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.
Nordengrahn A, Klingeborn B, Lindholm A, Merza M.A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to equine herpesvirus 2 in serum samples of horses. By measuring the binding to a single epitope, this blocking ELISA gives a good picture of the antibody status in the animal. The test is based on a monoclonal antibody with neutralizing activity and had a sensitivity of 94% and a specificity of 100%. Antibodies due to newly acquired infection in foals were successfully detected with this blocking ELISA.
Varrasso A, Drummer HE, Huang JA, Stevenson RA, Ficorilli N, Studdert MJ, Hartley CA.The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralizatio...
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...
McFarlane D, Sellon DC, Gibbs SA.To characterize age-associated changes in lymphocyte population subsets and immunoglobulin isotypes. Methods: 30 healthy young light-breed horses (5 to 12 years old) and 30 healthy aged light-breed horses (> 20 years old). Methods: Lymphocyte subset populations were identified, using monoclonal antibodies to cell surface markers CD5, CD4, CD8, and IgG. Subset populations were quantitated by use of flow cytometric analysis of antibody-stained cells. Serum immunoglobulin concentration was determined using single radial immunodiffusion. Results: Absolute cell counts of total lymphocytes, T cells,...
Hedges JF, Demaula CD, Moore BD, McLaughlin BE, Simon SI, MacLachlan NJ.Expression of E-selectin on activated endothelium is a critical initial step that leads to extravasation of leucocytes during inflammation, yet E-selectin is largely uncharacterized in several animal species including the horse. We have sequenced and compared E-selectin genes derived from activated cultures of purified equine (horse), cervid (black-tailed deer) and ovine (sheep) pulmonary artery endothelial cells (ECs). Phylogenetic and amino acid sequence comparisons indicate that bovine, cervid and ovine E-selectin are similar, whereas human and equine E-selectin are more closely related to ...
Takai S, Murata N, Kudo R, Narematsu N, Kakuda T, Sasaki Y, Tsubaki S.This report describes the discovery of two new virulence plasmid types from a crossbred foal with Rhodococcus equi pneumonia in Kumamoto died with severe R. equi pneumonia and ulcerative enteritis. R. equi was isolated in large numbers and isolates from the foal were investigated for the presence of virulence-associated 15-17 kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and by gene coding for VapA by PCR. Plasmid DNAs extracted from the isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII. The digestion patterns that resulted d...
Emenius G, Larsson PH, Wickman M, Härfast B.The objective was to establish an ELISA to detect horse allergen in ambient air and settled dust. Methods: Monoclonal antibodies (mAbs) were produced against extracts of horse antigen. Two mAbs were selected and used in a sandwich ELISA. By the aid of portable pumps, air samples were collected in one stable and in the ambient air surrounding this stable. Furthermore, settled dust was collected by wiping spots with pieces of fabric, at sites within 500 m of the stable. Results: Extracts of horsehair could be extensively diluted and still be positive. Extracts of cat and dog allergen failed to b...
Rhalem A, Sahibi H, Lasri S, Johnson WC, Kappmeyer LS, Hamidouch A, Knowles DP, Goff WL.A highly specific and sensitive competitive enzyme-linked immunosorbent assay for detection of specific antibody to Babesia equi in serum from equids was validated for use in Morocco. The assay is based on the specific inhibition of binding of a monoclonal antibody to a conserved epitope within a recombinant parasite peptide by serum from infected animals. The assay was compared to an established indirect immunofluorescence assay, with a concordance of 91%. The assay was used to determine seroprevalence for B. equi infections in donkeys and horses throughout Morocco. A total of 578 sera (163 h...
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...
