Serological activity against galactosyl-alpha(1-3)galactose in sera from patients with several kinetoplastida infections

Using rabbit erythrocyte-derived neutral glycosphingolipids enriched for a defined ceramide pentasaccharide as antigens, we have detected elevated anti-galactosyl-alpha(1-3)galactose (anti-G alpha G) antibody values in patients with American cutaneous leishmaniasis (ACL), chronic Chagas' disease, and Trypanosoma rangeli infections compared with normal subjects or with patients suffering from any of 15 other infectious diseases. The specificity of the G alpha G antibodies was determined by inhibition enzyme-linked immunosorbent assays, which revealed that several alpha-galactosyl- but not beta-galactosyl-bearing sugars blocked absorption of G alpha G antibodies to the specific antigen used. G alpha G antibodies were mainly distributed between immunoglobulin classes G and M in three Kinetoplastida infections studied, with a lower increase in reactivity detected in immunoglobulin A. Absorption of highly reactive G alpha G antibodies with purified murine laminin and nidogen, two basement membrane proteins, almost abolished G alpha G reactivity, suggesting the identity of anti-G alpha G with laminin and nidogen antibodies previously reported as elevated in Kinetoplastida infections. In ACL, G alpha G antibodies were detected in 71% of patients having skin lesions with a clinical evolution time of 0.5 month. This percentage increased with the time of evolution of skin lesions, reaching 93% in lesions older than 3 months, and tended to decrease inversely to the induration diameter in the skin leishmanin test. It is proposed that similar epitopes may exist on kinetoplast protozoa and that the determination of G alpha G antibodies may be a highly sensitive assay for the detection of humoral responses to Kinetoplastida infections.     

Monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus

Neutralizing monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus (LACV) were prepared. Two antibodies immunoprecipitated the 120 kDa virus attachment protein for vertebrate cells, G1, while five immunoprecipitated the 35 kDa G2 protein, whose function is currently unknown. Two monoclonal antibodies were obtained that specifically precipitated both G1 and G2 from [35S]cysteine labeled LACV infected cell lysates. The G2 specific monoclonal antibodies had high neutralizing titers when assayed in mosquito cells but limited ability to neutralize virus in mammalian cells. The G1/G2 specific antibodies neutralized virus infectivity in both vertebrate and invertebrate cells at high titers. These results suggest that G2 is involved in the interaction of virus with mosquito cells and that G1 and G2 may share a common structural epitope relevant to their role as attachment proteins in vertebrate and mosquito cells. Monoclonal antibodies directed against G2 or G1/G2 have not previously been reported and should be useful tools for characterizing the biological functions of these molecules in the divergent micro-environments of vertebrate and invertebrate hosts.     

Characterization of monoclonal antibodies with specificity for DNA and the synthetic polypeptide antigen (T,G)-A-L

Because of evidence for structural similarity of variable region genes of anti-DNA and anti-(T,G)-A-L antibodies, polyspecific interactions of monoclonal anti-DNA and anti-(T,G)-A-L antibodies were investigated. Of 20 monoclonal antibodies from C57BL/10 mice with (T,G)-A-L binding, two bound DNA as determined by ELISA. In contrast, two of five anti-DNA monoclonal antibodies from MRL-lpr/lpr mice bound (T,G)-A-L. For both sets of antibodies, antigen binding was shown to be the activity of the same antibody by cross-inhibition studies. To determine whether such polyspecific antibodies were expressed during autoimmune disease, sera of autoimmune MRL-lpr/lpr mice were tested for anti-(T,G)-A-L activity. This analysis demonstrated minimal elevations of anti-(T,G)-A-L in comparison to BALB/c controls. These studies thus confirm predictions about the binding activity of anti-(T,G)-A-L and anti-DNA antibodies based on structural analysis of variable region genes. They further indicate, that while anti-(T,G)-A-L and anti-DNA antibodies may have overlapping specificity, polyspecific antibodies of this kind are not preferentially expressed during autoimmunity.     

