Characterisation of monoclonal antibodies to haemocyte types of scallop (Chlamys farreri).
Four monoclonal antibodies (MAbs) (1E7, 1F12, 2H5, 2C6) against haemocytes of scallop (Chlamys farreri) were produced by immunising Balb/C mice. Analysed by the indirect immunofluorescence assay test (IIFAT), immunocytochemical assay, flow cytometry (FCM) and Western-blotting, they showed specificity for more than one haemocyte type (hyalinocyte and granulocyte) and various haemocyte components of scallop. Using IIFAT to detect monolayers separated from gradient density centrifugation, the four MAbs were positive with haemocytes at different interfaces. The percentage of positive cells (percent reactivity, PR) that MAb 1E7 reacted with at the 20-30%, 30-40% and 40-50% interfaces were 43.50%, 41.25% and 60.00% respectively, PR of MAb 1F12 were 31.00%, 63.50% and 41.00%, MAb 2C6 were 11.00%, 51.00%, 77.00%, and MAb 2H5 were 20.25%, 34.75%, 38.25%. For the immunocytochemical assay, MAb 1E7 1F12 and 2H5 was positive with the cytoplasm of both hyalinocyte and granulocyte, 2C6 was positive with the membrane and cytoplasm of hyalinocyte and granulocyte. Analysed by FCM, the PR of the four MAbs (1E7, 1F12, 2H5, 2C6) with haemocytes were 54.23%, 38.56%, 56.4%, and 79.7% respectively; moreover, the PR with different haemocyte types was variable. The results of Western-blotting showed that MAb 1E7 recognised an antigen of molecular weight 200 kDa, MAb 2C6 an antigen of 60 kDa, however, MAb 1F12 reacted with antigens of 70 kDa, 60 kDa and 45 kDa. There was no protein band that MAb 2H5 detected. In conclusion, 2C6 seems to be a very promising MAb to identify and differentiate granulocytes, and the four MAbs will be used in further studies on cellular defence mechanism research.
Jing
Xing
X
Laboratory of Pathology and Immunology of Aquatic Animals, LMMEC, Ocean University of China, Qingdao 266003, PR China.
Wenbin
Zhan
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006434:Hemocytes / Q000276:immunology*; D007150:Immunohistochemistry; D051379:Mice / Q000276:immunology*; D008807:Mice, Inbred BALB C; D008974:Mollusca
D000911:Antibodies, Monoclonal