Classification Of Hemocytes And Preparation Of Monoclonal Antibody Against Hemocytes Of Phascolosoma Esculenta

Four methods were used to classify the hemocyte of Phascolosoma esculenta, which are density gradient centrifugation, stained by the Wright's and Observed by fluorescent microscope and electron microscope.The two type hemocytes can be separated by density gradient centrifugation, with the hyalinocytes and granulocytes mainly above and below the interface. Under fluorescent microscope, haemocytes were classified into hyalinocyte and granulocyte, according the auto- fluorescence of hemocytes.Smears were stained by the Wright's and then observed under the light microscope. Hyalinocytes were stained light purple. They do not contain characteristic granules. Among granulocytes two subtypes were further distinguished: the small granular cells was smaller than hyalinocytes and contain numerous granules in cytoplasm. The big granular cells are filled with very obvious large granules. The granules were stained purple.Observed at the ultrastructural level by electron microscope, hyalinocytes were agranular and showed roundish nucleus surrounded by relatively small cytoplasm. Organelles are sparse. Compared with hyalinocytes, granulocytes possess relatively more granules and organelles in their cytoplasm. Two types of granulocytes can be recognized. Small granule cells contain a large centrally located irregularly shaped nucleus, mitochondria, small vesicles, lysosomes, golgi bodies, cisternae of the endoplasmic reticulum, osmiophilic membrane bodies, and numerous granules of varying electron density. Big granule cells are characterized by large osmiophilic cytoplasmic inclusions.The Balb\C mice were immunized by haemocytes of Phascoloso- ma esulen.Spleens from the immunized mice were dissected and cells fused with an Sp2/0 myeloma cell line using polyethylene glycol as fusogen. Hybridomaculture medium were assayed by means of enzyme-linked immunosorbent assays (ELISA). A lot of positive hybridomas were found and 8 of them which secret high titer antibody were reserved at -80℃. Eight positive hybridomas were cloned ( DB3,DB5,DC1,DD1,EB3,ED1,FA4,FC3) and analyzed by ELISA and immunomagnetic beads techniques. They were positive to haemocyte in diferent interfaces of gradient density centrifugation detected by ELISA. Mab EB3 was positive to of granulocyte, Mab DD1,ED1 was positive to plasma of hyalinocyte. Using immunomagnetic beads techniques, Mab DD1 was positive to plasma of hyalinocytes, Mab EB3 to plasma of granulocyte. In conclusion, Mab EB3 seems to be a very promising MAb to identify and differentiate granulocytes, and the three MAbs will be used in further studies on cellular defence mechanism research.