Search for cross-reactive idiotypes on monoclonal and myasthenia gravis acetylcholine receptor antibodies.

Myasthenia gravis patients have serum anti-acetylcholine receptor antibodies that compete with monoclonal antibodies for binding to epitopes on the human acetylcholine receptor. To investigate the presence of shared idiotypes we immunised syngeneic mice with each of ten well-characterised monoclonal antibodies, previously raised against purified human acetylcholine receptor, and tested the polyclonal antisera and seven monoclonal anti-idiotype antibodies, for binding to the antigen-combining site, to framework idiotopes, and by ELISA. The polyclonal sera were mostly directed against antigen-combining site idiotopes and cross-reacted only with monoclonal anti-acetylcholine receptor antibodies that bound to the same region on the acetylcholine receptor. In contrast, five of the seven IgM monoclonal anti-idiotypic antibodies raised, none of which demonstrated antigen-combining site specificity in solution, cross-reacted with mAbs binding to more than one region. None of the antisera showing reactivity with the antigen-combining site inhibited the binding of MG anti-acetylcholine receptor antibody.     

Heterogeneity of cross-reactive idiotypes. Serological and structural analysis of monoclonal anti-p-azobenzene-arsonate antibodies expressing major and minor idiotypes

Hybridoma-derived monoclonal anti-p-azobenzene-arsonate (ABA) antibodies were obtained from fusions of ABA-KLH primed A/J spleen cells with three different myeloma cell lines. Of the 156 antibody secreting hybridomas 24% carried the cross-reactive idiotype (CRI), which is known to be shared by 20-70% of anti-ABA serum antibodies in A/J mice. The isotypes, SDS-PAGE patterns and the partial amino acid sequences of the V-regions of one CRI negative and six CRI positive hybridoma proteins were determined. These antibodies were IgGl, kappa and IgG2b, kappa. Some idiotype carrying monoclonal antibodies appeared to be serologically identical. Although the partial VH amino acid sequences of these monoclonal antibodies showed great homology with each other and with serum antibody, several sequence variations in framework residues as well as in the first and second complementarily determining regions (CDRs) were found. The cross-reactive idiotype of the anti-ABA antibodies, therefore, exhibits structural microheterogeneity, i.e. it consists of a family of non-identical but closely related molecules as previously reported (Alkan et al., 1980; Estess et al., 1980; Marshak-Rothstein et al. 1980). Here the N-terminal sequence of the VH regions from 14 CRI+ and 8 CRI- antibodies as well as the VL regions from 11 CRI+ and 8 CRI- monoclonal antibodies are compared. Analysis of the available data demonstrated that there are pairs of hybridoma proteins (both CRI+ and CRI-) which have identical sequences for VH or VL. This suggests that there exist a minimum of 4 germ line genes coding for CRI+ VH, CRI+ VL, CRI- VH and CRI- VL respectively. In addition, CRI+ VL has always been found in association with a CRI+ VH.     

Inhibition by serum of encephalitogenic activity of myelin basic protein.

Basic protein of myelin from bovine brain (B-BPM) in Freund's complete adjuvant (FCA) is highly encephalitogenic for the guinea pig. However, when B-BPM is mixed with serum from various species it loses its encephalitogenic effect but not its immunogenic properties. When the synthetic tryptophan peptide matching residue 115-126 of human BPM is mixed with normal human serum it loses both its encephalitogenic and immunogenic effects. The time of exposure to serum necessary for complete inhibition of encephalitogenic activity of B-BPM varied: rat, horse, sheep and human sera produced their inhibitory effect immediately after mixing, whereas guinea pig serum required 8h. The capacity of serum to abrogate the encephalitogenic effects of B-BPM was not due to complete degradation of the molecule by proteinases in serum, because all animals injected with the B-BPM serum mixture gave cutaneous delayed hypersensitivity reactions to B-BPM. The inhibitory effect could be attributed to a factor in serum which is non-dialysable, thermostable at 56 degrees C, for 1 h, and present in high concentration in fetal calf serum. It may be an alpha2 macroglobulin which can act selectively on the main encephalitogenic determinant, around or within residues 115-126 of the basic protein of myelin.     

Solid-phase radioimmunoassay for detection of antibodies to myelin basic protein.

A solid-phase double-antibody radioimmunoassay was developed for the detection of anti-myelin basic protein (BP) in sera. Antigen was adsorbed to glass test tubes, reacted with rat anti-BP sera, followed by 125I-labeled rabbit anti-rat IgG. This assay was capable of detection of specific antibody at low nanogram per ml levels, was technically simple, and the results correlated well with established procedures.     

