B.B. Cohen

Subsets of Human D Locus Products Identified by a Series of Monoclonal Antibodies

Abstract A series of monoclonal antibodies, prepared against the Burkitts lymphoma line Daudi, define antigens predominantly expressed on cells of the B lymphoid series. Four antibodies which react with HLA-DR or DR-like antigens have enabled the serological definition of at least two and possibly three distinct subsets of molecules. Of these four antibodies, one (DA6.164) defines a DR allotype associated epitope common to all DR types tested except DR 7 homozygotes. A second (DA6.147) recognizes a DR-like antigen which is expressed in abundance on B lymphoblastoid cells, weakly on peripheral blood B cells and not at all on activated T cells. Two further antibodies (DA6.231 and DA6.121), with indistinguishable specificity, detect an epitope apparently common to more than one set of DR molecules.

The use of detergents in velocity sedimentation of cell culture IgM

The biosynthetically radiolabelled secreted proteins of human lymphoblastoid cell lines have been analysed by sucrose gradient velocity sedimentation. The addition of detergent to the gradient buffer dramatically improves the recovery of immunoglobulins from cell line supernatants, allowing the gradient fractions to be analysed further both serologically and biochemically. The results of these analyses show that IgM is synthesised in the 19S form and can be clearly separated from other components which include free light chains. Immunoglobulin appears to form a much larger portion of the proteins secreted by human lymphoblastoid lines than has formerly been recognised, a finding which may be of practical importance for the development and exploitation of human monoclonal antibodies.

Biochemical Analysis of Antigens Recognized by Workshop B Series Antibodies, Using “Western Blotting”

“Western blotting” is the name given to a technique whereby antigenic proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1), then transferred electrophoretically from the gel to a sheet of cellulose nitrate. The positions of the antigens, and hence their approximate molecular weights, can be determined by “probing” the sheet with the corresponding antibody which is either labeled itself (with a radioisotope, for example, or an enzyme) or which can be detected indirectly by a second (labeled) probe (2).

Methods of detecting mature human Ia-like antigen and their metabolic precursors

Ia antigens were shown to be present in the cell almost exclusively as mature alpha beta dimers which split into separate alpha and beta chains after boiling in SDS. In contrast metabolically labelling the cells with [35S]methionine resulted in only free alpha and beta chains being labelled. It is concluded that this widely used type of labelling, although useful for studying intermediate synthesis, should not be used for labelling mature cell surface molecules.

Monoclonal Anti-B Cell Antibodies: their Effects on Human Mixed Lymphocyte Reactions

Abstract Three monoclonal antibodies which recognise determinants on the surface of human B cells have been studied for their effect on mixed lymphocyte reactions (MLR). MLR inhibition was observed under a variety of conditions. While it was not possible to ascribe lymphocyte activation to a single determinant recognised by any of the antibodies, further analysis of the mechanisms involved in lymphocyte activation should be possible through the use of monoclonal reagents.

A reliable method for the analysis of human MHC class II α and β chains by western blotting of IEF gels

Abstract The solubility and ensuing transfer problems associated with IEF separation and blotting of membrane proteins have been eliminated by confining NP40 to the sample region of the gel and replacing it with octylglycoside in the separating gel. This permitted excellent separation of both the α and β chains of the human MHC antigens and their subsequent detection by Western blotting.

Analysis of Workshop “L” Series Antibodies: Radioimmunobinding and Biochemical Studies

Twenty-one of the Workshop “L” series antibodies (L5 was missing) were received in April, 1984. They were thawed and stored at 8°C throughout the study (April-August, 1984). Aliquots were diluted 1:20 for use in both serological and biochemical assays, the diluent being RPMI 1640 medium containing 5% fetal calf serum (FCS) and 5% horse serum (HS) plus 0.02% sodium azide. All the antibodies were tested for the ability to bind to a selected panel of cells comprising established human leukocyte lines and fresh tissue from cases of leukemia or lymphoma. Those which gave positive results were then examined by “Western Blotting” on solubilized membrane glycoproteins, derived from human leukemic cells, which had been run in polyacrylamide gel electrophoresis (PAGE) and electrotransferred to cellulose nitrate sheets.

TOWARD ANALYSIS OF THE HLA-DR SYSTEM WITH MONOCLONAL ANTIBODIES

Publisher Summary This chapter describes three monoclonal antibodies—DA6 231, 164, and 147— that precipitate glycoprotein dimers consisting of 34 K and 29 K subunits. 231 and 147 recognize DR-common epitopes, whereas 164 binds to all except DR7 homozygous cell lines. Protein transfer from sodium dodecyl sulfate (SDS) polyacrylamide gels to nitrocellulose paper, followed by probing with the monoclonal antibodies, has permitted assignment of the 231 and 147 epitopes to 29 K and 34 K subunits, respectively. Mutual binding inhibition between 231 and 164 is incomplete in one direction, suggesting that there is a major proportion of 231 + 164 + and a minority of 231 + 164 – 29K molecules expressed on all DR positive cells tested, except DR7 homozygous cells that have lost the 164 epitope. The serological expression of the 147 epitope at the cell surface is complex. The 231/147 ratio on peripheral and activated β lymphocytes is much higher than on β lymphoblastoid lines or on chronic lymphocytic leukemia cells. No significant binding of 147 can be detected on the strongly 231 + 164 + -activated cultured τ cells. Preliminary evidence suggests that molecules that can bind 147 are released on cell lysis even from 147 – activated τ cells.