Monoclonal antibodies (mAbs) are laboratory-produced molecules engineered to serve as substitute antibodies that can restore, enhance, or mimic the immune system's attack on cells. In equine research, monoclonal antibodies are utilized to study and influence immune responses, detect pathogens, and develop therapeutic interventions for various diseases. These antibodies are designed to bind to specific antigens with high specificity, allowing for targeted therapeutic and diagnostic applications. Research in this area focuses on the development, application, and effectiveness of monoclonal antibodies in treating infections, inflammatory conditions, and other health issues in horses. This page compiles peer-reviewed research studies and scholarly articles that explore the production, application, and impact of monoclonal antibodies in equine medicine.
Noronha LE, Harman RM, Wagner B, Antczak DF.The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the c...
Wagner B, Hillegas JM, Babasyan S.CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/...
Gómez-Almaguer D, Jaime-Pérez JC, Ruiz-Arguelles GJ.Antibodies have been the cornerstone of treatment of acquired aplastic anemia for more than 25 years. Treatment with antithymocyte globulin (ATG) is considered pivotal and the addition of cyclosporine improves the overall response rate. This antibody is heterogeneous and horse ATG is apparently more effective than rabbit ATG. Several issues remain unsolved in relation to the combination of ATG and cyclosporine: cost, toxicity and late clonal disorders. In recent years, alternative immunosuppressive therapy has been proposed and new antibodies have emerged: porcine ATG, alemtuzumab, daclizumab...
Ferris NP, Clavijo A, Yang M, Velazquez-Salinas L, Nordengrahn A, Hutchings GH, Kristersson T, Merza M.Two lateral flow devices (LFD) for the detection of vesicular stomatitis (VS) virus (VSV), types Indiana (VSV-IND) and New Jersey (VSV-NJ) were developed using monoclonal antibodies C1 and F25VSVNJ-45 to the respective VSV serotypes. The performance of the LFDs was evaluated in the laboratory on suspensions of vesicular epithelia and cell culture passage derived supernatants of VSV. The collection of test samples included 105 positive for VSV-IND (92 vesicular epithelial suspensions and 13 cell culture antigens; encompassing 93 samples of subtype 1 [VSV-IND-1], 9 of subtype 2 [VSV-IND-2] and 3...
Goodyear A, Troyer R, Bielefeldt-Ohmann H, Dow S.The Gram-negative bacterium Burkholderia mallei causes rapidly fatal illness in equines and humans when contracted by inhalation and also has the potential to be used as a bioweapon. However, little is known regarding the early innate immune responses and signaling mechanisms required to generate protection from pneumonic B. mallei infection. We showed previously that monocyte chemoattractant protein 1 (MCP-1) was a critical chemokine required for protection from pneumonic B. mallei infection. We have now extended those studies to identify key Toll-like receptor (TLR) signaling pathways, effec...
Iyer U, Kadambi VJ.Antibody drug conjugates (ADCs) consist of an antibody attached to a cytotoxic drug by means of a linker. ADCs provide a way to couple the specificity of a monoclonal antibody (mAb) to the cytotoxicity of a small-molecule drug and, therefore, are promising new therapies for cancer. ADCs are prodrugs that are inactive in circulation but exert their cytotoxicity upon binding to the target cancer cell. Earlier unsuccessful attempts to generate ADCs with therapeutic value have emphasized the important role each component plays in determining the efficacy and safety of the final ADC. Scientific adv...
Ibrahim S, Steinbach F.Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules f...
Kawai M, Kuwano A, Hiraga A, Miyata H.The myonuclear domain (MND) is the region of cytoplasm governed by a single myonucleus. Myonuclear domain size is an important factor for muscle fibre plasticity because each myonucleus has limitations in the capacity of protein synthesis. Previous studies have demonstrated that differences in MND size exist in different fibre types in several species, including horses. Objective: To understand the basic mechanism of muscle plasticity, the relationships between MND size, muscle fibre type population and metabolic properties of skeletal muscles throughout the whole body in Thoroughbred horses w...