Serum antibodies to structural proteins of Hantavirus arise at different times after infection.

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of serum antibodies against group-specific epitopes of the glycoproteins (G1, G2) and nucleoprotein (NP) of the genus Hantavirus. This assay was used to study the kinetics of the development of serum antibodies after natural infection with Puumala-like virus in humans. To this end a panel of 34 serum samples collected from individuals at different times after natural infection was tested by the ELISA. The samples were also tested for specific IgM and IgG levels against Puumala-like virus, which provided confirmatory data about the presumed timing of infection. It was shown that serum antibodies against the G1 epitope were present in the acute and early convalescent period just before antibodies to the NP epitope could be demonstrated. In contrast, antibodies to two G2 epitopes were present not earlier than in the convalescent and late convalescent period. Since all these categories of antibodies seem to persist for long periods, antibodies against the G1 epitope and the NP epitope may be of specific diagnostic value. Furthermore, levels of G1-specific antibodies and antibodies to either NP or G2 may allow estimation of the time elapsed following initial infection.     

Variable regions of antibodies to synthetic polypeptides--III. Antibodies arising in response to administration of anti-idiotope.

Antibodies elicited by the synthetic polypeptide antigen (T,G)-A-L are directed against two distinct epitopes. The majority of antibodies bind to a GT containing epitope and bear an idiotope defined by monoclonal antibodies I7 and I9. In this study, we have examined the effect of in vivo administration of the I7 and I9 antibodies to mice. Administration of anti-idiotope elicits anti-(T,G)-A-L antibodies in all strains of mice tested, including genetic non-responders to (T,G)-A-L. These antibodies bind to GT and express the idiotope. Additionally, idiotope expressing antibodies that fail to bind to antigen are also produced. Monoclonal anti-(anti-idiotope) antibodies were made. One antibody bound to (T,G)-A-L, the other did not. Sequence analysis was performed and the V-regions of (T,G)-A-L binding antibodies were compared to those of the antibody that failed to bind antigen. Both sets of antibodies are derived from the same germline V-genes as the anti-(T,G)-A-L antibodies. These results have implications for understanding the nature of network regulation of the immune system and for those attempting idiotypic vaccination.     

Fine specificity and idiotypic expression of monoclonal antibodies directed against poly(Tyr,Glu)-poly(DLAla)--poly(Lys) and its ordered analogue (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys).

In order to study the repertoire of poly(Tyr,Glu)-poly(DLAla)--poly(Lys) [(T,G)-A--L] specific antibodies, monoclonal antibodies were prepared by fusing myeloma cells with spleen cells from C3H.SW mice immunized with (T,G)-A--L and boosted with (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys)](T-T-G-G)-A--L]. Eleven clones which secreted homogeneous antibodies were obtained. In general, two families of monoclonal antibodies were detected: those which bind exclusively (T-T-G-G)-A--L and those which bind both (T-T-G-G)-A--L and (T,G)-A--L. Analysis for idiotypic expression revealed that only two antibodies (clones no. 103 and 160), which were found to be similar in their fine specificity, cross-reacted with antibodies against the major idiotypes of (T,G)A--L specific antibodies. Guinea-pig antibodies against clone no. 160 reacted with the polyclonal (T,G)-A--L specific antibodies, whereas antibodies against 103 monoclonal antibodies did not react with C3H.SW anti-(T,G)-A--L antibodies, but did cross-react with four other monoclonal antibodies. It appears that the idiotypic determinants expressed on polyclonal (T,G)-A--L specific antibodies are heterogeneous, and consist of at least two serologically different idiotypes detected by clones no. 103 and 160.     

