Identification,properties, and gene location of a novel glycoprotein specified by herpes simplex virus 1

We report the identification of a novel herpes simplex virus 1 (HSV-1) glycoprotein reactive with type specific monoclonal antibody H1379. The monoclonal antibody reacted with two broad bands with apparent mol wt of 60K to 68K and 44K to 48K formed by infected cell lysates subjected to electrophoresis in denaturing polyacrylamide gels and electrically transferred to a nitrocellulose sheet. Early in infection the H1379 reactive protein was found in the faster migrating band. The rate of accumulation was highest late in infection and only the slower migrating form incorporates significant amounts of glucosamine. The epitopic site recognized by H1379 was not uniformly distributed among strains. Analyses of HSV-1 × HSV-2 recombinants with monoclonal antibodies to HSV-1 and HSV-2 glycoproteins mapping in the S component of the HSV genomes and marker transfer experiments indicated that the gene specifying the H1379 reactive protein maps within BamH1 fragment J to the left of gD most probably within the open reading frame designated as US4 (D. J. McGeoch, A Dolan, S. Donald, and F. J. Rixon, 1985, J. Mol Biol. 181, 1–13). The gene specifying a recently discovered HSV-2 glycoprotein designated as gG-2 (B. Roizman, B. Norrild, C. Chan, and L. Pereira, 1984, Virology 133,242–247) maps in the corresponding domain of the HSV-2 genome and marker transfer experiments suggest that the H1379 reactive protein and gG-2 are collinear. We have therefore designated the novel HSV-1 glycoprotein as gG-1.     

The use of monoclonal antibodies in demonstrating the effect of antibody heterogeneity on immune complex size

The effect of antibody heterogeneity on immune complex size was investigated by using monoclonal antibodies. Five monoclonal antibodies which bind different antigenic determinants of human serum albumin were used to produce immune complexes. When one or two different monoclonal antibodies were used, under antigen-excess, antigen-antibody equivalent, and antibody-excess conditions, hardly any immune complexes larger than mouse IgM were produced. On the other hand, in a representative experiment, when five different monoclonal antibodies were used, immune complexes larger than mouse IgM comprised 21.5% of the total antigen under antigen-antibody equivalent conditions, 18.2% under antibody-excess conditions, and 4.3% under antigen-excess conditions. These results indicate that antibody heterogeneity is one of the important factors determining immune complex size.     

Interactions between extracellular Borrelia burgdorferi proteins and non-Borrelia-directed immunoglobulin M antibodies.

Previous work showed that outer surface protein A (OspA) and OspB of Borrelia burgdorferi may occur within an extracellular multiprotein complex, which was resolved by electrophoresis as an 83-kDa major extracellular protein band. To characterize the 83-kDa band, we sequenced the N terminus of the predominant peptide in the band and examined the interaction between the associated proteins. Peptide sequence and amino acid composition comparisons showed identity with the heavy chain of immunoglobulin M (IgM). Reduction sensitivity experiments and the recognition of the band by antibodies specific for rabbit mu chain indicated that the multiprotein complex contained pentameric IgM. Immunoelectron microscopy showed that anti-mu chain antibodies and monoclonal antibodies to OspA and OspB bound to extracellular amorphous material surrounding cells. Furthermore, the Osps coprecipitated with either nonspecific polyclonal rabbit IgM antibodies or with murine monoclonal anti-human serum albumin IgM antibodies, using insoluble anti-mu chain antibody conjugates. Although the apparent 83-kDa complex was stable under conditions of chelation and concentrated salts, it was disrupted by treatment with neuraminidase. These results indicate that extracellular B. burgdorferi proteins, including OspA and OspB, interact with IgM. The association is apparently not a classic antibody-antigen interaction but may result from other mechanisms.     

