Alexis E. Rose, Christopher D. Warner
The health and performance of the gut is influenced by a myriad of factors including diet, genetics, environment, stress, intestinal barrier, immune status, and the microbiome. The alteration of one or more of these factors can lead to enteropathy, which is characterized as an increase in intestinal permeability, impairment of gut immune function, and nutrient malabsorption¹. An imbalance in the gastrointestinal tract can lead to deteriorating intestinal barrier function allowing for the translocation of pathogens and induce circulating immune regulators, thus generating a cyclical cause and effect relationship of increased inflammation and aberrant cytokine production². The positive feedback loop of inflammation in the gut can ultimately cause chronic disease states such as IBS-D, IBD, or HIV-associated enteropathy. Therefore, restoring balance within the GI tract is critical to overall health.
Immunoglobulins have a positive role in gut homeostasis. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a spray-dried protein powder specially formulated to enrich the immunoglobulin content to help manage loose and frequent stools associated with gastrointestinal enteropathies. It is manufactured by fractionating edible grade bovine plasma to contain 90% protein, with most of the total protein comprised of immunoglobulins: 60% IgG, 5% IgM, and 1% IgA. The high concentration of immunoglobulins has shown to bind and neutralize opportunistic pathogens by immune and steric exclusion mechanisms using an in vitro cell culture model³. This binding of antigens helps limit the stimulation of immune regulators that modulate chronic intestinal inflammation when these microbial components translocate into the lamina propria.
The goal of this study was to expand the list of antigens to which SBI binds by adapting the ELISA used by Detzel et al³ to demonstrate binding of IgG to lipopolysaccharide, lipid A, and Pam3CSK4 antigens. New relevant antigens of interest were chosen based on the market needs for binding of gram-negative bacteria
components and mold mycotoxins.
The binding of C. ablicans lysate, Als3, H. pylori lysate, CagA, Shiga-like toxin 1, aflatoxin M1, and CDT antigens by IgG in SBI were initially screened by a dot blot method. All antigens except the H. pylori lysate and aflatoxin M1 exhibited binding by dot blot. No additional testing was performed on the H. pylori lysate, but aflatoxin M1 antigen-IgG binding tested negative by ELISA (Appendix).
Figure 1. SBI binding affinity to various antigens A) SBI-IgG binds to C. albicans antigens, B) SBI-IgG binds to CDT antigens, C) SBI- IgG binds to Shiga-like toxin 1, D) SBI-IgG binds to a H. pylori CagA antigen. Error bars represent ± one standard deviation from triplicate data. Absorbance units (A.U.) measured at 450 nm.
To demonstrate specificity of IgG binding, antigens were tested using a modified ELISA demonstrated in (Fig. 1, A-D). Absorbance of IgG bound to immobilized antigens increased proportionally to SBI concentration. SBI IgG specifically binds C. albicans lysate, C. albicans Als3 protein, CDT subunit A, CDT subunit B, Shiga-like toxin 1, and H. pylori CagA protein.
Specificity of immunoglobulin binding to each antigen was assessed by comparing the “Complete ELISA” curves shown in (Fig. 2, A-F) to control curves which excluded steps throughout the modified ELISA. SBI IgG binds specifically to antigens (Complete ELISA) when compared to the binding controls (Fig. 2, A-F). The complete ELISA displayed a higher absorbance compared to the blocking control (No antigen, Block), demonstrating IgG specificity for the antigen. Effective blocking is demonstrated by comparing wells treated with blocking buffer (No antigen, Block) to wells without blocking buffer (No antigen, No block). In summary, results from the specificity and binding controls indicate IgG binds specifically to each antigen in Table 1.
Figure 2. SBI IgG had a higher affinity for antigen protein (Complete ELISA) when compared to the blocking protein (-antigen, +block). Effective blocking is demonstrated by comparing wells treated with blocking buffer (-antigen, +block) to wells not treated with blocking buffer (-antigen, -block) A) SBI IgG binding to C. Albicans, B) SBI IgG binding to Als3, C) SBI IgG binding to CdtA, D) SBI IgG binding to CdtC, E) SBI IgG binding to Shiga Toxin 1, F) SBI IgG Binding to CagA. Error bars represent ± one standard deviation from triplicate data. Absorbance units (A.U.) measure at 450 nm.
Functional binding of SBI was assessed by comparing the complete ELISA curves shown in (Fig. 3, A-F) to the denatured SBI control, which displayed no binding activity for each ELISA. The background controls, no addition of the detection antibody or SBI, showed no significant absorbance compared to the Complete ELISA curve.
Figure 3. Specificity control experiments IgG binding to A) C. albicans lysate, B) C. albicans Als3 antigen, C) CdtA, D) CdtC, E) Shiga-like toxin 1, and F) H. pylori CagA antigen. Error bars represent ± one standard deviation from triplicate data. Absorbance units (A.U.) measured at 450 nm. Absorbance units (A.U) measured at 450 nm.