Antibodies to Deamidated Gliadin Peptides: An Accurate Predictor of Coeliac Disease in Infancy

Immunoglobulin G antibodies against deamidated gliadin peptides are now known to have diagnostic accuracy comparable to tissue transglutaminase and endomysium autoantibodies in patients with coeliac disease. However, little is known about their predictive value in infants with a suspected gluten enteropathy. We tested whether deamidated gliadin immunoglobulin G antibodies are more reliable than traditional tests for coeliac disease screening in infancy. Sixty-five children under 2 years of age (42 with malabsorption, 23 controls) were tested for deamidated gliadin immunoglobulin G, tissue transglutaminase and endomysium immunoglobulin A, and gliadin immunoglobulins A and G . Thirty-seven of the 42 children with malabsorption had deamidated gliadin antibodies, associated with tissue transglutaminase and endomysial antibodies in 33, and with gliadin immunoglobulins A and G in 21 and 29, respectively. Intestinal biopsy was performed in 34 of the 37 children positive for deamidated gliadin antibodies. Thirty-two/34 showed villous atrophy consistent with coeliac disease, while the remaining two had a Marsh 1 and a normal mucosa, respectively. Only gliadin immunoglobulins A (4.3 %) and G (39.1 %) were found in controls. The sensitivity of deamidated gliadin, tissue transglutaminase and endomysial antibodies for coeliac disease was significantly higher than that of gliadin immunoglobulins G and A. High titre deamidated gliadin antibodies correlated with severe intestinal damage. Deamidated gliadin antibodies showed a higher diagnostic accuracy for coeliac disease than gliadin antibodies in infancy. High titre deamidated gliadin antibodies predict a severe gluten-dependent duodenal damage.     

Cross-reactive,serotype- and monotype-specific neutralization epitopes on VP7 of serotype G3 and G5 porcine rotavirus strains

Summary VP7 specific monoclonal antibodies raised against serotype G5 porcine rotavirus strains isolated in Venezuela showed either a serotype G5- or monotype-specific pattern of reactivity by neutralization against a panel of 53 group A rotavirus isolates representative of all established G serotypes. Monoclonal antibodies raised against two G3 porcine strains were either specific for a subset of porcine G3 strains or reactive with another subset of porcine G3 strains and with most G5 strains. Neither were reactive with G3 strains from other species. Analysis of neutralization resistant mutants selected with these monoclonal antibodies indicated that epitopes defined by cross-reactive, serotype-and monotype-specific monoclonal antibodies overlap functionally and that binding and neutralization by these antibodies depended on specific amino acid residues in the region A or C of VP7. Results indicate that a high degree of monotypic variation occurs among G5 and G3 porcine rotavirus strains and the existence of at least one common epitope shared by G5 and G3 porcine strains, in the major neutralization domain of these VP7s.     

Specific immune response to vaccination with an inactivated flue vaccine depending on prevaccine status and age of the person vaccinated

Specific antibody immune response to vaccination with commercial inactivated trivalent vaccine A(H1N1) + A(H3N2) + B (IgA, IgG, IgG, subclasses G1, G3, G4, and accumulation of antiCD8) was studied in subjects aged 20-95 years. The initial immune status before vaccination is significant for a positive immune response to the vaccine. Subjects responding to immunization by an increment in specific IgG had a much lower prevaccination level of these antibodies than subjects without these Ig conversion. Antibody immune response to vaccination depended on patient's age. All vaccinees aged 20-25 years developed an increment in IgG to at least one of influenza antigens used. Specific postvaccinal immune response to inactivated influenza vaccine included accumulation of G1, G3, and A antibodies, but not G4 or E antibodies. This latter fact suggests the absence of sensitizing effect of vaccination. In elderly subjects an increment in G1, G3, and A antibodies may not involve an increase in the total level of IgG. In part of elderly subjects secretion of specific antibodies was observed in the presence of increased concentration of antilymphocytic antibodies (antiCD8), indicating a possibility of autoimmune reactions in subjects of this age after injection of inactivated influenza vaccine.     

