Colloidal carbon particles as a new label for rapid immunochemical test methods: quantitative computer image analysis of results.

Colloidal carbon particles can serve as label in sol particle immunoassays. The universal applicability of these particles in qualitative and (semi)quantitative immunoassays has been demonstrated. Sol particle and/or dipstick immunoassays, not yet optimized in terms of sensitivity, are discussed. The colloidal label has been used successfully in a mouse immunoglobulin isotyping kit. Human serum albumin spotted onto nitrocellulose in a concentration range of 7.8 to 1000 ng could be detected using anti-albumin antibody absorbed onto colloidal carbon particles. It was also possible to perform a competitive assay with this conjugate for a concentration range of free human serum albumin varying from 0.25 to 6.75 micrograms. The Kunitz-type trypsin inhibitor from soybean was determined by a colloidal carbon based immunoassay in a range of 2.5 to 160 ng. In this assay, free and colloidal carbon-bound inhibitor competed for binding specific antibodies spotted onto a nitrocellulose membrane. An image- and data-processing procedure has been developed that enables a rapid and simple quantification of colloidal carbon sol particle immunoassays. The average grey level of a spot is taken as a measure for quantitative purposes. This so-called Sol-particle Image Processed ImmunoAssay (SIPIA) procedure is equally well applicable to assays using other colloidal particles.

Activation of LFA-1 through a Ca2(+)-dependent epitope stimulates lymphocyte adhesion.

The leukocyte function-associated molecule-1 (LFA-1) plays a key role in cell adhesion processes between cells of the immune system. We investigated the mechanism that may regulate LFA-1-ligand interactions, which result in cell-cell adhesion. To this end we employed an intriguing anti-LFA-1 alpha mAb (NKI-L16), capable of inducing rather than inhibiting cell adhesion. Aggregation induced by NKI-L16 or Fab fragments thereof is not the result of signals transmitted through LFA-1. The antibody was found to recognize a unique Ca2(+)-dependent activation epitope of LFA-1, which is essentially absent on resting lymphocytes, but becomes induced upon in vitro culture. Expression of this epitope correlates well with the capacity of cells to rapidly aggregate upon stimulation by PMA or through the TCR/CD3 complex, indicating that expression of the NKI-L16 epitope is essential for LFA-1 to mediate adhesion. However, expression of the NKI-L16 epitope in itself is not sufficient for cell binding since cloned T lymphocytes express the NKI-L16 epitope constitutively at high levels, but do not aggregate spontaneously. Based on these observations we propose the existence of three distinct forms of LFA-1: (a) an inactive form, which does not, or only partially exposes the NKI-L16 epitope, found on resting cells; (b) an intermediate, NKI-L16+ form, expressed by mature or previously activated cells; and (c) an active (NKI-L16+) form of LFA-1, capable of high affinity ligand binding, obtained after specific triggering of a lymphocyte through the TCR/CD3 complex, by PMA, or by binding of NKI-L16 antibodies.

HLA-DR expression on human peritoneal macrophages in vivo and in vitro.

Qualitative, semi-quantitative (immuno-electronmicroscopy), and quantitative (radioimmunoassay) measurements were made of the in vivo and in vitro expression of HLA-DR on continuous ambulatory peritoneal dialysis (CAPD) patients' peritoneal macrophages (M phi) and on healthy persons' blood monocytes (MO). In vivo, great variation is seen in both the qualitative and (semi-) quantitative expression of HLA-DR in peritoneal M phi. After culturing for 5 to 20 h, CAPD patients' M phi with low to intermediate numbers of HLA-DR molecules per cell (25-80 x 10(3] showed a two- to threefold enhancement of HLA-DR expression. This enhancement was determined for the total peritoneal cell (PC) population and for the adherent subpopulation of peritoneal M phi and blood MO. CAPD patients whose cells initially had high numbers of HLA-DR molecules (80-110 x 10(3] showed no or only slight enhancement of HLA-DR expression when cultured.

Identification of a widely distributed 90-kDa glycoprotein that is homologous to the Hermes-1 human lymphocyte homing receptor.

