Xenogeneic antibodies with apparent public idiotypic specificity for anti-Ia.7 antibodies are directed in part against V kappa 21D and E subgroup marker

Public idiotypes (IdX) expressed on monoclonal antibodies (mAb) against a monomorphic alpha-chain determinant of the I-E molecule (Ia.7 epitope cluster I) have been studied by using xenogeneic anti-Id reagents derived from pig, rabbit, and rat. IdX+ anti-Ia.7 mAb were recently demonstrated to be structurally related by a high frequency expression of the V kappa 21E light chain subgroup. This raised the question of whether V region determinants of the IdX were related to V kappa 21E sequences or whether they were unique to hypervariable regions of Ia.7 binding antibodies. To clarify this question, the possible association between the expression of the public Id (IdX(s)Ia.7) and the presence of V kappa sequences (V kappa 21E and/or J kappa segment) was examined. The reactivity of the anti-Id reagents with a random panel of 28 myeloma products (each containing a light chain from one of the different V kappa 21 subgroups) was studied by assaying the ability of these mAb to inhibit the binding between the anti-Id and anti-Ia.7 mAb. This analysis demonstrates that what has previously been defined as IdX Ia.7 includes determinants shared by V kappa 21E and V kappa 21D light chain V regions. The structures recognized are expressed irrespective of the J kappa segment. In addition, this study demonstrates interspecies variation in immune responses to such V kappa 21E antigenic determinants. Additional IdX components are found on anti-Ia.7 mAb but not on other V kappa 21E or D proteins. Thus V region subgroup considerations have crucial implications for Id characterization. In addition, this work describes the first division of the V kappa 21 subgroup into component parts by a mAb.     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.     

Relationships between epitopes on IgE recognized by defined monoclonal antibodies and by the FC epsilon receptor on basophils

The present study investigated whether the sites on the FC region of the IgE molecule, recognized by different anti-IgE monoclonal antibodies (mAb), are identical to those recognized by the Fc receptor (Fc epsilon R). The anti-IgE mAb recognize different clusters of epitopes on the Fc region of IgE and could interfere to different degrees with the binding of IgE to mast cells and basophils, but still recognized cell-bound IgE. Analysis of the stoichiometry and affinity binding of 125I anti-IgE mAb Fab' to free IgE have revealed that anti-IgE mAb of one group (51.3) recognized three repetitive determinants on the IgE Fc portion, and another group (95.3) recognized only one determinant. When these stoichiometric studies were performed with cell-bound IgE, it was found that only one of the sites recognized by 51.3 mAb was involved in the Fc epsilon R binding site. On the other hand, the site recognized by 95.3 mAb was not the Fc epsilon R binding site. Such findings establish mAb 51.3 as a useful tool for isolating the IgE peptides involved in the binding site to the receptor.     

Autoantibodies to thyroglobulin are encoded by diverse V-gene segments and recognize restricted epitopes.

Murine experimental autoimmune thyroiditis has been used as a model for human autoimmune thyroiditis. Experimental autoimmune thyroiditis is induced in mice by immunization with mouse thyroglobulin (Tg) in CFA. To characterize the antibodies to this autoantigen, we have studied the binding specificities and determined the nucleotide sequences of monoclonal anti-Tg antibodies. The specificities of the mAb for determinants on Tg varied extensively. Seven of 16 mAb showed reactivity to only mTg, 4 reacted to Tg from more than one species and four reacted to a variety of Ag. Many of the mAb were competitively inhibited by thyroid hormones, suggesting that they recognize the hormonogenic sites on the Tg molecule. The mAb could be divided into at least seven reactivity patterns based on reciprocal competitive inhibition studies, indicating that mTg contains at least seven antigenic regions. DNA sequence analysis of the mAb showed that a large number of V region gene segments encoded the H and L chains. No evidence for preferential use of any V region family or gene segment was found. Gene segments from the VH 7183, Q52, J558, and VH10 families were used by heavy chains, and the V kappa 1, 4, 8, 9, 19, and 21 families were used by kappa-chains. The results indicate that the antigenic epitopes on mTg elicit a very diverse autoantibody response that is derived from a large number of V region gene segments. Many of these autoantibodies show specific reactivity with mTg indicating they recognize species specific epitopes. The results suggest that clonal deletion of autoreactive Ab to certain self-epitopes may not occur.     