Jensen TK, Boye M, Bille-Hansen V.Fluorescent in situ hybridization, immunohistochemistry, and Grocott's methenamine-silver nitrate staining were compared as diagnostic methods for Pneumocystis carinii pneumonia in formalin-fixed lung tissue from foals and pigs. An oligonucleotide probe targeting 18S ribosomal RNA of P. carinii was designed for in situ hybridization, and a commercially available monoclonal antibody was used for immunohistochemistry. Samples from six foals and 10 pigs with P. carinii pneumonia, as verified by Grocott's methenamine-silver nitrate staining, were examined concurrently with samples from seven anima...
The Journal of parasitologyApril 25, 2001
Volume 87, Issue 2 345-353 doi: 10.1645/0022-3395(2001)087[0345:COTOIO]2.0.CO;2
Dubey JP, Liddell S, Mattson D, Speert CA, Howe DK, Jenkins MC.Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical si...
Henderson K, Stewart J.A dipstick, competitive immunoassay for rapidly measuring serum oestrone sulfate (OS) concentrations in horses was developed to distinguish mares 100 or more days pregnant from non-pregnant animals. 6-Ketoestrone 6-carboxymethyloxime conjugated to bovine serum albumin (oestrone CMO-BSA) was 'dotted' 25 mm from the bottom edge of 45 x 5 mm strips of polyester-film-supported cellulose nitrate membrane, pore size 3 microm. The strips were blocked, dried and a 15 x 5-mm cellulose absorbent sink attached 10 mm from the top of each strip. The manufactured dipsticks were stored with desiccant at room...
Thougaard AV, Jaliashvili I, Christiansen M.The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca2+, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from mac...
Ozaki H, Shimizu-Nei A, Sugita S, Sugiura T, Imagawa H, Kida H.To provide information on the antigenic variation of the hemagglutinins (HA) among equine H 3 influenza viruses, 26 strains isolated from horses in different areas in the world during the 1963-1996 period were analyzed using a panel of monoclonal antibodies recognizing at least 7 distinct epitopes on the H 3 HA molecule of the prototype strain A/equine/Miami/1/63 (H 3 N 8). The reactivity patterns of the virus strains with the panel indicate that antigenic drift of the HA has occurred with the year of isolation, but less extensively than that of human H 3 N 2 influenza virus isolates, and diff...
Starik E, Ginter A, Coppe P.A monoclonal antibody (mAb) directed against the equine arteritis virus (EAV) nucleocapsid (N) protein was used for indirect enzyme-linked immunosorbent assays (ELISAs) using viral antigen from different sources. The same mAb was labelled with fluorescein isothiocyanate for direct immunofluorescence tests (DIFTs). The N-specific mAb appeared to be suitable for the detection in both ELISA and DIFT of different EAV strains and field isolates from semen and tissue samples after passage in lines of RK-13, Vero and fetal equine kidney cells. The ELISA described is an easy and fast method which can ...
Takai S, Ogawa K, Fukunaga N, Sasaki Y, Kakuda T, Tsubaki S, Anzai T.R. equi was isolated from soil samples obtained from the environment of seven native Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, Miyako and Yonaguni) and from fecal samples collected from three native horse breeds (Hokkaido, Kiso and Misaki). Virulent R. equi at various levels (ranging from 0.5 to 12.9%) was isolated from the feces or soil environment of Hokkaido, Kiso and Misaki horses. Isolates were investigated both for the presence of 15- to 17-kDa antigens (virulence-associated protein antigens; VapA) by colony blotting, using the monoclonal antibody 10G5, and the gene of...
Del Piero F.Two 5-year-old grade male horses presented with epiphora, rhinorrhea, conjunctival and nasal mucosal hyperemia, and dorsal and thoracic macropapular rash. Skin biopsies were collected from the affected areas, and serial sections were evaluated following hematoxylin and eosin and immunoperoxidase histochemistry staining by using a murine monoclonal antibody of the immunoglobulin G2A isotype recognizing the 30-kDa membrane protein of equine arteritis virus (EAV). In both horses, lesions consisted of mild to moderate diffuse superficial dermal edema and vasculitis with mild perivascular lymphocyt...
