Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup

Staphylococcal protein A (SPA) is a bacterial membrane protein that possesses, in addition to its Fc gamma-binding activity, a distinct specificity for the Fab region of some IgM, IgA, IgG, and IgE. The Fab site that binds to SPA has been localized to the V region of the Ig H chain. In a previous study of human monoclonal and polyclonal IgM, we demonstrated that binding to SPA was highly restricted to molecules of the VHIII subgroup, and that nearly all VHIII IgM were able to bind SPA. The present study examines the VH composition of SPA-binding and SPA-nonbinding fractions of purified human polyclonal IgA, and IgG F(ab')2 fragments. We found that 22% of the IgA and 15% of the IgG F(ab')2 bound to SPA-agarose. Analysis with VH subgroup-specific antisera indicated that the SPA-binding fraction of IgA was dominated by the VHIII subgroup, and the SPA-binding fraction of IgG F(ab')2 contained only VHIII molecules. Furthermore, substantial portions of the total VHIII protein in IgA and in IgG F(ab')2 bound to SPA. We conclude that Fab binding to SPA is both restricted to and highly prevalent among human VHIII molecules, regardless of Ig class. These results suggest that protein A is an Ig superantigen.     

Human IgM molecules that bind staphylococcal protein A contain VHIII H chains

Staphylococcal protein A (SPA) is a bacterial membrane protein which has distinct binding sites for Fc gamma and for the Fab region of some IgM, IgG, IgA, and IgE molecules. This study establishes a structure-function correlation responsible for the binding of Ig Fab regions to SPA. Binding of 24 isolated human monoclonal IgM proteins to SPA was measured in a solid phase RIA. VH and V kappa subgroups of each IgM were determined by SDS-PAGE, transfer blotting, and detection with antisera prepared against specific first framework region peptides. Binding to SPA was seen with 10 of 11 VHIII IgM, but none of the 7 VHI or 6 VHII. Similarly, polyclonal IgM fractionated on a SPA-Sepharose CL4B column showed nearly complete partition of VHIII molecules into the SPA-binding fraction, and VHI and VHII subgroup proteins into the fall-through. We conclude that SPA binding is a functional marker for VHIII H chains in human IgM molecules.     

Comparative conformational studies on the tryptic digestion fragments of human immunoglobulins M and G

IgM¹ immunoglobulins were cleaved into Fabμ and (Fc)5μ fragments by tryptic digestion. Comparative circular dichroism studies with the corresponding IgG fragments show that the Fab portions of IgG and IgM proteins have very similar CD spectral features, although the same is not true for their Fc fragments. These studies indicate the presence of higher amount of beta-structured regions in Fcμ than in Fcγ. Also, there are considerable differences in their pH-dependent structural transitions as measured by CD spectral changes. The conformational differences between IgG and IgM immunoglobulins are more pronounced in their Fc portions, which carry out class specific biological functions, rather than in Fab portions, which contain antigen combining sites.     

Modulation of human rheumatoid factor-specific lymphocyte responses with a cross-reactive anti-idiotype bearing the internal image of antigen

Rabbit anti-idiotypic antibodies to human rheumatoid factor (RF) autoantibodies were isolated by affinity chromatography on rabbit anti-human IgG Fc Sepharose 4B. The anti-idiotypic antibodies bore the "internal image" of the antigen, human IgG. They reacted specifically with multiple human monoclonal and polyclonal IgM-RF, independent of any particular light or heavy chain amino acid sequence. The anti-idiotypes did not react with IgM or IgG proteins lacking RF activity. The present experiments determined the potential of the "internal image" antibodies to modulate in vitro lymphocyte functions. The addition of anti-idiotypic antibody to peripheral blood mononuclear cell cultures from patients with rheumatoid arthritis elicited lymphocyte proliferation, but not RF synthesis. The antibody did not induce the proliferation of lymphocytes from a normal individual. Moreover, the anti-idiotype specifically suppressed IgM-RF secretory responses when preincubated with B cells before co-culture with autologous pokeweed mitogen-activated T cells. The data show that the anti-idiotypic antibodies with the "internal image" of antigen are capable of interacting with B cell receptors in an antigen-restricted manner, and possess specific immunomodulatory properties.     