IgM Reassociation In Vitro: No Influence of J Chain on the Amount of Polymers

Native IgM was reduced or reduced and alkylated in the presence or absence of urea, and the preparations were analyzed by electrophoresis in alkaline or alkaline-urea polyacrylamide gels. All methods easily showed J chain, although the J-chain pattern varied. Purification of J chain and of IgM depleted of J chain (H.L) was performed by preparative block electrophoresis of reduced IgM Reduction with mercaptoethanol of H.L. and of native IgM followed by reassociation experiments showed that H.L and native IgM had the same abilities to form polymers The reassociated polymers from native IgM sedimented in the ultracentrifuge more slowly than or at the same rate at 19S IgM (called a- and b-component, respectively). After purification of these reassociated polymers, polyacrylamide gel electrophoresis showed that the polymers contained less J chain than the native IgM. Reassociated H.L also contained a- and b-components, but polymers sedi men ting slightly faster than 19S IgM dominated (c-component). a-, b-, and c-components were also found in a native IgM lacking J chain. Reassociation studies performed on H.L added to the original amount of J chain again showed only a- and b-polymers It was concluded that J chain influences the mode of reassociation but should not be considered a requirement for the polymerization of IgM.     

Hormone control of autoantibodies to calf thymus nuclear extract (CTE) and DNA in MRL-lpr and MRL-+/+ mice

Because hormonal influences on autoimmune disease in MRL-lpr and MRL-+/+ mice have not been defined completely, we examined animals which had been castrated and implanted with the opposite sex hormone. Antibodies directed at non-DNA antigens in a calf thymus nuclear extract (designated CTE) and specific anti-DNA antibodies were increased in estrogen-treated males, testosterone-treated females, and sham-operated female controls compared to sham-operated males. Analysis by sucrose gradient ultracentrifugation revealed that gonadal hormones exerted marked differences in the distribution and nature of circulating IgM anti-CTE antibodies. Although 19 S IgM was the predominant form of anti-CTE antibodies in experimental groups showing elevated anti-CTE responses, estrogen-treated male MRL-lpr mice expressed a large additional population of anti-CTE IgM antibody released by acid dissociation of apparently cryptic complexes. An unexpected additional finding was the presence of cryptic anti-CTE IgG (7 S) in all groups of MRL-lpr and MRL-+/+ mice, revealed only in sucrose gradient analysis under acid conditions. It is suggested that sex-related factors may account, in part, for apparent differences in levels of circulating autoantibodies observed in MRL mice by influencing the degree to which autoantibody populations exist in circulating complexes.     

Human monoclonal antibodies produced in transgenic BABkappa,lambda mice recognising idiotypic immunoglobulins of human lymphoma cells

Clonal idiotypic immunoglobulins of follicular lymphomas can be isolated by somatic fusion procedures. Idiotypic IgMs (Id-IgM) were isolated from two patients and used to immunise a strain of mice, deficient in mouse antibody production and engineered with yeast artificial chromosomes (YAC) containing fragments of the human immunoglobulin (Ig) micro/delta heavy chain and kappa/lambda light chain loci. Sequence analysis showed that hybridomas prepared from spleen cells of immunised mice expressed exclusively one of the six VH genes (VH1-2) present in the YAC transgene with different D/J rearrangements, and secrete fully human monoclonal antibodies (mAb) that recognised the tumour-specific IgM proteins. Further studies of the reactivity of the monoclonal anti-human Id-IgM antibodies revealed that they are specific for the individual protein of each patient and probably react with idiotypic determinants. In one case studied, the antibody recognised specifically the lymphoma cell expressing the corresponding idiotypic IgM and lysed those cells in the presence of complement. This is the first example of a human monoclonal antibody with such characteristics and may be of further use in the therapy of patients with B cell malignancies.     

Monoclonal antibodies used to isolate IgM from chicken bile and avian sera and to detect specific IgM in chicken sera

Four hybridoma cell lines (designated M1, M10, M11, M31) were established which secrete antibody specific for chicken IgM. The specificity for the IgM heavy chain was shown by using ELISAs to screen for antibodies to IgM, IgA and IgG. Radioimmunoprecipitation tests confirmed that the four monoclonal antibodies reacted with IgM and also showed that they combined with Protein A. An immunoadsorbent was made using one (M1) of the monoclonal antibodies. IgM was purified in a single step by affinity chromatography from chicken bile and chicken, turkey and duck serum. This is the first unequivocal demonstration of chicken IgM in bile. The Ml monoclonal antibody was used in an ELISA to detect the specific chicken IgM response to the inoculation of bovine serum albumin. This anti-IgM reagent may also be used to detect the IgM response to other antigens.     

