الفهرس 
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Abstract
In Egypt, The losses caused by EHVs infections in Equine studs during last years increased more than ever, especially with the increase in Arabian horses’ trade and traveling of Egyptian horses to participate in international championships all over the world. Economic losses had reached millions of Egyptian pounds, especially for valuable Arabian breeds, indeed the suspect was EHV-1; however the epidemic had not subsided with the strict vaccination regimen for it. This initiated the present design of a protocol for isolation of EHV from field specimens to stand on the real cause, then prepare a seed isolate to develop a homemade ELISA kit for EHV antigen detection that is not offered commercially to assure rapid, accurate alternative with cost efficiency. A total of 287 samples were collected including 200 serum samples, 12 Nasal and 18 uterine swabs from suspected cases and 57 tissue samples from aborted feti and their dams. Rapid screening was performed using PCR on 87 samples of tissues and swabs. Four samples were found Positive for the presence of EHV-4 specific N.A, Sequencing and Phylogenetic based analysis were done. Viral isolation was carried out from PCR positive samples on MDBK cell line, followed by identification methods; AGID, IFAT, VNT and PCR. Virus titration was done on the sixth passage which revealed a titer of 106.75. Steps for antigen preparation were made starting with concentration using PEG-6000 followed by dialysis as initial purification ended with differential centrifugation using high speed centrifugation for final purification, then whole cell antigen preparation was done using sonication. First isolate was used for hyper-immunization of rabbits and guinea pigs. The produced antisera were titrated by ELISA for the specific EHV antibodies which revealed a titer of 1\1600 for rabbit antisera and 1\800 for guinea pig antisera, then purified and concentrated using ammonium sulfate precipitation followed with dialysis, the total protein concentration was measured using nanoDROP spectrophotometer which revealed a concentration of 12.51mg\ml for rabbit antisera and 9.61mg\ml for guinea pig antisera. Steps of development of homemade ELISA kit for EHV antigen detection were proceeded using guinea pigs antibodies for coating and rabbit antibodies for detecting, checkerboard titration was done for coating and detecting antibodies also to standard samples for test optimization, which revealed that the most suitable concentration to carry out the test is 7-10µg\ml for both types of antibodies used and 8-10% (W\V) for samples used.