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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.
Bupivacaine in the horse: relationship of local anaesthetic responses and urinary concentrations of 3-hydroxybupivacaine. Bupivacaine is a potent local anaesthetic used in equine medicine. It is also classified as a Class 2 foreign substance by the Association of Racing Commissioners International (ARCI). The identification of residues in postrace urine samples may cause regulators to impose significant penalties. Therefore, an analytical/pharmacological database was developed for this medication. The highest no-effect dose (HNED) for the local anaesthetic effect of bupivacaine was determined to be 0.25 mg by using an abaxial sesamoid local anaesthetic model. Administration of the HNED of bupivacaine to eight hor...
Analysis of myosin heavy chains at the protein level in horse skeletal muscle. Combined methodologies of enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting, traditional myofibrillar ATPase (mATPase) histochemistry and immunocytochemistry of whole biopsied samples were used to study myosin heavy chain (MHC) isoforms in the equine gluteus medius muscle. The ELISA technique allowed the quantification of the three MHC isoforms known to be present in different horse muscles: slow (MHC-I) and two fast (termed MHC-IIA and MCH-IIX). The SDS-PAGE method resolved MHCs in three bands: MHC-I, MHC-IIX and M...
Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA. The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the ...
Mepivacaine: its pharmacological effects and their relationship to analytical findings in the horse. Mepivacaine is a local anaesthetic drug that is widely used in equine medicine and is classified by the Association of Racing Commissioners International (ARCI) as a Class 2 foreign substance that may cause regulators to impose significant penalties if residues are identified in post-race urine samples. Therefore, an analytical/pharmacological database was developed for this agent and its metabolites. Using an abaxial sesamoid local anaesthetic model, it was determined that the highest no-effect dose (HNED) for its local anaesthetic effect was 2 mg. Using enzyme-linked immunosorbent assay (ELI...
Detection of equine antibodies to babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay. A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-termina...
Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats. To determine the infectivity and transmissibility of Hendra virus (HeV). Methods: A disease transmission study using fruit bats, horses and cats. Methods: Eight grey-headed fruit bats (Pteropus poliocephalus) were inoculated and housed in contact with three uninfected bats and two uninfected horses. In a second experiment, four horses were inoculated by subcutaneous injection and intranasal inoculation and housed in contact with three uninfected horses and six uninfected cats. In a third experiment, 12 cats were inoculated and housed in contact with three uninfected horses. Two surviving horse...
Detection of Trichinella infection in slaughter horses by ELISA and western blot analysis. In order to determine the presence of Trichinella infections in horses slaughtered at an abattoir in Mexico, 147 serum samples were examined by two immunoenzymatic methods. Specific antibodies were detected by ELISA in 7% of the serum samples at a dilution 1:400 and in 10% at lower dilutions (1:20, 1:40) using Trichinella spiralis muscle larvae (ML) excretory/secretory (E/S) products. Serum samples from four naturally infected horses (confirmed by direct methods) gave negative O.D. values in an ELISA at a 1:400 dilution and only two of them were positive at a 1:20 and 1:40 dilutions. Serum sam...
Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing G(L), M and N proteins expressed from recombinant baculoviruses. Indirect enzyme linked immunosorbant assays (ELISAs) utilizing the three major structural proteins (M, N, and G(L)) of equine arteritis virus (EAV) expressed from recombinant baculoviruses were developed. A large panel of sera collected from uninfected horses, and from animals experimentally and naturally infected with EAV or vaccinated with the modified live virus vaccine against equine viral arteritis, were used to characterize the humoral immune response of horses to the three major EAV structural proteins. The data suggest that the M protein was the major target of the equine antibody resp...
Lidocaine in the horse: its pharmacological effects and their relationship to analytical findings. Lidocaine is a local anaesthetic agent that is widely used in equine medicine. It is also an Association of Racing Commissioners International (ARCI) Class 2 foreign substance that may cause regulators to impose substantial penalties if residues are identified in post race urine samples. Therefore, an analytical/pharmacological database was developed for this drug. Using our abaxial sesamoid local anaesthetic model, the highest no-effect dose (HNED) for the local anaesthetic effect of lidocaine was determined to be 4 mg. Using enzyme-linked immunosorbent assay (ELISA) screening, administration...