Monoclonal antibodies to rabbit and pig zonae pellucidae distinguish species-specific and shared antigenic determinants

Monoclonal antibodies against rabbit or porcine zonae pellucidae (ZP) demonstrate species-specific and shared antigenic determinants. In addition, these antibodies are used to characterize the biochemical nature of these determinants. All of six monoclonal antibodies developed against porcine ZP react with porcine but not with rabbit ZP. Only one of seven monoclonal antibodies developed against rabbit ZP cross-reacts with porcine ZP. None of these antibodies recognized antigens associated with other tissues tested. High-resolution, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by immunoblotting was used to demonstrate that the cross-reactive antibody recognizes an antigenic determinant which is associated with the major low molecular weight glycoprotein of both the pig and rabbit ZP. Since this antibody recognizes all charge species of this glycoprotein, it is apparent that the antigenic determinant recognized by this antibody involves protein. Further studies demonstrate that proteolytic digestion of ZP will destroy the antigenic determinant while glycosidic digestion of ZP has no effect on antibody binding. Although polyclonal antibodies to this glycoprotein inhibit sperm from binding to the zona pellucida, this monoclonal antibody does not affect sperm binding. None of the species-specific antibodies recognize ZP glycoproteins following 2D-PAGE. This is a property typical of antibodies directed against conformational antigenic determinants. The presence of common as well as unique zona antigenic determinants could explain why ZP proteins induce heteroantibodies which result in infertility while alloimmunization has no effect on fertility.     

Monoclonal antibodies against hen's egg ovomucoid

Two monoclonal antibodies (mAb 23E5 and 32A8) to hen's egg ovomucoid (OM), which causes hen's egg allergy and has trypsin inhibitory activity, were prepared and purified. Their affinity to the three separate domains of the ovomucoid, which are homologous in primary structure and are designated as DI, DII, and DIII, was studied by a competitive radioimmunoassay. MAb 23E5 bound to OM more efficiently than to DI, DII, or DIII-2 (with carbohydrate), but reacted with DIII-1 (free from carbohydrate) more efficiently than with OM. Except for the binding to OM, mAb 32A8 bound to DIII-2 most efficiently and to DIII-1 least efficiently, suggesting that this antibody recognized the carbohydrate moiety of DIII. MAb 32A8 inhibited the trypsin inhibitory activity of OM, whereas mAb 23E5 had no effect on it. These monoclonal antibodies should be useful for analyzing the antigenic determinants and trypsin inhibitory activity of ovomucoid.     

Protective efficacy of IgM monoclonal antibodies in experimental group B streptococcal infection is a function of antibody avidity

We have produced and characterized six mAb directed against group B streptococci (GBS). All antibodies are IgM. We have previously shown that some of these antibodies are highly protective in the treatment of experimental infections in neonatal rats, whereas others do not appear to have any protective efficacy. Using an ELISA, we demonstrate the specificity of both protective and nonprotective antibodies. Two antibodies, binding different epitopes, are directed against antigenic structures present on all GBS; two are specific for type III carbohydrate determinants; one binds to a protein Ag present on all type I and II GBS; and one appears to bind to type Ia GBS only. Quantitative absorption assays provide evidence that the difference between protective antibodies and nonprotective antibodies is the avidity that the antibody demonstrates for the epitope recognized on the surface of the bacteria; 10 to 15 times as much protective antibody binds to GBS as does nonprotective antibody. Direct binding experiments with radiolabeled antibody confirm this conclusion.     

