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.
Yamanokuchi K, Tagami M, Nishimatsu E, Shimizu Y, Hirose Y, Komatsu K, Misumi K.Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Objective: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitope...
Lee HS, Heo EJ, Jeoung HY, Ko HR, Kweon CH, Youn HJ, Ko YJ.In this study, an enzyme-linked immunosorbent assay (ELISA) using glycoprotein and a monoclonal antibody (MAb) was developed for the detection of antibodies to vesicular stomatitis virus (VSV) serotype New Jersey (NJ). The glycoprotein to be used as a diagnostic antigen was extracted from partially purified VSV-NJ, and a neutralizing MAb specific to VSV-NJ was incorporated to compete with antibodies in a blocking ELISA using glycoprotein (GP ELISA). The cutoff of the GP ELISA was set at 40% inhibition, which corresponded to a virus neutralization test (VNT) titer of 32. With this threshold, th...
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...
Hobson-Peters J, Toye P, Sánchez MD, Bossart KN, Wang LF, Clark DC, Cheah WY, Hall RA.Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through ...
Figueiredo MD, Vandenplas ML, Hurley DJ, Moore JN.Our understanding of the innate immune response in the horse has been limited by a lack of definitive data concerning cell signaling in response to microbial products. Toll-like receptors (TLRs) recognize conserved molecular motifs of microbes and elicit immune responses through their coupling with intracellular adaptor molecules, particularly MyD88 and TRIF. To provide a more definitive characterization of TLR signaling in the horse, the objectives of this study were to: (1) characterize the responses of equine monocytes to TLR ligands that signal through MyD88, TRIF or both in other species,...
Kirsch MI, Hülseweh B, Nacke C, Rülker T, Schirrmann T, Marschall HJ, Hust M, Dübel S.Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results: In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different s...
Lin Y, Deng X, Shen N, Zhao L, Meng Q, Max J, Wang J, Shao Y, Zhou J.The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-gamma expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-gamma expression in different subtypes of T lymphocytes in EIAV-...
Wagner B, Hillegas JM, Flaminio MJ, Wattrang E.Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generatio...
Bai Y, Tong TG, Zhang WJ, Xu SL, Wang Q, Liu GL, Wu DL.To develop a quantitative ELISA by measuring interferon (IFN-gamma) of equine lymphocytes. Methods: Sandwich ELISA for equine IFN-gamma was developed using mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. Results: The detection limit of the sandwich ELISA for equine IFN-gamma was 1 microg/L and did not show cross-reactivity with recombinant equine IL-18. Equine IFN-gamma was detected by ELISA in culture medium of the peripheral blood mononuclear cells (PBMCs) stimulated with ConA or PMA/Ionomycin. Conclusions: This method can be used to help understand the role o...
de Mestre AM, Bacon SJ, Costa CC, Leadbeater JC, Noronha LE, Stewart F, Antczak DF.The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36-38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast-immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-fr...
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...
Agrícola R, Carvalho H, Barbosa M, Pereira M, Medeiros JA, Ferreira-Dias G.The aim of this study was to evaluate peripheral blood lymphocyte subpopulations, neutrophil phagocytic capacity and proteinogram characteristics in mares, during the last trimester of pregnancy and in postpartum. Measurement of phagocytosis and quantification of T-lymphocyte subsets were done by flow cytometry. Quantification of T-lymphocyte subsets was performed with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers. Natural killer and B-cell counts were estimated mathematically. Serum proteinogram was obtained by electrophoresis. No significant differences were observed ...
Xiao C, Liu Y, Jiang Y, Magoffin DE, Guo H, Xuan H, Wang G, Wang LF, Tu C.Four monoclonal antibodies (mAbs) were generated by immunizing BALB/C mice with recombinant nucleocapsid protein (N) of Nipah virus (NiV) and Hendra virus (HeV) expressed in E. coli. Two mAbs each were obtained for the HeV N and NiV N, respectively. All four mAbs displayed specific reactivity with the recombinant N proteins of both viruses by western blot, which was further confirmed by immunofluorescent antibody assay using fixed insect cells infected with recombinant baculoviruses expressing either the HeV or NiV N protein. Epitope mapping using a 12-mer random peptide phage display library ...