Studies on pulmonary and systemic Aspergillus fumigatus-specific IgE and IgG antibodies in horses affected with chronic obstructive pulmonary disease (COPD). Inhalant exposure to Aspergillus fumigatus (Asp. f.) antigens induces marked inflammatory and immunological alterations in the lungs of horses affected with chronic obstructive pulmonary disease (COPD). In this study we investigated the role of specific allergen(s) present in Asp. f. on systemic and pulmonary IgE and IgG responses in control and COPD-affected horses, using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting techniques. Compared with controls, horses affected with COPD had significantly higher levels of BALF IgE and IgG to somatic Asp. f. antigens as well as to the ...
Evaluation of equine infectious anemia virus core proteins produced in a baculovirus expression system in agar gel immunodiffusion test and enzyme-linked immunosorbent assay. Equine infectious anemia virus (EIAV) core proteins (Gag and p26) obtained from a baculovirus expression system were used in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) antigens to test seventy-six horse sera. Those sera showed false-positive reaction in AGID test using Nisseiken antigen. However, none of them showed false-positive reaction with both of the expressed antigens. The 76 horse sera were also tested by ELISA. The sera gave a high background in ELISA using Nisseiken antigen. Gag and p26 reacted strongly against positive sera from horses immunized wi...
Enzyme-linked immunosorbent assay (ELISA) for detection of anti-Trypanosoma evansi equine antibodies. The standardization of ELISA for the detection of anti-Trypanosoma evansi antibodies in naturally and experimentally infected horses is described. Bayesian analysis was used to establish the cutoff between positive and negative sera. In order to determine the assessment of the ELISA test, the results obtained were compared with those from an IFA. A relative sensibility of 98.39%, a specificity of 95.12% and a predictive value of 96.83% were determined. The standardized technique was used to evaluate the antibody production against trypanosome in an experimentally infected equine, in which the ...
Seroprevalence of equine herpesvirus 1 in thoroughbred foals before and after weaning. To investigate the seroprevalence of equine herpesvirus 1 in foals around weaning and after weaning on two large Thoroughbred farms using a type-specific enzyme-linked immunosorbent assay to determine exposure to infection. Methods: A longitudinal population study in groups of Thoroughbred weanling foals. Methods: Two hundred weanling Thoroughbred foals from a population of about 380 foals were enrolled on two adjacent stud farms in the Hunter Valley of New South Wales. Foals on both farms were weaned from February to May 1995 into randomly selected groups of 10 to 15 foals. Farms were selecte...
Diagnosis and sero-epizootiology of equine herpesvirus type 1 and type 4 infections in Japan using a type-specific ELISA. Recently, a type-specific ELISA using equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) glycoprotein Gs (gGs) was developed by Crabb and Studdert [1993]. To investigate the dissemination of EHV-1 and -4 among horses in Japan, we applied their ELISA as suitable for discriminating between EHV-1 and -4 infections serologically. Type-specificity of the ELISA was confirmed by using paired sera of infected horses with either EHV-1 or -4. Application of the ELISA to sera collected before and after the winter season of 1995-1996 from 80 racehorses revealed that 30 horses showed significant antibody...
Enzyme-linked immunosorbent assay for serological survey of equine arteritis virus in racehorses. To examine antibodies against equine arteritis virus (EAV), an enzyme-linked immunosorbent assay (ELISA) using purified virus antigen was developed. The results of ELISA were compared with those of serum neutralization (SN) tests. The ELISA absorbance values and the SN titers in sera collected weekly from EAV-infected horses showed a similar pattern. The ELISA could detect antibody to EAV in horses experimentally infected with not only a homologous virus strain, which was used as the ELISA antigen, but also a heterologous strain. Using the ELISA, serum samples collected in 1996 from racehorses...
