Ferritin-conjugated Antibody Research Articles

Trypanosoma lewisi: Ultrastructural observations of surface antigen movement induced by antibody

Cellular antigens of Trypanosoma lewisi have been located with ferritin-conjugated antibody (FCA) at the ultrastructural level. Incubation of live T. lewisi with antibodies from infected rats and ferritin-labeled rabbit anti-rat γ-globulin induced aggregation of surface antigens in the flagellar pockets, the desmosomal regions, and the flagellar membranes of parasites. Aggregation of surface antigens was not observed when the trypanosomes were incubated with γ-globulin and FCA at low temperatures (0–4 C). Sections of trypanosomes incubated at 37 C for 15 or 30 min after incubation at 0 C with FCA showed internalization of FCA in membrane-bound vesicles. Studying the movement and aggregation of these parasites' surface antigens may give information about the molecular dynamics of the plasma membrane and provide insights into the trypanosomes' antigenic modulation and the hosts' immunological responses.

Trypanosoma lewisi: Immunoelectron microscopic studies on the surface antigens of bloodstream forms

Cellular antigens of Trypanosoma lewisi bloodstream forms have been examined at the ultrastructural level with the direct and indirect ferritin-conjugated antibody (FCA) techniques. Ferritin was coupled to gamma globulins separated from the sera of uninfected rats and animals infected withh T. lewisi. In the direct method, trypanosomes were incubated with these conjugates and antigens were localized by the FCA on the surface of these parasites. There were no apparent differences between the distributions of antigens of live parasites incubated for either 1 or 2 hr in the ferritinconjugated gamma globulins of infected rats. Few ferritin particles were found on trypanosomes incubated with conjugated globulins from uninfected rats. Antisera for the indirect techniques were produced in rabbits by immunization with rat gamma globulin and Freund's complete adjuvant. Ferritin was coupled to gamma globulins isolated from unimmunized rabbits and those injected with rat gamma globulins. T. lewisi were incubated in unlabeled gamma globulins from uninfected or infected rats and washed and reacted with the ferritin-conjugated rabbit anti-rat gamma globulin. Appropriate controls were included in each experiment to ascertain the specificity of of the FCA reactions. Unlike the direct method which demonstrated FCA-antigen complexes on the surfaces of the parasites, trypanosomes treated with the indirect immunoferritin technique showed aggregates of FCA in the regions of desmosomal attachment, on the flagellar membrane surfaces, and in the flagellar pockets, and some scattered FCA on the surface coat matrices. Parasites fixed with formalin or incubated with sodium azide and subsequently treated by the indirect technique exhibited FCA complexed with unlabled antibody which had reacted with surface antigens, but these antigen-antibody-FCA complexes did not aggregate in the desmosomal regions, on the flagellar surfaces, or in the flagellar pockets. These observations suggested that movement and aggregation of the surface antigens occurred as an energy-dependent process in live T. lewisi. Movement and aggregation of the surface antigens were induced by a second ligand (antibody) present during treatment of the parasites with the indirect FCA technique. T. lewisi treated with both the direct and indirect immunoferritin techniques developed filopodial processes on their surfaces. FCA-antigen complexes were found on these structures. The locations, movements, and aggregation of antigens of T. lewisi and the formation of filopodia on the surfaces of the parasites described during these immunoelectron microscopic studies are discussed in terms of the direct and indirect FCA reactions, cell membrane structure and dynamics, and the host-trypanosomes interactions.

Immunochemical evidence for the transmembrane orientation of glycophorin A: Localization of ferritin-antibody conjugates in intact cells

Antibodies were raised in rabbits to a 51-amino acid cyanogen bromide-derived peptide of human erythrocyte glycophorin A which has been shown to represent the C-terminal end of the 131-residue polypeptide chain. Antibodies prepared by immunoadsorption were found to be directed against a chymotryptic-derived peptide (residues 102 to 118) of glycophorin A but were unreactive with either intact or proteolytically modified red blood cells. No cross-reactivity was observed with glycophorin B of human or sialoglycoproteins prepared from red blood cells of other mammalian species. Ferritin-antibody conjugates of such sera were applied to thin sections of intact red blood cells (frozen or protein embedded) and were found to localize exclusively to sites distributed uniformly along the inner surfaces of the membrane. No staining was seen on sections prepared from red blood cells from other species nor on sections of human red cells pretreated with unconjugated antisera. These results provide additional evidence in intact, fixed human erythrocytes that glycophorin A has a transmembrane orientation.

Proof for the Production of Cutinase by Fusarium solani f. pisi during Penetration into Its Host, Pisum sativum

Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this ferritin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.

