Polypeptide analysis of individual immunoprecipitates from crossed immunoelectrophoresis

The method presented makes it possible to analyze the polypeptides of the antigen of individual immunoprecipitates. Crossed immunoelectrophoresis of radioactively labeled human serum proteins, herpes simplex virus type 1 antigens, and human erythrocyte membrane proteins was performed using polyspecific antibody preparations. Individual immunoprecipitates were cut out of the agarose gel and the polypeptides were eluted with sodium dodecyl sulfate and 2-mercaptoethanol. The polypeptide composition and corresponding molecular weight of the antigenic material were determined by SDS-polyacrylamide-gel electrophoresis followed by autoradiography.     

Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: Evidence for two classes of Ia antigens

Previously published data suggest that both xenogeneic and allogeneic anti-Ia sera can recognize carbohydrate-defined antigenic determinants on the surface of lymphocytes. There is also evidence, based on studies with allogeneic anti-Ia sera, that protein-defined Ia antigens exist. In this paper the relationship between these two types of Ia antigen was examined. It was found that in capping studies, the allogeneic anti-Ia serum could cap off the antigens recognized by the xenogeneic antiserum, whereas the xenogeneic antibodies could, at least partially, clear the surface of lymphocytes of Ia antigens detected by the allogeneic antibodies. On the other hand, when immunoprecipitates of radioiodinated cell-surface antigens were examined by SDS-polyacrylamide-gel electrophoresis, it was found that the xenogeneic anti-Ia serum did not immunoprecipitate any labeled material. In contrast, the allogeneic antiserum immunoprecipitated a labeled molecule which corresponded to the protein-defined Ia antigen described by others. Finally, it was shown that serum Ia antigens could be bound by either mouse or rabbit anti-Ia antibody, and this binding blocked any further reactivity with either serum. These results were interpreted as suggesting that two separate classes of Ia antigen molecule appear on the lymphocyte surface-one class has carbohydrate-defined antigenic specificities and the other has protein-defined determinants. Allogeneic anti-Ia sera contain antibodies against both these antigenic systems, whereas xenogeneic sera recognize only the carbohydratedefined series. The genetic implications of this interpretation are discussed.     

Immunoelectrophoretic analysis of Streptococcus agalactiae serotype Ia antigens

Rabbit antibodies to heat-killed whole cells of Streptococcus agalactiae serotype Ia were used to establish an antigen map using Triton X-100 sonicates of homologous cells and crossed immunoelectrophoresis. A total of 11 antigens were identified but the density of immunoprecipitates was varied and only seven could be reliably detected, one of which dominated the immunoprecipitate pattern by its intensity. The antigens were partially characterized by immunological, chemical and cell-location methods. Five of the antigens contained carbohydrate and two of those were sensitive to trypsin and probably represent cell-wall compounds. Of the three most prominent antigens, one was surface located and represented the type and shared type antigens (Iabc), one was a cell-wall carbohydrate and very sensitive to periodate, and one was a protein/carbohydrate complex which was heat-labile and trypsin sensitive. Group B epitopes were detected in three immunoprecipitates. Cross-reactions between type Ia and other serotypes and streptococci were recorded.     

Antigenic specificity of opsonophagocytic antibodies in rabbit anti-sera to group B streptococci.

An opsonophagocytic assay has been developed which requires human polymorphonuclear leukocytes, immune serum, and complement for optimal killing of Group B streptococci. Only with all three of these components was killing of greater than 1.0 log10 of the initial inoculum achieved, using rabbit antisera directed to homologous strains of each of the five known serotypes of Group B streptococci. Titers of specific antisera which opsonized the strains and resulted in greater than 1 log 10 reduction of colony-forming units, ranged from 1:100 (serotype Ib) to 1:3200 (serotype Ia). Cross-reactions between serotype-specific sera and heterologous strains were seen in certain instances. Type Ic strain and serotype Ic antiserum demonstrated cross-reactions with types Ia and Ib which were explainable by known shared antigens among these types. The only other cross-reaction which resulted in greater than 1 log 10 reduction in colony-forming units was when unabsorbed antiserum to strain Ia was used to opsonize a strain of serotype III. Opsonization of 10 serotype III strains was demonstrated with a single type III antiserum. Killing of nine of these strains required polymorphonuclear leukocytes, complement, and antiserum, but one strain, D136C, the reference strain, could be killed (greater than 1 log 10 reduction in colony-forming units) without either complement or specific antiserum. Inhibition studies were performed utilizing large m.w. polysaccharide antigens extracted from each serotype. These antigens inhibited opsonization of homologous strains by homologous antisera with 50% inhibition points ranging between 0.5 and 4 mug.     

