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.
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...
Chopineau M, Maurel MC, Combarnous Y, Durand P.In order to localize the epitopes of equine chorionic gonadotropin (eCG) involved in interaction with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) receptors, we used 14 monoclonal anti-eCG antibodies (mAbs). Different effects of these mAbs on the bioactivities of eCG were observed in in vitro bioassays, but the effects of each mAb on the two bioactivities were similar for all but four mAbs. All mAbs were found to inhibit the binding of eCG to LH receptors except 3A3 mAb, in radioreceptor assay. Six mAbs, which were strong inhibitors of eCG binding to LH receptors and of both...
Gupta AK, Yadav MP, Uppal PK, Mumford JA, Binns MM.Two A/Equi-2 (H3N8) isolates were obtained during the 1987 Indian equine influenza epizootic. The sequence of the Ludhiana/87 HA1 gene revealed that this isolate was very similar to recent European and North American isolates of equine influenza. In contrast, the Bhiwani/87 HA1 gene was nearly identical to the Miami/63 prototype H3 sequence. These results support the antigenic analysis previously carried out on these isolates using monoclonal antibodies. However, the finding that Bhiwani/87 is so similar to Miami/63, coupled with the finding that equine H3N8 influenza viruses have previously b...
Ahmed SM, Broad SC, Edington N.Sera from two sibling groups of ponies experimentally infected with Equid herpesvirus 1 or 4 (EHV-1 or 4) were used to investigate which viral polypeptides (VPs) of EHV-1 and EHV-4 were recognised. Recognition was detected as early as 8 d.p.i. and thereafter. The polypeptides of EHV-1 (labelled with 35S-methionine) immunoprecipitated (IIP) by sera from both groups had Mr of 148, 138, 123, 117, 110, 77-79, 70, 55, 49-50, 47, 40 and 35-37 kDa respectively. Of these VP148K (VP9 nucleocapsid) gave the maximum precipitation, followed by 117 and 77-79 kDa. The latter were confirmed by monoclonal ant...
Lunn DP.Monoclonal antibody technology has allowed the recognition and study of numerous leucocyte antigens in man and laboratory animals for over a decade. Numerous advances in the understanding of immune responses and immunopathology have resulted. In recent years equine researchers have started to develop similar reagents, which now offer a powerful tool to investigators of equine immunology and disease.
Corstvet RE, Gaunt SD, Karns PA, McBride JW, Battistini RA, Mauterer LA, Austin FW.An enzyme-linked immunosorbent assay (ELISA) was used to detect antigen in plasma and antibody in serum of 3 horses inoculated with Ehrlichia equi. Clinical signs, including rectal temperature, were correlated with the antigen and antibody detection. ELISA was very efficient in detection of serum antibody. Antigen detection using monoclonal antibodies to E. equi and ELISA should be considered as a diagnostic method.
Ludwig H, Furuya K, Bode L, Klein N, Dürrwald R, Lee DS.Borna disease viruses (BDV) isolated from more than 20 naturally infected horses, 2 sheep and a possible feline isolate were included in these studies. Most of these wild-type viruses were grown in rabbit cells. Specifically rabbit-adapted viruses establish persistent infection in immortalized cell lines of various animal species. Brain-, tissue culture-, and cell-free released viruses could all be neutralized with antibodies from naturally and experimentally infected animals (horse; hamster, rat, rabbit, mouse, and chicken), with highest titres in birds. Splenectomized rabbits, which were sub...
van de Moer A, Rice M, Wilks CR.A type-specific monoclonal antibody was produced by immunizing mice with purified equid herpesvirus-1 (EHV-1). The EHV-1 specific mAb reacted with all the EHV-1 strains tested so far by indirect ELISA, immunofluorescence, and immunoperoxidase tests. No reactions were detected with the EHV-4, EHV-2, or EHV-3 strains tested. The indirect immunofluorescence and immunoperoxidase tests showed that the nuclei of infected cells were predominantly stained by this mAb. Triton treatment of the virus and immunogold labeling experiments indicated that the nucleocapsid of EHV-1 was the target antigen of th...
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...
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...
Couture L, Lemonnier JP, Troalen F, Roser JF, Bousfield GR, Bellet D, Bidart JM.The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primat...