Wagner B, Hillegas JM, Brinker DR, Horohov DW, Antczak DF.Interleukin-10 (IL-10) terminates inflammatory immune responses and inhibits activation and effector functions of T-cells, monocytes, macrophages and dendritic cells. IL-10 has also been found to be a key cytokine expressed by subpopulations of regulatory T-cells. In this report, we describe the generation and characterization of three monoclonal antibodies (mAbs) to equine IL-10. The antibodies were found to be specific for equine IL-10 using different recombinant equine cytokine/IgG fusion proteins. Two of the anti-equine IL-10 mAbs were selected for ELISA to detect secreted IL-10 in superna...
Figueiredo MD, Salter CE, Hurley DJ, Moore JN.Lipopolysaccharide-binding protein (LBP) is an acute phase protein that binds the lipid A moiety of lipopolysaccharide (LPS) and transfers LPS monomers to soluble CD14 in plasma or membrane bound CD14 on mononuclear phagocytes. The result of these interactions is activation of the TLR4 receptor complex, and the synthesis and release of inflammatory mediators. Inclusion of LBP in cellular assays increases the sensitivity of cells expressing CD14 to LPS. Therefore, the objectives of this study were to (1) compare differentially treated sera from cattle and horses as sources of LBP activity using...
Xu J, Cai J, Peek SF, Suresh M, Darien BJ.P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a critical role in regulating lymphocyte homing and leukocyte trafficking during inflammation. The lack of specific reagents for equine PSGL-1 (ePSGL-1) has prevented mechanistic studies regarding its function and regulation in the horse. We synthesized a ePSGL-1 peptide to generate a monoclonal antibody (mAb), ePL1. Using flow cytometry and Western blot, we showed that ePL1 binds specifically to ePSGL-1 in transfected mammalian cells. We also demonstrated that ePL1 binds to equine leukocytes and recog...
Morgan EE, Miller WH, Wagner B.Skin hypersensitivities (allergies) in horses are often diagnosed using clinical signs only. Intradermal testing or serological assays are diagnostic options to confirm the allergic nature of the disease and to identify the allergen(s). Our objective was to develop an allergen-specific enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for horse IgE and to examine its potential for allergen detection in serum in comparison to intradermal testing. Intradermal testing with 61 allergen extracts was performed on 10 horses affected with skin hypersensitivity. Their sera ...
Ibrahim S, Saunders K, Kydd JH, Lunn DP, Steinbach F.Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (di...
Ibrahim S, Steinbach F.In addition to the 379 monoclonal antibodies (mAbs) tested in the animal homologues section of HLDA8, another 155 mAbs were screened at the Institute for Zoo and Wildlife Research, Berlin for cross-reactivity with equine leukocytes. For this purpose, one colour flow-cytometric analysis was performed as screening test. This additional screening indicated further 16 mAbs as positive with staining homologous to human pattern, 1 mAb with weak (positive) reactivity, 11 mAbs with positive, but likely not valuable staining, 12 mAbs with alternate expression pattern from that expected from human immun...
Kitai Y, Shoda M, Kondo T, Konishi E.West Nile virus (WNV) is now widely distributed worldwide, except in most areas of Asia where Japanese encephalitis virus (JEV) is distributed. Considering the movement and migration of reservoir birds, there is concern that WNV may be introduced in Asian countries. Although manuals and guidelines for serological tests have been created in Japan in preparedness for the introduction of WNV, differential diagnosis between WNV and JEV may be complicated by antigenic cross-reactivities between these flaviviruses. Here, we generated a monoclonal antibody specific for the nonstructural protein 1 (NS...
