Anti-idiotype-induced, lipopolysaccharide-specific antibody response to Pseudomonas aeruginosa. II. Isotype and functional activity of the anti-idiotype-induced antibodies

We recently developed a murine anti-idiotypic mAb that functioned as a molecular mimic of the O-specific polysaccharide side chain (Ps) of Pseudomonas aeruginosa LPS in vitro, and which induced Ps-specific antibodies in syngeneic BALB/c ByJ mice. In the current studies, we demonstrate that these anti-Id-induced, Ps-specific antibodies fix complement to the bacterial cell surface, and protect neutropenic mice from fatal P. aeruginosa sepsis. The isotypic distribution of the anti-Id-induced antibodies, however, resembles the restricted pattern (IgM and IgG3) seen after administration of purified Ps to mice. The immunogenicity and number of isotypes of Ps-specific antibody produced could be enhanced by conjugating the anti-Id to keyhole limpet hemocyanin. Finally, the anti-Id administered before immunization with purified Ps, primed BALB/c ByJ mice for production of other Ps-specific antibody isotypes (IgG1). These studies show that this anti-Id induces functional anti-Ps antibodies in syngeneic mice, and when used in conjugate form or as a priming agent before Ps immunization, yields an antibody response consistent with "T cell dependence." These immunization strategies may be useful for the induction of polysaccharide-specific antibodies in man.     

Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72

The use of anti-idiotypic antibodies as immunogens represents one potential approach to active specific immunotherapy of cancer. Two panels of syngeneic monoclonal anti-idiotypic antibodies were generated. One panel was directed against mAb CC49 and the other to mAb COL-1. mAb CC49 recognizes the pancarcinoma antigen (Ag), tumor-associated glycoprotein-72 (TAG-72), and mAb COL-1 recognizes carcinoembryonic antigen (CEA). Seven anti-idiotypic (AI) antibodies (Ab2) designated AI49-1–7 were generated that recognize the variable region of mAb CC49. These mAb were shown to inhibit the interaction of mAb CC49 (Ab1) with TAG-72 (Ag). Five anti-idiotypic antibodies designated CAI-1–5 were also generated to the anti-CEA mAb, COL-1 (Ab1). These Ab2 were shown to inhibit the interaction between COL-1 (Ab1) and CEA (Ag). Immunization of mice, rats, and rabbits with Ab2 directed against CC49 or COL-1 could not elicit specific Ab3 humoral immune responses, i.e., antibody selectively reactive with their respective target antigens. However, immunization of mice with the CC49 anti-idiotypic antibody (Ab2), designated AI49-3, could induce a delayed-type hypersensitivity response (DTH) specific for tumor cells that express TAG-72. Similarly, immunization of mice with an anti-idiotypic antibody directed against COL-1, designated CAI-1, could induce specific DTH cell-mediated immune responses to murine tumor cells that express human CEA on their surface. These results thus demonstrate that while some anti-idiotype mAb may not be potent immunogens in eliciting Ab3 humoral responses, they are capable of eliciting specific cellular immune responses against human carcinoma-associated antigens. This type of mAb may ultimately be useful in active immunotherapy protocols for human carcinoma.Some of the studies described in this paper were in partial fulfillment of requirements for the completion of Dr. Irvine's dissertation at the George Washington University     

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200?d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.     

Induction of an anti-human type II collagen response by monoclonal anti-idiotypic antibody

Several distinct epitopes on human type II collagen were defined by using mAb. The presence of species-specific and species-nonspecific (common) epitopes was thus clarified. Anti-idiotypic mAb (Ab2) was developed against one of the antibodies (Ab1) reactive with species-specific epitopes. Thus Ab2 was demonstrated to recognize an idiotope expressed on the Ag-binding site (paratope) of Ab1, since the binding of Ab1 to human type II collagen was blocked by Ab2, and the binding of Ab2 to Ab1 was inhibited by soluble human type II collagen, but not by murine and bovine type II collagens. DBA/1 mice immunized with Ab2 coupled to keyhole limpet hemocyanin produced an antibody (Ab3) specifically reactive with human type II collagen. It was also demonstrated that Ab3 expressed an idiotype similar to that of Ab1. These findings indicate that anti-idiotypic antibody directed against mAb to human type II collagen mimics a species-specific epitope on human type II collagen. The anti-idiotypic antibody bearing internal image of type II collagen will open the way to isolation of the arthritogenic epitope on type II collagen.     

Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes.

We used flow cytometry to determine how LPS-binding protein (LBP) effects the binding of fluorescein-labeled LPS to human monocytes via receptor-dependent mechanisms. The addition of human, rabbit, mouse, or FCS strikingly increased the binding of LPS to monocytes compared with controls incubated in serum-free medium. This binding was totally prevented by preincubation of monocytes with MY4, an anti-CD14 mAb, or by enzymatic removal of CD14 from monocytes. Depletion of LBP from rabbit serum with anti-LBP antibodies also produced a similar suppression. Solutions of albumin did not support the enhanced binding observed in serum but the addition of purified rabbit LBP to albumin solutions resulted in binding similar to that observed in serum-containing medium. When type-specific anti-LPS mAb was added to human serum, LPS binding to monocytes occurred but was only partly inhibited by anti-CD14 mAb, suggesting that receptors other than CD14 (presumably Fc or complement receptors) were involved. Serum increased by 100- to 1000-fold the sensitivity of monocytes to the triggering by LPS resulting in TNF secretion. TNF secretion was inhibited by anti-CD14 mAb up to 100 ng/ml of LPS and by anti-LPS mAb up to 1 to 10 ng/ml. The inhibition of TNF secretion by anti-LPS mAb appeared to be the result of directing LPS to monocyte receptors other than CD14. In contrast, in medium containing normal as well as acute serum and in the absence of anti-LPS antibodies, the binding of LPS to monocytes and the triggering of TNF secretion appeared to be mediated mainly by interactions between CD14 and LBP-LPS complexes.     

Analysis of the immune response to lipopolysaccharide. Existence of an interspecies cross-reactive idiotype associated with anti-lipid A antibodies

LPS is the major surface glycolipid on gram-negative bacteria. In this work, we have idiotypically characterized the antibody response against LPS in different species. To do this, we have produced mAb against LPS. Binding of many of these antibodies to LPS could be inhibited by LPS and lipid A, indicating that the monoclonals are specific for lipid A, the toxic moiety of the LPS molecule. One anti-lipid A antibody, IC9, proved protective against gram-negative bacteremia and endotoxic shock in murine protection models. We generated anti-idiotypic antibodies against IC9. The binding of several of these anti-Id to IC9 was specifically inhibited by lipid A. We used these anti-Id to characterize the anti-LPS response, and the results revealed that the IC9 Id is conserved in different species. The importance of an interspecies cross-reactive Id in the response to endotoxin and its relevance in vaccine development for septic shock are discussed.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

The structure and serologic distribution of an extracellular neutral polysaccharide from Pseudomonas aeruginosa immunotype 3

Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.     

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

Rabbit anti-idiotypic antibodies (alpha Id Ab) were prepared against five murine monoclonal antibodies (mAb) specific for the rabies virus glycoprotein. Four of the mAb were directed against three known, type-specific, neutralizing sites on the glycoprotein, and the other mAb was directed against a topographically uncharacterized, nonneutralizing epitope. An absence of significant cross-reactivity among the alpha Id Ab for heterologous mAb suggested that the alpha Id Ab were highly specific for unique variable region determinants. The binding of three of the five alpha Id Ab to their homologous mAb could be inhibited by rabies virus-soluble glycoprotein, suggesting that the alpha Id Ab possessed subpopulations similar or adjacent to the antigen-binding site of the mAb. Two of the five alpha Id Ab injected into mice elicited a specific virus-neutralizing antibody response. Mechanisms to account for the induction of the virus-neutralizing antibody by alpha Id Ab are discussed.     