Monoclonal antibodies reactive with myelin basic protein

New monoclonal antibodies (MAbs) to myelin basic protein (BP) reveal epitopes to be in sequences 22–34, 75–82, 83–96, 118–131 and 125–131. Comparison of these results with those previously reported suggest that almost every sequence of about 10 amino acid residues may be sufficiently antigenic to make a single MAb but that certain regions are immunodominant, strong enough to make practically the same MAb repeatedly. One of these new MAbs (clone 3) has especially interesting reactivity, sharply limited to residues 75–82 in bovine and porcine BP: Lys-Ala-Gln-His-Gly-Arg-Pro. Whales presumably have the same sequence, since their BPs are fully reactive with clone 3 MAb, but all other species of BP, with known sequences of BP, have at least two changes in this sequence. Deletion of Lys⁷⁵ (as in a tryptic peptide of porcine BP) reduces reactivity with the MAb about 10-fold, whereas substitution of Ala⁷⁶ by Ser (as in all other species of BP) and either deletion of Gln⁷⁷ (as in human, monkey and rabbit BP) or His⁷⁸ (as in the guinea pig and rat BP) or substitution of Pro⁸² by Thr (as in human, monkey, rat and mouse BP) eliminates reactivity. We speculate that woodchuck and prairie dog BPs in this region closely resemble chicken BP, which has about 2% of the original reactivity. However, squirrel BP is unique, probably having only one of the changes in this region of BP, since it possesses 10–20 times the reactivity of chicken BP but still only 20–50% of the original reactivity with clone 3 MAb, a degree of reactivity not seen with any other species of BP.     

Expression of two cross-reactive idiotypes on mouse antibodies against bromelain-treated mouse erythrocytes.

Two cross-reactive anti-idiotype (Id) antibodies were previously prepared from sera of rabbits immunized with mouse monoclonal antibodies against bromelain-treated mouse erythrocytes (BrMRBC). Most of the anti-BrMRBC plaque-forming cells (PFC) were suppressed by either of the two anti-Id antibodies. The Id profiles of anti-BrMRBC PFC were almost identical among various cell populations in a strain, but different among various mouse strains. Mouse sera contained both of the Id-bearing immunoglobulins Ig, and a significant part of the Id-bearing Ig were eliminated by absorption with BrMRBC. Nude BALB/c mice were almost equal to normal BALB/c mice in the Id patterns of anti-BrMRBC PFC and in the concentrations of the Id-bearing Ig. The injections of anti-Id antibodies into suckling mice suppressed, specifically, the development of the B cells to produce the homologous Id-bearing Ig, but the injection of Id-bearing monoclonal antibodies barely affected Id expression. It is suggested that the two Id are encoded in germ-line genes of mice, and are expressed independently of each other and Id-anti-Id regulations by T cells or B cells.     

Immunoblot detection of antibodies to myelin basic protein in Alzheimer's disease patients.

Based on a suspected pathological relationship of autoimmunity in Alzheimer's disease (AD), antibodies to myelin basic protein (MBP) were investigated in the sera of AD patients, healthy controls and disease controls. As detected by the protein-immunoblotting technique at a screening serum dilution of 1:400, sera from 16 of 18 (89%) AD patients were positive for antibodies to MBP. In contrast, sera from only 7 of 90 controls (healthy adults and elderlies, Parkinson's disease patients, mentally-retarded children and Down's syndrome patients) showed a positive reaction. This approximately 11-times higher incidence of antibodies to MBP in AD patients was statistically significant (P < or = 0.0001) and may indicate an autoimmune process in the pathophysiology of the disease.     

Characterization of private and cross-reactive idiotypes associated with human antibodies to hepatitis B surface antigen.

Four mouse monoclonal anti-idiotypic antibodies (anti-Id) were generated against human monoclonal and polyclonal antibodies to hepatitis B surface antigen (HBsAg). These monoclonal anti-Id, along with a polyclonal anti-Id raised in rabbits, were used to characterize the idiotype (Id) specificity of the human antibody response to HBsAg (anti-HBs). The anti-Id reagents identified distinct private and cross-reactive Id expressed on monoclonal and polyclonal human anti-HBs preparations respectively. The anti-Id recognized both HBsAg combining site and non-combining site related private Id, and HBsAg combining site related cross-reactive Id. The Id specificities recognized by two of the monoclonal anti-Id were associated with the H chain alone, whereas two of the monoclonal anti-Id, along with the polyclonal anti-Id appeared to recognize Id determinants associated with both isolated H and L chains. These data suggest that Id heterogeneity exists within the human antibody response to HBsAg. The knowledge that Id heterogeneity exists is of importance in understanding the observed variability in the immune response during hepatitis B virus infection.     