Guanine polynucleotides are self‐antigens for human natural autoantibodies and are significantly reduced in the human genome

In the course of investigating anti‐DNA autoantibodies, we examined IgM and IgG antibodies to poly‐G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20‐mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo‐nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti‐G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170 000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain‐associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti‐G20 antibodies; so natural anti‐G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti‐G20 antibodies in human health and disease and of runs of G20 in the human genome.     

Detection of IgM and IgA HIV antibodies after removal of IgG with recombinant protein G

Indirect assays for IgM and IgA antibodies often lack sensitivity and specificity due to interference from IgG antibodies. To overcome this problem we have developed a simple procedure using recombinant protein G coupled to agarose beads to remove the interfering IgG. A series of HIV seroconversion panels was tested by IgM and IgA immunoblot after protein G treatment in order to evaluate IgG removal and to study appearance of IgM and IgA antibodies in early HIV infection. Protein G treatment removed 99.9% of the IgG and reduced IgG anti-HIV titers of over 1/100,000 to undetectable levels. Both IgM and IgA HIV antibodies were detected as early in seroconversion as were IgG HIV antibodies. IgA HIV antibodies persisted for a longer period of time, reacted with more HIV proteins, and showed more intense staining than IgM HIV antibodies.     

Antigenic analysis of Punta Toro virus and identification of protective determinants with monoclonal antibodies

Hybridomas producing monoclonal antibodies to the three major structural proteins of Punta Toro virus (PTV) were established by fusion of spleen cells with Sp2/0-Ag-14 mouse plasmacytoma cells. Thirty-six independently derived monoclonal antibodies were evaluated in neutralization, hemagglutination inhibition, and ELISA assays and the isotype, antigen specificities, and cross-reactivities were determined. These antibodies were also assessed for their ability to provide protection in a murine model. Both G1- and G2-specific antibodies were obtained which neutralized virus infectivity in vitro and inhibited hemagglutination, whereas nucleocapsid-specific antibodies exhibited neither activity. All of the anti-G1 antibodies were PTV-specific, whereas anti-G2 and anti-nucleocapsid antibodies exhibited varying patterns of cross-reactivity with heterologous phleboviruses. All of the G1-reactive monoclonal antibodies, which bound to epitopes in two distinct topological sites as determined by competitive binding assays, provided efficient protection to both immunocompetent and immunosuppressed mice. In contrast, of the 23 G2-reactive antibodies, only 8 were able to protect immunocompetent mice and only one was able to protect immunosuppressed animals. The degree of protection achieved in vivo did not correlate directly with the neutralization titers determined in vitro.     

Characterization of glycoproteins of viruses causing hemorrhagic fever with renal syndrome (HFRS) using monoclonal antibodies

Viruses causing hemorrhagic fever with renal syndrome (HFRS) encode two glycoproteins, G1 and G2. For determination of the biological functions of these glycoproteins, we isolated 15 hybridomas secreting monoclonal antibodies directed against the glycoproteins of the B-1 and Hantaan viruses (HV). From results of neutralizing and hemagglutination inhibition (HI) tests, and studies on the antigenic reactivities of the antibodies with other HV-related viruses by immunofluorescence, we classified these hybridoma clones into two groups producing antibodies to the G1 proteins of the B-1 virus, six groups producing antibodies to G2 proteins of the B-1 virus, and four groups producing antibodies to the G2 protein of HV. Of the antibodies to G2 produced by 12 clones, groups A and B had high HI activity with HV-related virus cross-reactivity and moderate neutralizing activity, group C had moderate HI activity with virus specificity but low neutralizing activity, group G had high neutralizing activity and low HI activity, and five other groups had little or no HI or neutralizing activity. Group A reacting with G1 protein had low level of both neutralizing and HI activity, while group B had no HI activity. One clone of monoclonal antibody had high neutralizing activity and no HI activity, but it did not react with either polypeptide by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by the immunoblotting method. These data suggest that both glycoproteins are the targets of neutralizing antibodies. Furthermore, the results indicate that the antigenic determinants with hemagglutination activity are mainly on the G2 protein, and that the domains related to neutralizing activity and to HI activity are separate.     

Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase

The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 micrograms enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with 125I-protein G, the antibodies were found to selectively bind to hormone-sensitive lipase and to a smaller extent to two minor contaminants, possibly proteolytic fragments of the lipase. The amount of 125I-labelled protein G bound to the lipase on the blot was quantitatively related to the amount of enzyme protein down to the detection limit 10 ng.     

A new assay uses monoclonal anti-Rh(D) antibodies to determine rheumatoid factor specificity: reactivity to a monoclonal antibody of the Gm allotype G3m(21) is more frequent in rheumatoid patients negative for G3m(21)

A new method has been developed to determine the specificities of polyclonal rheumatoid factors (naturally occurring antibodies which react with human Fc gamma) (RF) found in sera from patients with rheumatoid arthritis. In this method, monoclonal anti-Rh(D) antibodies of known IgG isotype and allotype are bound to erythrocytes and then act as the target IgG antigen for RF in a direct haemagglutination test. Using two monoclonal anti-D antibodies of the IgG3 isotype and G3m(21) allotype, which were cloned from different donors, we found that a large number of rheumatoid sera reacted with both these G3m(21) proteins. In contrast reactivity of rheumatoid sera with polyclonal anti-D of the G3m(21) allotype in the direct haemagglutination test was rare. A strong correlation was found between reactivities to both G3m(21) monoclonal anti-D antibodies but not with a monoclonal anti-D antibody carrying the alternative allele, namely G3m(5). Haemagglutination inhibition experiments using human paraproteins of known IgG isotype and allotype provided some additional evidence that this method can detect RF with specificity for the G3m(21) allotypic determinant or a related allotypic determinant in polyclonal rheumatoid sera. When each patient's autoantibody response was related to their Gm phenotype, we found that the frequency of reactivity for G3m(21) monoclonal anti-D antibodies was significantly greater in patients negative for G3m(21) than in patients positive for the G3m(21) allotype. IgM preparations from patients' sera were dissociated at acid pH but no 'hidden' antibodies were found. We suggest trans-placental sensitization as one of several possible interpretations of this finding.     

Binding Properties of Human Anti-DNA Antibodies to Cloned Human DNA Fragments

The DNA-anti-DNA antibody immune complexes were isolated from plasma of systemic lupus erythematosus (SLE) patients and DNA fragments separated from immune complexes were subjected to molecular cloning. The resulting recombinant DNA clones showed a molecular size of 37-79 base pairs, a high guanine and cytosine content, high frequencies of CpG dinucleotides, and palindromic sequences, and also clusters of G + C- and A + T-rich segments. These clones hybridized randomly to total human DNA. The reactivity of dsDNA antibodies, both monoclonal and polyclonal, from SLE was examined with a cloned SLE antigen DNA. A competitive inhibition assay showed that human monoclonal antibodies had at least one magnitude higher affinity to the cloned DNA than to the native DNA fragments. In order to characterize the factors that were recognized by antibodies, human G + C-rich and also A + T-rich 100 bp DNA fragments were cloned, and their base sequences determined. The antibody showed a higher affinity to the G + C-rich DNA fragment (71% G + C) than to the A + T-rich DNA fragment (46% G + C). When cytosines in CpG doublets in G + C-rich fragments were methylated (mCpG), the reactivity increased up to 100-fold. The native anti-DNA antibodies from SLE patients also showed preferential binding to G + C-rich fragments. These observations suggested that human anti-dsDNA antibodies may recognize some unique structures around the G + C regions or G + C clusters of DNA.     