Afable MG, Shaik M, Sugimoto Y, Elson P, Clemente M, Makishima H, Sekeres MA, Lichtin A, Advani A, Kalaycio M, Tiu RV, O'Keefe CL, Maciejewski JP.A combination of horse anti-thymocyte globulin and cyclosporine produces responses in 60-70% of patients with severe aplastic anemia. We performed a phase II study of rabbit anti-thymocyte globulin and cyclosporine as first-line therapy for severe aplastic anemia. Methods: Twenty patients with severe aplastic anemia treated with rabbit anti-thymocyte globulin were compared to 67 historical control cases with matched clinical characteristics treated with horse anti-thymocyte globulin. Results: Response rates at 3, 6 and 12 months were similar for patients treated with rabbit anti-thymocyte glob...
Ji Y, Guo W, Zhao L, Li H, Lu G, Wang Z, Wang G, Liu C, Xiang W.An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for the detection of the equine influenza virus (EIV), employing monoclonal and polyclonal antibodies against the A/equine/Xingjiang/2007 (H3N8) nucleoprotein (NP). Immunoglobulin G antibodies were purified and used as capture or detector antibodies. The specificity of the optimized AC-ELISA was evaluated using EIV, equine herpesvirus 1 (EHV-1), equine herpesvirus 4 (EHV-4), equine arteritis virus (EAV) and Japanese encephalitis virus (JEV), resulting in only EIV specimens yielding a strong signal. A minimal concentr...
Motoshima M, Okamatsu M, Asakura S, Kuribayashi S, Sengee S, Batchuluun D, Ito M, Maeda Y, Eto M, Sakoda Y, Sodnomdarjaa R, Kida H.A/equine/Kanazawa/1/2007 (H3N8), A/equine/Hokkaido/I828/2008 (H3N8) and A/equine/Mongolia/1/2008 (H3N8) were isolated from infected horses. A/equine/Yokohama/aq19/2009 (H3N8) and A/equine/Yokohama/aq13/2010 (H3N8) were isolated from horses imported from Canada and Belgium examined at the Animal Quarantine Service in Yokohama, Japan. In the present study, these five isolates were genetically and antigenically analyzed. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes showed that three isolates from horses in Japan and imported from Canada belonged to the same branch, cla...
Mujica FF, Perrone T, Forlano M, Coronado A, Meléndez RD, Barrios N, Alvarez R, Granda F.The main objective of this study was to demonstrate the occurrence of equine piroplasmosis (EP) in horses of Lara State, Venezuela, and to correlate it with the factors host's sex and age in order to know the epidemiology of this disease at the Venezuelan Centroccidental Region. Antibody levels to Babesia caballi and Theileria equi were assessed in 360 equine serum samples, collected from 9 municipalities of Lara State, using an ELISA technique with recombinant antigens and monoclonal antibodies (Mabs). Antibodies to B. caballi were found in 254 horses (70.6%), whereas 181 animals (50.3%) were...
De Schauwer C, Meyer E, Van de Walle GR, Van Soom A.Mesenchymal stromal cells (MSC) are a very promising subpopulation of adult stem cells for cell-based regenerative therapies in veterinary medicine. Despite major progress in the knowledge on adult stem cells during recent years, a proper identification of MSC remains a challenge. In human medicine, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) recently proposed three criteria to define MSC. Firstly, cells must be plastic-adherent when maintained under standard culture conditions. Secondly, MSC must express CD73, CD90 and CD105, and lac...
Faleiros RR, Nuovo GJ, Flechtner AD, Belknap JK.There is increasing evidence of involvement of inflammatory cells in acute laminitis. Objective: To immunolocalise monocytes/macrophages and B and T lymphocytes in the laminar tissue of normal horses and those with black walnut extract (BWE)-induced laminitis. Methods: Immunohistochemistry was used in archived laminar tissue samples from 20 horses divided equally into 4 groups: control animals (CON), and those administered BWE at 1.5 h (1.5H DTP group), at the onset of leucopenia (3H DTP group) and at the onset of lameness (LAM group). Antibodies against CD3, CD20 and CD163 were used to recogn...
