Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface.In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

DRw antisera react with activated T cells.

Nylon wool-purified T cells appear to be nonreactive in a lymphocytotoxicity assay with HLA-DRw antisera and complement before cell activation. However, after activation in mixed lymphocyte culture, responder cells express determinants that are strongly reactive with DRw alloantisera after 6 days and gradually disappear by 16 to 18 days. Restimulation of the primed cells resulted in re-expression of the blast determinants. Mitogenic stimulation with Con A or purified PHA (HA-17) also resulted in temporary expression of these determinants; reactivity usually conformed to DRw genetic restriction; however, occasional extra reactions occurred that were variable depending on the method of activation (i.e., MLC, Con A, or HA-17). The results suggest the presence of additional allospecificities within some of the DRw antisera that react with "Ia-like" antigens on activated cells from unique subsets of T cells. Whether these DRw antisera contain antibodies against T cells or agains activation or differentiation T cell antigens is not as yet clear.     

Identification of a tumor-associated antigen of the guinea pig L2C leukemia by using syngeneic antisera.

Inbred strain 2 guinea pigs immunized with L2C leukemia cells produced antibodies to L2C cells detected by 125I-protein A assay. L2C-associated tumor antigens were reacted with syngeneic antisera and analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. These sera recognized idiotypic determinants on surface IgM molecules of L2C cells but did not recognize any determinants on normal strain 2 spleen cells. Thus, determinants on IgM molecular act as tumor-associated antigens in the L2C system and can be detected by syngeneic sera.     

Isotachophoretic isolation of IgD and IgE and production of monospecific antisera against them]

The method of preparative isotachophoresis in acrylamide gel ensuring a high yield of IgD and IgE with insignificant admixtures of IgG, etc. was used for the isolation of IgD and IgE from the blood sera of myeloma patients. As a result of immunization with these antigens, monospecific IgD and IgE antisera were obtained. These antisera, alongside with specific antibodies, contained antibodies to admixtures; the latter were eliminated by the method of immune absorbtion carried out with the use of a sorbent based on Sepharose activated with bromo-cyanogen and conjugated with normal human blood serum. Ig D antisera were also shown to contain antibodies to idiotypical IgD determinants located in the Fab fragment of this immunoglobulin.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

Identification of exposed and buried determinants of the membrane-bound acetylcholine receptor from Torpedo californica

The ability of five rabbit anti-acetylcholine receptor antisera to recognize the membrane-bound receptor from Torpedo californica has been investigated. Two antisera, raised against affinity-purified native receptor, react extensively with purified receptor-rich membrane vesicles. Since the membrane vesicles are impermeable to macromolecules and are oriented right side out, these two antisera recognize predominantly extracellular determinants. Two antisera against sodium dodecyl sulfate denatured receptor and one against purified delta subunit react poorly with the membrane-bound receptor. Only 10-20% of the determinants recognized by these antisera are accessible to antibodies when the receptor is membrane bound. Many of the latent sites can be exposed by permeabilizing the vesicles with saponin, by alkaline extraction of the membranes to remove peripheral proteins, or by a combination of these two treatments. These treatments neither solubilize the receptors nor interfere with their ability to undergo agonist-induced affinity changes. Subunit analysis of the sites on the membrane-bound receptor that are accessible to antibodies indicates that the alpha, beta, and delta chains possess extracellular determinants. Buried sites are present on all four of the subunits. Saponin permeabilization makes latent sites accessible on alpha and delta while alkaline extraction uncovers determinants on alpha, gamma, and delta. Treatment of membranes by both procedures reveals sites on beta, gamma, and delta that are not uncovered by either treatment alone. This study, in conjunction with results from other laboratories demonstrating that the gamma chain is extracellularly exposed, suggests that all four subunits are transmembrane proteins.     

Evidence of a second polymorphic ELA class I (ELA-B) locus and gene order for three loci of the equine major histocompatibility complex

Two antisera, B-442 and R-2046, were produced by immunizing offspring with purified peripheral blood lymphocytes from a parent matched for the ELA-A specificity carried on the unshared haplotype. Absorption analysis demonstrated that these antisera contained at least two families of cytotoxic antibodies, one directed against antigens present on T and B cells, and a second directed preferentially against antigens present on surface Ig positive cells. Immunoprecipitation studies using these antisera demonstrated that both antisera contain antibodies specific for glycoproteins with molecular weights characteristic of class I and class II MHC antigens. In lymphocyte typing tests of unfractionated lymphocytes, only the class I activity was readily detectable since the class II activity killed less than 25% of the cells. Family studies demonstrated that these antisera recognize products of genes linked to the ELA system. Based on two recombinants in an extended family it became apparent that the specificities detected by B-442 and R-2046 are not products of the ELA-A locus, but rather they are products of at least one other locus, defined in this paper as ELA-B. In this family a third recombinant was found between the A blood group system and the ELA-A locus. Based on these three recombinants, the most probable linear relationship of the following genes is: A blood group system/ELA-A/ELA-B.     