抗人IgG Fc片段及Fab段单抗的鉴定及应用

本实验采用木瓜酶水解,SPA柱亲合层析等手段得到人IgGFc段及Fab段,以Sigma抗人IgGfFc段和抗人IgG Fab段单抗为标准品,鉴定了细胞库中抗人IgG系列的部分细胞株,得到特异性分泌抗人IgG Fc段和抗人IgG Fab段单抗的细胞各一株。 在上述实验基础上,用抗人IgG Fc及抗人IgG Fab单抗分别制备了Sepharose4B亲合层析柱,提纯了酶解人IgG Fc、Fab片段,经ELISA法鉴定,相互之间无交叉反应。同时用此方法制备了人抗HBe Fab片段,并将该片段进行了过氧化物酶标记,用来配制HBe ELISA诊断盒,证明其生物活性未受影响,而且消除了类风湿因子引起的HBe Ag假阳性现象。因抗HBe单抗来源困难,如采用HBe多抗制备ELISA试剂,本法将是提高质量的一个好方法。     

Potentiation of the rat delayed-type hypersensitivity reaction by the Fc portion of human IgG1

Fc fragments derived from a human IgG1 myeloma protein potentiate the rat delayed-type hypersensitivity (DTH) reaction to antigen challenge. Lewis rats immunized with heat-killed tubercle bacilli give augmented DTH reactions to the purified protein derivative of tuberculin when Fc fragments are included in the challenge dose. Similar potentiation of DTH by pFc' fragments indicates that the active site is located in the CH3 domain of IgG1. Histologic evaluation of the augmented reaction sites revealed predominantly mononuclear cell infiltrates characteristic of DTH reactions. Skin tests of tubercle bacilli-sensitized rats with an unrelated antigen and/or Fc fragments fail to elicit significant reactions. Augmentation of the DTH reaction to purified protein derivative is restricted to the Fc or pFc' region fragments since intact monomeric IgG1, Fab fragments, and bovine serum albumin were all shown not to be active potentiators. The DTH reaction of ovalbumin-sensitized rats was similarly augmented when Fc fragments were included with a challenge dose of ovalbumin, thus supporting the general nature of the phenomenon. These results support the concept of Ig molecules as multifunctional proteins that can not only serve effector functions but also participate in the regulation of immune responses.     

Solution structure of human and mouse immunoglobulin M by synchrotron X-ray scattering and molecular graphics modelling. A possible mechanism for complement activation

The pentameric 71-domain structure of human and mouse immunoglobulin M (IgM) was investigated by synchrotron X-ray solution scattering and molecular graphics modelling. The radii of gyration RG of human IgM Quaife and its Fc5, IgM-S, Fab'2 and Fab fragments were determined as 12.2 nm, 6.1 nm, 6.1 nm, 4.9 nm and 2.9 nm in that order. The RG values were similar for mouse IgM P8 and its Fab'2 and Fab fragments, despite the presence of an additional carbohydrate site. The IgM scattering curves, to a nominal resolution of 5 nm, were compared with molecular graphics models based on published crystallographic alpha-carbon co-ordinates for the Fab and Fc structures of IgG. Good curve fits for Fab were obtained based on the crystal structure of Fab from IgG. A good curve fit was obtained for Fab'2, if the two Fab arms were positioned close together at their contact with the C mu 2 domains. The addition of the Fc fragment close to the C mu 2 domains of this Fab'2 model, to give a planar structure, accounted for the scattering curve of IgM-S. The Fc5 fragment was best modelled by a ring of five Fc monomers, constrained by packing considerations and disulphide bridge formation. A position for the J chain between two C mu 4 domains rather than at the centre of Fc5 was preferred. The intact IgM structure was best modelled using a planar arrangement of these Fab'2 and Fc5 models, with the side-to-side displacement of the Fab'2 arms in the plane of the IgM structure. All these models were consistent with hydrodynamic simulations of sedimentation data. The solution structure of IgM can therefore be reproduced quantitatively in terms of crystallographic structures for the fragments of IgG. Putative Clq binding sites have been identified on the C mu 3 domain. These would become accessible for interaction with Clq when the Fab'2 arms move out of the plane of the Fc5 disc in IgM, that is, a steric mechanism exposing pre-existing Clq sites. Comparison with a solution structure for Clq by neutron scattering shows that two or more of the six globular Clq heads in the hexameric head-and-stalk structure are readily able to make contacts with the putative Clq sites in the C mu 3 domains of free IgM if if the Clq arm-axis angle in solution is reduced from 40 degrees-45 degrees to 28 degrees. This could be the trigger for Cl activation.     

Non-covalent interactions between Fab and Fc regions in immunoglobulin G molecules. Hydrogen-deuterium exchange studies

To reveal non-covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free-energy change (delta Go), associated with reversible micro-unfoldings, was measured by hydrogen-deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments. Hydrogen-deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5-kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non-covalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen-deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part.     

Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60- 90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.     

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.     