Demonstration of Ia antigens on mouse intestinal epithelial cells by immunoferritin labeling

Several kinds of epithelial cells that express H-2 antigens were studied by immunoferritin labeling with an antiserum reacting only with antigens of theI region of theH-2 complex. Spleen lymphocytes were used to test the labeling system and the effect of the epithelial cell dissociation procedure on Ia antigens. Immunoglobulin-positive B10.BR lymphocytes were labeled with an anti-la^k serum (A.TH anti-A.TL serum absorbed with BALB/c and B10.D2 cells), while congenic B10.D2 lymphocytes were unlabeled. The distribution of labeled Ia antigens on living B10.BR lymphocytes was patchy, while on cells fixed in periodate-lysine-paraformaldehyde before labeling, the distribution of label was continuous. Fixation evidently immobilized Ia antigens in the lymphocyte membrane. Trypsin and collagenase, as used in the epithelial cell dissociation procedure, had no discernible effect on the Ia antigens of lymphocytes. The epithelial cells studied included the columnar absorptive cells of the small intestine, uterine lining epithelium, tracheal brush cells, and pancreatic exocrine and duct cells. These cells were fixed before dissociation from their respective tissues. Ia antigens were detected only on the columnar absorptive cells of the small intestine. These cells labeled equally well with an antiserum reacting only with theK -end of theH-2 complex. In both cases, congenic control intestinal cells were unlabeled. Thus, intestinal epithelial cells appear to express theIa, K, and presumablyD regions of theH-2 complex, while the other epithelial cell types express only the K and D antigens. On fixed intestinal epithelial cells, Ia and H-2K antigens were continuously distributed on the lateral and basal cell membranes including the zonula adherens, but the antigens were absent from the apical microvillous membrane and the zonula occludens.     

Association of p21ras with cellular polypeptides

p21ras specific antiserum was used to immunoprecipitate p21ras polypeptides from human A431 cells. In addition to p21ras, this antiserum precipitated a series of polypeptides with relative molecular weights of 150,000, 120,000, 105,000, and 50,000. The precipitation of these polypeptides was prevented by preincubation of the antiserum with an excess of purified Ras protein. These polypeptides do not share an epitope with p21ras, and two of them (120 and 150 kDa) copurify with a fraction of p21ras. The co-precipitation of p21ras with these polypeptides was detected in a variety of cell types. The pattern of the immunoprecipitates was consistently different in normal and ras-transformed cells. The 120- and 150-kDa polypeptides are phosphorylated on serine and threonine in A431 cells. Serum treatment resulted in a 2-fold increase in the phosphoserine content of the 120-kDa polypeptides.     

Autoantibodies to the core proteins of hnRNPs

A novel autoantibody reacting the the core polypeptides of hnRNP particles has been detected in the serum of a patient with systemic lupus erythematosus (SLE) and Sj?gren's syndrome manifestations. Immunoblot analysis, using either rat liver or HeLa nuclear extracts as the antigen source, demonstrated that the autoantibody interacts with a specific subgroup of the core polypeptides of hnRNP particles, namely A2, B1 and B2, but not with A1, C1 and C2.     

Selective immunoprecipitate identification using monoclonal anti-rabbit IgG

A monoclonal mouse antibody directed against rabbit IgG has been conjugated with horseradish peroxidase and used to identify immunoprecipitates which contain rabbit antibodies. By combining a specific rabbit antisera with a general antiserum from another species (e.g., goat antiserum against human serum), immunoprecipitates containing the antigen(s) recognized by the rabbit antibodies have been selectively identified by colorimetric development of peroxidase activity. Since the monoclonal antibody is specific for rabbit IgG and nonprecipitating, the peroxidase conjugate can be included in the agarose with the primary antisera.     