Use of monoclonal antibodies to identify shared idiotypes on anticardiolipin and anti-DNA antibodies in human sera.

Using hybridoma technology we produced monoclonal antibodies (MoAb) to idiotypic determinants on human anti-cardiolipin antibodies purified from a patient with SLE. Hybridomas were screened by inhibition of cardiolipin binding activity of sera from patients with SLE. Seven hybridomas were selected, two of which were studied extensively. Sera from a number of patients with SLE were found to share idiotypic determinants. This cross-reacting idiotype was not detectable on anti-cardiolipin antibodies in syphilis sera. The cross-reacting idiotype was present in sera with anti-ssDNA antibodies even though some of these sera had no anti-cardiolipin antibodies. We propose that these MoAb may recognize a regulatory idiotype.     

Heterogeneity of rat encephalitogenic T cells elicited by variants of the myelin basic protein (68–86) peptide

By immunizing Lewis rats with myelin basic protein (MBP) peptide variants derived from the major encephalitogenic epitope of guinea pig (MBP(68–88) and then isolating encephalitogenic T cells from these animals, we demonstrated that the variant peptides do not elicit the same encephalitogenic T cell subsets as those induced by the wild-type peptide or by intact MBP. Rather, the pathogenic T cells differed in clonal composition as reflected by their heterogeneous responses to a panel of variant peptides and by their T cell receptor usage. Thus, molecules mimicking the MBP(68–88) autoantigen can elicit pathogenic T cell subsets without necessarily cross-reacting with T cells specific for the original autoantigen. This suggests that a more clonally diverse group of pathogenic T cells might be involved in EAE than has been apparent from studies with intact MBP or its unaltered peptides.     

Combined nasal administration of encephalitogenic myelin basic protein peptide 68-86 and IL-10 suppressed incipient experimental allergic encephalomyelitis in Lewis rats

Mucosal administration of low doses of myelin basic protein (MBP) peptide 68-86 (MBP 68-86) or anti-inflammatory cytokine IL-10 effectively prevented experimental allergic encephalomyelitis (EAE), but failed to suppress the disease if given after 7 days postimmunization (p.i.), i.e., after T cell priming had occurred. We anticipated that combined administration of autoantigen and IL-10 can treat incipient EAE. Lewis rats with EAE actively induced with MBP 68-86 and complete Freund's adjuvant received 120 microg MBP 68-86 + 200 ng IL-10 per rat per day from day 7 p.i. and for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss, and shorter duration of EAE than rats receiving MBP 68-86 or IL-10 only or PBS. EAE amelioration was associated with decreased infiltration of ED1(+) macrophages and CD4(+) T cells within the central nervous system and with decreased proliferative responses of lymph node cells, indicating that combined administration of MBP 68-86 and IL-10 induced immune hyporesponsiveness. IFN-gamma secretion as well as IFN-gamma, TNF-alpha, IL-4, and IL-10 mRNA expression by lymph node MNC was down-regulated in the treated rats. Immune hyporesponsiveness, rather than immune deviation or regulatory mechanisms, seems to be responsible for the protection of EAE after autoantigen + IL-10 administration by the nasal route.     

Blocking the encephalitogenic activity of the C-terminal part of the basic myelin protein in rabbits by treatment with serum.

Peptides HNB-92-169 and 116-169, tyr, derivatives of the C-terminal part of bovine myelin basic protein, sensitize rabbits for experimental allergic encephalomyelitis (EAE). Dissolving these peptides in heat-inactivated normal guinea-pig, human, or monkey serum immediately before emulsification with Freund's complete adjuvant (FCA) abrogated their disease-inducing activity. Dissolving them in homologous or autologous serum only slightly affected the encephalitogenicity.     

The isolation of cross-reactive monoclonal antibodies: hybridomas to streptococcal antigens cross-reactive with mammalian basement membrane

Based upon the assumption that post-streptococcal sequelae are the result of cross-reactive antibodies, hybridomas were prepared from the spleens of mice immunized with Group A type 12 streptococcal cell membranes (SCM) specifically to screen for such cross-reactive antibodies. One fusion produced a cell population displaying antibodies reactive to both SCM and glomerular basement membrane (GBM) antigens as demonstrated by ELISA technique. Ascites produced by this cell population also showed reactivity to lung basement membrane (LBM). Limiting dilution procedures have produced 15 monoclonal hybrids with both anti-SCM and anti-GBM activity. Confirmation of the cross-reactive and monoclonal nature of the antibody was accomplished by both direct and indirect competitive ELISA. These observations have established that unique cross-reactive antibody-secreting hybrid cells with reactivity to both SCM and basement membrane (BM) antigens can be isolated by standard cloning procedures.