Characterization of the antigenic determinants and host components in immune complexes from patients with secondary syphilis

Sera from patients with secondary syphilis were evaluated for abnormal levels of circulating immune complexes (IC), immunoglobulins (Ig), and complement components. Clq-solid-phase assays (Clq-SPA) that made use of monoclonal and polyclonal antibodies directed against IgG subclasses indicated that human IC were composed primarily of IgG3 and IgG1; these findings appeared consistent with subclass profile responses of electrophoretically transferred blots (Western blots) of Treponema pallidum reacted with syphilitic sera. Complexes were isolated from reactive sera by polyethylene glycol precipitation followed by either anti-Clq column chromatography or protein A-Sepharose chromatography. Although qualitative and quantitative differences were noted, all purified materials contained a treponemal polypeptide antigen with a m.w. of approximately 87,000. Subsequent analysis of this polypeptide, which was also present in purified IC from rabbits with experimental syphilis, suggests that it may represent the fibronectin receptor of the organism. The 76,000 and 66,000 materials, earlier identified in purified rabbit IC, appeared to represent C-terminal degradation products of fibronectin presumably of host origin, rather than treponemal antigens. Although fibronectin binds avidly to Clq and could represent a co-precipitable contaminant throughout the isolation procedure, anti-fibronectin antibodies in the sera of patients detectable by radioimmunoassay and the present of antibodies to 76,000 and 66,000 dalton fibronectin fragments in the globulin fractions of disassociated complexes argues against such a conclusion.     

Modification of simian virus 40 protein A.

The A protein of simian virus 40 is phosphorylated in both productive and transforming infection. The phosphorylated amino acid has been identified as serine and has been localized in a single tryptic peptide of the protein. Because the A protein synthesized in infection by A mutants is phosphorylated to the same extent and in the same peptide as in infection by wild-type virus, the functional defect of the A mutants is apparently unrelated to phosphorylation. At least three distinct forms of the A protein with apparent molecular weights of 85,000, 88,000, and 100,000 can be identified in extracts of cells infected by wild-type virus. After exposure of cells to Nonidet P-40, the 85,000- and 88,000-dalton proteins were found in varying amounts in extracts of permissive cells but not in extracts of transformed cells. This finding raised the question of the possible functional importance of the smaller proteins in productive infection. However, the virtual absence of the 85,000- and 88,000-dalton proteins in some extracts of the fully permissive CV-1 cell line indicates that a conversion of the larger to the smaller forms of the A protein is not required in significant quantity for productive infection. Furthermore, a study of extraction conditions shows that the smaller proteins are easily generated during extraction and provides an explanation for the appearance of these proteins in some cells after extraction under unfavorable conditions.     

The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immunologically distinct from the mouse NAD-dependent [corrected] bifunctional enzyme

Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2',5'-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase-cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.     

Human liver catalase: cloning,expression and characterization of monoclonal antibodies

We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.     

Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells.

During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.     

Further evidence that two unique subunits are essential for expression of hydrogenase activity in Rhizobium japonicum.

Eight strains of Rhizobium lacking hydrogenase uptake (Hup) activity and 17 transconjugant strains carrying the hup cosmids pHU1, pHU52, or pHU53 (G. R. Lambert, M. A. Cantrell, F. J. Hanus, S. A. Russell, K. R. Haddad, and H. J. Evans, Proc. Natl. Acad. Sci. USA, 82:3232-3236, 1985) were screened for Hup activity and the presence of immunologically detectable hydrogenase polypeptides. Crude extracts of these strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with affinity-purified antibodies against the two subunits of purified hydrogenase (Mr 60,000 and 30,000). Derepressed transconjugants carrying the cosmid pHU52 were Hup+ and contained detectable levels of both hydrogenase subunit polypeptides. Non-derepressed strains, Hup- parent strains, and strains carrying cosmids other than pHU52 did not express Hup activity and contained no immunologically detectable protein. These data provide further evidence for the essential involvement of the smaller (Mr 30,000) subunit in the expression of hydrogenase activity in Rhizobium japonicum and suggest that the determinants for hydrogenase subunit synthesis are present on pHU52.     

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.     

Presence of antibodies against the third intracellular loop of the m2 muscarinic receptor in the sera of chronic chagasic patients.

Patients in the chronic phase of Chagas' disease suffer from a slowly evolving inflammatory cardiomyopathy that can lead to severe cardiac dilatation, congestive heart failure, and death. This process appears to be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after infection with the parasite Trypanosoma cruzi. Recent evidence suggests that there are circulating antibodies in chronic chagasic patients that alter the physiological behavior of the heart on binding to G-protein-coupled cardiovascular receptors, including beta1-adrenergic and m2 muscarinic receptors. A 42 kDa fusion protein was constructed that contains the central part of the third intracellular loop (i3; Arg(267)-Arg(381)) of the human m2 muscarinic receptor, linked to glutathione S-transferase. This fusion protein was overexpressed in Escherichia coli and subsequently purified by affinity chromatography. Based on Western blots, the i3 loop is specifically recognized by the sera of chronic chagasic patients who have reached advanced stages of cardiac failure (according to the Los Andes classification). Analysis of the prevalence and distribution of these antibodies shows a strong association between seropositive patients and moderate (group II) to severe (group III) heart dysfunction.     