Mariotti F, Cuteri V, Takai S, Renzoni G, Pascucci L, Vitellozzi G.Two horses with Rhodococcus equi infection were examined post mortem by an immunohistochemical method (peroxidase-antiperoxidase; PAP) with a monoclonal antibody (Mab 10G5) to the 15-17 kDa antigen of R. equi. One of the horses was also examined bacteriologically, R. equi being isolated in culture. Immunolabelling with this Mab was marked and widespread. On the other hand, the immunohistochemical reactivity of infected macrophages with a polyclonal antibody specific for lysozyme was slight. Thus, Mab 10G5 would appear to be a useful diagnostic reagent in R. equi infection, with or without cult...
Dingboom EG, Dijkstra G, Enzerink E, van Oudheusden HC, Weijs WA.The fibre type composition of the deep gluteus muscle was studied in biopsies of Dutch Warmblood foals from birth until age 48 weeks. Half the foals were given box-rest, the other half received exercise consisting of an increasing number of gallop sprints. The muscle fibre types were determined using monoclonal antibodies discriminating against the following myosin heavy chain (MHC) isoforms: types I, IIa, IId, Cardiac-alpha and Developmental. During the first 48 weeks there was a consistent increase of fibres expressing types IIa MHC, replacing fibres expressing IId MHC. This change was refle...
Eagle RC, Tortonese DJ.Little is known about the neuroendocrine control of fertility in the horse. In this species, unusual features characterize the normal estrous cycle such as a prolonged preovulatory LH surge during the follicular phase and a distinctive FSH surge during the midluteal phase. This study investigated the distribution and hormonal identity of gonadotrophs in the pars distalis (PD) and pars tuberalis (PT) of the equine pituitary gland as possible morphological bases for the referred unusual endocrine characteristics. In addition, the proportion of gonadotrophs in relation to other pituitary cell typ...
Serrano AL, Rivero JL.Fourteen 4-year old Andalusian mares were used to examine the plasticity of myosin heavy chain (MHC) composition in horse skeletal muscle with heavy draught-exercise training and detraining. Seven horses underwent a training programme based on carriage exercises for 8 months. Afterwards, they were kept in paddocks for 3 months. The remaining seven animals were used as control horses. Three gluteus medius muscle biopsies were removed at depths of 20, 40 and 60 mm from each horse before (month 0), during the training (months 3 and 8) and after detraining (month 11). Myosin heavy chain compositio...
McCann ME, Moore JN, Carrick JB, Barton MH.The effect of intravenous administration of lipid emulsions enriched with omega-3 (n3) and omega-6 (n6) fatty acids on equine monocyte phospholipid fatty acid composition and the synthesis of inflammatory mediators in vitro was evaluated. In a randomized crossover design, horses were infused intravenously with 20% lipid emulsions containing n3 or n6 fatty acids. Monocytes were isolated from the horses before and 0 h, 8 h, 24 h, and 7 days after lipid infusion. Monocyte fatty acid analysis demonstrated incorporation of the parenteral n3 and n6 fatty acids in monocyte phospholipids immediately a...
Mazerbourg S, Zapf J, Bar RS, Brigstock DR, Monget P.We recently showed that insulin-like growth factor-binding protein-4 (IGFBP-4) proteolytic degradation in ovine preovulatory ovarian follicles is IGF-dependent and regulated by the heparin-binding domain (HBD) from IGFBP-3 and from connective tissue growth factor (CTGF), heparan/heparin-interacting protein (HIP), and vitronectin. The present study investigated regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine ovarian preovulatory follicles. Follicular fluid from such preovulatory follicles contains proteolytic activity, degrading exogenous IGFBP-4. An excess of IGF-I...