Luce R, Shepherd M, Paillot R, Blacklawst B, Wood JL, Kydd JH.An assay has been developed that measures EHV-1 specific interferon gamma synthesis (IFNgamma), a cytokine produced following the activation of memory T lymphocytes and therefore a measure of cell mediated immunity. The method requires validation in the field. Objective: To measure the frequency of EHV-1 specific, IFNgamma synthesising peripheral blood mononuclear cells (PBMC) in a population of Thoroughbred horses, and examine its relationship with age, gender, premises and history of vaccination or field infection with EHV-1. Methods: Lymphocytes from 200 Thoroughbred horses were stimulated ...
Helal IE, Misumi K, Tateno O, Kodama T, Ishimaru M, Yamamoto J, Miyakoshi D, Fujiki M.To evaluate changes in serum cartilage oligomeric matrix protein (COMP) concentrations in response to exercise in horses. Methods: 15 horses in experiment 1 and 27 horses in experiment 2. Methods: In experiment 1, 15 Thoroughbreds free of orthopedic disease underwent a standardized exercise protocol. Running velocity and heart rate (HR) were recorded, and blood samples were collected immediately before (baseline) and 1, 5, and 24 hours after a single episode of exercise. In experiment 2, 27 horses underwent 9 stages of a training program in which each stage consisted of 4 to 8 consecutive dail...
Gulati BR, Deepa R, Singh BK, Rao CD.Rotaviruses causing severe diarrhea in foals in two organized farms in northern India, during the period from 2003 to 2005, were characterized by electropherotyping, serotyping, and sequence analysis of the genes encoding the outer capsid proteins. Of 137 specimens, 47 (34.31%) were positive for rotavirus and exhibited at least five different electropherotypes (E), E1 to E5. Strains belonging to different electropherotypes exhibited either a different serotype/genotype specificity or a lack of reactivity to typing monoclonal antibodies (MAbs) used in this study. Strains belonging to E1, E2, an...
Choi KS, Ko YJ, Nah JJ, Kim YJ, Kang SY, Yoon KJ, Joo YS.A rapid immunoassay for detecting and quantifying West Nile virus (WNV)-neutralizing antibodies in sera was developed as an alternative to the plaque reduction neutralization test (PRNT), the gold standard test for WNV. The assay is a competitive, enzyme-linked immunosorbent assay using neutralizing monoclonal antibody 5E8 (NT-ELISA). A cutoff percent inhibition (PI) value of 35% (mean PI plus 3 standard deviations), with a specificity of 99%, was established based on analysis of 246 serum samples from horses free of WNV. The NT-ELISA detected neutralizing antibodies in all sera collected 7 or...
Borromeo V, Berrini A, Gaggioli D, Secchi C.Heterophile antibodies (HAs) present in serum recognize animal immunoglobulins and are one of the most unpredictable causes of false results in immunoassays. However, no study has yet reported their interference on the diagnostic reliability of immunochemical analyses on horse plasma. Recently, we developed a sandwich ELISA for detection of equine growth hormone (eGH) in plasma. In a pilot study to measure basal eGH levels (blood samples were drawn from 13 horses every 10 min for 1h), we noted one horse with abnormally high eGH (>100 ng/mL). We demonstrate here that this plasma eGH level wa...
Levy R, Forsyth CM, LaPorte SL, Geren IN, Smith LA, Marks JD.Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses that are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for vaccine development. Here, we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the...
Wagner B, Miller WH, Morgan EE, Hillegas JM, Erb HN, Leibold W, Antczak DF.In horses, allergies have been characterized by clinical signs and/or intradermal (i.d.) allergen testing. Our aim was to find the first direct evidence that immunoglobulin E (IgE) mediates equine allergy. In addition, we tested the hypothesis that immediate skin reactions in horses can also be mediated by IgG. Anti-IgE affinity columns were used to purify IgE from serum of one healthy horse and three horses affected with summer eczema, an allergic dermatitis which is believed to be induced by Culicoides midges. A modified Prausnitz-Küstner experiment was performed in four clinical healthy ho...
