Affinity of Streptococcal G Protein to Forest Musk Deer (Moschus berezovskii) Serum Immunoglobulin G Open Access

The forest musk deer (FMD; Moschus berezovskii) is a medium-sized mammal that dwells in alpine forests (Tian et al. 2017). It has great economic value because of the musk secreted by adult male FMD, which plays an important role in traditional Asian medicine and the international perfume industry (Sheng and Liu 2007). Currently, the FMD has been listed as critically endangered on the State Key Protected Wildlife List of China (Yan et al. 2016). However, it is worrisome that some diseases, including pneumonia, abscess disease, and diarrhea, continue to restrict its population increases, especially its high susceptibility to pneumonia, which was responsible for more than 50% of all deaths (Yan et al. 2016).

There is no method of serologic diagnosis for FMD disease, which raises an immunologic response. In addition, no reference intervals of physiologic and biochemical indices and early diagnosis methods have been established for FMD disease. Thus, sensitive and specific immunodiagnostic assays for the research and conservation of FMD species are needed.

Streptococcal G protein (SPG) is a cell surface component in the cell wall of Streptococcus groups A, C, and G that has high affinity for the fragment crystallizable region (Fc region) of immunoglobulin G (IgG) in a wide range of animals (Kronvall 1973; Reis et al. 1984). In addition, staphylococcal A protein (SPA) is a type I membrane protein derived from Staphylococcus aureus that also has binding affinity for the Fc region of IgG from a variety of animals and for some immunoglobulin M and immunoglobulin A molecules (Kangwa et al. 2019). Both SPG and SPA conjugated to horseradish peroxidase (HRP–SPG and HRP–SPA, respectively) have been used as antibodies to immunoglobulins for several wild animals in seroepidemiologic studies. Mendonça et al. (2019) reported that SPG and SPA could bind to West Indian manatee (Trichechus manatus manatus) immunoglobulin. Pelli et al. (2012) also found that SPG and SPA had high affinity for the immunoglobulins of many wild mammals.

We evaluated the potential use of HRP–SPG and HRP–SPA to serve as a good enzyme-labeled antibody substitute for the diagnosis of disease in FMD. Three healthy FMD venous blood samples were collected with vacuum tubes, and serum was collected and stored at –20 C until use. The process of blood collection was in accordance with the animal protection law of the People's Republic of China.

An agar double-diffusion test indicated that the immunoglobulin of FMD bound efficiently to SPG and bound weakly or not at all to SPA. Further verification was determined with enzyme-linked immunosorbent assay (ELISA) as previously reported (Pelli et al. 2012), and HRP–SPG and HRP–SPA were chosen as enzyme-labeled antibody substitutes. Sera of pigs (Sus scrofa) and Pekin Ducks (Anas platyrhynchos domestica) were used as positive and negative controls, respectively (Fischer and Hlinak 2000; Nielsen et al. 2008).

For the immunoblotting assay, we purified IgG from FMD serum by Protein G Agarose Prepacked Column (Fast Flow, 5 mL, Beyotime Biotechnology, Co., Ltd., Shanghai, China) based on the results of the agar diffusion test and ELISA. The purified IgG was run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis system. The immunoglobulin were treated with different protein loading buffers: sample A, used with prior heating, was treated with 250 mmol/L Tris-hydrochloride (pH 6.8), 10% (W/V) sodium dodecyl sulfate, 0.5% (W/V) bromophenol blue, 50% (V/V) glycerine, and 5% (W/V) β-mercaptoethanol; sample B was treated without prior β-mercaptoethanol and heating treatment. After electrophoretic separation, the gel was stained with Coomassie blue R-250, and another gel was electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane at 200 mA for 150 min using a Bio-Rad system (Bio-Rad Laboratories, Inc., Hercules, California, USA). Then, the PVDF membrane was incubated with HRP–SPG.

The ELISA results (Fig. 1) were consistent with the agar double-diffusion test. High optical densities for FMD serum immunoglobulins were detected. Compared with those of pigs and ducks, FMD serum immunoglobulins presented high affinity for SPG, but SPA binding was not observed in FMD serum. In previous studies, both SPA and SPG were proven to have high affinities for pig IgG, but no affinity for duck IgG (Fischer and Hlinak 2000; Nielsen et al. 2008). In this study, western blotting using HRP–SPG as an enzyme-labeled antibody was performed to determine whether the SPG actually bound to FMD IgG as observed in the agar double-diffusion test and ELISA. The FMD IgG samples A and B were separated on a 10% gel, and four bands with estimated molecular weights of approximately 25, 50, 100, and 150 kDa were found (Fig. 2a). Western blotting results showed that a band was observed on the PVDF membrane of the IgG sample B lane (Fig. 2b), which suggested that SPG bound to FMD IgG. A typical immunoglobulin consists of two heavy chains and two light chains, which are linked by four disulfide bonds (Hayashi and Yagihara 2016). In this study, in the sample A lane, under prior heating and β-mercaptoethanol treatment, the disulfide bonds and protein spatial structure of IgG were disrupted; thus, there were different bands found on the gel, and SPG binding was not observed on the PVDF membrane, whereas high affinity of FMD IgG to SPG was detected in sample B.

Research on FMD diseases includes mainly isolation and identification of pathogens, construction of pathologic models, and analysis of blood messenger RNA expression profiles (Zhao et al. 2017; Wang et al. 2018; Sun et al. 2018). Until now, serologic methods for FMD disease have not been reported, and the use of enzyme-labeled anti-FMD antibodies in the diagnosis of FMD disease need to be evaluated. Overall, the affinity of SPG to FMD IgG was determined by agar double-diffusion test, ELISA, and western blotting. Therefore, HRP–SPG should be considered as an important enzyme-labeled antibody substitute to develop serologic diagnosis methods for FMD disease. In addition, some reports have discussed the use of SPA as diagnostic biosensor for the diagnosis of some diseases in humans (Rigi et al. 2019). Thus, additional studies about SPG application in FMD disease need to be evaluated. The results provide the basis for the development of diagnostic methods and lay a foundation for immunologic research in FMD disease.