Small nuclear ribonucleoprotein antigens are absent from 10S translation inhibitory ribonucleoprotein but present in cytoplasmic messenger ribonucleoprotein and polysomes

A cytoplasmic 10S ribonucleoprotein particle (iRNP), which is isolated from chick embryonic muscle, is a potent inhibitor of mRNA translation in vitro and contains a 4S translation inhibitory RNA species (iRNA). The iRNP particle shows similarity in size to the small nuclear ribonucleoprotein (snRNP) particles. Certain autoimmune disease patients contain antibodies directed against snRNP antigenic determinants. The possibility that iRNP may be related to the small nuclear particles was tested by immunoreactivity with monospecific autoimmune antibodies to six antigenic determinants (Sm, RNP, PM-1, SS-A (Ro), SS-B (La), and Scl-70). By Ouchterlony immunodiffusion assays, the cytoplasmic 10S iRNP did not show any immunoreactivity. Also, a more sensitive hemagglutination inhibition assay for detecting Sm and RNP antigens failed to show reactivity with the 10S iRNP. Thus, the 10S iRNP particles are distinct from the similarly sized snRNP. However, free and polysomal messenger ribonucleoprotein (mRNP) particles and polysomes also isolated from chick embryonic muscle and analyzed by Ouchterlony immunodiffusion and hemagglutination inhibition for the presence of the antigenic determinants showed reactivity to Sm and RNP autoantibodies, but were not antigenic for the other four antibodies. Some of the Sm antigenic peptides of mRNP particles and polysomes were identical to those purified from calf thymus nuclear extract, as judged by Western blot analysis. The association of Sm with free and polysomal mRNP and polysomes suggests that Sm may be involved in some cytoplasmic aspects of mRNA metabolism, in addition to a nuclear function in mRNA processing.     

Paratope- and framework-related cross-reactive idiotopes on anti-acetylcholine receptor antibodies

Cross-reactive idiotopes are a possible target for therapeutical interventions in autoimmune diseases. To investigate their role in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) we analyzed the Id of rat anti-AChR mAb 6, 35, 61, 65 and a control myeloma protein IR27. Anti-Id 6, 35, 61, 65 bound in a direct binding assay with various affinity to all rat anti-AChR mAb that were tested. Anti-Id IR27 recognized none of the anti-AChR mAb. The specificity of these crossreactions was confirmed by inhibition studies with anti-AChR mAb and two control rat myeloma proteins (IR27 and IR241). In addition, the Id expression on mAb D6, a mouse anti-human AChR mAb was recognized by anti-Id 6, 35, and 65. Anti-Id, except anti-Id IR27, bound to affinity purified IgG from the sera of rats with EAMG, but not to preimmune Lewis IgG. These results suggest extensive sharing of idiotopes among anti-AChR mAb, which are also present in EAMG serum. Anti-AChR mAb against the main immunogenic region (6, 35, 65) from different rat strains, shared at least one paratope-related cross-reactive idiotopes. In the view of the fact that anti-main immunogenic region antibodies might form a predominant fraction of the polyclonal response against AChR, it is conceivable that an anti-Id recognizing these antibodies could have therapeutical applications as for example a selective immune absorbent or in immunotoxin therapy.     

Monoclonal antibodies directed against the galactose-binding lectin of Entamoeba histolytica enhance adherence.