Complement activating and opsonic capacity of monoclonal antibodies raised againstEscherichia coli O111 and its rough mutant J5

Six monoclonal antibodies raised againstEscherichia coli O111 and against its rough mutant J5 (chemotype Rc) were studied. One IgG2A, one IgM anti-J5, and one IgG2A anti-O111 monoclonal antibody did not bind to lipopolysaccharides of the homologous strain, but cross-reacted with heterologous gram-negative rods in an enzyme-linked immunosorbent assay. These three monoclonal antibodies activated complement when incubated with homologous or heterologous strains, but were opsonic neither in the presence nor in the absence of complement. The other three monoclonal antibodies were directed against lipopolysaccharide of the homologous strain, but showed no cross-reactivity. The IgG3 and one IgM anti-J5 monoclonal antibodies activated complement and were opsonic only in the presence of complement. The IgM anti-O111 monoclonal antibody activated complement and was opsonic both in the presence and absence of complement. Thus, the outcome of the interaction between bacteria, antibodies, and complement is influenced primarily by whether antibodies are directed against lipopolysaccharides or against other cell wall components.     

Non-covalent association of IgM subunits produced by reduction and alkylation

Purified IgM isolated from the serum of mice bearing the transplantable plasmacytoma MOPC 104E was reduced and alkylated and then analysed by sucrose density gradient centrifugation and by sodium dodecyl sulphate—polyacrylamide gel electrophoresis. On partial reduction a mixture of IgM subunits was obtained in the absence of covalently linked 19S IgM. When examined under dissociating conditions this mixture was found to consist of disulphide-linked 7S subunits (IgM_s), small amounts of HL subunits and oligomeric IgM of a size intermediate between monomeric and pentameric IgM. In the absence of a dissociating agent and on sucrose density gradient, however, the mixture resolved into a 19S and a 7S peak. The 19S peak consisted primarily of oligomeric IgM and IgM_s with small amounts of HL subunits. Thus alkylated IgM_s and HL subunits of IgM can associate through non-covalent forces to form a molecule sedimenting at 19S, providing oligomeric forms are present. In the absence of oligomeric forms, IgM_s, HL subunits and heavy and light chains sediment at about 7S. The products of partial reduction which sediment at 7S and 19S could also be isolated by preparative polyacrylamide gel electrophoresis. When this was done J chain was absent in the former and present in the latter, raising the possibility that J chain does not disulphide bond to each of the five IgM_s subunits, constituting an IgM molecule. Thus, within cells secreting IgM, J chain would be expected to mediate the formation of an oligomeric form of IgM. Once the oligomeric structure has been assembled, then non-covalent forces between this and IgM_s subunits will cause the formation of a 19S structure, thereby facilitating the final assembly through disulphide bonds.     

Clinical validation of an antibody-capture anti-rubella IgM-ELISA

An antibody-capture IgM-ELISA using monoclonal antibodies for conjugate was subjected to clinical validation with respect to sensitivity and specificity. In 103 serum specimens, known to contain anti-rubella IgM by a sucrose density gradient method, IgM was found by the ELISA in 99 sera. In a second study, 16 out of 17 acute rubella infections were detected by the IgM-ELISA. In 17 out of 17 vaccinees, a specific IgM response could be demonstrated.

Specificity of the antibody-capture ELISA was found to be high; no interference was seen in 60 rheumatoid-factor positive sera, in 100 highly positive IgG sera or 10 sera with anti-CMV IgM, Only one out of 100 sera with heterophile antibodies showed a positive response.

In acute rubella infections, IgM was shown to be detectable from 1 to 4 days after onset of illness up to about 12 wk, with peak values at about 1 wk after onset.     