Dai X, Hilsen RE, Hu WG, Fulton RE.An electrochemiluminescence (ECL) immunoassay, incorporating chemically biotinylated and ruthenylated antibodies down-selected from a panel of monoclonal and polyclonal reagents, was developed to detect and identify Venezuelan equine encephalitis virus (VEEV). The limit of detection (LOD) of the optimized ECL assay was 10(3)pfu/ml VEEV TC-83 virus and 1 ng/ml recombinant (r) VEEV E2 protein. The LOD of the ECL assay was approximately one log unit lower than that of a sandwich enzyme-linked immunosorbent assay (ELISA) incorporating the same immunoreagents. Repetition of ECL assays over time and...
Kabithe E, Hillegas J, Stokol T, Moore J, Wagner B.CD14 is a receptor for the complex of lipopolysaccaride (LPS) and LPS-binding protein. Binding of this complex to CD14 in association with Toll-like receptor 4 provides a major pathway for the initiation of innate immune responses to bacterial pathogens. We used a mammalian expressed extracellular region of equine CD14 (rCD14) derived from an IgG fusion protein to produce monoclonal antibodies (mAbs) to CD14. Eight mAbs were tested by flow cytometric analysis of equine leukocytes and by immunoblotting using rCD14 indicating that the mAbs recognized at least three different epitopes on equine C...
Brooks BW, Lutze-Wallace CL, Maclean LL, Vinogradov E, Perry MB.Lipopolysaccharides (LPSs) from Taylorella equigenitalis, the causative agent of contagious equine metritis, and T. asinigenitalis were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide profiles of 11 T. equigenitalis strains were similar, but different from the profiles of 3 T. asinigenitalis strains, and the profiles of 2 T. asinigenitalis strains were similar to each other. The serological specificities of the LPSs from these 14 strains were examined by immunoblotting and enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) ...
Tong T, Bai Y, Liu G, Wang Q, Zhang W, Xiao Y, Xu S, Liu N, Yang T, Wu D.IL-18 is a cytokine originally discovered as an important modulator of immune responses and subsequently shown to be pleiotropic. In this report, we expressed the recombinant equine mature interleukin-18 (rEMIL-18) in E. coli and purified it by nickel affinity gel column chromatography. Purified rEMIL-18 had biological activity commensurate with recombinant human IL-18, as determined by its synergistic effect with recombinant human IL-12 (rhIL-12) on the induction of IFN-gamma gene expression in equine peripheral blood mononuclear cells (PBMC). Following intraperitoneal (i.p.) immunization of ...
Sun WC, Berghaus LJ, Moore JN, Hurley DJ, Vandenplas ML, Thompson R, Linden J.Stimulation of adenosine A(2A) receptors results in anti-inflammatory effects in a variety of cell types. Lipopolysaccharide (LPS) and pro-inflammatory cytokines, such as TNF-alpha and IL-1, have been reported to up-regulate the expression of adenosine A(2A) receptors and thereby enhance the functional activity of adenosine A(2A) receptors in human and murine monocyte/macrophage cell lines and in monocytes/macrophages isolated from those species. In this study, we investigated the effects of LPS and TNF-alpha on the expression and functional activity of adenosine A(2A) receptors in isolated eq...
Fine DL, Jenkins E, Martin SS, Glass P, Parker MD, Grimm B.A multisystem approach was used to assess the efficiency of several methods for inactivation of Venezuelan equine encephalitis virus (VEEV) vaccine candidates. A combination of diverse assays (plaque, in vitro cytopathology and mouse neurovirulence) was used to verify virus inactivation, along with the use of a specific ELISA to measure retention of VEEV envelope glycoprotein epitopes in the development of several inactivated VEEV candidate vaccines derived from an attenuated strain of VEEV (V3526). Incubation of V3526 aliquots at temperatures in excess of 64 degrees C for periods >30 min i...
Retteg Pauls S, Jottini S, Takai S, Venner M, Wohlsein P.A 3-month-old female trotter foal was euthanized due to severe dyspnoea. Pathomorphologically a chronic granulomatous to necrotizing pneumonia was found and Rhodoccocus (R.) equi was isolated microbiologically. An immunohistological method using a murine monoclonal antibody against a 15-17 kDa antigen of virulent R. equi was established in formalin-fixed and paraffin-embedded tissue sections using various antigen retrieval techniques to optimize the staining results. Microwave treatment was most suitable for the demonstration of bacterial antigen localized predominantly in intralesional macrop...