Kurotaki T, Narayama K, Oyamada T, Yoshikawa H, Yoshikawa T.An immunohistochemical study was carried out on the kinetics of Langerhans cells (LCs) at various pathological stages of "Kasen". Skin lesions of "Kasen" that were collected by biopsy from May to October were classified histopathologically into three stages: initial (Group I, 31 cases), developing (Group II, 50 cases) and regressing (Group III, 13 cases). LCs showed a positive reaction with anti-equine thymocytes (EqT6) monoclonal antibody (MoAb) and anti-major histocompatibility complex (MHC) class II MoAb by immunohistochemical staining. The anti-EqT6 MoAb was intensely positive along the cy...
van Maanen C, Vreeswijk J, Moonen P, Brinkhof J, de Boer-Luijtze E, Terpstra C.Ten monoclonal antibodies (MAbs) were produced against equine herpes virus type 1 (EHV1). Two appeared type-specific, while the other eight were directed against epitopes common to both EHV1 and EHV4. Two MAbs directed against the glycoprotein gp2 recognized linear epitopes, as demonstrated by Western blotting. With pools of type-specific MAbs, 282 field isolates were typed in an immunoperoxidase monolayer assay (IPMA). From a total of 254 fetal or neonatal isolates, 244 (96%) were typed as EHV1, whereas 14 out of 15 (93%) respiratory tract isolates were typed as EHV4. Surprisingly, 3 out of 1...
Aggarwal N, Holmes MA.Despite the importance of IgG Fc receptors in the regulation of various immunological mechanisms, these receptors have not been well characterised in the domesticated animals including equines. This paper describes the production of two monoclonal antibodies (CVS 59 and CVS 61) that recognise equine IgG Fc receptors. Fusions were conducted using BALB/c mice hyperimmunised with equine peripheral blood mononuclear cells. Hybridoma supernatants were screened on the basis of their ability to inhibit the rosetting of equine antibody coated sheep erythrocytes with equine peripheral blood mononuclear...
Siedek EM, Honnah-Symns N, Fincham SC, Mayall S, Hamblin AS.Monoclonal antibodies (mAbs) recognizing equine macrophages are scarce. The present study compared the immunocytochemical staining of various equine tissues (lymphoid tissue, lung, liver, small intestine, skin and blood leucocytes) by an antibody, Ki-M6, which detects CD68 in human macrophages and dendritic cells, and by a new anti-equine mAb, JB10, with staining produced by two previously described anti-equine macrophage mAbs, CZ2.2 and CZ3.3. Ki-M6 was shown to identify equine macrophages, which had a distribution different from those identified by CZ2.2 and CZ3.3. JB10 identified equine mac...
Cho HJ, Entz SC, Deregt D, Jordan LT, Timoney PJ, McCollum WH.A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highl...
van Maanen C, de Boer-Luijtze E, Terpstra C.A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submi...
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...
Perryman LE, Wyatt CR, Magnuson NS, Mason PH.Monoclonal antibodies specific for equine T lymphocyte subpopulations were produced and procedures for the continuous culture of equine lymphocytes were developed. These reagents and procedures were used to analyse the appearance, maturation and functions of T lymphocytes in normal horses and in T lymphocyte deficient horses with severe combined immunodeficiency (SCID). T lymphocytes appeared as early as the 75th day of fetal development and were normally distributed prior to birth of normal foals. Analysis of thymic T lymphocyte differentiation in SCID foals revealed the presence of both prot...
Jensen TK, Boye M, Bille-Hansen V.Fluorescent in situ hybridization, immunohistochemistry, and Grocott's methenamine-silver nitrate staining were compared as diagnostic methods for Pneumocystis carinii pneumonia in formalin-fixed lung tissue from foals and pigs. An oligonucleotide probe targeting 18S ribosomal RNA of P. carinii was designed for in situ hybridization, and a commercially available monoclonal antibody was used for immunohistochemistry. Samples from six foals and 10 pigs with P. carinii pneumonia, as verified by Grocott's methenamine-silver nitrate staining, were examined concurrently with samples from seven anima...