Isolation of twelve monoclonal antibodies against Ia and H-2 antigens. Serological characterization and reactivity with B and T lymphocytes

The cell hybridization technique was used for the production of 12 monoclonal antibodies against H-2K^k, H-2D^b, I-A^k and I-E^k antigens. The strain distribution pattern indicated that three antibodies reacted with new H-2 and Ia determinants, respectively, while the majority of determinants defined by the monoclonal antibodies showed good correlation with H-2 and Ia determinants described by conventional alloantisera.Monoclonal Ia antibodies showed strong reactivity with about 90% of surface IgM positive B cells, but not with T cells. In double fluorescence studies, both I-A and I-E determinants were always found to be coexpressed on the same B cells. When the high sensitivity of the fluorescence activated cell sorter was utilized, about 30 to 40% of purified lymph node T cells were found to carry both I-A and I-E antigens, although in a much lower density than B cells. In conclusion, monoclonal Ia antibodies appear to display the same serological and cellular reactivity pattern as do conventional antisera.     

Detection of leishmania antigen in kala azar patients using monoclonal antibodies.

Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovani antigenic determinants ranging from 42-116 kDa were selected as 'capture antibodies' and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb.17 could effectively detect circulating leishmania antigen in 85.4%. The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.     

Distribution of the major histocompatibility complex antigens on different cellular components of human liver

Monoclonal mouse antibodies to the "framework" determinants of the class I and II molecules of the major histocompatibility complex (MHC) were used to demonstrate the presence of the MHC antigens in human liver. First, the localization of these antigens was demonstrated from frozen section histology with indirect FITC immunofluorescence and the cell component(s) binding the mouse antibody were identified by rabbit marker antisera and indirect TRITC immunofluorescence. Second, the antigen expression on the cell surface was analyzed by the Staphylococcus aureus rosette method from cytological cell smears. All antibodies reacted with cells in the liver sinusoids, both with the Kupffer cells and at least partially with the sinusoidal endothelial cells. The same antisera reacted also with the bile duct cells, though weaker, and with some stromal cells in close proximity of the blood vessels. The vascular endothelial cells of hepatic artery, hepatic vein, and portal vein displayed no reaction. Thus human liver differs strikingly from, e.g., human kidney, where the vascular endothelial cells contain large amounts of MHC antigens on the cell surface. This difference may be one explanation to why liver allografts are less promptly rejected than renal allografts in man.     

Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.     

Separation of neutralizing and hemagglutination-inhibiting antibody activities and specificity of antisera to sodium dodecyl sulfate-derived polypeptides of polyoma virions.

Antisera to the sodium dodecyl sulfate (SDS)-polyacrylamide gel-derived polyoma virion polypeptides were used in immunoprecipitation experiments with ethylene glycol-bis-N,N'-tetraacetic acid (EGTA)-dissociated polyoma virions and capsids to determine the specificity of the antipolyoma polypeptide sera. Additionally, a technique for applying 125I-labeled immunoglobulins to SDS-polyacrylamide gels was used to explore the antigenic specificities of the antisera. The results demonstrated that antisera directed against the SDS-gel-derived VP1, VP2, and VP3 did not react with native polyoma proteins, but would react with the appropriate antigens on denatured polyoma proteins. Antisera against the histone region of such gels reacted with native and denatured polyoma VP1. Separation of neutralizing antibodies from hemagglutination inhibition (HAI) antibodies to polyoma in antisera directed against the histone region of polyacrylamide gels was done by using a polyoma capsid affinity column. The antibodies eluted from this column which did not react with capsids possessed only neutralizing activity, whereas antibodies which bound to capsids possessed only HAI activity. These isolated immunoglobulin G fractions were then used in immunoprecipitation experiments to demonstrate that the antigenic determinants responsible for the HAI activity of the serum were contained on a 16,000-dalton polypeptide, whereas those antigenic determinants responsible for neutralizing activity were contained on a 14,000-dalton polypeptide. Both of these polypeptides present in the histone region of the SDS-gels appeared to be derived from the major virion protein VP1.     

Mapping antibody binding sites on cytochrome c with synthetic peptides: Are results representative of the antigenic structure of proteins?

