Immune Precipitation Assays Research Articles

Structural Polypeptides of Type II Avian Adenoviruses Analyzed by Monoclonal and Polyclonal Antibodies

Polypeptides of hemorrhagic enteritis virus (HEV) of turkeys and marble spleen disease virus (MSDV) of pheasants were analyzed by immune precipitation and immunoblot assays. A total of 11 polypeptides ranging in molecular weight from 14,000 to 97,000 were detected in lysates of HEV-infected turkey cells analyzed by immunoblot assay using a polyclonal antibody against HEV. Identical patterns were observed with preparations of MSDV. Five monoclonal antibodies (MAbs) against HEV were chosen based on their virus neutralization activity and used for identification of neutralizing epitopes of these two viruses. Three MAbs precipitated a single 97,000-molecular-weight hexon polypeptide in an immune precipitation assay.

Modulation, shedding, and serum titers of the chronic lymphatic leukemia-associated antigen: characterization and clinical correlations

The fate of the common chronic lymphatic leukemia antigen (cCLLa), a leukemia-associated antigen selectively expressed by clonal cells in chronic lymphatic leukemia (CLL) was examined in 31 patients. cCLLa was detected by immune precipitation assay in extracts of metabolically labeled CLL culture cells, in CLL cell culture supernatants, and in patients' sera. In vitro shed membrane cCLLa was comparable in all patients (n = 15) on a per cCLLa-positive cell basis but was independent of cCLLa density (r = .46), absolute lymphocyte count ([ALC] r = .46) and stage (r = .44). In contrast, serum cCLLa (n = 31) correlated with absolute cCLLa-positive cell count (r = .92) and to a lesser extent with stage (r = .67), but was independent of cCLLa density (r = .47). cCLLa modulation was assessed from changes in membrane density estimated by radioreceptor assay before and after in vitro exposure to anti-cCLLa monoclonal antibody (MoAb) CLL2. Immune precipitation studies of metabolically labeled CLL cells showed that modulated cCLLa was internalized as judged by its detection within modulated cells but not in their supernatants. Intact cCLLa-CLL2 complexes were not detected within the modulated cells nor in their supernatants. Regeneration of modulated cCLLa was rapid with return to baseline density levels within 24 hours of antibody removal. Modulation was specific and depended on exposure time, medium temperature, and on antibody titer; correlated with extent of disease (versus absolute lymphocyte count, r = .79; versus stage, r = .66; n = 22); was independent of cCLLa density or affinity (r = .44 to r = .57; n = 11); and was unaffected by T cells or by monocytes (n = 14).

Differential expression of HLA-DR, -DQ, and -DP antigens on malignant B cells.

HLA class II antigens mediate interactions among cells involved in the immune response and play an important role in the process of self recognition. We made use of conventional alloantisera and six well-characterized monoclonal antibodies (MoAb) to study the HLA class II antigens on CALLA-positive malignant B cell populations and autologous normal B cell lines. Forty additional HLA class II-specific MoAb were also tested for their ability to bind to these cells. By using indirect immunofluorescence and immune precipitation assays, we find that malignant B cells often fail to express one or more of the three known types of HLA class II antigens. Cell lines with the following five phenotypes have been identified: HLA-DR+, -DQ+, -DP+; HLA-DR+, -DQ-, -DP+; HLA-DR-, -DQ+, -DP+; HLA-DR-, -DQ-, -DP+; and HLA-DR-, -DQ-, -DP-. These cell lines have been used to characterize the subregion specificity of MoAb that react with HLA class II antigens. This work confirms the existence of complicated patterns of serologic cross-reactivity between the three different types of HLA class II molecules. It also increases our understanding of the specificity of individual MoAb, thereby facilitating future investigation of the distribution and function of individual antigens. Our studies are consistent with the proposal that altered expression of HLA antigens on tumors might impair recognition of these cells by the immune system of the host, thereby contributing to the proliferation of a malignant clone.

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity.

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gka and Gkb, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent Km values for MgATP2- and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.

Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.

5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs.