Grosenbaugh DA, Hood DM.In this study, we described water-insoluble proteins extracted from the germinative regions (stratum internum and coronary band epithelium) and the cornified outer surface (stratum medium) of the equine hoof wall. Two major types of polypeptides were identified: the intermediate filaments (IF) and the IF-associated proteins. The IF, including keratins, composed a major portion of this fraction, had electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the range of 40 to 80 kDa, and reacted with acidic or basic keratin-specific monoclonal antibodies. Differe...
Su X, Morris DD, Crowe NA, Moore JN, Fischer KJ, McGraw RA.We describe the production and purification of recombinant equine tumor necrosis factor alpha (rETNF alpha), generation and characterization of murine monoclonal antibodies (Mabs) and rabbit polyclonal antibodies (Pabs) against ETNF alpha, and development of a sensitive enzyme-linked immunosorbent assay (ELISA). Genomic-derived DNA sequences encoding mature ETNF alpha were reconstructed by the polymerase chain reaction (PCR) and oligonucleotide-directed mutagenesis and were cloned into the vector pFLAG-1 for expression in Escherichia coli. rETNF alpha was purified by anti-FLAG immunoaffinity c...
Victor S, Binnmyr J, Lampa E, Rask-Andersen A, Elfman L.Horses are an important source of allergens, but the distribution of horse allergens is poorly understood. Five horse allergens have been identified, Equ c 1-4 and 6. Equ c 4 seems to be an important allergen, with an IgE-binding frequency of 77% in horse-sensitized individuals. The aim of this study was to investigate levels of horse allergen Equ c 4 in dander, saliva and urine from ten horse breeds. The study population included 170 horses (87 mares, 27 stallions, 56 geldings) from ten breeds. Horse dander, saliva and urine samples were collected. Levels of horse allergen Equ c 4 were quanti...
Kuroda T, Kinoshita Y, Niwa H, Mizobe F, Ueno T, Kuwano A, Hatazoe T, Hobo S.We report the first case of methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a racehorse. A 5-year-old mare developed punctate keratitis after racing. The corneal ulcer continued to expand despite ophthalmic antimicrobial therapy. On day 6, a conjunctival graft surgery was performed. The mare was euthanized, following colitis and laminitis development on day 10. MRSA was isolated from the corneal swab taken at the time of euthanasia. Immunohistochemical analysis demonstrated gram-positive and anti-S. aureus monoclonal antibody-positive cocci infiltration of the corneal stroma; a...
Buechner-Maxwell V, Zhang C, Robertson J, Jain NC, Antczak DF, Feldman BF, Murray MJ.Leukemia is a neoplastic disease of one or more of the cell types of the hemopoietic system and is rarely diagnosed in the horse. This report describes a case of subleukemic acute myelomonocytic leukemia in an 11-year-old gelding. Preliminary cytological diagnosis was supported by two types of laboratory investigations. Cytochemical characterization of blood and bone marrow neoplastic cells was consistent with a myelomonocytic origin. Neoplastic blast cells in peripheral blood were labeled by monoclonal antibodies specific for cell surface molecules of horse granulocytes, but they were not lab...
Blach EL, Amann RP, Bowen RA, Frantz D.Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender w...
Starik E, Ginter A, Coppe P.A monoclonal antibody (mAb) directed against the equine arteritis virus (EAV) nucleocapsid (N) protein was used for indirect enzyme-linked immunosorbent assays (ELISAs) using viral antigen from different sources. The same mAb was labelled with fluorescein isothiocyanate for direct immunofluorescence tests (DIFTs). The N-specific mAb appeared to be suitable for the detection in both ELISA and DIFT of different EAV strains and field isolates from semen and tissue samples after passage in lines of RK-13, Vero and fetal equine kidney cells. The ELISA described is an easy and fast method which can ...
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...
Crump AL, Davis W, Antczak DF.A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23...