Antibodies to the scrapie protein decorate prion rods

Scrapie is a degenerative, transmissible neurologic disease of sheep and goats which occurs in the absence of any detectable host immune response. Antibodies to the scrapie agent have been produced after immunization of rabbits with either scrapie prions or the prion protein, PrP 27-30, purified from infected hamster brain. Immunoreactivity of the antisera was assessed by dot and Western immunoblots with purified prions and PrP 27-30. Antibodies raised against infectious prions were more immunoreactive with native than denatured preparations, whereas those raised against PrP 27-30 were more reactive with denatured prion preparations. As determined by second antibody-colloidal gold, both antisera were found to decorate scrapie prion rods in purified preparations. Antibodies to cellular filamentous proteins failed to react with PrP 27-30 or the scrapie prion rods; conversely, antibodies to PrP 27-30 did not exhibit immunoreactivity with cellular filamentous proteins. The monospecificity of the rabbit antiserum raised against PrP 27-30 was established by its reactivity after affinity purification. The purified antibodies reacted with PrP 27-30 on Western blots and with the prion rods. Considerable evidence indicates that the scrapie rods are aggregates of infectious prions; the findings presented here provide an immunologic demonstration that PrP 27-30 is a structural component of the prion rods.     

Purification and Partial Characterization of Immobilization Antigens from Ichthyophthirius multifiliis

ABSTRACT Ichthyophthirius multifiliis , a parasitic ciliate of freshwater fishes, was found to have surface antigens (Ag) which elieited immobilizing antibodies (Ab) when injected into rabbits. An effort was made to purify and characterize these Ag (referred to as immobilization Ag) because of their potential role in protective immunity in fishes. Mice immunized with theront cilia were used for production of immobilizing monoclonal antibodies (MAb). Hybridomas were screened by indirect immunofluorescent light microscopy and immobilization of live parasites. Six hybridomas producing immobilizing MAb were cloned. Immobilizing MAb were used to affinity purify Ag solubilized with Triton X-114 and Na deoxycholate. Two membrane protein Ag of approximately 48 and 60 kDa were identified. Immobilizing MAb failed to react with these Ag on Western blots and, conversely, MAb that reacted with the Ag on Western blots did not immobilize live organisms. These results suggest that immobilization required native conformational epitopes which were altered by Western blotting procedures. Individual MAb reactive on Western blots recognized both the 48- and 60-kDa proteins indicating the presence of common epitopes. Affinity purified Ag elicited immobilizing antisera when injected into rabbits, mice, and channel catfish.     

Purification and properties of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) from Streptomyces antibioticus.

Two forms of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) have been purified from Streptomyces antibioticus. The larger form has an M(r) of 88,000, while the M(r) of a smaller form is 47,000. Both synthetase forms are active in the formation of guanosine 5'-triphosphate, 3'-diphosphate in reaction mixtures containing methanol. Unlike the RelA protein from Escherichia coli, the synthetases from S. antibioticus do not use GDP efficiently as a substrate. Experiments using crude extracts of S. antibioticus mycelium and the 88,000-M(r) form of guanosine pentaphosphate synthetase strongly suggest that the 47,000-M(r) species is produced by proteolysis of the larger species. This conclusion is supported by the observation that antibody to either protein reacts with the other protein. Thus, the 88,000-M(r) species may be the catalytically relevant protein in vivo. Unlike the RelA protein, the 88,000-M(r) protein is not activated by ribosomes. Modest levels of guanosine pentaphosphate synthesis were observed in mycelial extracts derived from nine other actinomycetes.     

Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders

We have characterized the biochemical nature of the Ku protein, the antigen recognized by autoantibodies from certain patients with scleroderma-polymyositis overlap syndrome. From extracts of HeLa cells labeled with [32P]orthophosphate, anti-Ku antibodies precipitated a high molecular weight nucleic acid identified as DNA because of sensitivity to DNase I and resistance to RNase. From extracts of cells labeled with [35S] methionine, these antibodies precipitated two polypeptides of 70,000 and 80,000 Da. These proteins were purified using immunoaffinity column chromatography. In immunoblots most sera containing anti-Ku antibodies recognized both Ku proteins but one serum bound only to the 70,000-Da subunit. When nucleosomal segments of chromatin were used as antigen, anti-Ku antibodies precipitated dinucleosomes and larger forms of chromatin but not mononucleosomes. Thus, the Ku antigen is a novel DNA-binding protein that is at least partially exposed on nucleosomal segments of chromatin.     