Misumi K, Tagami M, Kamimura T, Miyakoshi D, Helal IE, Arai K, Fujiki M.To quantify the urinary concentration of cartilage oligomeric matrix protein (COMP), and to evaluate the relationship between urinary COMP concentration and the catabolic activity of synovial fluid (SF) in diseased horses. Methods: COMP in horse urine was detected by immunoblotting with a monoclonal antibody (mAb; 14G4) raised against equine COMP from articular cartilage. Urine and serum samples were obtained from 83 Thoroughbred horses with aseptic joint diseases (AJD, 79 horses) or septic joint diseases (SJD, four horses) at the time of anesthesia induction, and samples of SF were obtained d...
Okano S, Hurley DJ, Vandenplas ML, Moore JN.To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. Methods: Mononuclear cells from 18 horses and 3 dogs. Methods: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. Results: Addition of FBS to media significantly increased procoagulant activity; eq...
Murphy JE, Marsh AE, Reed SM, Meadows C, Bolten K, Saville WJ.Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses caused primarily by the protozoal parasite Sarcocystis neurona. Currently available antemortem diagnostic testing has low specificity. The hypothesis of this study was that serum and cerebrospinal fluid (CSF) of horses experimentally challenged with S neurona would have an increased S neurona-specific IgM (Sn-IgM) concentration after infection, as determined by an IgM capture enzyme linked immunoassay (ELISA). The ELISA was based on the S neurona low molecular weight protein SNUCD-1 antigen and the monoclonal an...
Braid LR, Hu WG, Davies JE, Nagata LP.: Mesenchymal stromal cells (MSCs) are being exploited as gene delivery vectors for various disease and injury therapies. We provide proof-of-concept that engineered MSCs can provide a useful, effective platform for protection against infectious disease. Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne pathogen affecting humans and equines and can be used in bio-warfare. No licensed vaccine or antiviral agent currently exists to combat VEEV infection in humans. Direct antibody administration (passive immunity) is an effective, but short-lived, method of providing immediate prote...
Choi KS, Ko YJ, Nah JJ, Kim YJ, Kang SY, Yoon KJ, Joo YS.A rapid immunoassay for detecting and quantifying West Nile virus (WNV)-neutralizing antibodies in sera was developed as an alternative to the plaque reduction neutralization test (PRNT), the gold standard test for WNV. The assay is a competitive, enzyme-linked immunosorbent assay using neutralizing monoclonal antibody 5E8 (NT-ELISA). A cutoff percent inhibition (PI) value of 35% (mean PI plus 3 standard deviations), with a specificity of 99%, was established based on analysis of 246 serum samples from horses free of WNV. The NT-ELISA detected neutralizing antibodies in all sera collected 7 or...
McGuire TC, Perryman LE, Davis WC.Nine monoclonal antibodies which reacted with equine immunoglobulin (Ig)M and not other equine Ig and serum proteins were prepared. Cells producing antibodies (C 1.9) which precipitated with IgM and bound to staphylococcal protein A were triple-cloned (C 1.9/3.2) and the antibodies further characterized. Monoclonal antibody C 1.9/3.2 reacted with an IgM determinant present on serum IgM from horses of several breeds. Studies with 125I-labeled IgM revealed the presence of this determinant on all IgM molecules. The monoclonal antibody enabled quantitation of IgM in presuckling foal and adult hors...
Bell CW, Boyle DB, Whalley JM.Transcript mapping of the equine herpesvirus 1 (EHV-1) glycoprotein B (gB) gene homologue by Northern blot, S1 nuclease and primer extension analyses indicated that two overlapping transcripts of 3.4 and 4.6 kb originated from the same strand and were transcribed from left to right between coordinates 0.40 and 0.43 of the EHV-1 genome. The 3.4 kb transcript encoded EHV-1 gB and the 5' RNA terminus was located approximately 30 bases downstream from a probable TATA element. The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis and inser...