Validation of ELISA for the detection of African horse sickness virus antigens and antibodies. The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y ...
Establishment of a monoclonal antibody (1/14/16H9) for detection of equine keratan sulfate. To establish a sensitive and specific monoclonal antibody (MAB) against equine keratan sulfate (KS) and to develop an enzyme immunoassay for measurement of the concentration of KS in serum and synovial fluid from horses. Methods: 18 synovial fluid and 48 serum samples were obtained from clinically normal horses and horses with arthritis. Methods: BALB/c mice were immunized with chondroitinase-ABC-digested proteoglycan monomer from equine joint cartilage, and MAB were raised, using Sp2/O cells as a fusion partner. A competitive ELISA was optimized, using one of the established MAB, and KS conce...
Screening of horse polyclonal antibodies with a random peptide library displayed on phage: identification of ligands used as antigens in an ELISA test to detect the presence of antibodies to equine arteritis virus. A random hexapeptide fusion-phage library was screened to isolate phages that bind to antibodies present in horse sera positive for equine arteritis virus (EAV). Analysis of the peptide sequences displayed by isolated phages identified seven groups. 25% of the isolated phages used as antigens in an ELISA test were specifically recognised by a pool of sera which was positive for EAV in virus neutralisation test (VN). Five of these, when used as antigen in ELISA, detected greater than 50% of sera (n = 30) containing antibodies to EAV as detected by VN. When these five phages were pooled together...
Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom. The objective of this study was the search for a suitable venom antigen to be used in an in vitro alternative immunoassay, to the standard antivenom neutralization assay using mice. Bothrops jararaca venom was fractionated in DEAE-Sephacel columns and the fractions were tested for a correlation between antibody capture enzyme linked immunosorbent assay (ELISA) absorbance values and the 'in vivo' antivenom potency. Individual antivenoms from 14 horses and 15 separate FUNED polyspecific Bothrops ampouled antivenoms (final product) were used. Fractions showing the higher correlations were further...
Neutralizing potency of horse antibothropic antivenom. Correlation between in vivo and in vitro methods. The correlation coefficients between in vivo neutralization of lethal toxicity (ED50), neutralization of the hemolytic activity (PLA2) and levels of antibodies measured by ELISA, was investigated to test the potency of horse anti-bothropic antivenom. Twenty six horses were hyperimmunized with Bothrops venoms (B. alternatus, B. jararaca, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, for neutralization of PLA2 activity and for determination of ED50 in Swiss mice, the whole Bothrops jararaca venom (reference venom for assessing the bothropic antivenom potency in Brazi...
Progesterone determination in equine plasma using different immunoassays. Several assay systems (3H radioimmunoassay (RIA) with and without extraction; microplate enzyme-linked immunoassay (ELISA); qualitative ELISA (tube test)] were used to measure plasma progesterone concentration in mare plasma. The direct RIA showed a close correlation (R = 0.94) with the extraction RIA. The direct RIA and the microplate ELISA were compared in two different studies. In the first study 1155 samples of postpartum mares were used for progesterone determination with both assays. The ELISA resulted in more elevated values both in oestrus and dioestrus (0.19+/-0.3 and 2.44+/-3.62 nmol...
An immunohistochemical investigation of the adult stage of the equine parasite Strongylus vulgaris. Adult Strongylus vulgaris, collected from the caecum of infected horses and embedded in paraplast using standard methods, were sectioned for immunohistochemistry (IHC) studies. Antibodies were raised in rabbit against the excretory-secretory product (ESP) and against two constituent protein bands (28-30 kDa). The use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting indicated the immunogenicity of ESP and of the subunits (28-30 kDa). In ELISA, both rabbit hyperimmune sera recognized the ESP and (28-30 kDa) ban...
Monoclonal antibody against Babesia equi: characterization and potential application of antigen for serodiagnosis. Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did n...