Dunowska M, Meers J, Johnson RD, Wilks CR.Representational difference analysis (RDA) was used to compare gene expression in equine mononuclear cells either infected with equine herpesvirus-2 (EHV-2) or adsorbed with inactivated EHV-2. Seven clones identified in non-infected cells after three rounds of selective subtraction and enrichment for differentially expressed genes contained sequences homologous to equine monocyte chemoattractant protein 1 (MCP-1). This suggested that EHV-2 may down-regulate MCP-1 transcription in infected cells. These findings correlate well with similar findings described for human cytomegalovirus and support...
Kendall A, Ekman S, Skiöldebrand E.Nerve growth factor (NGF) is a neurotrophin that has been implicated in pain signaling, apoptosis, inflammation and proliferation. The resultant effects depend on interaction with two different receptors; tyrosine kinase A (TrkA) and p75 . NGF increases in synovial fluid from osteoarthritic joints, and monoclonal antibody therapy is trialed to treat osteoarthritis (OA)-related pain. Investigation of the complex and somewhat contradictory signaling pathways of NGF is conducted in neural research, but has not followed through to orthopaedic studies. The objectives of this study were to compare t...
Turki I, Hammami A, Kharmachi H, Mousli M.Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protei...
Lunn DP, Holmes MA, Schram B, Duffus WP.Equine immunoglobulin G is currently classified as consisting of five sub-isotypes: IgGa, b, and c, IgG(T), and IgG(B). The study of the role of these immunoglobulins in antigen-specific responses, and the examination of their functional properties would be greatly facilitated by the availability of monoclonal antibodies (Mabs) that distinguish between them. The production and characterization of two Mabs that recognize an IgG sub-isotype with the characteristics of IgG(ab) is described. The immunoglobulin identified by these Mabs had a heavy chain weight of 53 kDa, was of rapid cathodal elect...
Amann B, Hirmer S, Hauck SM, Kremmer E, Ueffing M, Deeg CA.Colour vision in animals is an interesting, fascinating subject. In this study, we examined a wide variety of species for expression of S-opsin (blue sensitive) and M-/L-opsin (green-red sensitive) in retinal cones using two novel monoclonal antibodies specific for peptides from human opsins. Mouse, rat and hare did not express one of the investigated epitopes, but we could clearly prove existence of cones through peanut agglutinin labelling. Retinas of guinea pig, dog, wolf, marten, cat, roe deer, pig and horse were positive for S-opsin, but not for M-/L-opsin. Nevertheless all these species ...
Siedek E, Little S, Mayall S, Edington N, Hamblin A.Despite their important role in initiating T-cell responses in other species, dendritic cells have not been studied in the horse. A method for isolating blood dendritic cells by adherence and metrizamide gradients was adapted to equine cells. A number of monoclonal antibodies (mAbs), including some which label dendritic cells in other species, were tested for immunochemical reactivity with the isolated blood dendritic cells, and sections of lymph node and spleen. 62 +/- 6% of the isolated blood cells were MHC Class II positive and had typical dendritic cell morphology and only 4 +/- 2% contain...
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...
Cooper HM, Jemmerson R, Hunt DF, Griffin PR, Yates JR, Shabanowitz J, Zhu NZ, Paterson Y.Comparative binding studies with peptide fragments of the whole antigen, or with evolutionarily related intact proteins with varying degrees of sequence homology, have been used extensively to map antigenic sites on proteins to the resolution of single amino acid residues. These methods are limited, however, since high affinity antibodies will often not react with peptides and evolutionarily related proteins are available for only a few antigens. In this study we use site-directed chemical modification of horse cytochrome c to identify residues involved in the binding sites of four monoclonal ...