Wyatt CR, Davis WC, McGuire TC, Perryman LE.Six monoclonal antibodies reacting with equine T lymphocytes at different stages of maturation were selected from antibodies produced against lymphoid cell preparations. EqT12 and EqT13 antibodies identified subsets of cortical thymocytes with high terminal deoxynucleotidyltransferase (TdT) activity and no phytolectin responsiveness. EqT12+ thymocytes were scattered throughout the cortex while EqT13+ thymocytes were located in the subcapsular cortex. EqT12 bound to small numbers of bone marrow cells, splenocytes, and circulating lymphoid cells, but not to mature T lymphocytes. EqT13 bound to v...
Palmer JL, Bertone AL, Malemud CJ, Carter BG, Papay RS, Mansour J.The relevance of site and the influence of exercise on third carpal articular cartilage proteoglycan (PG) were assessed in 16 horses. Six horses were exercised (exercised group) for 30 minutes, 3 times/wk, for 6 weeks. The other 10 horses (nonexercised group) were housed in box stalls for the same 6-week period. At week 6, articular cartilage from the proximal surface of the right third carpal bone was harvested and cultured with radioactive sulfate to label newly synthesized PG. Endogenous PG was measured by use of a uronic acid assay. Newly synthesized and endogenous PG were characterized by...
Guptill JT, Raja SM, Juel VC, Walter EB, Cohen-Wolkowiez M, Hill H, Sendra E, Hauser B, Jackson P, Swamy GK.Botulism is a rare, life-threatening paralytic disease caused by Clostridium botulinum neurotoxin (BoNT). Available treatments, including an equine antitoxin and human immune globulin, are given postexposure and challenging to produce and administer. NTM-1632 is an equimolar mixture of 3 human IgG monoclonal antibodies, B1, B2, and B3, targeting BoNT serotype B (BoNT/B). This first-in-human study assessed the safety, tolerability, pharmacokinetics (PK), and immunogenicity of NTM-1632. This double-blind, single-center, placebo-controlled dose escalation study randomized 3 cohorts of healthy vol...
Lunn DP, Holmes MA, Duffus WP.This paper describes the characteristics of a monoclonal antibody (CVS10) that reacts with an equine leukocyte antigen. On the basis of tissue distribution and biochemical characteristics, this antigen is equine MHC II. The equine MHC II antigen was found on a large subset of T-lymphocytes in addition to all B-lymphocytes, as has been reported previously. In addition MHC II was found to be present on a large proportion of both the mutually exclusive equine T-lymphocyte subpopulations which express either the equine homologues of CD4, or CD8. In a study of changes in equine MHC II expression wi...
Wills JM, Watson G, Lusher M, Mair TS, Wood D, Richmond SJ.This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated i...
Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T, Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M, Harada H, Kaneko MK, Kato Y.Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized mice with synthetic horse PDPN peptides and developed anti-horse PDPN mAbs...
Ibrahim S, Steinbach F.In addition to the 379 monoclonal antibodies (mAbs) tested in the animal homologues section of HLDA8, another 155 mAbs were screened at the Institute for Zoo and Wildlife Research, Berlin for cross-reactivity with equine leukocytes. For this purpose, one colour flow-cytometric analysis was performed as screening test. This additional screening indicated further 16 mAbs as positive with staining homologous to human pattern, 1 mAb with weak (positive) reactivity, 11 mAbs with positive, but likely not valuable staining, 12 mAbs with alternate expression pattern from that expected from human immun...
Noronha LE, Harman RM, Wagner B, Antczak DF.The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize ...