Immune precipitation assays with antibodies specific for 2,2,7-trimethylguanosine (m2,2,7(3)G) have been used to study the accessibility of the 5'-terminal m2,2,7(3)G-containing caps of eucaryotic small nuclear RNAs (snRNAs) either as naked RNAs or in intact small nuclear ribonucleoprotein (snRNPs). The antibody selectively precipitates snRNA species U1a, U1b, U2, U4, and U5 from total deproteinized RNA isolated from Ehrlich ascites cells. Binding by the antibody occurs via the m2,2,7(3)G moiety of the snRNAs' caps, since complex formation with the antibody can be completely abolished by excess nucleoside m2,2,7(3)G. The specificity of the antibody is further demonstrated by the complete absence of reaction with deproteinized snRNA species U6, the 5' terminus of which does not contain m2,2,7(3)G. Most importantly, the cap structures of the snRNAs U1a, U1b, U2, U4, and U5 are also accessible for anti-m2,2,7(3)G IgGs when intact snRNPs are reacted with the antibody. In this case, snRNP species U6 is coprecipitated, suggesting that there are intermolecular interactions between this and other snRNPs. Our data demonstrate that the 5'-terminal regions of the above snRNAs are not protected by the snRNP proteins. This finding is of special interest for snRNP species U1, and is discussed in terms of a model which proposes that the 5'-terminal region of U1 participates in the proper alignment of splice junctions in eucaryotic pre-mRNAs (Lerner, M. R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224).

Antigenic cross-reactivities between mammalian hexokinases

Immunoelectrophoresis and gel diffusion techniques have demonstrated that, contrary to previous reports, antigenic cross-reactivity does exist between the three major rat hexokinase isoenzymes. Immune inhibition and immune precipitation assays performed in parallel generally failed to detect this cross-reactivity and possible reasons for this failure are discussed. The inability of the latter assay systems to detect cross-reactivity would seem to account for the discrepancies between the present results and those obtained in earlier studies.

Monoclonal antibodies against baboon endogenous virus and against host cell antigens.

Monoclonal antibodies were produced by murine hybridomas after immunization with semipurified baboon endogenous virus. In a solid-phase radioimmunoassay, two antibodies (F12-9 and B9-18) reacted with viral antigen only. The antibodies A6-8 and C9-12 also reacted with virus-producing cells but not with control cells, whereas antibodies E4-6 and D12-2 bound to virus-free cells as well. The cytofluorometry technique confirmed these results and showed a competition between antibodies A6-8 and C9-12 for binding to virus-producing cells as well as a competition between antibodies D12-2 and E4-6 for binding to virus-free human cells. An immune precipitation assay with disrupted virions indicated that antibodies A6-8, B9-18, and C9-12 were directed against the gp70 glycoprotein, and that antibody F12-9 reacted with a viral antigen with a molecular weight of 18,000. The syncytia induced in RSa cells by baboon molecular weight of 18,000. The syncytia induced in RSa cells by baboon endogenous virus could be inhibited either when antibody A6-8 or C9-12 was combined to the virus or when the RSa cells were treated with the anticellular antibody D12-2 or E4-6. These two effects were not observed with Mason-Pfizer virus. Thus, of three antibodies with specificities for viral gp70, two (A6-8 and C9-12) were directed at viral sites responsible for syncytium formation. Another antiviral antibody (F12-9) reacted with a protein of unknown function with a molecular weight of 18,000. The two anticellular antibodies were directed at similar or neighboring epitopes, which may be situated within the receptor to the virus.

Serological and biochemical analysis of the PlA1 alloantigen of human platelets.

A solid phase platelet antibody assay has been developed which rapidly and sensitively detects PlA1 antibodies. The three-step assay is performed by: (1) adhering platelets to the wheels of a microtitre plate, (2) incubating the platelets with test serum, and (3) adding radiolabelled Staphylococcal protein A which binds to the Fc domain of IgG antibodies. Immune reactions are detected by overnight autoradiography. Characterization of the PlA1 antigen was performed by using PlA1 antisera in immune precipitation assays. A 90 000 dalton molecular weight species was precipitated from PlA1 positive human and dog platelets.