A role for IgE in intestinal immunity. Expression of rapid expulsion of Trichinella spiralis in rats transfused with IgE and thoracic duct lymphocytes

In these experiments we characterize the protective antibodies in immune serum that interact synergistically with immune thoracic duct lymphocytes (TDL) to induce rapid expulsion (RE) of Trichinella spiralis in adult rats. Antibodies with both reaginic and nonreaginic activity mediated RE upon passive transfer to adult rats that had been adoptively transfused with immune TDL 7 days earlier. In serum collected 28 days after a primary infection, the most important antibody was homocytotropic IgE. Native IgE produced by active infection was isolated from 28-day immune serum by salt precipitation and/or by sequential affinity chromatography. The murine mAb A2 and B5 (anti-rat IgE) were conjugated separately to Sepharose 4B affinity columns for affinity separations. IgE was shown to be pure by gel electrophoresis and Western blots and its m.w. was estimated at approximately 190,000. As little as 183 micrograms of purified IgE could induce RE after passive transfer to adult rats. The IgE was shown to be functional by PCA activity, Ag-binding on Western blots, and skin sensitization; the latter could be blocked by pretreatment with 1R162, a rat myeloma IgE. Monoclonal IgG of any isotype transferred in amounts up to 35 mg/rat could not transfer RE to rats previously transfused with TDL cells. Immune serum collected 3 mo after the primary infection contained insufficient IgE to transfer RE, but complex non-IgE fractions were protective. The data thus demonstrate that IgE is a functional Ig in the rat capable of mediating the rejection of challenge nematode infections of the gut in the absence of other specific Ig. Secondly, other Ig may also play a role, in particular, several weeks after the primary infection when specific IgE levels in serum have declined.     

Humoral and cellular responses to homologous extracts of Nematospiroides dubius and Nippostrongylus brasiliensis

and 1986. Humoral and cellular responses to homologous extracts of Nematospiroides dubius and Nippostrongylus brosiliensis. International Journal for Parasitology 16: 601–606. Humoral and cellular responses to homologous parasite extracts were studied in C57BL mice infected with Nematospiroides dubius or Nippostrongylus brasiliensis, to determine whether these parasites induced specific immunosuppression which might facilitate their survival. IgG and IgM titres to adult, excretory-secretory, larval and egg antigens from N. dubius and adult antigen from N. brasiliensis increased progressively for several weeks, irrespective of parasite rejection. Delayed-type hypersensitivity responses to the same antigens peaked after about 2 weeks and then remained constant in N. dubius -infected mice, but declined after the rejection of N. brasiliensis. The specific lymphoproliferative responses of spleen cells to these extracts reached peak values 1–3 weeks after infection and were anti-Thy 1.2-sensitive. They then declined sharply, but remained above the levels seen in uninfected mice. These results should be considered in the interpretation of any investigations into the stable host-parasite relationship which exists during a primary N. dubius infection.     

Lack of Specificity of Commercially Available Antisera: Better Specifications Needed

The ideal antiserum for immunohistochemical (IHC) applications contains monospecific high-affinity antibodies with little nonspecific adherence to sections. Many commercially available antibodies are “affinity” purified, but it is unknown if they meet “hard” specificity criteria, such as absence of staining in tissues genetically deficient for the antigen or a staining pattern that is identical to that of an antibody raised against a different epitope on the same protein. Reviewers, therefore, often require additional characterization. Although the affinity-purified antibodies used in our study on the distribution of muscarinic receptors produced selective staining patterns on sections, few passed the preabsorption test, and none produced bands of the anticipated size on Western blots. More importantly, none showed a difference in staining pattern on sections or Western blots between wild-type and knockout mice. Because these antibodies were used in most studies published thus far, our findings cast doubts on the validity of the extant body of morphological knowledge of the whole family of muscarinic receptors. We formulate requirements that antibody-specification data sheets should meet and propose that journals for which IHC is a core technique facilitate consumer rating of antibodies. “Certified” antibodies could avoid fruitless and costly validation assays and should become the standard of commercial suppliers. (J Histochem Cytochem 56:1099–1111, 2008)     

Immunochemical detection of adenine nucleotide-binding proteins with antibodies to 5'-p-fluorosulfonylbenzoyladenosine

5'-p-Fluorosulfonylbenzoyladenosine (FSBA) is a useful reagent for the affinity labeling of adenine nucleotide binding proteins. We have developed an immunochemical approach to the detection of proteins that have been covalently modified with FSBA, which provides an alternative to the use of a radiolabeled ligand. Antibodies have been prepared against FSBA-modified glutamate dehydrogenase and purified by chromatography on ATP-agarose. The resulting affinity-purified antibodies react on Western blots only with proteins that have been labeled previously with the affinity reagent. The degree of immunoreactivity on Western blots correlates well with the extent of covalent modification as shown by studies on the modification and inhibition of the catalytic subunit of cAMP-dependent protein kinase. In crude cellular extracts, numerous proteins can be labeled with FSBA and then detected by using this approach. The labeling and subsequent detection of these proteins can be blocked by including an excess of MgATP, which competes with FSBA for nucleotide-binding sites. The labeling of specific proteins in crude mixtures is saturable, as shown by labeling studies of p56lck, a protein-tyrosine kinase that is abundantly expressed in membranes from the T lymphoma cell line LSTRA.     

