Topic:Enzyme-Linked Immunosorbent Assay (ELISA)
Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used analytical technique in equine research for detecting and quantifying specific proteins, hormones, and antibodies in horse biological samples. This method relies on antigen-antibody interactions and employs enzyme-linked antibodies to produce a measurable signal, typically a color change, indicating the presence and concentration of the target molecule. ELISA is applicable in various areas of equine health, including the diagnosis of infectious diseases, monitoring of immune responses, and assessment of physiological conditions. It is valued for its specificity, sensitivity, and ability to process multiple samples simultaneously. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and advancements of ELISA in equine science.
Equine piroplasmosis an update on diagnosis, treatment and prevention. Two haemoprotozoan parasites, Babesia caballi and Babesia equi, can cause equine piroplasmosis. Due to the presence of potential tick vectors in areas so far unaffected by equine babesias, import and export regulations often require the serum testing of animals for evidence of infection. Although the complement fixation test (CFT) has been recommended for detecting the presence of antibodies to Babesia spp., it has been demonstrated to have several disadvantages, including false-positive results and low sensitivity for detecting latent infections. An enzyme-linked immunosorbent assay (ELISA) m...
Antibody-mediated neutralization and binding-reversal studies on alpha-neurotoxins from Micrurus nigrocinctus nigrocinctus (coral snake) venom. 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...
Plasma, urine, and synovial fluid disposition of methylprednisolone acetate and isoflupredone acetate after intra-articular administration in horses. OBJECTIVE--To document plasma, urine, and synovial fluid disposition of 2 common intra-articularly administered steroid preparations, methylprednisolone acetate (MPA) and isoflupredone acetate (IPA). DESIGN--Descriptive investigation. SAMPLE POPULATION--100 mg of MPA or 4 mg of IPA was administered to 2 groups of 4 healthy sound radiographically normal female horses. PROCEDURE--Blood samples were collected at time 0 (before) and 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours after administration of the designated steroid. Complete urine collection for measurement of designated steroid was ac...
Use of enzyme-linked immunosorbent assay and radioimmunoassay to determine serum and urine dexamethasone concentrations in thoroughbreds after intravenous administration of the steroid. To develop a simple and sensitive ELISA for detection of dexamethasone in horse serum and urine. Methods: Blood and urine samples from 3 thoroughbred mares. Methods: A dexamethasone oxime was prepared and conjugated to hemocyanin, bovine serum albumin and to horseradish peroxidase. One- and two-step double-antibody ELISA methods, as well as a radioimmunoassay method, were performed. The one-step ELISA was used to test urine from 3 Thoroughbred mares injected with 5 mg of dexamethasone, IV. Results: The ELISA could detect dexamethasone in the range of 0.01 to 50 ng/ml, with intra- and interassa...
Use of excretory/secretory antigens for the serodiagnosis of Anoplocephala perfoliata cestodosis. Whole worm extract (WWE) and excretory/secretory (E/S) antigens of Anoplocephala perfoliata were characterised by SDS-PAGE and their use in the serodiagnosis of equine cestodosis was evaluated. An enzyme-linked immunosorbent assay (ELISA) was used to compare WWE and E/S antigen as the capture layer in an antibody capture ELISA. E/S antigen gave the best differentiation between sera from tapeworm-positive and tapeworm-negative horses. The E/S-ELISA was optimised and validated against sera from horses of known tapeworm status. This assay gave a diagnostic sensitivity of 68% (n = 38) and a specif...
Nationwide serological survey of equine influenza in Nigeria. The objective of this work was to examine the incidence of equine influenza viruses in the equine population of an area of tropical Africa where equine influenza virus activity has recently been reported for the first time. A serological survey of sera from horses and donkeys from regions of Nigeria taken from 1990 to 1993 was carried out and the results obtained were com-pared with equine sera from Western Europe (Ireland). The sera were assayed for presence of antibodies by both haemagglutination inhibition (HI) and ELISA using a monoclonal antibody to the prototype H3 equine influenza virus...