Wagner B.Since the initial characterization of IgE by Ishizaka et al. (1966), IgE was described in several mammalian species. In horses, a single gene encoding the IgE heavy chain constant region (IGHE gene) exists per haploid genome and several allelic variants of the equine IGHE gene were found. IgE occurs in its soluble form in equine serum and physiological concentrations of total IgE are around 1000-fold higher in normal horse than in normal human serum. Maternal IgE is enriched in the colostrum and transferred to the neonatal foal after birth. Foals do not produce detectable concentrations of end...
Wagner B, Miller WH, Erb HN, Lunn DP, Antczak DF.IgE antibodies are mediators of mast cell degranulation during allergic diseases. The binding of IgE to its high-affinity IgE receptor on mast cell surfaces is called "sensitization" and precedes the development of clinical allergy. Previously, intradermal injection of anti-IgE or the anti-IgG(T) antibody CVS40 induced immediate skin reactions in horses. This suggested that both IgE and IgG(T) sensitize equine skin mast cells. Here, we investigated sensitization to allergen and with IgE or IgG(T) in clinically healthy horses of different age groups. In addition, immediate skin reactions to Cul...
Kawai M, Minami Y, Sayama Y, Kuwano A, Hiraga A, Miyata H.We examine the muscle fiber population and metabolic properties of skeletal muscles from the whole body in Thoroughbred horses. Postmortem samples were taken from 46 sites in six Thoroughbred horses aged between 3 and 6 years. Fiber type population was determined on muscle fibers stained with monoclonal antibody to each myosin heavy chain isoform and metabolic enzyme activities were determined spectrophotometrically. Histochemical analysis demonstrated that most of the muscles had a high percentage of Type IIa fibers. In terms of the muscle characteristic in several parts of the horse body, th...
Sprague LD, Zachariah R, Neubauer H, Wernery R, Joseph M, Scholz HC, Wernery U.The internationally mandatory complement fixation test (CFT) for testing of equine sera for the absence of glanders has repeatedly led to discrepant results. Not only do "false positive" sera pose a problem for the diagnostician and the animal health authorities but they can also result in significant financial losses for the animal owners.Due to the very low prevalence of glanders in the horse population it is of major importance to use tests with a high specificity to overcome unreliable predictive values. We have compared formalin-fixed B. mallei whole cell antigen and a well characterised ...
Bacigalupo MA, Meroni G, Secundo F, Scalera C, Quici S.Highly luminescent Eu(3+) and Tb(3+) complexes of 10-[4-(3-isothiocyanatopropoxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane-1,4,7 triacetic acid Eu(3+) is a subset of 1 and Tb(3+) is a subset of 1 were conjugated with a goat anti-rabbit IgG and a rabbit anti-mouse IgG, respectively, and applied as markers in a time resolved immunoassay for simultaneous quantitative determination of anabolic compounds clenbuterol (CL) and hydrocortisone (HC). The assay was performed in horse urine, using a monoclonal antibody specific to CL and a rabbit polyclonal antibody specific to the free HC. These lant...
Satué K, Hernández A, Lorente C, O'Connor JE.Assessment of lymphocyte subsets is an effective method for characterizing disorders such as leukemia, lymphomas, autoimmune and infectious diseases. In order to clinically interpret these parameters, normal reference values should be set, estimating age- and gender-related variations. This research aimed to: (1) characterize lymphocyte subpopulations in Andalusian horse, and (2) evaluate age and gender-related variations of lymphocyte subsets. Jugular blood samples were obtained from 159 animals, 77 males and 82 females, belonging to four age groups-1: 1-2 years (N=39; 21 males and 18 females...
Nizoli LQ, Conceição FR, Silva SS, Dummer LA, Santos AG, Leite FP.The equine piroplasmosis caused by Theileria equi is one of the most important parasitic diseases of the equine, causing damage to animal health and economic losses. In T. equi, 2 merozoite surface proteins, equi merozoite antigen EMA-1 and EMA-2, have been identified as the most immunodominant antigens. This suggests that these antigens might be used as immunobiological tools. The EMA-1 of Theileria equi was cloned and expressed in the yeast Pichia pastoris. The transformed yeast was grown at high cell density, expressing up to 389 mg x L(-1) of recombinant protein. The protein was concentrat...
de Mattos Carvalho A, Alves AL, Golim MA, Moroz A, Hussni CA, de Oliveira PG, Deffune E.The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique...