Corstvet RE, Gaunt SD, Karns PA, Burgermeister D, McBride JW, Nicholson SM, Battistini RA.Four horses were inoculated with Ehrlichia risticii contained in either infected murine P388 D1 cells or heparinized blood from an infected horse. All 4 horses produced serum antibody, plasma antigen, and clinical signs of the disease. An enzyme-linked immunosorbent assay was used to detect antibody in the serum and was also used in conjunction with an anti-E. risticii monoclonal antibody to detect antigenemia. These laboratory and clinical findings were correlated to determine the efficiency of the antigen detection method for discerning E. risticii infection.
Knowles DP, Kappmeyer LS, Stiller D, Hennager SG, Perryman LE.Horses infected with Babesia equi were previously identified by the presence of antibodies reactive with a merozoite surface protein epitope (D. P. Knowles, Jr., L. E. Perryman, L. S. Kappmeyer, and S. G. Hennager. J. Clin. Microbiol. 29:2056-2058, 1991). The antibodies were detected in a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) by using monoclonal antibody 36/133.97, which defines a protein epitope on the merozoite surface. The gene encoding this B. equi merozoite epitope was cloned and expressed in Escherichia coli. The recombinant merozoite protein, designated equ...
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...
Miller CC, Fayrer-Hosken RA, Timmons TM, Lee VH, Caudle AB, Dunbar BS.This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubiliz...
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...
Aida Y, Okada K, Kageyama R, Amanuma H.Tumor-associated antigens that are expressed in lymphosarcoma B cells of cattle with enzootic bovine leukosis had been analyzed in terms of their reactivity with 13 monoclonal antibodies (MAB). By use of flow cytometry and radioimmunoprecipitation, 1 of the MAB (c143) that recognized a tumor-associated antigen cross-reacted with blood lymphocytes (BL) from various mammalian species. By use of flow cytometry, the c143 MAB reacted with 10 to 49% of BL derived from human beings, mice, dogs, horses, pigs, llamas, sheep, goats, and cattle. Titer of the c143 MAB with BL from horses, pigs, human bein...
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...
Sellon DC, Perry ST, Coggins L, Fuller FJ.In situ hybridization of tissues from two horses infected with the wild-type Wyoming strain of equine infectious anemia virus (EIAV) identified the liver, spleen, lymph nodes, kidney, lung, and adrenal gland as the primary host tissue sites for viral transcription during acute infection. Combined immunohistochemistry, with a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes, and in situ hybridization for viral RNA identified most infected cells as mature tissue macrophages. In contrast, in situ hybridization of adherent peripheral blood mononuclear cells co...
Maurel MC, Ban E, Bidart JM, Combarnous Y.In the present study we have established an immunochemical mapping of equine Chorionic Gonadotropin (eCG/PMSG) using three monoclonal antibodies (mAbs), namely the antibodies ECG01, E10 and D7, raised against the native hormone. These antibodies do not bind to reduced, alkylated hormone, suggesting that they recognize discontinuous rather than continuous epitopes. We have also assessed the reactivity of mAbs towards human CG, and ovine, porcine, equine and bovine LH and FSH. The antigenic determinant recognized by ECG01 is localized on the alpha-subunit of equine gonadotropins and of human CG ...
Sinclair R, Mumford JA.An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine herpesvirus type-1 (EHV-1) antigens in nasal swab specimens. The test was designed as a solid phase, amplified sandwich assay in which an EHV-1 specific monoclonal antibody was used to capture virus antigen and polyclonal antisera used to detect antigen bound to the test plates. Eight monoclonal antibodies were tested for their ability to capture virus antigen and one was selected for routine use. The sensitivity and specificity of the ELISA was compared with that of virus isolation using swa...
Geyer C, Hafner A, Pfleghaar S, Hermanns W.Six canine, one feline and one equine granular cell tumours (GCTs) were investigated electron microscopically and immunohistochemically. The tumours were tested for reactivity with monoclonal antibodies against vimentin and desmin and with polyclonal antibodies against cytokeratin, S-100 protein, glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE). All GCTs were characterized by their PAS positive cytoplasmic granules in light microscopy, which in electron microscopy appeared as lysosome-like granules. In each case two canine GCTs were stained by the antibody against cytok...
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...