Surface Coat of Meloidogyne incognita

The nematode surface coat is defined as an extracuticular component on the outermost layer of the nematode body wall, visualized only by electron microscopy. Surface coat proteins of Meloidogyne incognita race 3 infective juveniles were characterized by electrophoresis and Western blotting of extracts from radioiodine and biotin-labeled nematodes. Extraction of labeled nematodes with cetyltrimethylammonium bromide yielded a principal protein band larger than 250 kDa and, with water soluble biotin, several faint bands ranging from 31 kDa to 179 kDa. The pattern of labeling was similar for both labeling methods. Western blots of unlabeled proteins were probed with a panel of biotin-lectin conjugates, but only Concanavalin A bound to the principal band. Nematodes labeled with radioiodine and biotin released ¹²⁵I and biotin-labeled molecules into water after 20 hours incubation, indicating that surface coat proteins may be loosely attached to the nematode. Antiserum to the partially purified principal protein bound to the surface of live nematodes and to several proteins on Western blots. Differential patterns of antibody labeling were obtained on immuno-blots of extracts from M. incognita race 1, 2, and 3; Meloidogyne hapla race 2; and Meloidogyne arenaria cytological race B.     

Purification and characterization of phosphofructokinase in bovine parotid gland.

1. Phosphofructokinase (PFK) was purified from bovine parotid gland to 750-fold with the specific activity of 67.5 units/mg protein by Cibacron Blue F3GA affinity chromatography, and TSK DEAE-5PW ion-exchange and TSK G4000SW size exclusion chromatographies on HPLC. 2. On gel-filtration, molecular weight of the native PFK was estimated to 400,000. 3. PFK was a heterotetramer composed of three kinds of subunit with molecular weights of 92,000 (C-type), 88,000 (M-type) and 86,000 (L-type), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometrically, relative amounts of C-, M- and L-type subunit were 1:1:2. 4. Under the physiological conditions of fructose 6-phosphate (Fru-6-P) and ATP concentrations and pH, PFK activity was suppressed and hardly detectable. 5. Fru-6-P relieved PFK from the ATP inhibition. 6. Fructose 2,6-bisphosphate (Fru-2,6-P2) and AMP activated PFK with a reduction of S0.5 for Fru-6-P and subunit cooperativity. Fru-2,6-P2 was more effective than AMP.     

Immunodetection of spectrin antigens in plant cells

The occurrence of spectrin in plant cells was studied by immunoblotting of extracts, and its localization by immunolabelling of cells, using two polyclonal antibodies raised against spectrin from human and chicken erythrocytes. A variety of plant cells were studied. The two antibodies gave the same results on blots as well as on cells. Western blots of extracts showed weak immunolabelling at 220 kD, where spectrin can be expected, but bands at 85 kD stained more heavily. Because the latter bands were also seen on blots with commercially purified spectrin, we conclude that they were breakdown products of spectrin. Native plant extracts on blots from IEF gels showed a band at pI 4.8, where the purified animal spectrin is also found. Immunolocalizations done on whole cells, PEG-, BMM-, or cryo-sections gave similar data. In most cells the labelling was localized predominantly at the plasma membrane, especially of thin-walled cells. Labelling was also seen in the periphery of a particular class of organelles, probably plastids. This labelling was tissue specific in maize somatic embryos. During carrot somatic embryogenesis cytoplasmic labelling was observed depending on the developmental stage. Many cells with cytoplasmic labelling also had stained nuclei. Labelled nuclei had more condensed chromatin than non-labelled nuclei.     

Two active forms of RD-114 virus DNA polymerase in infected cells.

Two forms of DNA polymerase are present in RD-114-infected human, dog, and mink cells, but are not detectable in uninfected cells. The two enzymes are indistinguishable catalytically and immunologically, but differ with respect to molecular weight and elution position from (dT)12-18-cellulose and phosphocellulose. The large enzyme (equivalent 95,000 daltons) is found in the infected cells, but not the virions produced by these cells. The virions contain only the smaller enzyme (equivalent 70,000 daltons). The larger form may represent a mammalian viral equivalent to the beta subunit of avian RNA tumor virus DNA polymerase.