Jean-François MJ, Poskitt DC, Turnbull SJ, Macdonald LM, Yasmeen D.We have developed an in vivo passive transfer assay using mice to identify monoclonal antibodies (mAbs) which offer protection against Streptococcus equi infection. The assay was developed using serum antibodies collected from horses convalescing from strangles. In this study, we show that a preparation of M-like protein, acid-extracted from S. equi, affords 80% protection to mice immunized with it. A number of mouse mAbs directed against a preparation of M-like protein were then assessed for their ability to passively protect mice against challenge with a lethal dose of the bacteria. Two mAbs...
Enck KM, Lee KW, McKinney BH, Blankenship KD, Montesano C.It has been demonstrated that immunoglobulin (Ig)E specific for cross-reactive carbohydrate determinants (CCD) is present in the serum of sensitized humans, dogs and cats, and that these CCD-specific antibodies might confound serological testing. Objective: The objective was to determine whether or not CCD-reactive antibodies occur in horses and to investigate the prevalence of CCD-reactive IgE antibodies in equine sera using a monoclonal cocktail-based enzyme-linked immunosorbent assay designed to detect allergen-specific IgE in horses, and to evaluate a means for successful inhibition of the...
van Maanen C, de Boer-Luijtze E, Terpstra C.A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submi...
Williams H, Richards CM, Konfortov BA, Miller JR, Tucker EM.In a study of 35 horse-mouse heterohybridoma cell lines, synteny in the horse was found between LDHB, PEPB and IGF1 and between NP, MPI and IDH2. A synteny between ADA and PEPC was also indicated. The loci for horse immunoglobulin light chain (IgL) genes and for LDHA were independent.
Dart AJ, Little CB, Hughes CE, Chu O, Dowling BA, Hodgson DR, Rose RJ, Johnson KA.Recombinant equine growth hormone (reGH) has recently been evaluated for effects on body condition and wound healing. It has the potential to influence articular cartilage via stimulation of IGF-1. Objective: To investigate effects of administration on synovial joint metabolism. Methods: Six mature horses were given 20 microg/kg bwt reGH daily for 8 weeks by i.m. injection. Three control horses were injected with sterile water. Serum and synovial fluid samples were collected at 6, 8, 11 and 16 weeks for GH and IGF-1 assays. Articular cartilage harvested at week 16 was evaluated by Western anal...
Nordengrahn A, Klingeborn B, Lindholm A, Merza M.A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to equine herpesvirus 2 in serum samples of horses. By measuring the binding to a single epitope, this blocking ELISA gives a good picture of the antibody status in the animal. The test is based on a monoclonal antibody with neutralizing activity and had a sensitivity of 94% and a specificity of 100%. Antibodies due to newly acquired infection in foals were successfully detected with this blocking ELISA.
Madarame H, Takai S, Morisawa N, Fujii M, Hidaka D, Tsubaki S, Hasegawa Y.Rhodococcus equi was isolated from the lungs of six foals with bronchopneumonia. All isolates expressed 15-17-kd antigens by immunoblot analysis and contained a virulence-associated plasmid of 85 or 90 kb. Immunohistochemically, R. equi from all pulmonary lesions showed the expression of 15-17-kd antigens mainly in the phagocytic cells. The specific monoclonal antibody to 15-17-kd antigens of R. equi (MAb 10G5) may be an aid in the diagnosis of R. equi-induced pneumonia.
Song J, Song R, Wang P, Zhang Y, Yan Y, Zhou J, Chahan B, Liao M.The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from...
Hook RR, Riley LK, Franklin CL, Besch-Williford CL.A monoclonal antibody based competitive inhibition assay was used to detect antibodies in horse sera to purified flagellar antigens from distinct Clostridium piliforme isolates. Sequential absorption of hyperimmune rat serum to C. piliforme isolate E (horse-origin isolate), a positive C. piliforme-immune horse serum, and other suspected immune horse sera with unrelated bacteria or C. piliforme isolates E or isolate R1 (rat-origin isolate) alone demonstrated the specificity of this assay for C. piliforme. This specificity was associated with the inhibition of monoclonal antibody binding to C. p...
