Leon Wofsy

Affinity targeting of membrane vesicles to cell surfaces

Abstract A general methodology has been developed for immunospecific attachment of sealed erythrocyte ghosts or liposomes to selected cell surface antigens. Antibody or avidin is coupled to a red cell or liposome membrane by one of several simple chemical procedures. Membrane vesicles bearing anti-hapten antibody or avidin form clusters exclusively around cells that have been labeled with first-layer hapten- or biotin-modified antibodies. Such targeting methods should be useful in studies aimed at facilitating vesicle-cell fusion and microinjection of selected molecules into cells. The new protein-membrane coupling procedures are also readily applied to the preparation of selectively modified red cells for use in plaque assays to detect cells secreting immunoglobulin or a specific anti-protein antibody.

Hapten-sandwich labeling. IV. Improved procedures and non-cross-reacting hapten reagents for double-labeling cell surface antigens

New procedures are presented for preparation of hapten-antibody conjugates with the bifunctional amidinating reagent methyl-p-hydroxybenzimidate (HB). Conjugates with improved solubility are effective for hapten-sandwich labeling of cell surface antigens with high sensitivity and specificity. Several non-cross-reacting hapten-antihapten antibody systems are described which are well-suited for simultaneous labeling of different surface antigens.

Specific purification of equine anti-SII antibodies by precipitation with A hemocyanin-glucuronide conjugate☆

Abstract Approximately 95 per cent of the antibodies in equine anti-SII serum can be precipitated specifically with an extensively coupled azo-conjugate of KL hemocyanin and ortho -diazoniumphenyl-β-glucuronide, thus showing an almost exclusive specificity for determinants in which glucoronic acid is the main component. The hemocyanin-glucuronide conjugate has been utilized in a simple procedure for the isolation and specific purification of anti-SII antibodies in high yield. A general method for specific purification of antihapten antibodies is proposed which takes advantage of the special properties of hemocyanin-azohapten conjugates.

Affinity labeling of anti-group a streptococcal polysaccharide antibodies☆

Abstract The active sites of rabbit antibodies to the Group A streptococcal polysaccharide have been affinity labeled with a reagent ( p -diazoniumphenyl- β -N-acetylglucosaminide) based on the terminal immunodominant monosaccharide of the Group A polysaccharide. Isoelectric focusing of radiolabeled antibodies showed that two of six antipolysaccharide antisera contained small amounts of restricted individual antibody components which affinity labeled with very high specificity. Most of the antibodies, including major restricted components which stand out from a heterogeneous background in isoelectric focusing, were affinity labeled with low specificit. The antipolysaccharide antibodies were purified by affinity chromatography on Sepharose conjugated with p -aminophenyl- β -N-acetylglucosaminide.

Fluorescence visualization of Ia antigens on T cells

Abstract Visualization with the fluorescence microscope of Ia antigens on T cells has previously not been successful. In this paper we have used fluorescence hapten-sandwich labeling to examine cells for the simultaneous display of Ia antigens and T-cell-specific antigens. In contrast to small unstimulated T cells which bear only small amounts of Ia antigens, we find that concanavalin A-stimulated T-cell blasts exhibit amounts that are clearly recognizable. The predominant specificity expressed is encoded by the I -A subregion and appears to be indistinguishable from that on B cells. Smaller subpopulations exhibit products coded by the I -C and I -J subregions. Separation of subpopulations of T cells on the basis of selective display of Ia antigens appears only to be feasible with stimulated cells.

Converting Sendai virus into a specific fusogen whose cell target can be selected

Covalent intermolecular hybrids of Fab anti-hemagglutinin-neuraminidase (HN) monoclonal antibody and avidin were prepared and characterized. These conjugates were used to block and redirect the fusion activity of Sendai virus (SV). After incubation of SV with Fab anti-HN: avidin conjugate on ice for 1-2 h, the SV fused only those P815 or BW5147 cells which were labeled with biotin-modified anti-cell surface immunoglobulin. The levels of cell-cell fusion obtained were at least as high as those achieved with unmodified SV and unlabeled P815 or BW5147 cells. These results demonstrate that it is possible to block the normal agglutinating activity of the HN molecules of SV and to introduce a new cell recognition feature without negating the fusogenic potential of the virus. Such an approach may be useful in harnessing the fusion activity of SV to a targeted delivery system for microinjection of macromolecules into selected cell populations.