Detection and isolation of an Ia glycoprotein antigen from mouse serum

Ia antigenic activity is present in mouse serum and exists predominantly (>90 percent) as a dialyzable oligosaccharide which can be purified readily. This paper describes experiments which indicate, however, that a small proportion (<10 percent) of the Ia antigenic activity in serum, particularly from mitogen-injected mice, exists in a high molecular-weight form. Furthermore, this high molecular-weight Ia antigen, like the Ia oligosaccharide, appears to be secreted by T lymphocytes, particularly by T cells which have been activated recently by mitogens. The high molecular-weight Ia antigen was isolated from mouse serum by a procedure which utilized salt fractionation, gel filtration, density separation, and sucrose gradient sedimentation. This procedure gave a 190–260-fold increase in specific Ia antigenic activity, and up to 80 or 90 percent of high molecular-weight Ia antigenic activity detected in the original serum was recovered in the purified fraction. On the basis of sucrose gradient sedimentation studies, the high molecular-weight Ia antigen was estimated to have a molecular weight of approximately 500,000 daltons, if it is assumed that it exists as a globular protein. In addition, the protein has an ₂-macroglobulin-like electrophoretic mobility, contains little or no lipid, and, based on its ability to bind to Concanavalin A columns, is a glycoprotein. The relationship between this Ia glycoprotein antigen and other molecular forms of Ia antigen is discussed.     

Isolation or group B streptococci defective for the type antigen Ia by transposon-mediated mutagenesis

Transposon-mediated mutability has been used to isolate the isogenic strains of the group B serotype Ia streptococci with the mutations in the genes coding for the production of capsular antigen. The transconjugants have lost the ability to bind type Ia antiserum as demonstrated by immunoblotting technique. The loss of type-specific antigen by the strains has resulted in a dramatic decrease in virulence for mice while the absorbtion indexes of transconjugants increased 2-3 fold. The mutant clones deficient in capsule expression had the increased buoyant density in the percoll gradient as compared with the parent strains of group B. The stable mutants impaired in ability to produce the polysaccharide capsule on the cell surface were obtained as a result of site-specific insertion of transposon Tn916 into the genome of group B serotype I streptococcus.     

Immunological Studies on Viral Polypeptide Synthesis in Cells Replicating Murine Sarcoma-Leukemia Virus

Antibodies to disrupted murine sarcoma-leukemia virus (MSV[MLV]) were used to study the synthesis of viral polypeptides in the transformed, virus-producing rat cell line 78A1. When cultures were labeled for 10 min with radioactive amino acids, about 9% of the total labeled proteins were precipitated with antiserum against purified MSV(MLV), and 3 to 4% were precipitated with the same antiserum after it had been absorbed with an extract from uninfected rat cells. The difference is due to the presence in the unabsorbed antiserum of antibodies to cellular proteins that are present in purified virus preparations. Intracellular viral proteins labeled with radioactive amino acids were isolated by immunoprecipitation and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The mobilities of intracellular viral polypeptides were identical to those of the purified virion. However, labeled polypeptides having electrophoretic mobilities lower than that of the major virion polypeptide, the group-specific antigen of molecular weight 31,000, were present in higher proportion in the total cell extract and in the membrane fraction than in the virion. These polypeptides appear to be of cellular origin for they were present only in minute amounts in the immunoprecipitates obtained with the absorbed serum. After a 10-min labeling period, radioactive proteins were assembled into extracellular virions rapidly for the first 4 hr followed by a slower rate. More than 2% of the total proteins of the cell labeled in a 10-min pulse were assembled into virions at the completion of a 24-hr chase. The high-molecular-weight polypeptides with the same mobilities as those detected in the immunoprecipitate of intracellular proteins were found in virions released from cells after a 10-min pulse. A larger proportion of these high-molecular-weight proteins was detected in virions released after short chase periods (30-120 min) than after longer chase periods (6-24 hr). Two possible interpretations of these data are that the high-molecular-weight cell-derived polypeptides (i) have a turnover rate higher than that of the major virion polypeptides or (ii) are cleaved proteolytically from the virions during long incubation in the culture media.     

Characterization ofShigella dysenteriae type 1 capsular polysaccharide by immunochemical methods

We have isolated the capsular polysaccharide from the strain ofShigella dysenteriae type 1 8337. The product was purified by ultracentrifugation, treated with enzymes (proteinase K, DNA-RNAase) and analyzed by immunochemical methods. Polyclonal antibodies were obtained from rabbits immunized by whole cell antigens prepared fromShigella by ultrasonic treatment and by purified capsular polysaccharide. Crossed immunoelectrophoresis, PAGE and Western blot analysis showed that this product containing mainly the polysaccharide component also contained glycoprotein and lipopolysaccharide. Double diffusion in agarose gel confirmed that the capsular preparation contained at least three antigens reacting with rabbit polyclonal antiserum.     