Ulrich R, Eydner M, Grün A, Haydn J, Baumgärtner W.This report describes the macroscopic, histologic, immunohistologic and ultrastructural characteristics ofa biphasic malignant mesothelioma in the peritoneal and pleural cavity of a 13-year-old Icelandic pony mare, which exhibited recurrent ascites clinically. Immunohistology was performed employing multiple monoclonal antibodies against cytokeratins (CK) and vimentin. The ultrastructural examination included the quantitative evaluation of the length to diameter ratio of the microvilli. Post mortem examination revealed a severe ascites and hydrothorax. The serosal surfaces of the peritoneum an...
Misumi K, Vilim V, Hatazoe T, Murata T, Fujiki M, Oka T, Sakamoto H, Carter SD.This study was designed to assay and compare cartilage oligomeric matrix protein (COMP) in horse sera, in samples from normal and joint diseased horses, and to investigate the relationships between COMP in sera and synovial fluids (SF) with keratan sulphate (KS) data. Sera from 38 horses free of any joint pathology (controls) and from horses with aseptic joint disease (AJD horses, n = 40) were assayed for COMP and KS concentrations. Of the 78 horses in the study, 53 were also assayed for COMP and KS concentrations in SF. COMP and KS were measured by inhibition ELISA, using monoclonal antibodie...
Caplazi P, Ehrensperger F.Borna disease (BD) has been recognized as a virally induced T-cell dependent immunopathological disorder of the central nervous system (CNS), as shown by experimental infection of rats with Borna disease virus (BDV). In contrast to the rat model, little is known about the pathogenesis of spontaneous BD in sheep and horses. The present study describes the brain lesions of 12 ovine and 11 equine cases of naturally occurring BD. A set of monoclonal and polyclonal antibodies was used in order to determine the cells operative in encephalitic lesions and to detect expression of MHC-I and MHC-II prod...
Duggan JM, Coates DM, Ulaeto DO.Venezuelan equine encephalomyelitis (VEE) virus is an important human and veterinary pathogen of Central and South America. The virus can cause widespread epidemics, affecting hundreds of thousands of horses, and thousands of humans. Detection of the virus early in infection and in mosquito populations may allow epidemics to be predicted such that suitable prophylaxis, such as vaccination, can be used to reduce disease severity and transmission. The sensitivity and specificity of current immunoassays, based on conventional monoclonal and polyclonal antibodies, needs to be improved for the diag...
Sironi G, Riccaboni P, Mertel L, Cammarata G, Brooks DE.The expression of p53 protein was investigated in eight formalin-fixed, paraffin-embedded conjunctival squamous cell carcinomas of five horses and one cow, dog and cat each by an immunohistochemical procedure in order to evaluate protein overexpression. Anti-human p53 protein mouse monoclonal antibodies known to be cross-reactive with p53 protein of the animal species examined were used. Positive p53 nuclear immunostaining was detected in five equine, one bovine and one feline cases. Conversely, no p53 immunostaining was found in the only canine case examined. These results demonstrate a frequ...
Ghim SJ, Rector A, Delius H, Sundberg JP, Jenson AB, Van Ranst M.Equus caballus papillomavirus type 1 (EcPV-1) was isolated from a cutaneous papilloma, the most common neoplasm in horses. The complete EcPV-1 nucleotide sequence and genomic organization were determined. Phylogenetic analysis showed that EcPV-1 is a close-to-root papillomavirus, with only distant relationships to the fibropapillomaviruses and the benign cutaneous papillomaviruses. To produce EcPV-1 virus-like particles (VLPs), the EcPV-1 L1 major capsid protein was expressed in insect cells using a recombinant baculovirus vector. The self-assembled EcPV-1 VLPs were morphologically indistingui...
Netolitzky DJ, Schmaltz FL, Parker MD, Rayner GA, Fisher GR, Trent DW, Bader DE, Nagata LP.The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5' end). A 5' RACE reaction was used to sequence the 5' terminus from WEE strain CBA87. The deduced WEE genome was 11508 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28% A, 25% C, 25% G and 22% U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation; a total of 149 nucl...
