The reversible binding of the haptens 2,4-dinitrophenyllysine (DNP-lysine) and 2,4,6-trinitrophenyllysine (TNP-lysine) to either the nitrophenyl-binding myeloma protein MOPC-315 or to pooled mouse anti-DNP or anti-TNP antibodies produces large and characteristic extrinsic Cotton effects (in the circular dichroic spectra). Despite the similarities in binding characteristics of the three proteins, the circular dichroic spectra produced by the haptens bound to the active sites of these proteins were markedly different. Extrinsic Cotton effects, therefore, provide a powerful new probe of the structure of the reversible complex formed between a hapten and an antibody active site.

Animals↗

PubMed1971

Ferritin-conjugated plant agglutinins as specific saccharide stains for electron microscopy: application to saccharides bound to cell membranes.

An electron microscopic stain for specific saccharides was prepared by the conjugation of ferritin to concanavalin A, a plant agglutinin that specifically binds to oligosaccharides containing terminal d-glucose, d-mannose, or sterically related sugar residues. A technique was developed to allow topological visualization of erythrocyte and other membranes by means of transmission electron microscopy, and the distribution of the binding sites for ferritin-concanavalin A on such membrane preparations was determined. The conjugate was found to bind specifically to the outer, but not the inner, surface of erythrocyte membranes. The number of conjugate molecules bound per unit area of the membrane was larger for rabbit than for human erythrocytes.

Animals↗

PubMed1971

Extrinsic Cotton effects in hapten--carrier and hapten--antibody interactions.

Two homogeneous univalent hapten-protein conjugates, prepared by the covalent attachment of a single 2,4-dinitrophenyl (DNP-) or 2,4,6-trinitrophenyl (TNP-) side chain to the cysteine-SH in the active site of the enzyme papain, have been found to exhibit large Cotton effects in the wavelength region of the absorption bands of the DNP or TNP groups. This indicates that the DNP or TNP groups are noncovalently bound to some asymmetric region(s) of the papain molecule. These homogeneous papain conjugates are antigens that can elicit anti-DNP or anti-TNP antibody production in mice or rabbits. It is suggested that the noncovalent binding of a hapten to the surface of its carrier protein may profoundly affect the characteristics of the anti-hapten antibodies that are elicited. It has also been observed that the specific binding of these papain conjugates, and of the simple haptens DNP-lysine or TNP-lysine, to anti-hapten antibodies produces characteristic extrinsic Cotton effects.

Antibody Formation↗

PubMed1971

Quantitative two-dimensional ultrastructural distribution of Rh o (D) antigenic sites on human erythrocyte membranes.

A method is described for determining the two-dimensional distribution of specific antigens on cell surfaces, and is applied to the D antigen of the Rh antigenic system. Rh-positive human erythrocytes are allowed to react with purified (125)I-labeled human anti-Rh(o)(D) gamma-globulin antibodies, and the sensitized cells are then lysed at an air-water interface. The residual cell membranes are spread flat by surface forces, and are picked up on a carbon-strengthened collodion-coated electron microscope grid. The membranes are then stained with ferritin-conjugated goat antibodies directed against human gamma-globulins. Only Rh-positive cells sensitized with anti-Rh(o)(D) antibodies bind the ferritin-conjugated antihuman gamma-globulins. The ferritin particles are found in small clusters on the membrane surface, and the number of such clusters per unit area agrees with the number of (125)I-labeled anti-Rh(o)(D) antibodies bound per unit area. The Rh(o)(D) antigenic sites appear to be molecularly dispersed on the membrane surface, but in a random two dimensional array.

Adult↗

PubMed1971

The two-dimensional topographic distribution of H-2 histocompatibility alloantigens on mouse red blood cell membranes.

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Animals↗

PubMed1971

The localization of spectrin on the inner surface of human red blood cell membranes by ferritin-conjugated antibodies.

Spectrin, a major protein constituent of mammalian red blood cell membrane preparations, has been localized on the inner surface of human red blood cell membranes by techniques that utilized specific ferritin-conjugated antibodies and fixation of membranes shortly after hemolysis so as to allow penetration of the ferritin-antibody labels. The labeling of spectrin was shown to be specific by the following criteria. (a) Nonhomologous ferritin-conjugated antibodies did not specifically bind to either membrane surface. (b) Blocking the membrane-bound spectrin with excess unconjugated antispectrin antibodies prevented ferritin-antibody labeling. (c) Removal of spectrin by treating the membrane preparation with a low ionic strength buffer containing ethylenediaminetetraacetate and beta-mercaptoethanol prevented labeling by specific ferritin-conjugated antibodies.

Animals↗

PubMed1971