Homing of recirculating lymphocytes from the blood into the lymphoid tissues is mediated by 90-kDa homing receptors on the lymphocyte cell surface, allowing selective binding to specialized endothelium lining high endothelial venules. This study describes two novel mAb, NKI-P1 and NKI-P2, directed against functional epitopes of a human lymphocyte homing receptor, gp90. Biochemical studies demonstrated that these antibodies recognize a 90-kDa glycoprotein which is similar to the Ag recognized by the mAb Hermes-1. This notion was confirmed by immunohistochemical studies showing identical reaction patterns. Furthermore, it was observed that NKI-P1 and NKI-P2 blocked adhesion of lymphocytes to high endothelial venules. Immunohistochemical, immunofluorescence, and immunoprecipitation studies revealed that gp90 is widely expressed on hemopoietic cells including lymphocytes, macrophages/dendritic cells, myeloid cells, and erythrocytes. The gp90 is also expressed on a number of nonhemopoietic cells such as endothelial cells, certain epithelial cells, and fibroblasts. In addition to its expression on normal cells, gp90 is present on a spectrum of tumor cell lines of lymphoid, monocytic, epithelial, glial, and melanocytic origin. In addition to the 90-kDa product, the antibodies immunoprecipitate several polypeptides in the range of 120 to 200 kDa. Interestingly, it was observed that certain mamma tumor cell-line cells lack the 90-kDa polypeptide indicating the heterogeneous expression of the molecules recognized by the antibodies. These results indicate that the 90-kDa glycoprotein homologues of the Hermes-1 human lymphocyte homing receptor are expressed on hemopoietic tissues as well as on a number of nonhemopoietic tissues and tumor cell lines. Although the function of these molecules in nonlymphoid cells is presently unknown, they might play a role in cell-cell or cell-matrix adhesion.

Evidence that leukocyte function-associated antigen-1 is involved in recirculation and homing of human lymphocytes via high endothelial venules.

We report the results of mAb inhibition studies of human lymphocyte-high endothelial venule interaction in vitro. These studies in which T cells from both normal donors and from a LFA-1-deficient patient were used indicate that in addition to a system of organ-specific 90-kDa "homing" receptors on lymphocytes, LFA-1 is also involved in lymphocyte recirculation and homing.

A monoclonal antibody (NKI-L16) directed against a unique epitope on the alpha-chain of human leukocyte function-associated antigen 1 induces homotypic cell-cell interactions.

In the present study a unique antibody (NKI-L16) reacting with the alpha-chain of the human leukocyte function-associated Ag-1 (LFA-1) is described, which stimulates homotypic cell-cell interactions in a manner very similar to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), in contrast to other anti-LFA-1 mAb which inhibit cell aggregation. The induction of aggregate formation of EBV-transformed B cells (JY) and CTL clones by TPA or NKI-L16 is not accompanied by an increase in the expression of LFA-1. Nevertheless, this cluster formation is LFA-1 dependent, inasmuch as anti-LFA-1 antibodies, other than NKI-L16, completely abrogate aggregation. Simultaneous addition of NKI-L16 and TPA did not result in a further increase of the speed of cluster formation, suggesting that a similar pathway is activated. Immunoprecipitation and enzyme digestion studies revealed that NKI-L16 recognizes a unique epitope on the alpha-chain of LFA-1, most likely situated close to the transmembrane segment of the molecule. It is hypothesized that NKI-L16 or TPA can cause the LFA-1 molecule to convert from an inactive to an active configuration, thereby permitting binding of LFA-1 to its natural ligand.

Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of human monocytes.

The leukocyte function-associated antigen-1 (LFA-1), the C3bi receptor (CR3) and the p150,95 antigen belong to a family of leukocyte surface molecules consisting of bimolecular complexes with alpha chains of 170 kDa, 165 kDa and 150 kDa, respectively, and a common beta subunit with a mol. mass of 95 kDa. In order to determine the function of the p150,95 antigen on human monocytes and U937 cells, and to study the functional relationship between this antigen and LFA-1 or CR3, we investigated the influence of monoclonal antibodies (mAb) directed against these cell surface molecules on the adhesive properties of these cells. The observation that anti-beta chain mAb strongly inhibited migration, chemotaxis, adhesion and phagocytosis of monocytic cells indicates a major role for LFA-1 family antigens in monocyte functions. Detailed analysis with a panel of anti-alpha chain antibodies demonstrated that both p150,95 and LFA-1 mediate random migration whereas in contrast, p150,95 and CR3 were shown to be involved in the directed migration of monocytes to f-Met-Leu-Phe. Furthermore, adhesion of monocytes to plastic surfaces or monolayers of endothelial cells as well as phagocytosis of latex particles was mediated by p150,95. The results demonstrate that, in spite of its relative low expression, the p150,95 glycoprotein is a major adhesion-associated molecule expressed by human monocytic cells.

