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We have characterized a panel of 6 monoclonal antibodies raised against human platelet talin by Western blotting, immune precipitation, and immunofluorescence, and shown that antibodies TA205 and TD77 disrupt actin stress fibers and focal adhesions, and inhibit cell motility when microinjected into human fibroblasts. Using a series of chick talin fusion proteins spanning the entire length of the molecule, we have mapped the epitopes recognized by these antibodies to the conserved N- and C-terminal regions of the protein. TA205 bound to an epitope contained within residues 139-433, a region which overlaps an F-actin binding site, and which shows homology with the ezrin/radixin/moesin family of cytoskeletal proteins. The epitope recognized by TD77 was located within the C-terminal region of the protein (residues 2269-2541) which also contains an F-actin binding site homologous to that in the yeast actin-binding protein SIa2p. To investigate the possibility that TD77 disrupts actin stress fibers by binding directly to the C-terminal actin binding site, additional talin fusion proteins were generated and analyzed for TD77 and actin binding. Fusion proteins containing residues 2269-2541, 2304-2541, and 2304-2463 all cosedimented with F-actin, whereas TD77 did not recognize the latter fusion protein. These results show that the C-terminal actin-binding site is distinct from the region recognized by the anti-functional antibody TD77, raising the possibility that it binds to a novel functionally important ligand-binding site in the talin molecule.

The rabbit CD11c molecule has been characterized by use of new monoclonal antibody, mAb 3/22. Expression of the p150,95 integrin (CD11c/CD18) has been shown by flow cytometry and immunohistochemistry to be restricted to monocytes, macrophages, dendritic cells and a small population of lymphocytes in peripheral blood. No expression on neutrophils could be demonstrated. Incubation of the newly derived CD8+ T cell line, BJ/873, with mAb 3/22 causes homotypic aggregation, which has been shown to be cell surface event that is not dependent on intracellular signaling or on receptor cross-linking. Inhibition studies show that the ligands responsible for this aggregation are CD11c/CD18 and ICAM-1, both of which are expressed on BJ/873. One other rabbit T cell line, K34, that also expresses p150,95 and ICAM-1, shows a similar aggregation response when stimulated with 3/22. Cell lines that express p150,95 but not ICAM-1 do not aggregate. These observations suggest that ICAM-1 is a ligand for activated p150,95.

The CD44 adhesion receptor is a hyaluronan-binding cell surface glycoprotein whose major isoform is a 95 kDa molecule with a wide tissue distribution. We have characterized two MAbs, W4/86 and RPN3/24, by immunochemistry, flow cytometry and fluorescence staining and demonstrated that they recognize the rabbit CD44 molecule. The results show that CD44 in the rabbit has a similar molecular size and cell distribution to human CD44. These antibodies have demonstrated for the first time, the presence of CD44 on synoviocytes. Preincubation of the rabbit synovial cell line HIG-82 with W4/86 inhibited its synthesis of PGE2 in response to autocrine synovial cytokines. These results indicate that the CD44 molecule may help to modulate synoviocyte metabolism.

Monoclonal antibodies raised to a number of microfilament-associated proteins were shown to recognize the appropriate proteins in extracts from human colon tissue. They were then used in an immunohistochemical study of normal colonic mucosa, adenomas, and adenocarcinomas. A strong reaction was seen in stromal cells within the tumours (both adenomas and adenocarcinomas) when frozen sections were stained with antibodies to filamin and caldesmon. In addition, a similar reaction was seen in the adenocarcinomas when stained with antibodies to talin and gelsolin. We believe that immunohistochemical staining with these antibodies reveals a tumour-induced process in the surrounding cells, possibly related to a host response to tumours.

The monoclonal antibodies, L13/64 and RCN1/21, raised against rabbit leucocytes, have been shown, by sequential immunoprecipitation and immunoblotting, to react with the rabbit CD18 molecule. They recognise not only surface-expressed CD18 but also an intracellular form which appears to be partially glycosylated. The expression of the CD11 and CD18 glycoproteins on a wide variety of rabbit leucocyte populations has been investigated by flow cytometry, using these two monoclonal antibodies (Mabs), together with others which recognise human CD11 and CD18 proteins but cross-react with rabbit tissues. The distribution of these leucocyte integrin molecules has been shown to be similar to that observed in humans and determination of the N-terminal sequence of rabbit CD11b shows strong homology with human and mouse sequences. Of four anti-rabbit CD18 Mabs tested, only one, L13/64, has been shown to be capable of inhibiting the adhesion of fMLP-stimulated neutrophils to gelatin coated plastic and the homotypic aggregation of PMA-stimulated T cells, both of which assays have been shown to be CD18-dependent. RCN1/21 causes aggregation of unstimulated neutrophils, but it is not known whether this is due to cellular activation or agglutination.

Monoclonal antibody PM6/248, which recognizes the GPIIb-IIIa complex on human platelets, causes platelet aggregation in platelet-rich plasma or in gel-filtered platelet suspensions. Aggregation follows a concentration-dependent lag phase and reaches a maximum at 8 micrograms/ml. High concentrations of antibody (less than 30 micrograms/ml) produce complete inhibition of the aggregation response. Aggregation is accompanied by serotonin secretion and thromboxane A2 synthesis, neither of which are inhibited by high concentrations of antibody, and by the mobilization of intracellular Ca2+. The F(ab')2 fragment of PM6/248 does not cause platelet activation and pre-incubation of platelets with this fragment inhibits all platelet responses stimulated by the whole antibody. Pre-incubation with the F(ab')2 fragment of the anti-Fc gamma RII Mab, IV. 3, also inhibits all responses to PM6/248. These data indicate that platelet activation stimulated by PM6/248 is caused by cross-linking of GPIIb-IIIa to the Fc gamma RII which stimulates signal transduction across the plasma membrane through a conformational change in the Fc gamma RII.

The structural characteristics of antigens recognized by a panel of monoclonal antibodies prepared against a rabbit T-lymphocyte cell line have been investigated. Those antigens which could be isolated using immunoadsorbents prepared from the monoclonal antibodies had mol. wts of 42,000, 90,000 and 120,000. The 42,000 mol. wt molecule is similar or identical to a rabbit class I major histocompatibility complex antigen and its characterization has been reported elsewhere. Three different 90,000 mol. wt proteins can be distinguished by their reactivity with lectins and by sequential immunoprecipitation. The 120,000 mol. wt protein is a very abundant surface glycoprotein that appears to be a specific marker for T-cells in the rabbit. It is the immunodominant antigen in a lentil lectin bound glycoprotein pool. Over half of the antibodies were directed against this antigen. All antigens detected by the panel of monoclonal antibodies have been detected on normal lymphoid cells.

To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.

A monoclonal antibody (mAb) termed ITI-Pl 1 has been prepared by the hybridoma procedure. Using immuno-absorption and crossed immunoelectrophoresis of Triton X-100 extracts of untreated and EDTA-treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP-stimulation and the presence of Ca2+-ions. It saturates at around 870 ng/10(8) cells corresponding to approximately 35,800 molecules/platelet. ITI-Pl 1 did not significantly inhibit GP IIb-IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI-Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno-electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I-labelled ITI-Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb-IIIa complex. The epitope on GP IIb detected by ITI-Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase-treated GP IIb-IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb-IIIa from untreated internal membranes isolated by continuous flow electrophoresis.