Sinha AK, Rose RJ, Pozgaj I, Hoh JF.The histochemical ATPase method for muscle fibre typing was first described by Brooke and Kaiser in 1970. However, problems have been found with the subdivision of type II fibres using this technique. To determine whether indirect myosin immunocytochemistry using anti-slow (5-4D), anti-fast (1A10) and anti-fast red (5-2B) monoclonal antibodies with cross reactivity for type I, II and IIa fibres, respectively, in a number of species, could identify three fibre types in equine skeletal muscle, data on fibre type composition and fibre size obtained using the two different techniques were compared...
Richards CM, Aucken HA, Tucker EM, Hannant D, Mumford JA, Powell JR.Studies were carried out to determine the optimum conditions for the production of equine monoclonal antibodies (MAbs). Lymphocytes from ponies immunised with influenza A equine 2 virus, isolate A/Equine/Newmarket/79 (H3N8) were fused with mouse myeloma (NSO) cells and with horse-mouse heterohybridomas made aminopterin-sensitive by selective growth in 8-azaguanine. Although all fusions initially resulted in heterohybridoma colonies that secreted equine immunoglobulin, many of these were unable to maintain secretion for longer than a few weeks. Increasing the time between immunisation and the b...
Appleton JA, Gagliardo LF.A large panel of mouse monoclonal antibodies was produced and tested against field isolates of the equine H7N7 influenza A virus subtype. Only a limited degree of H7 haemagglutinin variation was detected. At least four antigenic sites were identified by selecting variant viruses in eggs. The limited variation in the field did not correlate with the frequency of variant viruses detected in eggs; this frequency was similar to those reported for other influenza viruses. We sought to determine whether the limited amount of variation could be correlated with an epitope-restricted antibody response ...
Studdert MJ, Crabb BS, Ficorilli N.The restriction endonuclease DNA fingerprints of 57 isolates of equine herpesvirus 1 (EHV1; equine abortion virus) from abortion, perinatal foal mortalities and encephalitis from 15 epidemics that occurred in Australasia between 1975 and 1989 were examined using the enzymes Bam HI, EcoRI and Bgl II. There was a remarkable degree of uniformity in the restriction patterns; mobility differences were observed in only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located within the repeat structures that bracket the unique short region of the genome or were located at the left ...
Ball JM, Rushlow KE, Issel CJ, Montelaro RC.We describe here a detailed analysis of the antigenic determinants of the surface unit glycoprotein (gp90) of equine infectious anemia virus (EIAV), using a comprehensive panel of synthetic peptides in enzyme-linked immunosorbent assays with immune serum from naturally and experimentally infected horses and with a panel of gp90-specific neutralizing and nonneutralizing monoclonal antibodies. The results of these studies identify immunoreactive segments throughout the conserved and variable domains of gp90 but localize immunodominant (100% reactivity) determinants to the amino and carboxyl term...
Browning GF, Chalmers RM, Fitzgerald TA, Snodgrass DR.Ten cultivable equine rotavirus isolates, two of North American, six of British, and two of Irish origin, were compared with standard rotavirus strains and with each other by cross neutralization, neutralization with a panel of monoclonal antibodies (MAbs), hybridization to a simian rotavirus (SA-11) VP7 gene probe, and reaction with rotavirus subgrouping and serotyping MAbs in enzyme-linked immunosorbent assays. Six isolates, two of which had previously been serotyped as G3 by other workers, were found to be serotype G3; one was confirmed to be G5, and three were not related to serotypes G1 t...
Schuberth HJ, Anders I, Pape U, Leibold W.The cells of 60 randomly selected Hannoveranian warm-blooded horses were subjected to one-dimensional isoelectric focusing and immunoblotting with a cross-reacting monoclonal antibody (Bo 1) recognizing bovine class I antigens. The banding patterns were correlated with the serologically defined specificities of the ELA-A locus. ELA-A2 was correlated with four bands, while ELA-A5, ELA-W18, ELA-A6, ELA-A14 and ELA-A9 were correlated with a single band each. The complexity of the pattern and additional polymorphic bands which could not be correlated to any of the known ELA specificities may indic...
Akens MK, Holznagel E, Franchini M, Bracher V.In the present study, two methods of lymphocyte preparation, whole blood lysis and Ficoll-Paque separation, prior to FACS analysis were compared. The comparison was done with single and dual-colour staining techniques. Monoclonal antibodies (mAb) against eCD4, eCD5, eCD8 and eMHC class II were used. There was no significant difference in the results obtained by these two methods.