Ellison SP, Omara-Opyene AL, Yowell CA, Marsh AE, Dame JB.A gene encoding a major 29 kDa surface antigen from Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis (EPM), was cloned, sequenced, and expressed as a recombinant protein. A cDNA library was prepared in the expression vector lambda ZAP from polyA+mRNA isolated from S. neurona merozoites cultivated in vitro. Random sequencing of 96 clones identified a clone of an abundant transcript having a translated amino acid sequence with 30% identity to the 31-kDa surface antigen of Sarcocystis muris cyst merozoites. Southern blot analysis indicated that the correspond...
Lunn DP, Holmes MA, Duffus WP.The aim of this study was to produce monoclonal antibodies (mAb) recognizing equine lymphocyte surface antigens. Fusions were conducted using BALB/c mice hyperimmunized with equine thymocytes. Hybridoma supernatants were screened by flow cytometry and positive hybridomas were cloned twice by limiting dilution. These mAb were then characterized for tissue distribution by immunohistology and flow cytometry, and by precipitation and analysis of the lymphocyte antigens which they recognized. Three mAb (CVS5, CVS4 and CVS8) are described which recognize only T lymphocytes in peripheral blood. Two-c...
Wagner B, Miller WH, Erb HN, Lunn DP, Antczak DF.IgE antibodies are mediators of mast cell degranulation during allergic diseases. The binding of IgE to its high-affinity IgE receptor on mast cell surfaces is called "sensitization" and precedes the development of clinical allergy. Previously, intradermal injection of anti-IgE or the anti-IgG(T) antibody CVS40 induced immediate skin reactions in horses. This suggested that both IgE and IgG(T) sensitize equine skin mast cells. Here, we investigated sensitization to allergen and with IgE or IgG(T) in clinically healthy horses of different age groups. In addition, immediate skin reactions to Cul...
Lunn DP, McClure JT, Schobert CS, Holmes MA.Severe combined immunodeficiency (SCID) is a fatal autosommal disease of Arabian horses that leads to failure of maturation of T- and B-lymphocyte populations, although natural killer (NK) cells are unaffected. Thymic and lymph node tissues from two foals suffering from SCID were examined in an immunohistological study using a panel of monoclonal antibodies recognising equine leucocyte differentiation antigens. In both foals, the majority of cells in lymphoid tissues had an EqCD3-EqCD4-EqCD8+ phenotype, although rare EqCD3+ cells were also detected. The EqCD3-EqCD4-EqCD8+ cells may represent a...
Martínez-Torrecuadrada JL, Langeveld JP, Venteo A, Sanz A, Dalsgaard K, Hamilton WD, Meloen RH, Casal JI.African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a ...
Leigh JA.A protein capable of activating bovine, equine and ovine plasminogen, but not that from human or porcine plasma, was purified from culture filtrates of Streptococcus uberis (strain 0140J). Purification was achieved by ammonium sulphate precipitation followed by molecular exclusion chromatography. The elution position of the native molecule was equivalent to a molecular mass of approximately 57 kDa. However, the molecular mass, as determined by SDS-PAGE, was 29 kDa, suggesting the existence of a dimeric structure. Purified immunoglobulin from three out of five monoclonal antibodies raised to th...
Guerriero V, Raynes DA.Cultured bovine, equine, ovine and chicken lymphocytes responded to heat stress by the increased synthesis of a specific set of proteins known as heat stress proteins (HSP). Proteins with molecular weights of 70 and 90 kDa were synthesized in all species. Additional proteins were found in bovine, ovine and chicken lymphocytes. A time course of induction showed an increased synthesis of some of these proteins with only 30 min of heat stress and of several proteins with 60 min of heat stress. A specific monoclonal antibody was used to identify HSP70 as one of the stress proteins in bovine lympho...
McGuire TC, O'Rourke KI, Baszler TV, Leib SR, Brassfield AL, Davis WC.Cells infected with vaccinia viruses expressing the equine infectious anaemia virus (EIAV) gag gene (VGag) or gag plus the 5' pol encoding protease (VGag/PR) were evaluated with monoclonal antibody to a p26 capsid protein linear epitope (QEISKFLTD). Both recombinant viruses expressed Gag precursor protein (55K) whereas only VGag/PR expressed a detectable Gag-Pol fusion protein (82K) with a functional protease, shown by subviral particles containing processed p26. Horses inoculated with VGag/PR produced antibodies reactive with EIAV Gag proteins.
