA, complete Freund adjuvant; CRP, C-reactive protein; MPV, mouse parvovirus; SAA, serum amyloid A; SAP, serum amyloid PSophisticated technologies including serology, culture, histology, and PCR are available to evaluate laboratory animals for the presence of infectious disease.⁴⁸ These analyses, albeit expensive and labor-intensive, are necessary to ensure that laboratory rodents are free from infectious agents that can interfere with research. In both human and veterinary medicine, the quantitation of acute phase proteins (APP) has been proposed to have diagnostic and prognostic utility to study disease and infection.^{2,11,14,21,26,40} APP are blood proteins primarily synthesized by hepatocytes as part of the complex systemic response termed the acute phase response. The acute phase response is part of the early defense or innate immune system, which is triggered by various stimuli, including trauma, infection, stress, neoplasia, and inflammation. The acute phase response has been referred to as the ‘molecular thermometer,’ whereby quantitation of specific APP might reflect the response to the triggering event.^10,14,40,44 To this point, several studies have been conducted in companion, laboratory, and large animals profiling changes in APP after experimental and natural infection.^{17,18,38,40,45,50}Mice have several major APP that may reflect acute and chronic inflammatory processes including C-reactive protein (CRP), haptoglobin, serum amyloid P (SAP), and serum amyloid A (SAA).^20,47 ELISA assays for these proteins are commercially available. A broader view of the sum of APP changes and the overall acute phase response is obtained through the use of protein electrophoresis.³³ This technique uses an agarose gel to separate protein fractions into albumin, α1 globulins, α2 globulins, β globulins, and γ globulins. Protein electrophoresis does not quantitate single proteins but rather groups of proteins that are mediators of acute inflammatory process. α1 globulins include α1 antitrypsin and α1 acid glycoprotein; α2 globulins include α2 macroglobulin and haptoglobin; β globulins include transferrin, SAA, and CRP, and γ globulins are composed primarily of IgG.³³ Many diagnostic and prognostic uses of protein electrophoresis in veterinary medicine have been reported.^1,4,12,25,33 Although rarely diagnostic of a particular disease, protein electrophoresis is helpful for the detection of acute and chronic inflammatory processes and stimulation of humoral immunity.^12,15,33APP have been proposed to be valuable biochemical markers of stress, infection, and pain in laboratory animals.^14,42 Previously, we established normal reference ranges for the protein fractions of several laboratory strains of mice by using a commercially available agarose system of protein electrophoresis.⁵⁴ The primary goal of the current project was to study the potential changes in APP and protein fractions in laboratory mice after experimental infection with viral pathogens. These data were compared to those generated by using traditional means of inducing acute inflammation with the injection of LPS and complete Freund adjuvant (CFA). In addition, we addressed the possible application of protein fractionation and quantitation of APP by using samples from sentinel mice from colonies with endemic infection.