Cell surface-localized alkaline phosphatase of Escherichia coli as visualized by reaction product deposition and ferritin-labeled antibodies

When cells of a wild-type Eschericia coli O8 strain bearing a complete lipopolysaccharide were incubated for alkaline phosphatase reaction product and examined by electron microscopy, the depostion of lead salts was to be observed primarily within the periplasmic space. A similar treatment of cells derived from this strain, which bears a highly abbreviated lipopolysaccharide, showed a mixed cell surface and periplasmic localization of reaction product, suggesting a surface association of a portion of the enzyme. To further explore this possibility, ferritin-antibody conjugates against the active enzyme and its irreversibly dissociated subunits were prepared and allowed to react with cells of both strains. The results obtained from these experiments revealed the presence of both the active enzyme and inactive subunits of the enzyme at the cell surface of the mutant strain. The evidence obtained offers further proof of the validity of the reaction product deposition technique and indicates that alkaline phosphatase may be associated with some component of the outer membrane in this organism. The observation of enzyme subunits at the cell surface further suggests that an association of these subunits with structural components of the cell envelope may provide a locus at which they may dimerize to form active enzyme.

Separation of ferritin labeled antibody from free antibody

A procedure for the separation of ferritin—antibody conjugates from free antibody has been developed using isokinetic sucrose density gradient sedimentation. The gradient (5% w/w sucrose at the top to about 35% at the tube bottom) provides optimal separation of 7S free immunoglobulin from the ferritin (65S) and ferritin—antibody conjugate. Recovery of the conjugate band is simple and avoids problems of aggregation found by centrifugal pelleting and resuspension procedures. The method was tested by labeling human globulin and fractionating the gradient. Free antibody was detected at the top one-fifth of the gradient by immunodiffusion. Ferritin and the labeled antibody were found one-third to one-half of the way down the gradient. Immunoelectrophoresis was also used to demonstrate the separation of unconjugated ferritin and ferritin labeled antibody from free antibody.

Lipid vesicle-cell interactions. III. Introduction of a new antigenic determinant into erythrocyte membranes.

The introduction of a new antigenic determinant, 2,4-dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE), into the surface membranes of intact human erythrocytes is described. Fresh cells were incubated in the presence of liposomes composed of 10% DNP-Cap-PE, 5% stearylamine, 20% lysolecithin, and 65% lecithin. Such liposome-treated erythrocytes are shown to be susceptible to immune lysis by anti-DNP serum in the presence of complement. Uptake of DNP-Cap-PE by erythrocyte membranes is also demonstrated by immunofluorescence using indirect staining with rabbit anti-DNP serum followed by fluroescein-conjugated goat anti-rabbit IgG and by electron microscopy using ferritin-conjugated antibody. Antigen uptake did not occur at low temperatures or from vesicles lacking lysolecithin and stearylamine. Fluorescence microscopy shows that the antigen-antibody complexes are free to diffuse over the cell surface, eventually coalescing into a single area on the cell membrane. Electron microscopy suggests that a substantial proportion of the lipid antigen is incorporated by fusion of vesicles with the cell membrane. There are indications that vesicle treatment causes a small proportion of cells to invaginate.

Immunochemical and immunoelectron microscope studies on localization of NADPH-cytochrome c reductase on rat liver microsomes.

By the use of ferritin-conjugated antibody (conjugate) indirect immunoelectron microscopy, NADPH-cytochrome c reductase was localized on rat liver microsomes. Most microsomes in the sections had from 1 to 12 conjugates on their outer surfaces. Among the conjugates, 83% was estimated to bind to NADPH-cytochrome c reductase at a molecular ratio of 1:1, 12% at the ratio of 2:1, and 5% at the ratio of 3 or 4:1. The correlation between immunochemical and morphological data confirmed that most of the NADPH-cytochrome c reducatase reacted with the conjugates. Subsequent morphological analyses have revealed that the enzyme is distributed homogeneously on the outer surfaces of microsomes but heterogeneously within microsomes in groups of three to five enzyme molecules.

Micropinocytosis of Transferrin by Developing Red Cells: An Electron-microscopic Study Utilizing Ferritin-conjugated Transferrin and Ferritin-conjugated Antibodies to Transferrin

Abstract Electron-microscopic examination of rat reticulocytes and normoblasts incubated with transferrin conjugated to ferritin or ferritin-labeled antitransferrin revealed binding of ferritin conjugates to the surface membrane, and uptake of ferritin conjugates in micropinocytotic vesicles. No binding or endocytosis of ferritin was visualized when rat reticulocytes or normoblasts were incubated with ferritin alone or ferritin conjugated to nonspecific rabbit IgG. These observations support the concept that transferrin binds to a surface membrane receptor and is subsequently internalized by the developing red cell. Time course and temperature dependence studies suggest the endocytosis of transferrin may be an important mechanism in delivery of iron to the developing red cell.