Laviada MD, Babín M, Dominguez J, Sánchez-Vizcaíno JM.A sandwich enzyme-linked immunsorbent assay (ELISA) for rapid detection of African horsesickness virus (AHSV) in infected spleens or cell culture supernatant was developed. This method uses two monoclonal antibodies (MAbs) which recognize two non-overlapping epitopes of the major core protein (VP7) to coat the solid phase, and one labeled with biotin as second antibody. This ELISA was evaluated for its ability to detect AHSV in infected spleens resulting in a sensitivity of 97.4% and a specificity of 100% compared with virus isolation in cell culture, and can be used for the detection of the n...
Arai K, Misumi K, Carter SD, Shinbara S, Fujiki M, Sakamoto H.Cartilage oligomeric matrix protein (COMP) is abundant within cartilage; its turnover and/or degradation have been investigated in various equine joint diseases and it has been suggested that COMP fragmentation might be useful for monitoring such conditions. Objective: To determine whether COMP metabolism is compromised in equine osteoarthritis (OA) and whether COMP degradation is a useful joint marker representing cartilage destruction. Objective: A monoclonal antibody (mAb) with a higher affinity for degraded COMP allows discrimination of diseased joints by quantifying COMP levels and fragme...
Avarzed A, Igarashi I, De Waal DT, Kawai S, Oomori Y, Inoue N, Maki Y, Omata Y, Saito A, Nagasawa H, Toyoda Y, Suzuki N.Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did n...
Flaminio MJ, Nydam DV, Marquis H, Matychak MB, Giguère S.Susceptibility of foals to Rhodococcus equi pneumonia is exclusive to the first few months of life. The objective of this study was to investigate the immediate immunologic response of foal and adult horse antigen-presenting cells (APCs) upon infection with R. equi. We measured the activation of the antigen-presenting major histocompatibility complex (MHC) class II molecule, costimulatory molecules CD40 and CD86, the cytokine interleukin-12 (IL-12), and the transcriptional factor interferon regulatory factor 1 (IRF-1) in monocyte-derived macrophages (mMOs) and dendritic cells (mDCs) of adult h...
Wyatt CR, Magnuson NS, Perryman LE.Six monoclonal antibodies, designated EqT2, EqT3, EqT6, EqT7, EqT12, and EqT13, which identify T lymphocyte antigens present at different stages of T cell maturation were used to examine T lymphocyte development in foals with severe combined immunodeficiency (SCID). Flow microfluorimetry demonstrated the presence of EqT12+ and EqT13+ prothymocytes and a few phenotypically mature EqT2+ and EqT3+ thymocytes within the thymic remnants of SCID foals. However, very few EqT6+ and EqT7+ resident cortical thymocytes were detected. The near absence of EqT6+ and EqT7+ cortical thymocytes was confirmed b...
Bártek J, Viklický V, Franĕk F, Angelisová P, Dráber P, Jarosíková T, Nĕmec M, Verlová H.Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG1 subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitati...
Keggan A, Freer H, Rollins A, Wagner B.Horses have 11 immunoglobulin isotypes: IgM, IgD, IgA, IgE, and seven IgG subclasses designated as IgG1-IgG7, each of which are distinguished by separate genes encoding the constant heavy chain regions. Immunoglobulin (Ig) isotypes have different functions during the immune response and pathogen-specific isotypes can be used as indicators for immunity and protection from disease. In addition to existing monoclonal antibodies to various equine Igs, quantification of the individual isotypes requires pure isotype standards. In this report, we describe a fusion between X63-Ag8.653 mouse myeloma ce...
Binns MM, Daly JM, Chirnside ED, Mumford JA, Wood JM, Richards CM, Daniels RS.The haemagglutinin (HA) gene from the equine influenza H3N8 isolate Suffolk/89 has been cloned by reverse transcription and polymerase chain reaction amplification. The nucleotide sequence of the HA gene was determined from two independently cloned copies of the gene and was found to be most closely related to recent American isolates supporting the idea that most isolates of equine H3N8 are evolving as a single lineage. When the predicted amino acid sequence of the Suffolk/89 HA was examined, changes had taken place in at least four of the major antigenic sites, A, B, C, and D when compared t...