Serological differentiation of the potato-cyst nematodes Globodera pallida and G. rostochiensis: II. Preparation and characterization of species specific monoclonal antibodies

Hybridomas producing antibodies which react with thermostable protein antigens isolated from the potato cyst nematode species Globodera rostochiensis (TSRoP) and G. pallida (TSPaP) were isolated. Three of the isolated hybridomas (WGP 1, WGP 2 and WGP 3) produce antibodies that react with preferent affinity with protein antigens isolated from G. pallida, and two (WGR 11 and WGR 12) produce antibodies which bind preferentially to G. rostochiensis. Binding constants were determined to quantitate the differences in affinity of WGP 1, WGP 2, WGP 3, WGR 11 and WGR 12 for the protein antigens from both nematode species, and to asses the similarity in affinity for either protein antigen with respect to the other non-specific antibodies. In immunoblotting experiments a binding could be demonstrated, for most antibodies, to two thermostable proteins with apparent molecular weights of 20.6/20.8 kD for G. rostochiensis and 20.5/21.0 kD for G. pallida. the reactivity of the monoclonal antibodies with thermostable protein antigens from other common cyst nematodes was also investigated. All monoclonal antibodies, which are not specific for TSRoP or TSPaP, bind to thermostable proteins of these cyst nematode species. The use of some of the isolated monoclonal antibodies to improve the diagnosis of potato cyst nematodes in soil samples is discussed.     

Enzyme-linked immunosorbent assay for determination of antibodies to the envelope glycoprotein of rabies virus.

The envelope glycoprotein G of rabies virus induces neutralizing antibodies, which are important in protection against rabies. This protein was solubilized from purified virus and isolated by differential and sucrose density gradient centrifugation followed by high-performance liquid chromatography. Conditions for solubilization and purification of G were optimized by using immunoblotting and enzyme-linked immunosorbent assay techniques. The reaction with conventional antisera and monoclonal antibodies indicated that purified G protein was essentially devoid of internal viral proteins. Microdilution plates were coated with purified G protein, and sera from humans vaccinated against rabies were tested for the presence of antibodies. Results were compared with those of the rapid fluorescent focus inhibition assay, which is the standard neutralization assay for antibodies to rabies virus. The results of this comparison indicate that the enzyme-linked immunosorbent assay for G is a reliable and simple alternative to the neutralization test.     

Specificity of genes controlling immune responsiveness to (T,G)-A--L and (Phe,G)-A--L

Mice possessing the H-2b haplotype are high responders to the cross-reactive antigens (T,G)-A--L and (Phe,G)-A--L whereas mice with the H-2k haplotype respond only to (Phe,G)-A--L. On the level of cross-immunization we have demonstrated that either (Phe,G)-A--L or (T,G)-A--L primed high responder C3H.SW (H-2b) mice could be boosted with both antigens. On the other hand, low responder C3H/DiSn (H-2k) mice which were primed to (Phe,G)-A--L and thus possess (T,G)-A--L specific antibodies, could not be boosted with (T,G)-A--L to mount a secondary response. Only (Phe,G)-A--L primed and boosted H-2k mice produced high levels of (T,G)-A--L reactive antibodies. Furthermore, the binding of the anti-(Phe,G)-A--L antibodies of either C3H/DiSn or C3H.SW mice to 125I-(T,G)-A--L was better inhibited by guinea-pig anti-idiotypes than the binding of C3H.SW anti-(T,G)-A--L antibodies which are the homologous idiotypes (T,G)-A--L was found to be an equally efficient tolerogen in both high and low responder mice. Thus, when C3H.SW and C3H/DiSn mice were injected with a tolerogenic dose of (T,G)-A--L and then immunized with (Phe,G)-A--L, they were found to be tolerant to (T,G)-A--L antigenic determinants, since they produced only the unique antibodies to (Phe,G)-A--L. These results suggest that the H-2 linked Ir genes controlling antibody response to (T,G)-A--L are not involved in the induction of tolerance to (T,G)-A--L.