How SJ, Lloyd DH, Lida J.A monoclonal antibody (McAb) to Dermatophilus congolensis was produced from murine hybridoma cultures and purified by affinity chromatography. Species specificity was demonstrated using indirect immunofluorescent staining; the McAb was shown to react with 10 D congolensis isolates but not with 10 Nocardia species isolates, a Rhodococcus and a Streptomyces species isolate. The McAb was used to demonstrate D congolensis in clinical material from confirmed bovine and ovine cases and presumptive equine cases of dermatophilosis by indirect immunofluorescent staining.
Appleton JA, Gagliardo LF.A large panel of mouse monoclonal antibodies was produced and tested against field isolates of the equine H7N7 influenza A virus subtype. Only a limited degree of H7 haemagglutinin variation was detected. At least four antigenic sites were identified by selecting variant viruses in eggs. The limited variation in the field did not correlate with the frequency of variant viruses detected in eggs; this frequency was similar to those reported for other influenza viruses. We sought to determine whether the limited amount of variation could be correlated with an epitope-restricted antibody response ...
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 ...
Rahnama S, Spence R, Vathsangam N, Baskerville CL, Bailey SR, de Laat MA, Anderson ST, Pollitt CC, Sillence MN.Although it is understood that equine endocrinopathic laminitis can be triggered by high concentrations of insulin, it is unclear whether this represents a direct action on lamellar tissue via insulin receptors (InsR), an interaction with IGF-1 receptors (IGF-1R), or some other, indirect action. This uncertainty is because of the reported scarcity of InsR in lamellar tissue and the low affinity of insulin for equine IGF-1R. In the present study, the effects of insulin and IGF-1 (as a positive control) were examined using lamellar explants isolated from the hooves of healthy horses and incubate...
Jaspers U, Thiele D, Krauss H.A competitive ELISA system for the detection of antibodies against Coxiella (C.) burnetii in cattle, sheep, goats, horses and humans is described. The ELISA is based on a biotinylated monoclonal antibody with specificity for C. burnetii lipopolysaccharide in combination with streptavidin peroxidase. For evaluation and statistical analysis, 413 sera from cattle, sheep, goats, horses and humans were tested in parallel in the indirect immunofluorescence test (IFT). Furthermore, a total of 448 bovine and human sera were also tested with an indirect ELISA and 47 sheep sera were investigated using t...
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...
Kamhieh S, Hodgson J, Bode L, Ludwig H, Ward C, Flower RL.Borna disease virus (BDV) is a unique RNA virus that is a cause of neurological disease in horses, sheep and cats. The finding that BDV also infects humans has raised concern related to the impact of infection with this virus. The extent to which BDV may be endemic in geographical regions outside Europe is of interest in management of international movement of animals including horses. Sera from Australian horses (N = 553) sampled in Sydney, New South Wales (NSW), were analysed for BDV antigen, circulating immune complexes (CICs), and antibodies by monoclonal antibody-based ELISAs. One-tenth o...
Maurel MC, Ban E, Bidart JM, Combarnous Y.In the present study we have established an immunochemical mapping of equine Chorionic Gonadotropin (eCG/PMSG) using three monoclonal antibodies (mAbs), namely the antibodies ECG01, E10 and D7, raised against the native hormone. These antibodies do not bind to reduced, alkylated hormone, suggesting that they recognize discontinuous rather than continuous epitopes. We have also assessed the reactivity of mAbs towards human CG, and ovine, porcine, equine and bovine LH and FSH. The antigenic determinant recognized by ECG01 is localized on the alpha-subunit of equine gonadotropins and of human CG ...
Monos DS, Wolf B, Radka SF, Rifat S, Donawick WJ, Soma LR, Zmijewski CM, Kamoun M.Six mouse and 13 rat monoclonal antibodies (mAb) recognizing HLA-DR, DQ and DP antigens were used for the detection of cell surface class II MHC antigens of equine lymphocytes. The monoclonal antibodies were tested against peripheral blood lymphocytes (PBL) from a panel of thoroughbred horses, using two-color fluorescence flow cytometry. Seven of these mAbs reacted with both surface immunoglobulin positive (sIg+) and surface immunoglobulin negative (sIg-) lymphocytes. sIg+ cells stained consistently brighter than sIg- cells. The fluorescence pattern did not vary from donor to donor for each of...
van den Heuvel LP, van den Born J, Veerkamp JH, Janssen GH, van de Velden TJ, Monnens LA, Schröder CH, Berden JH.1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had...