Antibody response against a possible arthritogenic epitope on human type II collagen induced by anti-idiotypic antibody

We reported the presence of three distinct epitopes commonly present on murine and human type II collagen (CII), observed using mAb. To investigate the possible involvement of these epitopes in collagen-induced arthritis, we raised rabbit anti-idiotypic antibodies that may bear the internal image of these epitopes. Anti-idiotypic antibodies developed against three anti-CII mAb designated as 1-5, 2-14, and 2-15 were demonstrated to recognize idiotype expressed on Ag-binding site (paratope) of their related mAb. Anti-CII antibody response specific for a given epitope could be induced in DBA/1J mice upon immunization with anti-idiotypic antibodies coupled to keyhole limpet hemocyanin. Anti-idiotypic antibody to 1-5 antibody in particular could stimulate all DBA/1J mice for production of anti-CII antibody possessing Ag specificity and idiotype similar to those of 1-5 antibody. Although the mice immunized with anti-1-5 antibody alone did not develop arthritis, they did show a much more enhanced antibody response against a given epitope than did control mice non-treated with anti-idiotypic antibody upon the subsequent immunization with human CII. Some of the mice immunized with anti-1-5 antibody and challenged with human CII developed arthritis, whereas the control mice did not. These findings strongly suggest that a common epitope recognized by 1-5 antibody might be involved in the induction of arthritis.     

Collagen-Induced Arthritis Suppressed with Monoclonal Anti-Idiotypic Antibody

In foregoing work, we identified at least 5 distinct epitopes on human type II collagen (CII), using 8 murine monoclonal antibodies (mAb) against human CII, and suggested that a species-nonspecific epitope on CII recognized by anti-CII mAb termed 1-5 is an arthritogenic epitope. We also found that antibody response against a selected epitope of human CII could be induced by immunization with rabbit anti-idiotypic (Id) antibody against anti-CII mAb. The author developed and characterized monoclonal anti-Id antibodies against 1-5 mAb recognizing a putative arthritogenic epitope. The author also investigated whether the anti-Id mAb could regulate antibody response directed against a selected epitope recognized by 1-5 mAb, and the induction of collagen-induced arthritis in DBA/1J mice. DBA/1J mice intravenously preinjected with anti-Id mAb to 1-5, did not produce anti-CII antibody expressing 1-5 Id upon immunization with human CII. Furthermore, as the development of collagen-induced arthritis (CIA) in DBA/1J mice pretreated with anti-Id mAb to 1-5 was significantly suppressed, anti-Id mAb will be a useful tool for studying the regulation of antibody response to a selected epitope. This study lends support to our hypothesis that the 1-5 epitope is an arthritogenic epitope.     

High idiotypic connectivity of the VH7183-encoded antibodies directed to a murine embryonic carbohydrate antigen, Lewis Y, as ascertained by syngenic anti-idiotype monoclonal antibodies

The Lewis Y Ag is a carbohydrate Ag which is closely related to a well-known murine embryonic Ag, the stage-specific embryonic Ag-1 (SSEA-1), in its biochemical structure. It is expressed at the surface of murine embryonic cells as well as many murine cancer cells. For the analysis of idiotopes carried by the anti-Lewis Y antibodies, we generated two syngenic anti-idiotypic mAb, Id-A1 and Id-B4 (both BALB/c IgG1), which are directed to the idiotypic determinants carried by the anti-Lewis Y mAb, AH-6 (BALB/c IgM). Both Id-A1 and Id-B4 (Ab2) recognized paratope-related idiotopes carried by the AH-6 antibody (Ab1); they specifically inhibited the binding of AH-6 to the Lewis Y Ag. The high idiotypic connectivity of anti-Lewis Y antibodies was noted; the polyclonal anti-idiotype antibody, produced in the sera of BALB/c mice by immunizing AH-6 antibody, cross-reacted with several anti-Lewis Y mAb which has been established in different laboratories. Id-B4 and Id-A1 seem to represent such cross-reactive anti-idiotypic antibodies. Id-A1 recognized an idiotope carried by two out of six panel Ab1 mAb directed to the Lewis Y Ag. Id-B4 reacted with four out of the six panel antibodies, and was considered to recognize a recurrent idiotope of anti-Lewis Y antibodies which occurs more commonly than the idiotope recognized by Id-A1. All of the anti-Lewis Y antibodies which carry idiotopes that react with Id-A1 or Id-B4 were encoded by the VH genes of the VH7183 family; the most D-J proximal VH gene family in BALB/c mice, which is known to be preferentially expressed in embryonic B cells. Immunization of BALB/c mice with keyhole limpet hemocyanin-conjugated Id-B4 and/or Id-A1 induced a significant titer of anti-Lewis Y antibodies (Ab1-like Ab3) in the sera.     