Micropinocytosis of transferrin by developing red cells: an electron- microscopic study utilizing ferritin-conjugated transferrin and ferritin-conjugated antibodies to transferrin

Electron-microscopic examination of rat reticulocytes and normoblasts incubated with transferrin conjugated to ferritin or ferritin-labeled antitransferrin revealed binding of ferritin conjugates to the surface membrane, and uptake of ferritin conjugates in micropinocytotic vesicles. No binding or endocytosis of ferritin was visualized when rat reticulocytes or normoblasts were incubated with ferritin alone or ferritin conjugated to nonspecific rabbit IgG. These observations support the concept that transferrin binds to a surface membrane receptor and is subsequently internalized by the developing red cell. Time course and temperature dependence studies suggest the endocytosis of transferrin may be an important mechanism in delivery of iron to the developing red cell.

Ultrastructural visualization of virus-induced surface antigens. Comparative studies of three immunohistochemical techniques.

Three immunohistochemical techniques used for ultrastructural localization of cell surface antigens were compared with respect to ease of reagent preparation and qualitative aspects of cell surface staining. The techniques compared were: (a) ferritin-conjugated antibody, (b) "hybrid" antibody and (c) a modification of the "bridging" technique which employs soluble immune complexes. Used in conjuction with strain MC29 avian oncornavirus-infected chick embryo cells and chicken antiviral immune serum, the results of all three techniques were comparable. In terms of ease of preparation, the "bridging" technique employing soluble complexes was found to be the most satisfactory.

Ultrastructural study of the reversion of protoplasts of Bacillus licheniformis to bacilli.

The reversion of protoplasts of Bacillus licheniformis 6346 His- on a medium containing 2.5% agar has been studied in sectioned material after reaction with a ferritin-conjugated antibody specific to the peptidoglycan isolated from the walls of the bacilli. Freeze etching has also been used. Fibrils of material reacting with the antibody have been detected emerging from isolated areas of the protoplasts after 3 h of incubation. This material gradually covers the cell and can eventually (at 6 h) be seen in freeze-etched preparations as a fringe of up to 400 nm around the cells and covering the surfaces with particles that can be removed by lysozyme. At later stages the wall begins to take on a compact, well-defined appearance that can be seen in sections; however, the cells are still grossly deformed. A transitory emergence, beyond the wall of long fibers of 6 nm in diameter, takes place after about 12 h of incubation. These fibers react with the conjugated antibody and after freeze etching show a regular banded structure. They are probably indentical with the fibers isolated elsewhere (Elliott et al., 1975) and shown to contain all the wall constituents (i.e., peptidoglycan, teichoic acid, and teichuronic acid). These fibers are not detectable in the final stages of reversion.

Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.

Three distinct antiprocollagen preparations were characterized and used in immunocytochemical staining of chick embryo corneal and tendon cells. The several ferritin-conjugated antibody preparations permitted similar location of procollagen in the cisternae of the rough endoplasmic reticulum and in Golgi elements in both cell types. The ability to demonstrate and interpret specific ferritin staining was dependent on the extent of membrane breakage in each of those organelles, coupled with adequate retention of cell morphology. Corneal fibroblasts appeared to suffer more extensive intracellular membrane damage under controlled conditions of homogenization than tendon fibroblasts, facilitating the identification of procollagen in Golgi vacuoles of these cells. None of the labeled material appeared to by cytoplasmic in origin since ferritin was observed in the cytoplasm only in the vicinity of Golgi elements that were extensively broken. This study extends previous immunological evidence for the presence of procollagen in the Golgi complex and calls attention to the problems to be encountered in locating the antigen in small Golgi vesicles and lamellae.

Electrophoresis of ferritin-conjugated antibody into fixed tissue sections as a method for immune electron microscopy

A technique was developed to stain cells intracellulary with immunoferritin reagents, using disc electrophoresis. The cells were fixed in formalin and embedded in agarose for electrophoresis, then post-fixed in glutaraldehyde and osmium. EM sections were examined using standard methods.

T antigen binds to simian virus 40 DNA at the origin of replication.