PROBING LYMPHOCYTE MEMBRANE ORGANIZATION

We discuss here approaches to some problems of lymphocyte membrane organization. By randomly modifying lymphocyte surfaces with a hapten, we have been able to demonstrate that all exposed membrane protein can be capped without effecting an equivalent aggregation of intramembranous particles (IMP). A versatile and highly specific method is described for simultaneous labeling of multiple cell surface antigens for fluorescence and for transmission and scanning electron microscopy.

Sixteenth Midwinter Conference of Immunologists: Structure and specificity: Antibodies and receptors

Abstract The Sixteenth Midwinter Conference of Immunologists was held January 22–25, 1977, at Asilomar Conference Grounds, Pacific Grove, California, with Joel W. Goodman and Leon Wofsy serving as Cochairmen. The subject of the conference, “Structure and Specificity: Antibodies and Receptors”, was discussed during four half-day sessions by invited speakers, and in six concurrent working sessions. Topics of the individual workshops were (1) identification and significance of surface immunoglobulin on B and T lymphocytes, (2) isolation and molecular characterization of surface antigens, (3) use of surface markers in lymphocyte differentiation, (4) separation of lymphocyte subpopulations and functional analysis, (5) molecular techniques for detecting antibody genes, and (6) how membranes transmit signals. The Third Annual Dan H. Campbell Memorial Lecture was given by Dr. Elvin A. Kabat (Columbia University, New York).

Meeting reportSixteenth Midwinter Conference of Immunologists: Structure and specificity: Antibodies and receptors☆

Abstract The Sixteenth Midwinter Conference of Immunologists was held January 22–25, 1977, at Asilomar Conference Grounds, Pacific Grove, California, with Joel W. Goodman and Leon Wofsy serving as Cochairmen. The subject of the conference, “Structure and Specificity: Antibodies and Receptors”, was discussed during four half-day sessions by invited speakers, and in six concurrent working sessions. Topics of the individual workshops were (1) identification and significance of surface immunoglobulin on B and T lymphocytes, (2) isolation and molecular characterization of surface antigens, (3) use of surface markers in lymphocyte differentiation, (4) separation of lymphocyte subpopulations and functional analysis, (5) molecular techniques for detecting antibody genes, and (6) how membranes transmit signals. The Third Annual Dan H. Campbell Memorial Lecture was given by Dr. Elvin A. Kabat (Columbia University, New York).

CHEMICAL APPROACHES TO THE CELL RECEPTOR PROBLEM

Evidence is mounting for the presence, and critical function in the immune response, of antibody receptor protein on lymphocyte membranes. For many investigators, sufficient proof has accumulated to elevate the “clonal selection” scheme from the level of hypothesis to that of fact. The two main lines of evidence are: (1) studies14 which use various antiimmunoglobulin antisera to show that a substantial portion of lymphocytes have immunoglobulin determinants on cell surfaces, that this is not accounted for by cytophillic antibody, and that specific anti-immunoglobulin antisera (e.g., antiallotype) can stimulate blast transformation; (2) studies5--’* which show that there are lymphocytes which specifically bind a particular antigen, and that when the specific antigen-binding cells are removed or destroyed, the capacity for in vivo or in vifro response to the antigen is selectively suppressed. Though compelling, this evidence is indirect, a situation somewhat analogous to that which prevailed in the world of bacteriological repressors and permeases prior to their actual isolation in recent years. The prospects for direct characterization of cell receptor antibody and for examination of cellular triggering mechanisms are complicated hy the problem of antibody heterogeneity. By the precepts of clonal selection, there must be a clone of cells with a unique set of receptor antibody for each idiotypic immunoglobulin specificity within the immune potential of the individual. Moreover, the form of expression on or by a cell of antibody specificity, whether as receptor or as secreted immunoglobulins of several classes, varies with multiple stages of differentiation occurring within a clone. Inevitably, investigators in this area seek simplifying approaches: (1) the search for a pathological phenomenon, e.g., a lymphocyte tumor, which might provide copious quantities of identical receptor immunoglobulins; (2) the search for common features that might distinguish all, or at least a substantial set, of heterogeneous cell receptor antibodies from other immunoglobulins; and (3) the search for means of focusing on a set of receptors on cells competent to respond to a given antigen or, better, to those determinants related by a common hapten specificity. One advantage of the third approach is that it is possible to correlate ultimate structural findings with recognizable stages and features of an immune response. To facilitate this approach, our laboratory has set its sights on developing practical methods for isolating pure populations of cells competent for, or involved in, response to antigens with chemically distinct (hapten)