Watson JL, Stott JL, Blanchard MT, Lavoie JP, Wilson WD, Gershwin LJ, Wilson DW.The alterations in lymphocyte subsets in chronic obstructive pulmonary disease (COPD) in the horse were investigated by using monoclonal antibodies to identify CD3+, CD4+, CD8+, and surface immunoglobulin positive (sIg+) lymphocytes in peripheral blood, bronchoalveolar lavage fluid (BALF), and pulmonary biopsy frozen tissue sections. COPD-affected horses (n = 5) and normal controls (n = 5) were sampled prestabling and 14 days poststabling, at which time the COPD-affected horses wee exhibiting clinical signs of COPD. The peripheral blood absolute CD4+ lymphocyte count was significantly elevated...
McGorum BC, Dixon PM, Halliwell RE.Phenotypic analysis of lymphocytes in peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) of control and chronic obstructive pulmonary disease (COPD) affected horses, both before and after 'natural (hay and straw) challenge', were performed using immunofluorescent labelling with monoclonal antibodies and flow cytometry. BALF lymphocytes were shown to be predominantly EqCD5+ cells, approximately half of which were also EqCD8+, with a smaller proportion of B cells. In comparison with PB, BALF contained higher proportions of EqCD5+ cells and EqCD8+ cells and a lower proportion of B cell...
Yamano S, Eto D, Kasashima Y, Hiraga A, Sugiura T, Miyata H.To determine the growth-related changes in metabolic and anatomic properties in equine muscle fiber type, including hybrid fibers identified with immunohistochemical analysis. Methods: 24 2-, 6-, 12-, and 24-month-old female Thoroughbreds. Methods: Samples were obtained from the gluteus medius muscle of all horses. Expression of myosin heavy chain (MHC) isoforms MHC-I, -IIa, -IIb, and -IIx in each muscle fiber was detected by use of 4 primary monoclonal antibodies: BA-D5, SC-71, BF-F3, and BF-35, respectively. Five muscle fiber types (types I, I/IIA, IIA, IIA/IIX, and IIX) were immunohistochem...
Antczak DF, Oriol JG, Donaldson WL, Poleman C, Stenzler L, Volsen SG, Allen WR.Monoclonal antibodies raised against horse placenta were tested using an indirect immunoperoxidase-labelling technique for reactivity with a panel of tissues from adult horses and conceptuses of various gestational ages. The pattern of reactivity of 4 of the antibodies (F67.1, F71.3, F71.7, F71.14) on trophoblastic tissues described unique antigenic phenotypes for the non-invasive trophoblast of the allantochorion, the invasive trophoblast of the chorionic girdle, and the mature endometrial cup cells, which are derived from the chorionic girdle. Two of the monoclonal antibodies (F67.1 and F71....
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...
Tavernor AS, Kydd JH, Bodian DL, Jones EY, Stuart DI, Davis SJ, Butcher GW.An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T-lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50-65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the ...
Tomlinson JE, Wagner B, Felippe MJB, Van de Walle GR.Immune phenotyping of equine peripheral blood mononuclear cells (PBMC) is commonly described by single or double marker labeling, which limits complex phenotypic descriptions and subpopulation identification. Our objective was to develop a new multispectral flow cytometry protocol to identify and sort equine lymphocyte subpopulations using commercially available, pre-conjugated monoclonal antibodies to cell surface markers. Two clones of anti-equine CD8 (CVS8 and CVS21) were compared in combination with CD3. Clone CVS21 bound non-T CD3 cells in addition to CD8 T cells. Further analysis using c...
Marsh AE, Hyun C, Barr BC, Tindall R, Lakritz J.Equine protozoal myeloencephalitis (EPM), caused by a protozoal parasite infection of the central nervous system, is the most commonly diagnosed neurologic disease of horses in North America. In specific regions of the United States approximately 50% of the horse population is seropositive to Sarcocystis neurona. However, not all seropositive horses develop clinical signs. Detailed clinical examination, along with cerebrospinal fluid antibody evaluation are often used to diagnose EPM. Postmortem evaluation of the brain stem and spinal cord for histopathologic lesions compatible with nonsuppura...