Kawaoka Y, Webster RG.A severe influenza outbreak occurred in horses in South Africa in 1986. The causative agent was identified as an influenza virus [A/Equine/Johannesburg/86 (H3N8)]. Antigenic analyses of the hemagglutinin (HA) with ferret antisera and monoclonal antibodies showed that the Eq/Johannesburg/86 virus is similar to recent equine H3 viruses. The nucleotide sequence analysis on the HA genes of Eq/Johannesburg/86 and other equine H3 influenza viruses, together with the epidemiological data, clearly demonstrated that the Eq/Johannesburg/86 virus was derived from a virus that had been circulating in hors...
Wagner B, Glaser A, Hillegas JM, Erb H, Gold C, Freer H.West Nile virus (WNV) is a zoonotic pathogen of global importance. In horses with neurological signs, detection of WNV-specific immunoglobulin M (IgM) in serum is widely used to identify clinical cases of WNV encephalitis. Here, we describe the development of two monoclonal antibodies (mAbs) to equine IgM which were used in a WNV IgM-specific enzyme-linked immunosorbent assay (ELISA). Their performance was compared to an established assay based on polyclonal anti-IgM. Check test serum samples from the National Veterinary Service Laboratory (NVSL) were used to evaluate the performance of the th...
Rivero JL, Talmadge RJ, Edgerton VR.In adult horses, three myosin heavy chain (MyHC) isoforms can be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunohistochemistry using specific anti-MyHC monoclonal antibodies. This report studies the suitability of a consistent SDS-PAGE technique for quantifying MyHC profiles in homogenized cryostate sections of equine gluteus medius muscle biopsies (n = 18). The method used (previously described by R. J. Talmadge and R. R. Roy; J. Appl. Physiol. 1993, 75, 2337-2340) resolved MyHCs in three bands: I, IIB or IIX, and IIA from the fastest to the slowe...
Karàdi I, Kostner GM, Gries A, Nimpf J, Romics L, Malle E.Earlier studies demonstrated that lipoprotein (a), a lipoprotein of high atherogenicity, possesses proteolytic activity. In this report, we provide evidence that the lipoprotein (a)-specific antigen, apoprotein (a) is immunochemically related to plasminogen. This was demonstrated by polyclonal antisera from rabbit, sheep and horse, and with three monoclonal antibodies from mouse. Using immunospecific adsorbers against lipoprotein (a), all plasminogen could be adsorbed from lipoprotein (a)-positive and apparently lipoprotein (a)-negative plasma. As an additional similarity to plasminogen, lipop...
Bullido R, Doménech N, Alvarez B, Alonso F, Babín M, Ezquerra A, Ortuño E, Domínguez J.A set of five monoclonal antibodies (mAb) against porcine major histocompatibility complex (MHC), or swine leukocyte antigens (SLA), class II molecules has been characterized. These mAbs appear to recognize monomorphic determinants on SLA-DR (2F4, 1F12 and 2E9/13) and SLA-DQ (BL2H5 and BL4H2) molecules, as assessed by flow cytometry and immunoprecipitation. By Western blot, the 2F4, 1F12, BL2H5 and BL4H2 epitopes were located on the beta-chains of these molecules. mAbs 2F4 and 1F12 crossreact with leucocytes of dog, cattle, horse and human; mAbs 2E9/13, BL2H5 and BL4H2 bind leucocytes of cattl...
Ibrahim S, Steinbach F.Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules f...
Steinbach F, Stark R, Ibrahim S, Gawad EA, Ludwig H, Walter J, Commandeur U, Mauel S.The myeloid cell system comprises of monocytes, macrophages (MPhi), dendritic cells (DC), Kupffer cells, osteoclasts or microglia and is also known as the mononuclear phagocytic system (MPS). Essential cytokines to differentiate or activate these cells include GM-CSF or IL-4. Important markers for characterization include CD1, CD14, CD68, CD163 and CD206. All these markers, however, were not cloned or further characterized in equids by use of monoclonal antibodies earlier. To overcome this problem with the present study, two approaches were used. First, we cloned equine cytokines and markers, ...
