Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay.

The research article is about the development and characterization of two new monoclonal antibodies (mAbs) used to quantify the concentration levels of immunoglobulin A (IgA) in horse serum and mucosal secretions, using a fluorescent bead-based assay. The study confirms the predominant presence of IgA in mucosal surfaces such as the nasopharynx and saliva in horses.

Development and Characterization of Monoclonal Antibodies

• Two new monoclonal antibodies (mAbs), designated as clones 84-1 and 161-1, against equine IgA were developed and thoroughly tested.
• These IgA mAbs were able to specifically bind to both monomeric and dimeric equine IgA in different applications including Western blots and fluorescent bead-based assays.
• There was no observed cross-reactivity with other equine immunoglobulin isotypes, proving the specificity of the newly developed mAbs.

Establishing a Fluorescent Bead-Based Assay

• The research utilized the new IgA mAb 84-1 in conjunction with a previously characterized anti-equine IgA mAb BVS2 to create and validate a fluorescent bead-based assay.
• This assay was designed to quantify the total IgA present in equine serum and various secretions like nasal wash fluid, saliva, and colostrum.
• The assay had a linear detection range of 64pg/ml to 1000ng/ml, allowing for a wide span of IgA concentration measurements.

IgA Quantification and Results

• The fluorescent bead-based assay was used to quantify IgA in different equine samples from healthy adult horses.
• Given the different signal intensities of monomeric and dimeric IgA in the bead-based assay, different standards for IgA quantification were developed from serum and nasal wash fluid accordingly.
• Results revealed that the median total IgA in serum was 0.45mg/ml in Thoroughbred horses and 1.16mg/ml in Icelandic horses. Additionally, the median total IgA was 0.13mg/ml in the nasopharyngeal secretions of Thoroughbred horses and 0.25mg/ml for Icelandic horses.
• IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum.

Significance of the Study

• The research provides a significant contribution to the field of equine immunology by developing effective tools to measure and evaluate mucosal immunity in horses.
• The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA could help to further understand and monitor the immune response in horses, particularly the mucosal immune defense.