| Myeloperoxidase (MPO) is a major component of azurophil granules in polymorphonuclear leukocytes (PMN) and plays an important role in phagolysosomal killing functions (Klebanoff, 1991 ). Like human (Agner, 1941; Andrews and Krinsky, 1981; Pember et al., 1983; Nauseef et al., 1988 ) and canine MPO(Agner, 1958; Harrison et al., 1977), mature bovine MPO is also a haem-con-taining glycoprotein composed of heavy and light subunits (Cooray et al., 1993 ). although there were small differences in the EPR g values of high spin derivatives (Ikeda-Salto and Prince,1985; Ikeda-Saito, 1985). Identified three forms of MPO (I, II and III ) in bovine peripheral blood neu-trophils from a single donor using CM-Sepharose fast flow column chromatography. Although the amino acid compositions of the three forms of bovine MPO were rather similar to one another, there were indications of charge heterogeneity, probably arising from variations in the carbohydrate content. Resting neutrophils do not release MPO, whereas neutrophils allowed to ingest bacteria can release large quantities of MPO. At sites of infection MPO is released extracellularly or into phagocytic vacuoles. Thus, the level of MPO at an infection site should serve as a suitable (noncellular) marker of the neutrophil activity stimulated by microorganisms. Thus, MPO measurements will provide insight into the state of neutrophil activation ,which will be valuable in studying the pathophysiology of infectious diseases in the bovine species.The results clearly show that the quantitative analysis of the amount of MPO in mastitic milk can be used to detect intramammary infections in dairy cattle.Monoclonal antibodies against bovine MPO should therefore be useful reagents for assessing the involvement of MPO in inflammatory reactions in cattle.PMN were isolated from venous bovine blood of healthy animals. MPO was purified by the Sephadex G-75 gel filtration and ConA-Sephroseaffinity chromatograph.The extracted protein was identified as MPO based on its absorption spectrum , peroxidase activity and polypeptide structure. Bovine neutrophils contained more different forms of MPO. When subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis each of the purified form showed three distinct bands corresponding to bovine MPO Intermediates of approximately 89,80, and 74kDa ,84-80, 59, 45, 30, and 15 kDa were found, approximately 92000, 52000 and 31000 molecular radius. Gel lamellar scanning method result the MPO purity is 89%. Absorption Spectra of MPO result that there are two character peaks(λ280 andλ430) inλ200～560nm light scanning. MPO activity reaction system; 6.7%DMF,1.34mM TMB,0.22mM H2O2,66.9mM NaAc (pH5.6,37℃). detect average activity is 0.052 OD/min by UV-260spectrophotometer withλ655.Mice were immunized with extracted MPO for 4 times. Anti-MPO monoclonal antibodies were produced by conventional cell fusion technique. The spleen cells of immunized Blab/c mice were fused with SP2/0 myeloma cells using PEG, and selected with HAT and HT medium. Tow hybridoma cell lines of C1 and D11 were obtained using extracted MPO by indirect ELISA, and cloned by limited dilution method for three times, whose titers of cultivate medium and ascites were 1:600, 1:1000 and 1:1×104, 1:4×104respectively. The subtype of them were both IgG1.SDS-PAGE showed that C1 and D11 all had two strips in about 50 000 and 25 000 respectively.Analysized by prohibitive ELISA , Anti-MPO monoclonal antibodies showed good sensitivity. Both of them have not across response with other bovine proteins.In the present paper describe the preparation of anti-MPO mouse monoclonal antibodies (AM Mo abs) by immunization with the purified bovine MPO. The specificity of the AM Mo abs was demonstrated with the help of sensitive biochemical techniques. Our data suggest that these AM Mo abs could be used tomonitor neutrophil activity at sites of infection and to monitor the involvement of MPO in the primary defense mechanisms in bovine species. |
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