Differential function of LFA-1 family molecules (CD11 and CD18) in adhesion of human monocytes to melanoma and endothelial cells.

Human peripheral blood monocytes from normal, healthy donors express the leucocyte function-associated antigen (LFA)-1, CR3 and p150,95. These heterodimeric antigens are members of a glycoprotein family sharing a common beta subunit but endowed with distinct alpha chains. They have been shown to play an important role in cell-cell interactions. In the present study we have investigated the role of these molecules in the interaction of monocytes with endothelial cells and melanoma (tumour) cells. Heterotypic cell-cell interactions were studied in single cell conjugate assays and by adhesion of monocytes to monolayers of cells. The results demonstrate that monoclonal antibodies directed against LFA-1 alpha, CR3 alpha, p150,95 alpha and the common beta chain strongly reduce the number of conjugates (71, 50, 60 and 89% inhibition, respectively), formed between monocytes and melanoma or endothelial cells in a single cell assay. In contrast, adhesion of monocytes to monolayers of the same cells seems only to depend on p150,95, since only antibodies directed to the alpha chain of this molecule and to the common beta chain inhibited adhesion. Interestingly, the number of conjugates formed with melanoma cells in single cell assays was at least twice the number of conjugates formed between monocytes and endothelial cells, whereas no differences were observed in the adhesion of monocytes to monolayers of these cells. However, the basis for this phenomenon is not yet clear. These results indicate that not only LFA-1 but also CR3 and p150,95 can mediate adhesion to target cells in suspension, but that monocyte adhesion to monolayers is caused by a different mechanism in which the p150,95 molecule seems to play a prominent role.

Membrane glycoprotein p150,95 of human cytotoxic T cell clone is involved in conjugate formation with target cells.

The p150,95 heterodimer, one of three members of the leukocyte function associated antigen (LFA) family, is expressed by monocytes, granulocytes, NK cells, and a small percentage of lymphocytes. We now report that the p150,95 glycoprotein is expressed by some cytotoxic T cell clones and that it is involved in cell-mediated cytolysis by these clones. Two CTL clones, clone JS-93 (CD3+ CD4+ CD8-) and clone JS-102 (CD3+ CD4- CD8+) expressed high levels of p150,95 and were shown to be specifically directed against HLA-DR and HLA-A2, respectively. Immunoprecipitations followed by two-dimensional gel electrophoresis demonstrated no heterogeneity in the p150,95 molecule isolated from both clones. Furthermore, we demonstrated that monoclonal antibodies (moab) directed against p150,95 could inhibit the cytotoxic activity of both clone JS-93 and clone JS-102 (50% and 47%, respectively). Single cell assays revealed the inhibition to occur at the level of conjugate formation rather than at the level of the lethal hit. Similar results were obtained with moab directed against LFA-1 (p170,95). The capacity of the moab directed against LFA-1 and p150,95 to inhibit CTL activity and conjugate formation were additive, resulting in a similar percentage of inhibition as found with moab directed against the common beta-chain of these molecules. It is concluded that at least some CTL clones express the p150,95 antigen at their cell surface, and that this molecule, like LFA-1, acts at the level of conjugate formation between effector and target cells.

Sensitive and quantitative determination of monocyte adherence.

A new sensitive and highly reproducible one-step ELISA is described to quantitatively determine the adherent capacity of monocytes and related cell lines. Cells were labelled with a monoclonal antibody/peroxidase conjugate which did not affect the adhesive properties of these cells. The labelled cells were allowed to adhere for 1 h and subsequently stained by the addition of substrate. The results demonstrate that there is a good correlation between the number of peroxidase-labelled adherent cells and the absorbance measured at 450 nm. Furthermore the assay permits the use of very low cell numbers since adherent cells could be measured efficiently at a level of only 100-500 cells/well. The method may be very useful in the selection of hybridomas that secrete antibodies which inhibit adherence of cells. In addition it can be applied to study the adhesive properties of any cell type, provided that appropriate monoclonal antibodies are available.