Identical D region sequences expressed by murine monoclonal antibodies specific for a human tumor-associated antigen

Sequence analysis has revealed significant structural similarities among murine mAb specific for the human tumor-associated Ag GA733. Antibodies generated in independent immunizations, either with Ag-positive tumor cells or with anti-idiotypic antibodies produced by immunization of goats with a GA733-specific antibody, all use members of the same gene families; remarkably, they also express identical amino acid sequences in their H chain CDR3. Inasmuch as this region normally exhibits considerable sequence variability, the identity displayed by the antibodies indicates a requirement for this particular sequence in the generation of their specificity for the GA733 antigen. Moreover, this homology suggests that the antibodies recognize a common determinant on the Ag, and that the polyclonal anti-idiotypic antibodies can functionally mimic the Ag at the level of the structure of the Ag-specific antibody that is induced.     

A syngeneic monoclonal anti-idiotypic antibody with internal image properties restricted to a single aldosterone-binding system

Immunization with a murine anti-aldosterone mAb (AAC) resulted in the isolation of a syngeneic monoclonal anti-idiotypic antibody, LH9G4. LH9G4 bound to Fab fragments of AAC and was affinity-purified on an AAC column. LH9G4 inhibited the binding of aldosterone to AAC in a dose-dependent manner with an apparent dissociation constant of 0.5 nM as determined by competitive inhibition assays in ELISA and RIA. LH9G4 and aldosterone have similar relative affinities for AAC. Kinetic studies and Scatchard plot analysis support a reversible and reciprocal competitive inhibition mechanism between LH9G4 and aldosterone for the paratope of AAC. The possibility of a steric hindrance mechanism was eliminated. No cross-reactivity was seen with six other murine anti-aldosterone mAb, with a rabbit polyclonal antibody or with aldosterone receptor. The anti-idiotypic antibody, defined as a "restricted" internal image of aldosterone, is apparently directed at a private idiotope present in the paratope of AAC but not in binding sites of other aldosterone-binding proteins. Biophysical considerations involving characteristics of nonbonded attractive forces can explain these findings. An advantage of the one-step auto-anti-idiotypic procedure for the generation of Ab2-beta or internal image antibodies is discussed.     

Mimicry of human IgE epitopes by anti-idiotypic antibodies

According to Jerne's network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunization, and confirm Jerne's hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.     

Human monoclonal antibodies to Pseudomonas aeruginosa produced by EBV-transformed cells

Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD50) values were 0.5, 2.6, and 3.1 micrograms immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.     

Syngeneic monoclonal anti-idiotype antibodies that bear the internal image of a human cytomegalovirus neutralization epitope

Two glycoproteins have been identified on human CMV that induce neutralizing antibody; an 86,000-Da glycoprotein and a 130,000-, 92,000-, and 50,000-Da glycoprotein coimmunoprecipitating complex that appears to be the gB homologue of HSV. We have produced syngeneic monoclonal anti-Id antibodies (mAb2) of the IgM isotype to a CMV-neutralizing monoclonal antibody (mAb1) that is known to bind to the 86,000-Da glycoprotein on the virion envelope. These mAb2 bear the internal image of the original viral antigen as shown by their ability to 1) recognize an interspecies idiotype in CMV-positive human antisera, 2) block mAb1 binding to CMV antigen, and 3) block CMV neutralization by mAb1 in vitro. Immunization of mice with both of these affinity chromatography-purified mAb2 stimulated the production of anti-anti-Id monoclonal antibodies (which we termed mAb3), which bound to the mAb2 by ELISA and neutralized CMV infectivity.     

