Assays for cellular antigens in the presence of detergents

The monoclonal antibody (F 10-89-4) described in this study recognizes an antigen which by quantitative absorption analysis is absent from human brain, kidney, liver, heart, erythrocytes, platelets and normal serum, but is present on spleen, lymph node, chronic lymphatic leukemia cells, bone marrow, thymus and granulocytes at a ratio of 1:1:0.8:0.3:0.1, respectively. Analysis with the fluorescence-activated cell sorter showed that 100% of thymocytes, lymph node lymphocytes, blood mononuclear cells and granulocytes carry the antigen, while 83% of bone marrow cells are positive. There was marked heterogeneity in the amount of labeling of thymocytes, with 3 major peaks. There was also heterogeneity of labeling of blood mononuclear cells and lymph node lymphocytes, with a weakly staining hump containing approximately 20% of the cells in the case of lymph node lymphocytes. Double labeling experiments demonstrated that the weakly staining cells of blood and lymph node were B lymphocytes, while frozen sections of thymus showed that the antigen was expressed most weakly in subcapsular cortical thymocytes, and most strongly on medullary thymocytes. Biochemical studies established that the antigen bound to lentil lectin columns, and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies using NaB3H4-labeled blood mononuclear cells established that the antigen was a major glycoprotein of lymphocytes, and that its molecular weight was in the region of 190000 to 215000.

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“…The targets in the binding assays were human spleen cells fixed for 5 min in 0.25% glutaraldehyde and stored at -40°C in 5% BSAiPBS [13].…”

Section: Cell Suspensionsmentioning

confidence: 99%

Monoclonal antibody to a human leukocyte‐specific membrane glycoprotein probably homologous to the leukocyte‐common (L‐C) antigen of the rat

1980

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“…In this way the relative amounts of antigen can be measured in different tissue homogenates. These absorption or inhibition assays can also be extended to studying antigens solubilized in detergents if glutaraldehyde-fixed target cells are used to assay residual antibody (Williams, 1973;Letarte-Muirhead et al, 1974).…”

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confidence: 99%

The kinetics of antibody binding to membrane antigens in solution and at the cell surface

Mason¹,

Williams²

1980

Biochemical Journal

522

194

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The reaction kinetics of 251I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/13 and W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+ ), dissociation rate constants (k-) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-i antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-, for the univalent interaction, in which case all binding was bivalent.(ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 105-1.1 x 106M-1s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently.However, the k-, values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.

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“…Since these proteins are probably recognized by rabbit anti-mouse lymphocyte antibodies, it may be possible to prepare, by absorption, monospecific antisera directed against the marker proteins. We could then use the double antibody assays described by Williams (26)(27)(28) …”

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confidence: 99%

Differences in the surface proteins of mouse B and T cells.

Trowbridge¹,

Ralph²,

Bevan³

1975

Proc. Natl. Acad. Sci. U.S.A.

122

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We have selectively labeled the surface of mouse thymus-dependent (T) and thymus-independent (B) lymphocytes by means of lactoperoxidase-catalyzed iodination. Examination of the labeled proteins by electrophoresis in the presence of sodium dodecylsulfate followed by autoradiography revealed differences in the cell surface proteins of B and T cells. Proteins from labeled thymocytes, T cell lymphomas, and both normal and activated peripheral T cells give a broad band of radioactivity corresponding to at least two protein components with apparent molecular weights of 170,000 to 190,000. This band is absent from autoradiographs of iodinated B cell proteins, which instead have another band corresponding to a protein with an apparent molecular weight of about 220,000. We have shown that these T and B cell marker proteins are synthesized by the cell and are not serum components selectively bound to the cell surface. We have also established that these proteins are the major iodinated species precipitated from Nonidet P-40 cell lysates by rabbit antimouse lymphocyte serum, suggesting that they are the major antigens recognized by rabbit antibodies to mouse lymphocytes.The physiological role of the immune system is to discriminate between self and nonself. In mammals and birds this task is shared by two classes of lymphocytes: T cells, which differentiate in the thymus, and B cells, which are generated independently of the thymus. After Serologically detectable cell surface differences between T and B cells have been exploited to identify each class of lymphocyte and to study their physiological roles. Antigen recognition, the characteristic migratory properties of both B and T cells, and the effector reactions of T-cell-mediated immunity are all believed to be associated with the lymphocyte plasma membrane. As a consequence, further detailed studies of the lymphocyte cell surface are warranted not only to provide additional means of discriminating between T and B cells, but as an approach directed towards understanding their different immunological roles.We have selectively labeled the cell surface of mouse T and B cells by lactoperoxidase-catalyzed iodination and examined the labeled proteins by gel electrophoresis in the presence of sodium dodecylsulfate. The resolution of our analysis has been enhanced by using a high pH discontinuous buffer system, polyacrylamide gel gradients, and by identifying the iodinated proteins by autoradiography. We Preparation of Activated T and B Cells. Concanavalin A (Con A) twice crystallized was from Miles-Yeda, Ltd., and used without further purification. Lipopolysaccharide (LPS) from Salmonella typhosa 0901 (Difco) was boiled 60 min at 1 mg/ml in 0.1 M sodium phosphate buffer pH 8.0 before use (1). Pea lectin was purified by affinity chromatography (4).Spleen cell suspensions from B6D2F1 mice (2-to 6-monthsold) were prepared by teasing spleens in balanced salt solution with forceps. In most experiments red cells were removed by selective lysis with Tris-buffered ammonium...

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Assays for cellular antigens in the presence of detergents

Cited by 58 publications

References 12 publications

Monoclonal antibody to a human leukocyte‐specific membrane glycoprotein probably homologous to the leukocyte‐common (L‐C) antigen of the rat

Monoclonal antibody to a human leukocyte‐specific membrane glycoprotein probably homologous to the leukocyte‐common (L‐C) antigen of the rat

The kinetics of antibody binding to membrane antigens in solution and at the cell surface

Differences in the surface proteins of mouse B and T cells.

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