Characterization of melanoma-associated surface antigens involved in the adhesion and motility of human melanoma cells.

The functional properties of the melanoma-associated antigens detected by monoclonal antibodies (MAbs) AMF-6 and AMF-7 were investigated. These MAbs were selected previously because of their capacity to block the anti-melanoma reactivity of cytotoxic T-lymphocyte clones AMF-6 and AMF-7 detect a melanoma-associated proteoglycan (MW greater than 450-250 kDa) and a molecular complex, which under reducing conditions consists of 4 compounds of 120, 95, 29 and 25 kDa respectively. AMF-6 reacted strongly with all 30 cultured melanomas and all 41 melanomas in frozen tissue sections. Significant cross-reactivity was only observed with nevi and perineurium, whereas normal skin melanocytes were negative. AMF-7 reacted with all 25 cultured melanomas and all 34 melanomas in frozen sections. AMF-7 cross-reacted with a proportion of nevi and endothelial cells from small vessels. The antigen detected by AMF-6 and AMF-7 could not be modulated by retinoic acid or recombinant gamma-IFN, which induced or enhanced the expression of HLA-DR, HLA-DQ and Class-I MHC antigens. In addition, the antigens were not readily modulated when cells were incubated in excess amounts of AMF-6 and AMF-7. Interestingly, the antigen detected by AMF-7 was strongly associated with the adhesion and cytoplasmic spreading of melanoma cells to plastic surfaces and monolayers of vascular endothelial cells. AMF-6 did not block the adhesion of melanoma cells but delayed cytoplasmic spreading. Both AMF-6 and AMF-7 blocked fibronectin-induced chemotaxic motility and chemokinesis of melanoma cells. In addition to their membrane localization, the antigens detected by AMF-6 and AMF-7 were also abundant in extracellular adhesion plaques deposited by cultured melanoma cells. Our results indicate that the high-MW melanoma-associated proteoglycan and the antigen detected by AMF-7 are associated with adhesion and/or cytoplasmic spreading and motility of human melanoma cells, suggesting that these antigens are associated with the (hematogenic) dissemination of human melanoma. This is supported by the finding that AMF-7 stained primary tumors heterogeneously, whereas metastases were homogeneously stained.

Biochemical and functional characteristics of the human leukocyte membrane antigen family LFA-1, Mo-1 and p150,95.

The human leukocyte function-associated (LFA-1) antigen, the monocyte differentiation antigen Mo-1 which is characterized as the C3bi receptor and the glycoprotein p150,95 are characterized biochemically. Immunoprecipitations carried out with 6 different monoclonal antibodies (mAb) against LFA-1 indicated that four mAb (SPV-L1, SPV-L5, SPV-L7 and SPV-L11) were directed against the alpha chain, whereas mAb CLB54 and MHM-23 were found to react with the common beta chain of LFA-1, Mo-1 and p150,95. LFA-1 and Mo-1 expressed on KG-1 cells or lymphocytes, monocytes and granulocytes from one donor were homogeneous. Interestingly the alpha chain of p150,95 showed heterogeneity. The molecular weight of the alpha chain expressed on monocytes was consistently higher than that of the alpha chain on granulocytes. The beta subunits of LFA-1 and Mo-1 (as detected by mAb Bear-1) are not only similar in molecular weight and isoelectric focusing patterns, but it is demonstrated here that they are also identically glycosylated and have similar protein backbones as judged by tryptic peptide mapping. In spite of their structural similarities. LFA-1 and Mo-1 differ completely in some of their biological functions. Anti-LFA-1 mAb strongly inhibited monocyte-dependent T cell proliferation induced by tetanus toxoid or Helix pomatia hemocyanin and pokeweed mitogen-driven specific antibody production in vitro, whereas the anti-Mo-1 antibody Bear-1 was ineffective. These results suggest that the differences in these biological functions of LFA-1 and Mo-1 may be related to their different alpha subunits, which may recognize specific counter structures.