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...
Fontanals AM, Becú T, Polledo G, Gaskin CK, Braun M.An R. equi vaccine, prepared under conditions which induce the expression of many antigens, and which has given encouraging results in field trials, was analyzed by SDS-PAGE and immunoblots and compared with other R. equi preparations: a preparation made in with the same technique from a nonvirulent isolate (virulence associated protein negative, VapA-negative); a whole cell preparation of a VapA-positive R. equi, prepared as a standard bacterin; and a semipurified VapA preparation (APTX). The antigens in these preparations were analyzed using hyperimmune sera (from adult horses vaccinated wit...
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...
Simonin EM, Wagner B.IgE-binding monocytes are a rare peripheral immune cell type involved in the allergic response through binding of IgE on their surface. IgE-binding monocytes are present in both healthy and allergic individuals. We performed RNA sequencing to ask how the function of IgE-binding monocytes differs in the context of allergy. Using a large animal model of allergy, equine Culicoides hypersensitivity, we compared the transcriptome of IgE-binding monocytes in allergic and non-allergic horses at two seasonal timepoints: (i) when allergic animals were clinical healthy, in the winter "Remission Phase", ...
Timms M, Ganio K, Forbes G, Bailey S, Steel R.CJC-1295 is a 30 amino acid peptide-based drug that stimulates the release of growth hormone (GH) from the pituitary gland. It is unique among performance-enhancing peptides due to the presence of a reactive maleimidopropionic acid group that covalently links the peptide to free thiols on the surface of plasma proteins. Once conjugated, CJC-1295 remains active in the bloodstream for significantly longer than non-conjugated peptide-based drugs that are rapidly excreted. Conjugation of CJC-1295 to plasma proteins prevents its detection by top-down mass-spectrometry-based peptide screening protoc...
Miller CC, Fayrer-Hosken RA, Timmons TM, Lee VH, Caudle AB, Dunbar BS.This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubiliz...
Schuberth HJ, Anders I, Pape U, Leibold W.The cells of 60 randomly selected Hannoveranian warm-blooded horses were subjected to one-dimensional isoelectric focusing and immunoblotting with a cross-reacting monoclonal antibody (Bo 1) recognizing bovine class I antigens. The banding patterns were correlated with the serologically defined specificities of the ELA-A locus. ELA-A2 was correlated with four bands, while ELA-A5, ELA-W18, ELA-A6, ELA-A14 and ELA-A9 were correlated with a single band each. The complexity of the pattern and additional polymorphic bands which could not be correlated to any of the known ELA specificities may indic...
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 ...
Byrne KM, Davis WC, Holmes MA, Brassfield AL, McGuire TC.Two monoclonal antibodies (MAb), HB65A (IgG2a) and HB86A (IgGI), recognize a unique cell surface molecule on equine T-lymphocytes. The molecule, designated EqWC4, identified by these MAbs is present on a subpopulation of CD4+ equine lymphocytes (6.3-10.2% of Arabian lymphocytes CD4+ WC4+) and a smaller population of CD8+ lymphocytes (0.5% to 1.2% of Arabian lymphocytes CD8+ WC4+). EqWC4 is absent from B-lymphocytes, granulocytes, and macrophages. Both MAbs bound to a 46-kDa protein following immunoprecipitation reactions with lysates of surface labeled thymocytes. Immunoaffinity purification u...
Lunn DP, Schram BR, Vagnoni KE, Schobert CS, Horohov DW, Ginther OJ.Lymphokine activated killing (LAK) is an example of natural cytotoxicity, and as such is a critical means of defense against diseases such as viral infection and neoplasia. Despite this important role, the specific molecular interactions involved in LAK or other forms of natural cytotoxicity are only partially understood. In some species, cells capable of mediating natural cytotoxicity express the CD8 molecule, although no specific role has been demonstrated for CD8 in non-MHC restricted cytotoxicity. In this study the role of the EqCD8 equine homolog of CD8 in LAK cell activity was examined. ...
