Production and characterization of monoclonal antibodies specific to haemocytes of the black tiger prawn Penaeus monodon

Abstract

Monoclonal antibodies (MAbs) specific to haemocytes of black tiger prawn were generated from mouse immunized with a mixture of SDS-treated and formalin-fixed haemocytes. Hybridoma clones were selected by immunohistochemistry against fixed haemocytes, heart, lymphoid organ, haemopoietic tissue, and Western blot against haemocyte extract and haemolymph. Two parts of MAbs were produced ; MAbs specific to haemocyte and MAbs specific to other tissues. MAbs specific to haemocytes were obtained and could be divided into 6 groups according to their binding capacities to various haemocyte proteins in Western blot analyses and their differences in recognition of haemocyte sub-populations. The first group of antibodies consists of 4 MAbs (HC47d, 126d, 178d and 425d) reacted with 102 kDa haemocyte protein and strongly recognized a small subset of semi-granulocytes (SG) and hyalinocytes (H) but occasionally stained lightly a very small population of granulocytes (G). The antibodies also bound to a group of cells in haemopoietic tissue as well as cells located at the end of the tubules in the lymphoid organ but not in the spheroid. The second group of antibodies consists of 3 MAbs (HC54, 58d and 249d) strongly bound to 43 kDa and recognized a large sub-population of G and SG with coarse granules but did not bind to most of the H. This group of antibodies also cross-reacted with cells in the outer layer of the tubules in the lymphoid organ. The third group of antibodies (HC111, 114, 245 and 327) reacted with ~20 kDa and recognized all G and only a small portion of SG. The fourth (HC201d), fifth (HC136, 200 and 239) and sixth groups (HC55) reacted with 61 kDa, 175 kDa and ~230 kDa, respectively. They bound to sub-populations of G, SG and H in similar proportions. None of the antibodies showed any cross-reactivity to other components in haemolymph. MAbs specific to other tissues could be divided into 3 groups. First, HC32 recognized cells of the tubules in lymphoid organand group of cell in haemopoietic tissue. It is also bound to small granule of SG and H. The second groups (HC39, 62) reacted with spheroid cell and tubule of lymphoid organ, cell in haemopoietic tissue, neuron, hepatopancreas and gill. They also recognized all G, SG and H. The third group (HC31, 179d) bound to 116 kDa haemocyte protein. The MAbs bound to spheroid cell and tubule of lymphoid organ and neuron cell. They also recognize all G, SG and H as well. The common antigens recognized by MAbs in the haemopoietic tissue and the lymphoid organ may reflect relationships among these organs in the development of the sub-populations of G and SG. Haemopoietic tissue may be the site for haemocyte production and the lymphoid organ may be the site for further differentiation of at least two different lines of haemocytes.