Production and Characterization of Monoclonal Antibodies against Horse Immunoglobulins Useful for the Diagnosis of Equine Diseases

Abstract

Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELISA versus purified immunoglobulins from donkey, cow, buffalo, sheep, pig, and chicken. Three MAbs (1B10B6C9, 1B10B6C10, 1B10B6E9) reacted only with horse and donkey IgG and IgM and, in western blotting, were specific for the Fc fragment of equine IgG. MAb 1B10B6E9 was used in chemiluminescent immunoblotting assay for the diagnosis of dourine and in indirect immunofluorescence assay (IFA) for the diagnosis of African horse sickness and dourine.

Analysis of Trypanosoma equiperdum Recombinant Proteins for the Serological Diagnosis of Dourine

“…One-hundred μL/well of serum dilution buffer was added in two wells of each microplate as blank. Plates were then washed and incubated at RT for 30 min (A0A1G4I8N3 and A0A1G4I464 proteins) or 1 h (A0A1G4I740 protein) with 100 μL/well of MAb-HRP 1B10B6E9 anti horse-IgG (IZSAM) [ 13 ] diluted 1:100,000 in PBS-T. After further washes, 100 μL of 3-3′-5-5′ tetramethylbenzidine (TMB, Surmodics) were dispensed into each well, and the plates were incubated at RT for 30 min. The reaction was stopped by adding 50 μL/well of 0.5 N sulphuric acid.…”

“…For the protein A0A1G4I464, the sera were diluted 1:20 in PBS-T containing 2.5% skimmed milk. After three washes with PBS-T for 10 min, membranes were incubated for 1 h at RT with Mab-HRP 1B10B6E9 anti horse-IgG (IZSAM) [ 13 ] diluted 1:100,000 in Roti Block 1x. After three washes with PBS-T, and one final wash with PBS for 10 min, membranes were detected by chemiluminescence (ECL Western blotting detection kit, GE Healthcare, Chicago, IL, USA) using the Chemidoc MP (Bio-Rad, Hercules, CA, USA).…”