Rapid diagnosis of African horse sickness. The rapid diagnosis of African horse sickness (AHS) during the incubation period using virus antigens in peripheral blood mononuclear cells (PBMC) and red blood cells (RBC) in a sandwich indirect enzyme-linked immunosorbent assay (ELISA) is reported. PMBC consistently gave higher positive ELISA results than RBC from blood collected during viraemia from clinically affected horses. The potential of the method described for wider application in rapid diagnosis and virus surveillance in susceptible equine populations, particularly in AHS-free and in enzootic areas, for effective control strategies...
Evidence for a high rate of false-positive results with the indirect fluorescent antibody test for Ehrlichia risticii antibody in horses. The original objective was to determine seroprevalence of Ehrlichia risticii antibody among horses in California. On the basis of the unexpected results of the survey, an investigation into the accuracy and reproducibility of results of the indirect fluorescent antibody (IFA) test for E risticii was carried out. Methods: Prospective, seroprevalence study. Methods: Healthy horses (n = 655) and horses with clinical signs of equine monocytic ehrlichiosis (EME; n = 514) from various regions of California. Methods: The IFA test was performed. Results were compared with results of an ELISA and with ...
Serial measurement of peripheral oestrogen and progesterone concentrations in oestrous mares to determine optimum mating time and diagnose ovulation. Rapid enzyme-based immunoassays were used to measure concentrations of oestradiol-17 beta and progesterone in daily blood samples recovered throughout oestrus and for a few days after ovulation from 34 Thoroughbred and 8 pony-type maiden, barren and foaling mares. The first detectable fall in oestradiol-17 beta levels occurred in 88% of the mares within the interval -72 to 0 h with respect to ovulation and in 65% of mares within the interval of -48 to 0 h. The results indicated that serial daily hormone assays of this type could, in a high proportion of animals, predict a correct time for a si...
Sensitivity of antigen ELISA test for detecting Trypanosoma evansi antigen in horses in the subtropical area of Argentina. The sensitivity of an antigen detection enzyme immunoassay (Ag-ELISA) based on a Trypanosoma brucei group-specific monoclonal antibody was evaluated to detect circulating Trypanosoma evansi antigen in horse sera. Three horses and 2 mules were experimentally infected with T. evansi. Circulating antigens were detected on 7 and 21 days postinfection. Antigen levels increased during the course of the illness and remained high even when parasitemia was low or when parasites could not be detected. Antigens were cleared from serum when drug treatment was effective but persisted when it was not. In 6 ...
Sandwich enzyme-linked immunosorbent assay for quantitative measurement of serum amyloid A protein in horses. To measure the concentration of serum amyloid A (sAA) protein in horses, a sensitive and highly reproducible sandwich (ELISA) was established, using affinity purified SAA antibody. Results of the ELISA were found to have a high correlation (r = 0.95) with those of the single radial immunodiffusion test. Equine SAA concentration was measured by use of this ELISA. In clinically normal horses, the concentration of SAA was high immediately after birth to 2 weeks of age. After that, SAA concentration had periodic fluctuations in the range of approximately 1.0 to 30 micrograms/ml. Mean (+/- SD)) con...
Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anoplocephala perfoliata in horse sera. A scolex antigen of the horse tapeworm Anoplocephala perfoliata containing at least 14 different proteins was employed in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to A. perfoliata in equine sera. The assay was applied to sera from 426 slaughtered horses with different numbers of worms and with varying degrees of intestinal lesions. As measured by the ELISA, there was a very strong effect on the antibody levels both from the number of tapeworms present and from the intestinal lesion score. However, considerable individual variation was observed between horses wit...
An assessment of mucosal immunisation in protection against Streptococcus equi (‘Strangles’) infections in horses. The ability of mucosally administered antigen to provide protection against Streptococcus equi ('Strangles') infections in horses was examined. First, an enzyme linked immunosorbent assay (ELISA) was developed to detect the immune status of horses to S. equi. This assay was used to select Strangles-naive horses for the study and also to monitor their response to immunisation. Potential vaccine candidates were: (a) orally administered paraformaldehyde killed S. equi; (b) intraperitoneally (IP) administered paraformaldehyde killed S. equi in a non-inflammatory adjuvant; (c) orally administered l...
Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus. A recombinant glutathione-S-transferase fusion protein expressing amino acids 55-98 of equine arteritis virus (EAV) GL (rGL 55-98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1...
Application of an equine herpesvirus 1 (EHV1) type-specific ELISA to the management of an outbreak of EHV1 abortion. Sera from 33 Australian thoroughbred mares were tested during an outbreak of equine herpesvirus 1 (EHV1) abortion with an enzyme-linked immunosorbant assay (ELISA) for the presence of EHV1-specific antibodies. The ELISA used a recombinant EHV1 antigen derived from glycoprotein G (gG) and distinguished antibodies to EHV1 from those of the antigenically related and widespread herpesvirus EHV4. Sera were obtained from most of the mares on three occasions, three, 13 and 67 days after the first abortion. Mares which were negative in the ELISA were kept separate from mares which were positive. A sec...
Detection of clenbuterol (Ventipulmin) in the horse. An enzyme linked immunosorbent assay (ELISA) was developed to detect the beta 2-agonist clenbuterol in equine blood and urine. The antiserum was raised in rabbits, employing clenbuterol-diazo-BSA as antigen. Clenbuterol-diazo-horseradish peroxidase served as enzyme conjugate. The concentration of clenbuterol to decrease tracer binding by 50% (IC50 value) was found to be 27.50 +/- 4.20 pg/well (1.37 ng/ml). The antibody cross-reacted with salbutamol (30%), terbutaline (14%) and cimaterol (1%). Horse serum was used directly to screen for clenbuterol, while urine was employed diluted. Positive sc...
Development of an ELISA using a universal method of enzyme-labelling drug-specific antibodies. Part I: Detection of dexamethasone in equine urine. The development, validation, and application of an ELISA for dexamethasone in equine urine is described. The drug-protein conjugate was immobilised in microtitre plate wells and antiserum raised against the same drug-protein conjugate was allowed to compete with sample or standard drug and the immobilised drug-protein conjugate. The proportion of antiserum binding to the immobilised drug-protein conjugate was detected using a biotinylated protein G/extravidin-alkaline phosphatase complex in situ and measurement of the substrate product. The method was used to detect the presence of drug-derive...
Polyclonal antibody-based antigen-detection immunoassay for diagnosis of Trypanosoma evansi in buffaloes and horses. An enzyme-linked immunosorbent assay (ELISA) was employed for the detection of Trypanosoma evansi antigens in serum samples of field cases of buffaloes and horses in northern India. In 323 naturally infected/suspected buffaloes, circulating antigenaemia was detected in 180 (55.72%), whereas parasitaemia by wet blood smear examination was found in 62 (19.19%) only. The antigen-ELISA was positive in 47 of the 62 parasitologically proven cases and in 86 of the 116 cases with anti-trypanosome antibodies detected by ELISA. Of the 80 horses examined antigen-ELISA was positive in 45 (56.75%) sera. Th...
Cloning and expression of two genes from Babesia equi merozoites and evaluation of their diagnostic potential. High-titre equine immune sera were used to screen a lambda gt 11 expression library of Babesia equi cDNA fragments. Two cDNA clones which did not cross-hybridize to each other were studied. Both clones hybridized specifically to DNA from B. equi but not to DNA from B. caballi, B. divergens or B. ovis. Recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins with apparent molecular weights of 40 kDa and 75 kDa. Polyclonal antibodies directed against the 40 kDa and 75 kDa recombinant proteins detected native antigens of 55 kDa and 50 kDa respectively in crude lysate...
Continuing prevalence of African horse sickness in Nigeria. Equine sera collected from 10 widely separated regions throughout Nigeria were tested for antibodies against African horse sickness viruses (AHSV) using a competitive enzyme-linked immunosorbent assay (ELISA). The animals sampled included imported, exotic horses, indigenous and locally cross-bred (local) horses and African donkeys. A high percentage of the sera (79.8%) were positive, confirming the continued prevalence of AHSV antibodies in Nigerian horses and donkeys.