Identification and characterization of a monoclonal IgM reacting with disialylated gangliosides recognizing the CANOMAD syndrome

We reported the laboratory phenotype of a monoclonal IgM-lambda against disialylated gangliosides, in a 81-year-old man admitted to a neurological department because of the progressive development of distal paresthesias, gait unsteadiness, difficulty to walk and having falls. Serological studies revealed an IgM monoclonal protein with lambda light chain component of MGUS type. IgM level was 4?g/L. The positive laboratory studies showed high titers of IgM antibodies in excess of 1/10(5) against specific disialylated gangliosides including GD1b, GD3, GT1b and GQ1b. There was no serum IgM binding to MAG and SGPG/SGLPG. Clonality by in-house immunodot of ganglioside antibodies was demonstrated using kappa and lambda light chain specific antibodies. Light chain subtype of the anti-ganglioside antibody activity and monoclonal IgM was lambda subtype. The reactivity at high titers was against gangliosides containing the disialosyl epitope. The clinical and laboratory features have been described under the acronym CANOMAD: Chronic Ataxic Neuropathy with Ophthalmoplegia, M proteins, cold Agglutinins and Disialosyl antibodies. Administration of IVIg produced a significant neurological improvement during six years. Then the neuropathy became refractory in the IVIg and worsened in severity, a cure by Rituximab? was established. The patient died from a pneumopathy only two months later. Monoclonal IgM binding to disialylated gangliosides have high level of specificity for diagnosis of the CANOMAD syndrome.     

A monoclonal antibody with specificity for murine mu heavy chain which inhibits the formation of antigen-specific direct IgM plaques

A panel of monoclonal rat antibodies binding to mouse mu heavy chain were tested for their ability to inhibit the formation of antigen-specific plaques in the hemolytic plaque assay. Nine antibodies inhibited SRC-specific direct IgM plaques at high concentrations (greater than 20 micrograms/ml). In contrast to all others, however, one antibody inhibited these plaques at much lower concentrations (down to 0.4 microgram/ml) when added to the assay. This antibody also inhibited plaques formed by cells secreting antibodies against trinitrophenyl or phosphorylcholine determinants. IgG plaques with any of the above specificities were not inhibited. IgM secretion was unaffected by the monoclonal anti-mu antibody. Its inhibitory effect on plaque formation rather appears to be a consequence of its ability to inhibit complement dependent, IgM mediated lysis of erythrocytes. This monoclonal anti-IgM antibody therefore provides a convenient reagent to distinguish specific direct IgM plaques from indirect IgG plaques.     

Monoclonal antibodies specific for murine IgM I. Characterization of antigenic determinants on the four constant domains of the μ heavy chain

Seventeen monoclonal rat antibodies with specificities for mouse μ heavy chain recognize seven distinguishable determinants that are located on the four constant region domains. All determinants are present on secreted, intracellular and membrane bound IgM, and all but two are expressed on isolated μ heavy chain. One antibody, specific for a site in the first constant region domain, recognizes a determinant that is present on IgM of AKR, C3H/HeJ, C57BL/6J, SJL, DBA/2, BALB/c, NZB and CBA/J mouse strains, but not on IgM of A.TH, A/J and A.CA strains of mice.     

Antibody response of goldfish to a protein immunogen and to haptenic determinants

The serum antibody response of goldfish (Carassius auratus) to protein immunogen (bovine serum albumin, BSA) and haptenic determinants was studied. The goldfish antibody response was compared with rabbit antibody response to similar antigens. The goldfish received BSA only or BSA with one of three haptens coupled to it. Arsanilic acid, sulphanilic acid and para-amino-benzoic acid were used as haptens. Precipitation and passive haemagglutination were used to demonstrate the presence of antibody. Only one size of antibody could be demonstrated to all antigens (13.2S₂₀). Two populations of antibody could be demonstrated by the use of electrophoresis, and both populations of antibody reacted with BSA in fish receiving only BSA. In fish receiving BSA-hapten, the BSA antibody was found in the slower migrating population, while the haptenic antibody was found in the faster migrating population. The fish antibodies to all immunogens were poor precipitins, but gave titres similar to rabbit antibody when tested by passive haemagglutination. The specificity of fish protein and haptenic antibody is similar to rabbit antibody.     

Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]₂G_D3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]G_T3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]₂G_D3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.     

Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]₂G_D3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]G_T3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]₂G_D3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.     

Immunochemical Studies on Free and Bound J Chain of Human IgA and IgM

J chains purified from colostral IgA. polymeric (mainly dimeric) polyclonal IgA from serum, and monoclonal IgM appeared to be immunochemical identical in tests with antisera produced against the two latter antigens. None of the antisera precipitated with the native immunoglobulins, but J chain antibodies became bound to all three types of Ig polymer. On a molar basis polymeric IgA was almost twice as efficient as IgM and 17 times more efficient than colostral IgA in antibody inhibition tests. The J-chain antibodies were able to link two or more IgA dimers and two or more IgM pentamers. After incubation in large antibody excess, the sedimentation properties of most IgM molecules were altered, whereas about 40% of the IgA dimers were unaffected by the treatment. Thus, although the J chain on the whole was slightly more exposed in polymeric IgA than in IgM, it was more homogeneously available in the latter polymer After denaturation in acid urea, the J chain became markedly more exposed in all three types of Ig polymer, and they were all precipitated by the most potent antiserum. Immunochemical quantitation of J chain released after reduction with 20 mM dithiothreitol indicated that on the average there are about 2 mol of J chain per mol of polymeric IgA, and 3 to 4 mol per mol of IgM.     

On the structure of polymeric IgM

The cysteine at position 575 of the immunoglobulin mu heavy chain is thought to provide the only disulfide bonds joining the monomer subunits of mouse polymeric IgM. The importance of this cysteine in the assembly of polymeric IgM was investigated by using site-directed mutagenesis to produce mu chains with serine at position 575. Thirty percent of the secreted mutant IgM was covalently assembled polymer implying that cysteines other than Cys575 can form inter-subunit disulfide bonds. The polymeric IgM lacked J chain, mediated complement-dependent cytolysis and appeared to have a higher molecular weight than conventional IgM pentamers, as judged by sucrose gradient sedimentation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility. Electron microscopy revealed that the mutant IgM molecule contained six subunits. Wild-type IgM, while synthesized predominantly as a pentameric molecule, was assembled in at least two other forms, which were distinguished by their electrophoretic mobility. The apparently higher molecular weight forms of wild-type IgM include hexameric molecules which, like the hexameric mutant IgM, contained much less J chain that the pentameric form and were 20-fold more efficient at activating complement-dependent cytolysis.     

Identification of a low molecular weight IgM antibody with treponema pallidum specificity in sera of patients with chronic syphilis

Summary A low-molecular (8S) treponema-specific IgM antibody was isolated by means of Sephadex 200 G gel filtration and/or sucrose gradient ultracentrifugation in 78 of 4,120 sera of patients who had been identified to have had syphilis. The IgM specificity can be shown by indirect immunofluorescence using a -chain specific antiserum. The low molecular IgM (LMW-IgM) antibodies are not identical with the 19S-IgM as demonstrated in studies with gel filtration and sucrose gradient methods. They are not identical neither with 7S IgG or IgA. Neither the presence of antinuclear nor rheumatic factor could be shown in the LMW-IgM fraction. In most of the patients with LMW-IgM antibody, there existed a treponema infection of late latency.     

Neutralizing serum IgM antibodies in infections withHerpes simplex virus hominis

Forty-two patients with herpes simplex virus infections were tested for neutralizing serum IgM antibodies. A technique in which serum antibody fluorescein staining was combined with sucrose gradient centrifugation facilitated the isolation of the serum IgM fraction for the use in neutralization tests. In nearly all cases with primary infection, especially those presenting heavy clinical signs (encephalitis/meningitis) the IgM tests were positive. In one case we could detect the IgM antibodies for 11 weeks after the onset of the illness, in another case in cerebrospinal fluid samples for 6 weeks. In localized herpes infections, which were mostly due to reactivations, serum IgM antibodies could only rarely be demonstrated. Among the serologic tests used in this study (NT, CFT, IFT, ACIFT), only the CFT beside the NT (with certain reservations) can be applied for subtyping HSV serum antibodies.The work was supported by the Deutsche Forschungsgemeinschaft, SFB 31.