Sengupta PP, Rudramurthy GR, Ligi M, Jacob SS, Rahman H, Roy P.Trypanosoma evansi, a haemoflagellated protozoan parasite, is responsible for chronic as well as the acute debilitating disease called surra in a wide range of herbivores and carnivores including domestic and wild animals. Since the parasite is having wide host range, there is a need for diagnostic test which can detect the T. evansi specific antibody in different species of animals for generating sero-surveillance data. In the present study we developed and evaluated competitive enzyme immunoassay using monoclonal antibodies (MAbs) raised against recombinant variable surface glycoprotein (rVS...
Qi T, Hu Y, Hu Z, Zhao S, Cullinane A, Lyons P, Gildea S, Wang X.Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference stan...
Whyte A, Ockleford CD, Wooding FB, Hamon M, Allen WR, Kellie S.The establishment of a monolayer culture of cells derived from the girdle region of a 34-day-old donkey conceptus is described. These cells have had over 100 repeated passages in culture. Low levels of pregnant mares' serum gonadotrophin (PMSG, eCG) could be detected in the cells by indirect immunofluorescence using some monoclonal anti-eCG antibodies, but the cells did not secrete eCG as measured by radioimmunoassay or inhibition of haemagglutination. There was marked nuclear polymorphism with binucleate and occasional multinucleate cells. The cells were strongly reactive with wheatgerm agglu...
Fedele C, Berens D, Rautenfeld V, Pabst R.Monoclonal antibodies against Desmoplakin and Plakoglobin were tested for their suitability as specific markers of lymphatic vessels. The tissue samples were taken from horse skin in an attempt to establish the horse as a model for human lymphatic diseases. To obtain a clear, positive identification of blood and lymphatic vessels, immunohistochemical staining with antibodies against vascular endothelial growth factor receptor 3 (VEGFR-3) and platelet endothelial adhesion molecule (PECAM-1, CD31), was compared with Desmoplakin and Plakoglobin. Because anti-VEGFR-3 is specific for lymphatic vess...
Messier PE, Drouin R, Richer CL.We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. ...
Rahnama S, Vathsangam N, Spence R, Anderson ST, de Laat MA, Bailey S, Sillence MN.Prolonged hyperinsulinemia is thought to be the cause of equine endocrinopathic laminitis, a common and crippling disease of the foot, for which there are no pharmacologic treatments other than pain relief. It has been suggested that insulin causes its effects on the lamellae by activating IGF-1 receptors (IGF-1R), as insulin receptors (InsR) are scarce in this tissue, whereas IGF-1R are abundant and become downregulated after prolonged insulin infusion. As a first step toward confirming this mechanism and beginning to develop a therapeutic anti-IGF-1R monoclonal antibody (mAb) for horses, it ...
Truax RE, Powell MD, Montelaro RC, Issel CJ, Newman MJ.A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples b...
Barrey E, Valette JP, Jouglin M, Picard B, Geay Y, Robelin J.The content in slow and fast myosin heavy chains (MHC 1 and MHC 2) of 5 equine muscles was determined using an enzyme-linked immunosorbent assay. The results obtained with this immunoenzymatic method were compared with complementary techniques: electrophoresis and immunohistochemistry. Slices of masseter, diaphragm, tensor faciae latae, semitendinosus and cutaneus trunci were obtained from a 12-year-old saddle horse after slaughter. Muscular proteins were specifically extracted to be analysed by ELISA. The technique used 2 complimentary monoclonal antibodies (MAb). MAb 1 was prepared from a hu...
Wagner B, Siebenkotten G, Leibold W, Radbruch A.Due to characteristic clinical signs, immunoglobulins of isotype E (IgE) are believed to be involved in several allergic diseases of the horse. To date, closer investigations have been hampered by the fact that neither purified equine IgE nor anti-equine IgE monoclonal antibodies were available for IgE isotype determination. As an approach to solve this problem, we constructed a stable cell line (EqE6) that expresses recombinant equi-murine IgE specific for 4-(hydroxy-3-nitro-phenyl) acetyl (NP). Biochemical analysis of the purified protein revealed a highly glycosilated IgE monomer of approxi...
