In the mouse alloimmunization has revealed the Ly series of lymphocyte differentiation antigens Which have been used to show the existence of T-cell subpopulations with distinct surface phenotypes (1, 2). Furthermore, cytotoxic analysis using anti-Ly antisera has shown that T cells exhibiting different functions, e.g. helper activity or suppressor activity, can be distinguished on the basis of their surface markers (3-5). In other species such alloantigenic markers are not available, and their identification would be time-consuming and haphazard since the existence of serologically detectable polymorphisms is unpredictable. A method (6, 7) is now available for producing monoclonai antibodies to cell surface antigens using xenogeneic immunization in which most cell surface molecules should be antigenic. Mice are immunized with cells or subcellular fragments from another species, and their spleen cells are subsequently fused with a mouse myeloma cell line. Hybrid cells are selected for and screened for the production of antibodies to cell surface antigens. Monoclonal antibodies can then be obtained by cloning the antibody-secreting hybrids.The application of this system to iymphocytes was investigated using antibodies produced by hybrids formed with spleen cells from mice immunized with rat thymocyte membrane (7). One of these antibodies, W3/25, labeled 80% of rat thymocytes but only 52-58% of thoracic duct lymphocytes. This subset in thoracic duct lymphocytes (TDL) 1 was independent of the Ig + cells, but contained only 71-75% of the remainder. W3/25 antibody thus appeared to be labeling a T-lymphocyte subset, and this could be clearly identified using the fluorescenceactivated cell sorter (FACS II), thus allowing the separation of lymphoid cell populations into W3/25 positive and W3/25 negative fractions. The isolation of subsets of lymphocytes on the basis of antibody binding using the cell sorter rather than by complement-mediated cytolysis has the great advantage that both the labeled and unlabeled cell populations can be assayed for their immune functions.In the present study, rat TDL labeled by W3/25 antibody were separated from the unlabeled cells, and both populations were assayed for graft-versus-host activity and helper or suppressor functions. Materials and MethodsRats. Inbred strains PVG.I-a (10th backeross) and PVG/c (l-b) congenic for the Ig-1 light chain allotype were used with F1 hybrids between DA (l-a) and PVG/c. The strain PVG. l-a (8) was developed by Dr. S. V. Hunt (University of Oxford, England) who kindly provided the rats for these experiments. All rats were maintained at the MRC Cellular Immunology Unit, t Abbreviations used in this paper: BGG, bovine gamma globulin; DAB, phosphate-buffered saline containing 0.1 g calcium chloride and magnesium chloride per liter; DNP, 2A-dinitrophenol; FACS II, fluorescenceactivated cell sorter model If: FCS, fetal calf serum; FITC, fluorescein isothiocyanate; PBS, phosphatebuffered saline; PFC, plaque-forming cell; TDL, thoracic duct lymphocytes. 664J. ...

The development of the monoclonal antibody YC5/45 HLK (YC5/HLK) against a 5HT-bovine seroalbumin immunogen and its application for immunocytochemistry is described. The YC5/HLK antibody is the product of a rat x rat hybrid myeloma, producing a heavy chain and two light chains. In hemagglutination tests, the antibody cross-reacts to entirety with dopamine, serotonin, and tryptamine at high concentrations. The serotonin-albumin conjugate is 20,000 times more effective in displacing the binding antibody, while albumin itself goes unrecognized by the antibody. In fixed preparations of brain tissue, immunofluorescence is observed only in neurons known to contain serotonin, while no reaction is observed in dopamine-rich neurons. All immunofluorescence is extinguished by the use of agents that inhibit the biosynthesis of 5HT, but not of the catecholamines.

Monoclonal antibodies were produced following immunization of mice with either [Leu5)enkephalin-bovine serum albumin or [Met5jenkephalin-keyhold limpet hemocyanin conjugates. Two monoclonal antibodies coded NOC1 and NOC2, respectively, were derived. These monoclonal antibodies did not discriminate between Leu-and Metenkephalin in either radioimmunoassay or immunocytochemistry. NOC1 was characterized in detail. In radioimmunoassay NOC1 displayed about 40% crossreactivity with C-terminal extended Met-enkephalin hexapeptides and 7% with the extended heptapeptide (-Arg-Phe-OH), but did AGAINST ENKEPHALINS 957 28. Miller RJ, Chang K-J, Cooper B, Cuatnecasas P: Radioimmu-enkephalin: immunohistochemical mapping in the rat central nernoassay and characterization of enkephalins in rat tissue.

An anti-peroxidase-anti-biotin hybrid hybridoma rat cell line, capable of producing a bispecific monoclonal antibody, has been derived to explore its use in conjunction with a luminol immunodetection system. Luminescence was detected using x-ray film. The method was sufficiently sensitive and effective, but was less sensitive than autoradiographic methods using high-specific-activity 32P-labeled probes. Exposure times, on the other hand, were of the order of seconds rather than days. The direct binding of both peroxidase and biotin by the bispecific monoclonal antibody is simpler but less sensitive than the more conventional indirect method using a commercial peroxidase coupled with anti-rat antibody as a developing antibody.

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