Seroprevalence of Babesia equi among horses in Israel using competitive inhibition ELISA and IFA assays. Sera from 361 horses were tested by indirect immunofluorescence antibody test (IFA) and by competitive inhibition ELISA (cELISA), to detect antibodies to Babesia equi. The concordance between the assays was 95.7%. Application of a cutoff based on a calculated percent inhibition of 20% inhibition was used. Approximately one-third of all the horses tested were found serologically positive to B. equi, with more horses testing positive from northern Israel. Among horses raised with access to pasture there was a significant difference in the percentage of seropositive reactors (76.6% in the north ...
Monoclonal antibodies to subclass-specific antigenic determinants on equine immunoglobulin gamma chains and their characterization. This paper describes the production of a panel of monoclonal antibodies (mAbs) identifying the four recognised equine IgG subisotypes IgG, IgGa, IgGb, IgGc and IgG(T). Pure preparations of the subisotypes for use in immunisations and testing were produced using a combination of gel filtration, salt precipitation, ion exchange chromatography and protein A and Protein G affinity chromatography. The specificity of mAbs for the IgG subisotypes was confirmed using ELISA assays, by characterisation of affinity purified proteins recognised by the mAbs, and by Western blotting of equine serum proteins...
Inhibition by CaNa2EDTA of local tissue damage induced by Bothrops asper (terciopelo) venom: application in horse immunization for antivenom production. The ability of the chelating agent CaNa2EDTA to inhibit local tissue damage induced by Bothrops asper venom was studied in mice and in horses used for polyvalent (Crotalinae) antivenom production. CaNa2EDTA was devoid of toxicity when injected i.m. or s.c. inducing only a mild edema. Preincubation of B. asper venom with CaNa2EDTA inhibited hemorrhagic and dermonecrotic activities, but did not reduce edema-forming and myotoxic effects. A group of horses initially immunized with native venoms developed less severe local tissue reactions when injected with booster doses of venom and CaNa2EDTA tha...
Development and use of an enzyme-linked immunosorbent assay to monitor serum and urine acepromazine concentrations in thoroghbreds, and possible changes associated with exercise. To develop an ELISA that is sensitive and suitable for measurement of immunoreactive acepromazine (ACP) in horse serum and urine and to determine the acute effects of exercise on immunoreactive ACP values in Thoroughbreds. Methods: 12 healthy Thoroughbreds (5 mares, 5 geldings, 2 stallions), aged 2 to 8 years. Methods: A commercially available antibody and a horseradish peroxidase-conjugated oxime derivative of immunoreactive ACP were used to develop a one-step ELISA. Horses were used in a crossover design study to evaluate possible effects of treadmill exercise on serum and urine ACP concentr...
Epidemiology of Anoplocephala perfoliata infection in foals on a stud farm in south-western Sweden. The egg output and humoral antibody response to scolex antigens of the equine tapeworm Anoplocephala perfoliata were monitored in naturally infected foals by an egg flotation/centrifugation method and an indirect enzyme-linked immunosorbent assay (ELISA). The study was performed on a stud farm in south-western Sweden between May 1994 and April 1995. Sequential blood and faecal samples were taken from 21 foals during their first summer on pasture and until tapeworm eggs were detected. Results were expressed separately for 10 and 11 foals born before and after the end of April 1994, respectively...
Detection and determination of theobromine and caffeine in urine after administration of chocolate-coated peanuts to horses. The objective of this study was to determine the urinary excretion of methylxanthines in horses following ingestion of chocolate over eight days. The study was performed in response to gas chromatography-mass spectrometry (GC-MS) confirmation of the presence of caffeine in a positive urine test in a racehorse. The trainer of the horse alleged that he often administered chocolate-coated peanuts as treats to his horses, and he believed that the ingestion of chocolate was responsible for the positive urine test. The urinary excretion of theobromine and caffeine after the ingestion of chocolate-co...
Determination of butorphanol in horse race urine by immunoassay and gas chromatography-mass spectrometry. An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC-MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC-MS system. The ELISA test (20 microl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC-MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC-MS method in the SIM mode using nalbup...