Caulobacter crescentus pili: Analysis of production during development

The pili of Caulobacter crescentus are structures whose appearance is regulated during the development of the swarmer cell pole. Pili are assembled during the predivisional and swarmer cell stages, at the same time as the flagellum, and disappear as the swarmer cell differentiates into a stalked cell. Pilin is the protein which polymerizes to form the pilus. An immune precipitation assay, developed to examine the periodicity of pilin synthesis during the cell cycle, demonstrated that pilin synthesis begins in the early stalked cell and is probably completed before cell division. Thus, the entire period of synthesis occurs before the pili are clearly visible at the differentiated cell pole. Likewise, the functional stability of the pilin mRNA is relatively short, further suggesting that the protein monomers accumulate prior to assembly. Unlike the case of the flagellins, experiments with the DNA replication inhibitor hydroxyurea did not establish a correlation between the DNA replication and the onset of pilin synthesis. In addition to pilin, several other developmentally regulated proteins, including the flagellins, are reproducibly precipitated in the pilin immunoassay. Their presence in the precipitate is a specific consequence of the antipilin antibody. Analysis of the antibody preparations yielded conflicting results; electron microscopic studies with ferritin-coupled antibody and double diffusion analysis indicated no binding activity to any cell components other than pilin. However, an assay based on filter transferred preparations of electrophoresed cell proteins indicated that at least one additional class of proteins in the immune precipitate may bind pilin antibody. These results are discussed in the context of the possible formation of a discrete membrane complex in the polar region of the cell which may be involved in the regulation of spatial development in Caulobacter.

Subgenomic fragment of molecular cloned Friend murine leukemia virus DNA contains the gene(s) responsible for Friend murine leukemia virus-induced disease.

Friend murine leukemia virus (G-MuLV) is a helper-independent, type C retrovirus isolated from stocks of Friend virus complex (spleen focus-forming virus plus MuLV). In cell culture, F-MuLV has an ecotropic and NB-tropic host range and causes XC cells to fuse. When injected into newborn NIH Swiss mice, F-MuLV produces hepatosplenomegaly, severe anemia, and numerous circulating hematopoietic precursors in the peripheral blood with normal thymus and lymph nodes after 3 to 6 weeks. Recently, we molecularly cloned an 8.5-kilobase pair (kbp) form of F-MuLV DNA from which we could recover the pathogenic F-MuLV virus by DNA transfection of NIH 3T3 cells. From this molecularly cloned F-MuLV DNA, we have now subcloned in pBR322 a 4.1-kbp HindIII fragment which contains in continuity 3.0 kbp from the 3' terminus (env and c region), 0.6 kbp of the terminal repeat sequences, and 0.5 kbp from the 5'terminus of the viral RNA (genome). NIH 3T3 fibroblasts were transfected with this DNA fragment an then infected with the wild mouse amphotropic retrovirus (cl 1504-A). In cell culture, 1504-A is a helper-independent type C virus which has an N-tropic host range and does not cause fusion of XC cells. When injected into newborn NIH Swiss mice, 1504-A does not produce splenomegaly or thymic enlargement in mice held for up to 8 months. The transfection with the F-MuLV fragment and the infection with 1504-A consistently yielded virus preparations that were XC positive. From such virus stocks we were able to isolate both helper-independent and replication-defective XC-positive viruses. The helper-independent virus was shown to be a recombinant virus since it contains a gp70 molecule derived at least in part from F-MuLV and a specific gag precursor derived from 1504-A as determined by radioactive immune precipitation assays. When injected into newborn Swiss mice, the recombinant helper-independent virus caused hepatosplenomegaly in approximately 50% of the mice in 6 to 8 weeks. The histology of the diseased splenic tissue was indistinguishable from that seen in the disease caused by the whole F-MuLV. The replication-defective virus could be pseudotyped with new 1504-A virus, and this viral complex also caused the F-MuLV disease picture when the complex was injected into newborn Swiss mice. We conclude that the genetic information responsible for the pathogenicity of F-MuLV is contained within the 4.1-kbp DNA fragment, which includes env gene sequences, the terminal repeat sequences, and the c region sequences of the F-MuLV genome.

Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes

We describe here two new monoclonal antibodies that react with surface antigens of human lymphocytes. Antibody 7.2 identified a determinant on the framework region of the human Ia antigen. It was cytotoxic for all cultured B-cell lines, normal B cells, and monocytes. The antibody was not cytotoxic for normal T cells or for established T leukemic cell lines. In immune precipitation assays, the 7.2 antibody reacted with a bimolecular complex of two chains that resolved in polyacrylamide gels as polypeptides with molecular weights of 29000 and 34000 daltons. These precipitation results were analogous to those achieved with a rabbit antiserum prepared against human Ia antigens. Antibody 9.3 identified a determinant on the framework region of a T-cell antigen. It was cytotoxic for 50–80% of peripheral T cells and for 20–50% of thymocytes. It was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the 9.3 antibody reacted with a single polypeptide with a molecular weight of 44000 daltons. Due to the expression of this antigen on a limited subpopulation of human T cells, we have designated the antigen HuLyt-1.

Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p 15(E) and gp70 envelope proteins.

Hybrid cells that produced monoclonal antibodies against the envelope proteins of murine leukemia virus (MuLV) were prepared by the polyethylene glycol-mediated fusion of a mouse myeloma cell line with lymphocytes from mice immunized with allogeneic MuLV-producing leukemia cells. Twenty-three independent cell lines were cloned and inoculated into syngeneic mice for the production of ascites fluids that contained high-titered (20–75 mg/ml) monoclonal antibodies. Six serologically distinct specificities were detected when these ascites fluids were tested on a broad panel of MuLV and non-murine retra iruses. Prototype cell lines producing monoclonal antibodies that were representative of each pattern of reaction were selected for further study. In immune precipitation assays each of the prototype antibodies reacted with viral envelope proteins; three of these identified antigenic determinants on p15(E), while three others identified antigenic determinants on gp70. The p15(E) antigenic determinants were shared by a diverse panel of MuLV. One of these p15(E) antigenic determinants was also found in feline leukemia virus. The gp70 antigenic determinants, on the other hand, had a more restricted distribution and were found in only selected isolates of MuLV.

Biosynthesis and metabolism of viral proteins expressed on the surface of murine leukemia virus-infected cells

Viral proteins expressed on the surface of the Gross virus-induced leukemia E♂G2 were detected by immune precipitation assays from detergent lysates of cells that were labeled by the [ 125I]lactoperoxidase method. These proteins included gp70 and two glycosylated polyproteins that contained antigens coded by the gag gene. The 95,000-dalton polyprotein (gpP95 gag ) reacted with antisera against p30, p12, and p10, while the 85,000-dalton polyprotein (gpP85 gag ) reacted with antisera against p30 and p12, but not against p10. Neither polyprotein reacted with antiserum against reverse transcriptase. GpP95 gag and gpP85 gag contained glucosamine, but not fucose, while gp70 contained both sugars. Tryptic peptide maps of the iodinated polyproteins indicated that gpP95 gag and gpP85 gag were highly related. Viral polypeptides, having identical electrophoretic mobilities as the cell surface gpP95 gag and gpP85 gag , were also detected by immune precipitation of lysates from cells that were metabolically labeled with radioactive methionine or arginine. In kinetic studies, gpP95 gag was labeled rapidly following a 10-min pulse; in contrast, gpP85 gag was not detected until late in the chase period, suggesting that this glycosylated polyprotein was a processed product of gpP95 gag . Mapping of arginine-labeled tryptic peptides of different gag polyproteins supported this conjecture. In fact, peptide maps with isolated viral proteins further demonstrated that gpP95 gag and gpP85 gag contained peptides of p30,p15/p12, and p10. The presence of p10 peptides in both polyproteins was an unanticipated finding since the gpP85 gag did not react in immune precipitation assays with anti-p10 serum. By peptide mapping, both of the glycosylated polyproteins also appeared highly related to Pr75 gag , the intracellular precursor to virion gag proteins. Our data suggest that gpP95 gag is a glycosylated cell surface form of the primary gag precursor Pr75 gag , and that processing of the gpP95 gag results in the production of gpP85 gag .

Synthesis and distribution of H-2 antigens in preimplantation mouse embryos.

Synthesis of H-2 antigens by preimplantation mouse embryos is first detectable at the late blastocyst stage. These antigens were detected using immune precipitation assays of extracts of embryos labeled by incorporation of radioactive amino acids but not by surface iodination. Experiments using isolated inner cell massess and trophoblast vesicles indicate that it is the cells of the inner cell mass that synthesize these antigens. H-2 antigens were not detected in either early blastocysts or at earlier cleavage stages.

Identification of the Gross cell surface antigen associated with murine leukemia virus-infected cells.