Bovine respiratory syncytial virus antibodies in non-bovine species. To study the role of non-bovine species in the epidemiology of bovine respiratory syncytial virus (RSV) infections, sera obtained from 9 non-bovine animal species and from humans were examined for bovine RSV specific antibodies. Sera were mainly from animals and humans which had been in contact with cattle. Forty sera of each species were tested in an RSV specific whole virus ELISA as well as in a peptide based ELISA, that was developed to measure antibodies specific for bovine RSV. Antibodies directed against RSV were detected in over 50% of sera obtained from sheep, goat, cattle and human be...
Enzyme-linked immunosorbent assay for myosin heavy chains in the horse. 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...
A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1. We describe a type-specific ELISA, which distinguishes antibody to equine herpesvirus 4 (EHV4; equine rhinopneumonitis) and EHV1 (equine abortion virus) thereby identifying horses that have been infected with either or both of these antigenically related viruses. The antigens used are parts of the EHV4 and EHV1 glycoprotein G (gG) homologues expressed in E. coli as fusion proteins [Crabb and Studdert, 1993: J Virol 67: 6332-6338). The expressed proteins comprise corresponding regions of the gG molecules that are highly divergent and encompass strong, typespecific epitopes. Plasma samples from ...
Generic immunoassay of corticosteroids with minimum pre-treatment of urine samples. A generic, rapid and sensitive enzyme linked immunosorbent assay (ELISA) test has been developed which allows large-scale simultaneous testing of synthetic corticosteroids viz., flumethasone, dexamethasone and betamethasone. This assay can be directly applied to diluted urine samples (1 + 9) without hydrolysis of glucuronide or sulfate conjugates or any other treatment of samples. The polyclonal antibody was obtained by immunizing sheep with a flumethasone derivative linked to human serum albumin. This polyclonal antibody displayed high-reactivity with several synthetic corticosteroids whilst ...
Investigations on naturally occurring Trypanosoma evansi infections in horses, cattle, dogs and capybaras (Hydrochaeris hydrochaeris) in Pantanal de Poconé (Mato Grosso, Brazil). The prevalence of Mal de Cadeiras--Portuguese for Trypanosoma (T.) evansi infections in horses--as well as the prevalence of T.evansi infections in cattle, dogs and free-ranging capybaras (Hydrochaeris hydrochaeris) was investigated in Pantanal de Poconé (Mato Grosso, Brazil). In 0.3, 8.6 and 8.0% of the horses, dogs and capybaras, respectively, infection was detected using standard parasitological methods. A seroprevalence of 4.1, 2.3, 7.1 and 22.0% was found in horses, cattle, dogs and capybaras, respectively, using an enzyme-linked immunosorbent assay for the detection of T.evansi antigen ...
Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms. A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately...
Hosts of Lutzomyia shannoni (Diptera: Psychodidae) in relation to vesicular stomatitis virus on Ossabaw Island, Georgia, U.S.A. Hosts of Lutzomyia shannoni Dyar, a suspected biological vector of the New Jersey serotype of vesicular stomatitis (VSNJ) virus, were determined using an indirect enzyme-linked immunosorbent assay (ELISA) of 333 blood-fed female sandflies collected from their diurnal resting shelters on Ossabaw Island, Georgia, U.S.A. Sandflies had fed primarily on white-tailed deer (Odocoileus virginianus) (81%) and to a lesser extent on feral swine (Sus scrofa) (16%), two species of host infected annually with VSNJ. Other hosts were raccoons (Procyon lotor) and horses (Equus caballus) or donkeys (E. asinus),...
Rapid diagnosis of equine influenza by the Directigen FLU-A enzyme immunoassay. The Directigen FLU-A enzyme immunoassay was tested for its ability to detect equine-2 influenza viruses in nasopharyngeal fluids from horses and ponies. A total of 125 swabs from experimental infections and from different sources of natural infection in the USA and Hong Kong were examined. The assay results were compared with the results of standard virus culture in embryonated chicken eggs or Madin-Darby canine kidney cells, and with the serology of the horses sampled. In comparison with virus culture the enzyme immunoassay exhibited 83 per cent sensitivity, 78 per cent specificity, 70 per ce...