Crystallographic work on antigen-antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies. On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain. Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti-protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen. We have addressed this question using yeast iso-1 cytochrome c as a protein antigen against which antisera were raised in rabbits. The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA. For the assay, anti-cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso-1 cytochrome c and on apo-cytochrome c; the latter is a random coil. It was found that only antibodies retained by the apo-cytochrome c affinity column react with synthetic peptides. These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c-specific antibodies. The majority of antigenic determinants, which seem to consist of strongly conformation-dependent topographic epitopes, could not be uncovered by the peptide approach. Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.     

Antiserum inhibition of the mixed lymphocyte culture (MLC) interaction. Inhibitory effect of antibodies reactive with HLA-D-associated determinants.

Two antisera, procured by immunization within HLA-A- and HLA-B-identical and HLA-D-incompatible unrelated combinations, were cytotoxic to B lymphocytes from the immunizing donor and from persons sharing his HLA-D-incompatible phenotype(s). The sera strongly and specifically inhibited lymphocytes from these donors when used as stimulating cells in mixed lymphocyte culture (MLC) reactions, while specific responding cell inhibition was less evident. The inhibitory effect was retained in the immunoglobulin G (IgG) and the F (ab)′₂ fractions of these antisera. Inhibition was observed when the antisera were added within 48 hr after initiating the MLC. We conclude that these antisera contain antibodies reactive with structures closely associated with HLA-D determinants and that these may be human analogs of the mouse Ia antigens.     

Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina

Basal lamina (BL) ensheathes each skeletal muscle fiber and passes through the synaptic cleft at the neuromuscular junction. Synaptic portions of the BL are known to play important roles in the formation, function, and maintenance of the neuromuscular junction. Here we demonstrate molecular differences between synaptic and extrasynaptic BL. We obtained antisera to immunogens that might be derived from or share determinants with muscle fiber BL, and used immunohistochemical techniques to study the binding of antibodies to rat skeletal muscle. Four antisera contained antibodies that distinguished synaptic from extrasynaptic portions of the muscle fiber's surface. They were anti- anterior lens capsule, anti-acetylcholinesterase, anti-lens capsule collagen, and anti-muscle basement membrane collagen; the last two sera were selective only after antibodies binding to extrasynaptic areas had been removed by adsorption with connective tissue from endplate-free regions of muscle. Synaptic antigens revealed by each of the four sera were present on the external cell surface and persisted after removal of nerve terminal. Schwann cell, and postsynaptic plasma membrane. Thus, the antigens are contained in or connected to BL of the synaptic cleft. Details of staining patterns, differential susceptibility of antigens to proteolysis, and adsorption experiments showed that the antibodies define at least three different determinants that are present in synaptic but not extrasynaptic BL.     

Antigenic properties of the electrophoretic fractions of the causative agent of melioidosis

Antisera to the antigens of 5 fractions, isolated as the result of the separation of P. pseudomallei aqueous saline extract by continuous electrophoresis in the vertical block of granulated gel, have been obtained. Immunoelectrophoresis with the use of P. pseudomallei aqueous saline extract has revealed that antisera to electrophoretic fractions contain antibodies mainly to the antigens of the corresponding fractions, which shows that this technique ensures the effective separation of P. pseudomallei biopolymers by their electrophoretic motility and molecular weight. These antisera differ in their species specificity. Thus, antisera to antigens with anode motility have been found to contain antibodies mainly to P. pseudomallei antigens and antisera to electroneutral antigens or to those with cathode motility, to P. pseudomallei and P. mallei antigens.     

Further polymorphism of the Tla locus defined by monoclonal TL antibodies

Six new monoclonal TL antibodies are described. At least one new TL antigen is defined (TL.7), and at least one more Tla allele, bringing the total number of known Tla alleles to six. Five of the monoclonal antibodies, and probably all six, identify distinct TL antigenic specificities. Four of these antigens conform in strain distribution and expression on leukemia cells to antigens defined by conventional antisera. The data contain a hint that monoclonal TL antibodies like TL.m6 may serve to identify a region of the Tla gene, which determines whether or not prothymocytes will respond to physiological induction by expressing TL, and thus may provide a means to study the regulatory mechanism that determines whether mouse strains are phenotypically TL⁺ or TL^– The nomenclature TL.m4–9 for the six monoclonal antibodies described follows McIntyre and coworkers (1980). The serial numbers 4–9 do not imply any correspondence with numbers assigned to TL antigens defined by conventional antisera. The corresponding hybridoma lines are available to interested investigators.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence micro cytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence microcytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.