The immune response towards beta-adrenergic ligands and their receptors. VI. Idiotypy of monoclonal anti-alprenolol antibodies

Four murine monoclonal antibodies specific for alprenolol, a synthetic beta-adrenergic ligand, with different binding properties towards alprenolol and other beta-adrenergic antagonists and agonists (as described in a previous report) were used to induce anti-idiotypic responses in rabbits and mice. Three of the rabbit anti-idiotypes inhibited, and one increased the binding of tritiated dihydroalprenolol to the Ab1 against which they were induced. The syngeneic mouse anti-idiotypes all had an inhibitory effect on the ligand binding to their corresponding Ab1. Cross-reactivity tests of the xenogeneic and syngeneic anti-idiotypes were positive only for two monoclonal anti-alprenolol antibodies. Cross-reaction could be shown neither on a panel of 15 other monoclonals, nor on polyclonal anti-alprenolol antibodies of the BALB/c and the C57BL/10 mice. These results suggest that the immune response against alprenolol results in antibodies with mostly private idiotypic determinants. Moreover, the properties of the anti-idiotypic response against the same monoclonal antibody seem to be different according to the species used for anti-idiotypic induction.     

Idiotypic determinants of monoclonal antibodies that bind to 3-fucosyllactosamine

Many monoclonal antibodies that react with the lacto-N-fucopentaose III (LNF III) antigenic determinant, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc, have been described recently. The terminal trisaccharide of this determinant, fucosyllactosamine, is present on glycolipids and glycoproteins and on the surface of granulocytes, monocytes, and other cells. To study the structural and genetic diversity of these antibodies, syngeneic anti-idiotypic monoclonal antibodies were produced in BALB/c mice against PMN 6, a monoclonal antibody directed against this sequence. Anti-idiotypic antibodies 6B1 and 6C4 reacted with 50% of a panel of 20 anti-LNF III monoclonal antibodies, whereas 6A3 reacted strongly only with PMN 6. This indicates that the determinants recognized by 6C4 and 6B1 represent major cross-reactive idiotopes of this family of antibodies. The binding of idiotypic antibodies to a glycolipid bearing this antigenic determinant was completely inhibited by the three anti-idiotypic antibodies, 6A3, 6B1, and 6C4. The idiotopes could be demonstrated on the heavy chain of the monoclonal antibodies by an antibody transfer technique when mild reducing conditions were employed, but a high concentration of reducing agent destroyed the idiotypic determinants. This suggests that the anti-idiotypic antibodies recognize conformational structures expressed on the heavy chain molecules. The binding of 18 monoclonal antibodies to two glycolipid antigens and to a fucosyllactosamine-bovine serum albumin conjugate was compared. Antibodies that possessed the 6C4 cross-reactive idiotope bound to fucosyllactosamine-bovine serum albumin more weakly than idiotype-negative antibodies (p = 0.001). This suggests that the 6C4-positive antibodies might represent germline structures.     

A monoclonal antiidiotypic antibody with rheumatoid factor activity defines a cross-reactive idiotope on murine anticollagen antibodies.

A syngeneic antiidiotypic mAb, C1C3, was characterized as to its binding to monoclonal anti-collagen II (-CII) auto-antibodies reactive with different epitopes of the native CII molecule. Both by direct binding and by inhibition ELISA studies, the anti-idiotypic antibody was shown to react with a cross-reactive idiotope present on Fab fragments of most, but not all, tested anti-CII mAb, whereas the binding to Fab fragments from normal mouse IgG was low. As previously described, C1C3 bound to isolated Fc fragments from normal mouse IgG. The binding to intact normal mouse IgG was, however, weak, and only isolated Fc-gamma fragments, not intact IgG, competed efficiently with Fab fragments of anti-CII antibodies for binding to the antiidiotypic antibody. The antibody was shown to self-associate, i.e., to behave similarly to certain IgG rheumatoid factors obtained from patients with rheumatoid arthritis. The presented data indicate that the described anti-anti-CII mAb may be representative of antibodies involved in the physiologic regulation of autoimmunity to CII and, consequently, may be used as a tool for further studies on idiotypic regulation in CII-induced arthritis.