A technique employing ferritin-conjugated antibody has been developed to visualize specific protein-DNA complexes in the electron microscope and has been used to demonstrate the preferential binding of simian virus 40 (SV40) T antigen at or near the origin of replication of SV40 DNA, 0.67 fractional length clockwise from the EcoRI restriction endonuclease cleavage site. urified covalently closed supercoiled circles of SV40DNA were treated with partially purified T antigen and the complex was stabilized by crosslinking with glutaraldehyde. Hamster antiT antigen gamma-globulin, ferritin-labeled goat anti-hamster gamma-globulin, and glutaraldehyde were then added sequentially. The location of the bound ferritin cores was measured with respect to the EcoRI cleavage site and the orientation of the cores relative to the ends of the DNA was determined with respect to the locations of Escherichia coli DNA unwinding protein, which binds to covalently closed supercoiled SV40 DNA at either of two preferred sites, 0.46 or 0.90 fractional length clockwide from the EcoRI cleavage site.

Two improved methods for preparing ferritin-protein conjugates for electron microscopy.

Two improved procedures were developed for activating ferritin so that the ferritin could be covalently linked to antibodies. One procedure involved use of a water-soluble carbodiimide and N-hydroxysuccinimide to prepare ferritin-containing activated esters. The other involved activation of the ferritin with excess glutaraldehyde. The ferritin-antibody conjugates prepared with the two procedures were shown to have a number of properties which made them suitable for locating antigenic components in cells.

Asymmetrical distribution and artifactual reorientation of lipopolysaccharide in the outer membrane bilayer of Salmonella typhimurium.

Labelling of cell walls or outer membranes from Salmonella typhimurium with ferritin-conjugated antibodies directed against the polysaccharide moiety of the lipopolysaccharide gave the following results: 1. Cell walls or outer membranes from which the mucopeptide had been removed by lysozyme digestion at 0 degrees C carried the label on the outer face of the membrane. 2. When the murein layer was removed by either lysozyme or trypsin at physiological temperature (25-37 degrees C) subsequent labelling showed the lipopolysaccharide to be present on both membrane faces. 3. This reorientation could be achieved by a 1-min treatment of the membranes at 37 degrees C. 4. Glutaraldehyde fixation of the outer membranes did not entirely prevent but somewhat inhibited the temperature-induced reorientation process. 5. The same reorientation phenomenon was observed in lysozyme spheroplasts, which were prepared at 37 degrees C and were subsequently lysed in hypotonic medium at 0 degrees C. These observations are discussed as evidence for a transmembrane movement of lipopolysaccharide, which only takes place in areas where the mucopeptide layer is defective, and only when the temperature is sufficiently high to allow such movement.

Antigenic Difference Between Polar Monotrichous and Peritrichous Flagella of Vibrio parahaemolyticus

Polar monotrichous and peritrichous flagella of Vibrio parahaemolyticus were isolated and purified separately. On hydroxylapatite column chromatography, the flagellins of polar monotrichous flagella were eluted with a higher concentration of phosphate than those of peritrichous flagella. Gel diffusion tests showed an antigenic difference between the flagellins of polar monotrichous and peritrichous flagella. Electron microscope observations on cells stained with ferritin-conjugated antibodies demonstrated that polar monotrichous and peritrichous flagella reacted specifically with antimonotrichous flagellin and antiperitrichous flagellin antisera, respectively.

Ferritin-conjugated antibodies used for labeling of organelles involved in the cellular synthesis and transport of procollagen.

An improved procedure was used to conjugate ferritin to antibodies specific for the NH(2)-terminal extensions on the precursor form of collagen known as procollagen. The ferritin-antibody conjugates were then used to determine the localization of procollagen in fibroblasts isolated from chick-embryo tendons. Procollagen was found in the cisternae of the endoplasmic reticulum, indicating that the protein passes into this compartment early in its biosynthesis. Specific labeling of large Golgi vacuoles was also observed, suggesting that this compartment is also involved in the secretory process. When cells were incubated with colchicine so that the secretion of procollagen was delayed, there was an increase of large, smooth-surfaced vacuoles in the cells and these vacuoles were labeled with the ferritin-antibody conjugate.

A two-stage method for cross-linking antibody globulin to ferritin by glutaraldehyde II. Antigen binding and agglutinating capacity

Immunoferritin conjugates produced in a glutaraldehyde (GA) two-step reaction were compared with regard to antigen binding and agglutinating activity with conjugates which had been produced with m-xylylene-diisocyanate (XC), toluene 2,4-diisocyanate (TC) and in a GA one-step reaction. The agglutinating activity of all the conjugates was greatly reduced to 1–6% that of the unconjugated antibody. In contrast to this approximately half the ferritin-conjugated antibody was still able to react specifically with the antigen. We obtained the best results with conjugates formed in a GA two-step reaction and with TC as coupling reagent. The formation of aggregates in the GA one-step reaction may result in a considerable reduction of antibody activity.