Shankarappa B, Dutta SK, Sanusi J, Mattingly BL.Competitive enzyme-linked immunosorbent assay (CELISA), mediated by a monoclonal antibody designated HybI, was developed for the diagnosis of equine monocytic ehrlichiosis. Inhibition of binding of HybI by the horse antibodies to Ehrlichia risticii was optimum at dilutions of 1:20 for serum and 1:10,000 for HybI. Mean optical densities (ODs) of positive and negative sera were 0.158 and 0.855, respectively. A comparison of ODs obtained by CELISA and indirect enzyme-linked immunosorbent assay (ELISA) indicated a marked tendency of positive and negative samples to cluster separately with respect ...
Ranz AI, Miguet JG, Anaya C, Venteo A, Cortés E, Vela C, Sanz A.A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the non-structural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 microliters. The NS1-speci...
Jemmerson R.To gain a better understanding of the diversity of epitopes on a protein, the specificities of 103 monoclonal antibodies to a model antigen, horse cytochrome c(cyt c), were analyzed. The antibodies were generated in in vitro monoclonal, secondary antibody responses against horse cyt c coupled to hemocyanin in splenic fragment cultures. For this assay, horse cyt c-primed murine B lymphocytes were transferred to irradiated, hemocyanin-primed recipients. A panel of seven mammalian cyts c differing at one to six residues out of 104 and cyanogen bromide-cleaved fragments of horse cyt c containing r...
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...
Moyo NA, Marchi E, Steinbach F.Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both ph...
Barone A, Benktander J, Whiddon C, Jin C, Galli C, Teneberg S, Breimer ME.Pericardial tissue from various animal species is utilized for the production of the bioprosthetic heart valves (BHV) used clinically. Experimental data show that the eventual breakdown of BHV is partly due to immunological interactions with carbohydrate tissue antigens. To understand these processes, we have examined the glycolipid-based carbohydrate antigens in naïve porcine, bovine, and equine pericardia. Total non-acid and acid glycosphingolipid fractions were isolated from porcine, bovine, and equine pericardia, and individual glycolipid compounds were characterized by thin-layer chromat...
Alape-Giron A, Stiles BG, Gutierrez JM.An ELISA based, non-radioactive acetylcholine receptor (AchR) binding assay was used to detect the alpha-neurotoxins present in Micrurus nigrocinctus nigrocinctus venom. Sera from horses hyperimmunized against M. nigrocinctus venom contain antibodies which inhibit the binding of M. n. nigrocinctus alpha-neurotoxins to AchR and reverse the binding of toxins already complexed with the receptor. This result supports the importance of using antivenom therapeutically in M. n. nigrocinctus envenomations even after the onset of neurological symptoms. M. nigrocinctus antivenoms cross-reacted in an ELI...
Hu Z, Chang H, Chu X, Li S, Wang M, Wang X.The equine infectious anemia virus (EIAV) capsid protein (p26) is one of the major immunogenic proteins during EIAV infection and is widely used for the detection of EIAV antibodies in horses. However, few reports have described the use of EIAV-specific monoclonal antibodies (MAbs) in etiological and immunological detection. Previously, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the quantification of the EIAV p26 protein level. However, the epitopes recognized by the MAbs were not identified, and the utilization of the MAbs needs to be evaluated. In this ...
Takai S, Morishita T, Nishio Y, Sasaki Y, Tsubaki S, Higuchi T, Hagiwara S, Senba H, Kato M, Seno N.We recently generated a monoclonal antibody immunoglobulin G1 (MAb 10G5), which can recognize 15- to 17-kDa antigens, virulence-associated antigens of Rhodococcus equi, and developed a colony blot enzyme-linked immunosorbent assay with MAb 10G5 for the rapid identification of virulent R. equi. In this epidemiologic study, we evaluated the results of the colony blot test in the identification of virulent isolates of R. equi from feces of horses and soil and compared them with those from a conventional procedure (plasmid profiles of isolates by agarose gel electrophoresis). Environmental isolate...