Low-molecular-weight Ia antigens in normal mouse serum

Low-molecular-weight Ia antigens can be detected in mouse serum. A procedure for isolating these antigens from serum in high yield is described. The Ia antigen preparation was found to be rich in carbohydrate, low in protein, and strongly bound to Concanavalin A andLotus lectins. Furthermore, the Ia antigenicity was destroyed by periodate oxidation and neuraminidase treatment, but was unaffected by pronase. These observations strongly suggest that the Ia antigens in serum are oligosaccharide in nature. Such a conclusion implies that at least some of the genes in theI region of theH-2 gene complex code for glycosyl transferase enzymes.     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.     

Expression and function of I region determinants on immunocompetent cells. I. Selective expression of I-C region determinants on immune cells.

We have examined the cytotoxic activity of anti-Iak serum and complement on various immune functions of BALB/c (H-2d) cells. Since the cytotoxic action of this antiserum on H-2d cells defines specificity Ia.7, an I-C region product, we have looked at the selective expression of this antigen. We have mainly used the in vitro anti-Lac2 response to study the cells involved in the induction and regulation of antibody. The data presented here show that Ia.7 is present on both IgM and IgG precursor B cells and in lesser amounts on plaque-forming cells. The antiserum also recognizes with less efficiency a product on specific T suppressor cells, which is possibly coded for by the adjacent I-J subregion. Both fluorescence and functional tests indicate the absence of Ia.7 on macrophages. It is also lacking on T helper cells. When we tested the antiserum on the in vitro cytotoxic responses to alloantigens, we found that neither T effector cells nor their precursors were affected.     

Biochemical characterization of Ia antigens. I. Characterization of the 31K polypeptide associated with I-A subregion Ia antigens

Procedures are presented for the preparative isolation of murine Ia antigens directly from splenocyte detergent extracts with monoclonal immunoadsorbents. Utilizing these procedures, three Ia (I-A subregion) polypeptides (alpha, 31K, beta) were isolated and their m.w. and pI values characterized. Evidence is presented that indicates that: 1) the 31K polypeptide probably does not associate with the Ia alpha and beta chain complex during the Ia isolation procedure; 2) the 31K polypeptide is not tightly bound to the alpha/beta Ia complex and can be selectively removed by freezing and thawing and by washing the Ia-immunoadsorbent with buffers containing pyrrolidinone (a polar solvent); and (3) unlike the alpha and beta chains, the 31K polypeptide is not intrinsically radiolabeled with 3H fucose and 3H glucosamine, indicating that the 31K polypeptide either contains a carbohydrate structure that is different from that of the alpha and beta chains or it is not a glycopeptide. These data suggest that although Ia antigens are probably comprised of three polypeptides in the intact cell, only two (alpha and beta) are required to maintain alloantigenic determinants.     

Synthesis of cytochrome c oxidase polypeptides in an Escherichia coli cell-free system directed by Saccharomyces cerevisiae mitochondrial DNA.

Summary Using purified yeast mitochondrial DNA as a template forE. coli RNA polymerase (holoenzyme) complementary mitochondrial RNA has been synthesized in vitro. This RNA has been used to direct a low backgroundE. coli S-30 protein-synthesizing system. The synthesis of mitochondrial polypeptides has been detected by using antiserum raised against purified cytochromec oxidase holoenzyme and shown to be specific for this antigen. The antiserum-antigen complex was dissociated and subject to SDS-polyacrylamide gel electrophoresis and the presence of 3 polypeptides of 39, 31, and 26×10³ daltons molecular weight demonstrated, which correspond to the subunits synthesized by mitochondria in whole cells which are inhibited with cycloheximide.     

Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus

The preparation of antisera to the three purified sodium dodecyl sulfate (SDS)-treated polypeptide components (VP1, VP2, VP3) of adenovirus-associated virus (AAV) type 3H is described. In immunofluorescence tests (FA), these antisera stained heat-stable antigens with distinct morphologies in cells co-infected with either adenovirus or herpes simplex virus. Kinetic studies of antigen formation showed that VP1 antiserum first stained the cytoplasm (14 hr) and later (by 18 hr) stained both cytoplasmic and intranuclear areas. VP2 antiserum stained only discrete intranuclear areas, and VP3 antiserum stained nearly the entire nucleus. All three VP antigens appeared at about the 14th hr postinfection, about 2 hr prior to the appearance of whole virion antigen. The VP antisera cross-reacted in FA with AAV types 1 and 2 (all at one-eighth of the homologous titer), but did not react with other parvoviruses, i.e., rat virus, hemadsorbing enteric virus of calves, minute virus of mice, or H-1 virus. These non-neutralizing antisera reacted specifically with SDS-treated AAV virion antigens in complement fixation and immunodiffusion tests, and antiserum prepared against SDS-treated helper adenovirus structural polypeptides reacted with adenovirus polypeptide antigens. All antisera to SDS-treated polypeptides were specific for new antigens revealed on the dissociated peptides and did not react with whole virions, whereas whole-virion antisera did not cross-react with the polypeptide antigens. These findings suggest that antigens unique to the polypeptides of AAV are revealed by SDS treatment and that these antigens can be detected in cells prior to the folding of the polypeptides into the molecular configuration they possess as virion subunits. These results also indicate that at least one AAV polypeptide component is synthesized in the cell cytoplasm.     

Demonstration of carbohydrate- and protein-determined Ia antigens by monoclonal antibodies

Ten monoclonal alloantibodies were examined by submitting each antibody to five independent tests in order to determine whether they reacted primarily with the glycoprotein or glycolipid class of Ia antigens. The tests employed were as follows: (1) the ability to precipitate an la-like protein from the cell surface as detected by SDS-PAGE; (2) inhibition by protein-la extracts free of CHO-Ia; (3) inhibition by CHO-Ia extracts free of protein-la; (4) neuraminidase sensitivity of the antigen and (5) inhibition by simple sugars. Using these tests, three of the ten monoclonal antibodies were shown to recognize a CHO-Ia antigen while seven recognized the protein class of Ia antigens. The three CHO-Ia-specific monoclonal antibodies recognized Ia specificities 2,9 and 17. Monoclonal antibodies recognizing protein-defined Ia.2 and 17 specificities were also characterized. These results imply that some Ia specificities, as defined by genetic testing, can occur both as carbohydrate-defined and protein-defined determinants.— Sugar inhibition studies showed that CHO-Ia.2 has D-glucosamine as its immunodominant sugar while CHO-Ia. 17 shows preference for a- linked galactose. Furthermore, studies with neuraminidase demonstrated that sialic acid plays a role in the antigenic determinants of CHO-Ia.9 and CHO-Ia.17. Finally, it is noteworthy that CHO-Ia.2, the private specificity of thek haplotype, appears to be expressed only on cells and not in serum. These studies clearly demonstrate the existence of the two Ia antigen classes and emphasize the complexity of the murineI region.     

Identification of the MT3 molecule using two-dimensional gel electrophoresis and alloantisera

The MT3 antigen is defined serologically as a DR supertypic specificity and is strongly associated with DR4, DR7, and DRw9. To determine whether the MT3 molecule is distinct from the DR molecule, DR4 and MT3 antigens were immunoprecipitated from 125I-labeled plasma membrane glycoproteins of a DR4-homozygous, MT3-homozygous B lymphoid cell line, Wa, and compared by two-dimensional (2-D) gel electrophoresis. The precipitates with two different anti-DR4 alloantisera and with three different mouse antibodies against human Ia monomorphic determinants gave the same 2-D gel pattern consisting of one heavy chain with a molecular weight of 34 000 and a set of light chains with a molecular weight of 30 000, indicating that these polypeptides are the components of the DR4 molecule. On the other hand, all three anti-MT3 alloantisera used precipitated an identical set of anti-MT3 alloantisera specific light chains with a molecular weight of 30 000, and one heavy chain with a molecular weight of 34 000. The pI of the MT3 light chain was more acidic than that of the DR4 light chain. The amount of MT3 light chains was much smaller than that of DR4 light chains in unlabeled plasma membrane glycoproteins. Thus, we have demonstrated directly using 2-D gel electrophoresis and anti-MT3 alloantisera that the MT3 antigen is a new human Ia molecule distinct from DR4.