The Entamoeba histolytica galactose-binding lectin is a surface glycoprotein composed of 170- and 35-kDa subunits. Inhibition of this lectin with galactose or anti-170 kDa subunit polyclonal antibody blocks amebic adherence to target cells and colonic mucin glycoproteins. We describe the properties of 10 mAb with specificity for the 170-kDa subunit. Based on competitive binding studies, six nonoverlapping antigenic determinants on the lectin were identified. The effect of the mAb on adherence of amebic trophozoites to both Chinese hamster ovary (CHO) cells and human colonic mucins was measured. Antilectin antibodies directed against epitopes 1 and 2 enhanced adherence, with the number of amebae having at least three adherent CHO cells increasing with the addition of epitope 1 mAb from 26 +/- 9 to 88 +/- 2% and the binding of colonic mucins increasing from 34 +/- 1 to 164 +/- 3 pg/10(5) amebae. Antibody-enhanced adherence remained 90 to 100% galactose inhibitable, occurred at 4 degrees C and was not Fc mediated. Univalent Fab fragments of epitope 1 mAb augmented mucin binding by 238% and CHO cell adherence by 338%. The binding of purified lectin to CHO cells was increased from 1.1 +/- 0.1 to 2.4 +/- 0.3 ng/10(3) CHO cells by mAb directed to epitope 1, demonstrating that enhanced adherence was due to direct activation of the lectin. mAb to epitope 3 bound to the lectin only upon its solubilization from the membrane and had no effect on adherence. Adherence to CHO cells and mucins was inhibited from 50 to 75% by mAb to epitopes 4 and 5; epitope 6 mAb inhibited amebic adherence to CHO cells but not mucins. The pooled sera from 10 patients with amebic liver abscess blocked the binding to the 170-kDa subunit of mAb directed to all six epitopes. Striking individual variations in the effects of immune sera on adherence were observed. Although the sera of all 44 South African patients with amebic liver abscess had high titer anti-lectin antibodies, 16 patients' sera significantly (more than 3 SEM) enhanced adherence whereas 25 patients' sera significantly inhibited adherence. Antilectin antibodies exert profound functional effects on the interaction of E. histolytica with target cells and human colonic mucins. Exploration of the clinical consequences of adherence-enhancing and inhibitory antibody responses may give insight into the role of antilectin antibodies in immunity to invasive amebiasis.     

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.     

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.     

Discrimination of human placental alkaline phosphatase allelic variants by monoclonal antibodies.

Eighteen monoclonal antibodies were produced by the mouse hybridoma method using purified placental alkaline phosphatase (ALP) as antigen. The ability of the various antibodies to discriminate among allelic variants of the enzyme was tested using a large panel of placental ALPs that had been typed electrophoretically. The panel included sets of samples of each of the six common polymorphic phenotypes as well as a series of rare variants. The reactivity of each antibody with each placental ALP (binding ratio) was determined relative to a single standard placental ALP (type 1) in a quantitative binding assay. The findings for six of the antibodies have already been reported. The results on the other 12 antibodies are presented here, and the combined data on the total series of 18 antibodies are analyzed and discussed. Six of the 18 antibodies showed significantly reduced binding to one or another of the products of the three common alleles. In three cases, the discrimination was reflected by essentially "all-or-none" binding reactions. In the other three cases, the binding differences were less marked but could be demonstrated by quantitative comparisons of the binding ratios. Quantitative binding ratio comparisons also enabled heterozygotes to be differentiated from homozygotes in each case. Some of the antibodies showed reduced binding with certain of the rare variant ALP electrophoretic phenotypes. It is estimated that at a minimum this unselected series of 18 antibodies is directed to at least nine different antigenic determinants on the surface of the placental ALP molecule. The results illustrate the power of monoclonal antibodies to discriminate among allelic variants of enzymes.     

Epitope clusters of Qa-2 antigens defined by a panel of new monoclonal antibodies

A recently derived intra-MHC recombinant mouse strain, the C3H.KBR was found to produce a surprisingly high titer of anti-Qa antibodies when immunized with C3H.SW lymphocytes. By using this immunization combination, a panel of 10 mAb with specificity for determinants encoded by the Q region was produced. These reagents were analyzed for strain distribution by microcytotoxicity, immunofluorescence, and flow cytometry assays. Competitive inhibition analyses, performed by using fluorescein-labeled antibodies and normal spleen cells, defined at least three epitope clusters, or groups of spatially related determinants, detected by this panel. One epitope cluster was unique to this new series of antibodies in that it was not detected with seven previously described anti-Qa mAb. These antibodies also have been analyzed for reactivity with products of isolated Q-region genes by using transfected cell lines. The data indicate that the Q6d, Q7d, and Q10d genes encode determinants reactive with one or more mAb and that two of the three epitope clusters defined with normal cells map to the N and/or C2 domains of these molecules. The third epitope cluster is presumed to map to the C2 domain. These reagents should be useful in determining the number of Q-region genes expressed and in analyses of Q gene expression in subpopulations of normal cells, in transfected cell lines, and during differentiation and ontogeny.     

Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis

Sixteen species-specific monoclonal antibodies were produced against membranes of Leishmania donovani. These antibodies only reacted with determinants present on L. donovani. No cross-reactions were found with any other species of Leishmania or with membranes of Trypanosoma cruzi. An extensive analysis of the binding specificities of selected antibodies was carried out by using whole promastigote homogenates as antigen. Monoclonal antibodies D-1, D-2, D-3, and D-4 correctly identified all 44 L. donovani stocks from a cross-panel of 84 New and Old World Leishmania stocks. Antibodies D-1 and D-2 were also useful for species classification by immunofluorescence. No cross-reactions were observed with any other Leishmania species examined. Based on either Western blot and/or radioimmunoprecipitation analyses, five distinct groups of molecules associated with L. donovani-specific antigenic determinants were identified. These molecules range in m.w. from 18 to 84 kilodaltons. The antigenic molecules recognized by antibodies D-2, D-10, and D-13 are also recognized by antibodies present in sera from patients with visceral leishmaniasis (kala-azar). Kala-azar sera obtained from cases in both the Old and New World specifically compete with these monoclonal antibodies for the appropriate antigenic determinants in Western blot analysis. These monoclonal antibodies and/or the purified protein antigens may be useful in the development of a serologic assay for the clinical diagnosis of visceral leishmaniasis caused by L. donovani and in epidemiologic studies of leishmaniasis.     

Characterization of antigenic properties of horseradish peroxidase with monoclonal antibodies

Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO ₁ and 36F ₉ were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.     

Unusual amino acid usage in the variable regions of mercury-binding antibodies

Monoclonal antibodies (mAb) specific for mercuric ions were isolated from BALB/c mice injected with a mercury-containing, hapten-carrier complex. The antibodies reacted by enzyme-linked immunosorbent assay with bovine serum albumin-glutathione-mercuric chloride (BSA-GSH-HgCl) but not with BSA-GSH without mercury. Nucleotide sequences from polymerase chain reaction products encoding six of the antibody heavy-chain variable regions and seven light-chain variable regions revealed that all the antibodies contained an unpaired cysteine residue in one hypervariable region, which is unusual for murine antibodies. Mutagenesis of the cysteine to either tyrosine or serine in one of the Hg-binding antibodies, mAb 4A10, eliminated mercury binding. However, of two influenza-specific antibodies that contain cysteine residues at the same position as mAb 4A10, one reacted with mercury, although not so strongly as 4A10, whereas the other did not react at all. These results suggested that, in addition to an unpaired cysteine, there are other structural features, not yet identified, that are important for creating an appropriate environment for mercury binding. The antibodies described here could be useful for investigating mechanisms of metal-protein interactions and for characterizing antibody responses to structurally simple haptens.     

A rabbit antiserum detects a VH J558 subgroup marker highly expressed among anti-alprenolol antibodies

A rabbit antiserum raised against anti-alprenolol mAb 14C3 detects common antigenic determinants (ADC3) in 10 out of 14 anti-alprenolol mAb that use different germ-line VH and/or Vk genes. The anti-14C3 antiserum binds only to H chains in immunoblots, therefore suggesting that at least part of the ADC3 determinants may be encoded by H chain V region genes. Analysis of VH gene family usage among the anti-alprenolol mAb reveals that the expression of ADC3 correlates with utilization of VH genes that belong to the J558 gene family, regardless of the JH, Vk, and Jk genes. To determine whether the ADC3 determinants are general V region markers or whether they are unique to anti-alprenolol antibodies, we have extended our analysis to a random panel of antibodies that also use VH genes of the J558 family. Among 23 mAb of various specificities, 14 react with the anti-14C3 antiserum in immunoblot and in ELISA, irrespective of antibody specificity. Adsorption of the antiserum on one of these positive antibodies results in a loss of reactivity toward both anti-alprenolol and unrelated antibodies. Therefore, several but not all antibodies that use a J558 VH gene also express the complete set of epitopes defining ADC3. These results strongly suggest that ADC3 are markers of a subset of J558 VH gene products. The anti-14C3 antiserum may thus constitute a "serologic probe" for identification of a VH gene subgroup from the J558 gene family.