The Gross cell surface antigen (GCSA) is produced by cells that are either exogenously infected with murine leukemia virus (MuLV) or are expressing endogenous MuLV genomes. In immune precipitation assays, GCSA was resolved into two serologically distinct 85,000- and 95,000-dalton viral proteins. These antigenic components are glycosylated forms of the polyprotein precursors of the MuLV internal structural proteins.

Detection of measles antibodies in cerebrospinal fluid and serum by a radioimmunoassay.

Evidence that different structural components of the measles virus may act as antigens has been provided by the serologic methods of hemagglutination inhibition hemolysin inhibition, and nucleocapsid complement fixation. Using radioiodinated measles viral antigens, and immune precipitation assay has been designed that is capable of discriminating among various reactivities to measles viral structural components in serum or cerebrospinal fluid (CSF) and of distinguishing whether IgG and IgM antibody is involved. This technique has been applied to the study of measles antibodies in CSF and sera of patients with multiple sclerosis (MS) and other neurologic diseases. From data presented here, it was found that both groups of patients have individual reactivity to measles proteins, present in CSF and serum, whereas three normal CSF samples were found not to have such antibodies. It appears that oligoclonal immunoglobulins in CSF of MS patients may be detected by this method, and one patient with MS was found to have CSF IgM anti-measles antibodies.

Detection of Measles Antibodies in Cerebrospinal Fluid and Serum by a Radioimmunoassay

Evidence that different structural components of the measles virus may act as antigens has been provided by the serologic methods of hemagglutination inhibition hemolysin inhibition, and nucleocapsid complement fixation. Using radio‐iodinated measles viral antigens, an immune precipitation assay has been designed that is capable of discriminating among various reactivities to measles viral structural components in scrum or cerebrospinal fluid (CSF) and of distinguishing whether IgG and IgM antibody is involved. This technique has been applied to the study of measles antibodies in CSF and sera of patients with multiple sclerosis (MS) and other neurologic diseases. From data presented here, it was found that both groups of patients have individual reactivity to measles proteins, present in CSF and serum, whereas three normal CSF samples were found not to have such antibodies. It appears that oligoclonal immunoglobulins in CSF of MS patients may be detected by this method, and one patient with MS was found to have CSF IgM anti‐measles antibodies.

Synthesis of Uridine Diphosphate Glucose Pyrophosphorylase during the Development of Dictyostelium discoideum

Abstract Uridine diphosphate glucose pyrophosphorylase (EC 2.7.7.9) from Dictyostelium discoideum has been purified to apparent physical and immunochemical homogeneity. The molecular weight estimates from gel diffusion and equilibrium sedimentation analyses are 390,000 and 384,000, respectively. The enzyme is polymeric apparently composed of a single monomeric species with a molecular weight of 55,000 as shown by dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity is 48,000 units per mg. The turnover numbers are 16,500 moles of UDP-glucose and 18,700 moles of glucose-1-P per min per mole of enzyme. The following Km values were obtained: glucose-1-P, 2.6 x 10-4 m; UTP, 1.1 x 10-4 m; UDP-glucose, 1.7 x 10-4 m; pyrophosphate, 4.4 x 10-4 m. UTP inhibits pyrophosphorylase activity both as a substrate at excess concentration and as a product, the latter inhibition being competitive with UDP-glucose. Pyrophosphate is a potent product inhibitor, not competitive with either glucose-1-P or UTP. Mixtures of the purified enzyme with crude extracts of cells harvested at different stages of fruiting body construction demonstrated that the assay of enzyme activity employed is an accurate reflection of the concentration of enzyme in the extracts. A high titer antiserum has been obtained which yields a single band in double diffusion assays with either purified enzyme preparations or crude extracts. Quantitative complement fixation and immune precipitation assays revealed no serological differences between the basal enzyme found in the vegetative cells and that which accumulates during fruiting body construction and demonstrated that the 10-fold increase in the specific activity of the enzyme which occurs during specific stages of fruit construction is correlated with a proportionate increase in a single antigenic component. The enzyme was also immune precipitated from crude extracts of cells labeled with [35S]methionine during the period of enzyme accumulation. Polyacrylamide gel electrophoresis of the enzyme antibody complex solubilized with dodecyl sulfate showed a single labeled peak at the position of the enzyme monomer. Thus at least part if not all of the enzyme that accumulates during fruiting body construction is composed of newly synthesized monomers.