Site-specific N-glycosylation of chicken serum IgG

Avian serum immunoglobulin (IgG or IgY) is functionally equivalent to mammalian IgG but has one additional constant region domain (CH2) in its heavy (H) chain. In chicken IgG, each H-chain contains two potential N-glycosylation sites located on CH2 and CH3 domains. To clarify characteristics of N-glycosylation on avian IgG, we analyze N-glycans from chicken serum IgG by derivatization with 2-aminopyridine (PA) and identified by HPLC and MALDI-TOF-MS. There were two types of N-glycans: (1) high-mannose-type oligosaccharides (monoglucosylated 26.8%, others 10.5%) and (2) biantennary complex-type oligosaccharides (neutral, 29.9%; monosialyl, 29.3%; disialyl, 3.7%) on molar basis of total N-glycans. To investigate the site-specific localization of different N-glycans, chicken serum IgG was digested with papain and separated into Fab [containing variable regions (VH + VL) + CH1 + CL] and Fc (containing CH3 + CH4) fragments. Con A stained only Fc (CH3 + CH4) and RCA-I stained only Fab fractions, suggesting that high-mannose-type oligosaccharides were located on Fc (CH3 + CH4) fragments, and variable regions of Fab contains complex-type N-glycans. MS analysis of chicken IgG-glycopeptides revealed that chicken CH3 domain (structurally equivalent to mammalian CH2 domain) contained only high-mannose-type oligosaccharides, whereas chicken CH2 domain contained only complex-type N-glycans. The N-glycosylation pattern on avian IgG is more analogous to that in mammalian IgE than IgG, presumably reflecting the structural similarity to mammalian IgE.     

Isolation of an Fcgamma-binding protein from the cell membrane of a macrophage-like cell line (P388D1) after detergent solubilization

Lactoperoxidase-catalyzed iodination, NP-40 lysis, and subsequent affinity chromatography on IgG-Sepharose were used in an attempt to define some of the molecular properties of the Fc receptor of P388D1, a macrophage-like mouse tumor line. Radioiodinated material retained on columns of Sepharose coupled either to monomeric mouse IgG2a or monomeric human IgG1 appeared on SDS polyacrylamide gel electrophoresis to contain principally three labeled components, a major band of about 57,000 m.w. and two minor bands of 28,000 and 24,000 m.w. The mobilities of these components changed little on reduction, which suggested that they represented single polypeptide chains, An identical pattern was obtained with Sepharose-linked Fc fragments of human IgG1, but neither Fab fragments of IgG1 nor IgM appeared to bind these components. Since the specificity of binding to the immobilized proteins is the same as that observed in vivo, it is postulated that these proteins represent either all or some portion of the P388D1 Fc receptor.     

Noncovalent association of heavy and light chains of human immunoglobulins. IV. The roles of the CH1 and CL domains in idiotypic expression

A rabbit anti-idiotypic antiserum was raised against a monoclonal human IgM kappa(Me) in order to analyze the possible modulation of idiotypic expression by Fab constant domains. IgM(Me) fragments, subunits, and domains were prepared by chemical and enzymatic cleavages. All molecular species were shown to have a well-defined secondary and tertiary structure by circular dichroism. Full recombination between domains and subunits was ascertained by difference spectroscopy. The expression of the idiotype on native and recombined fragments, domains, and subunits was quantitated in a competitive enzyme-linked immunosorbent assay (ELISA). Reduced and alkylated Fab, isolated H and L chains, purified Fv(Me), intact VH and VL domains and H-L, VH-L, VL-H, and VH-VL recombinants were compared on a molar basis to native Fab(Me) for idiotypic expression. VH-specific determinants were found, whereas the L chains were virtually devoid of idiotypic activity. Both the peptic FV(Me) fragment, which is composed of intact VH and VL domains, and the recombined VH-VL heterodimer were found to be fourfold less active for idiotype expression than native Fab(Me). However, full inhibition was achieved at high molar concentrations, suggesting that all the idiotopes present on Fab(Me) were expressed on FV(Me) but with a reduced antigenicity. Comparison of VH-L and VL-H hybrid molecules revealed that the presence of the C mu 1 domain was sufficient to restore full idiotypic expression as compared with native Fab(Me). These data support the hypothesis that the first constant domain of the mu heavy chain alters the quaternary interaction between the variable domains, and therefore modulates the expression of the idiotype through longitudinal interactions that are not affected by reduction of the inter-H-L chain disulfide bond.     

The controlling effect of carbohydrate in human, rabbit and bovine immunoglobulin G on proteolysis by papain

About two-thirds of the hexose of human and rabbit immunoglobulin G (IgG) was located in the Fc fragment and one-third in the `hinge' region of the γ (heavy) polypeptide chain at the junction of the Fab and Fc fragments. In contrast, bovine IgG contained more hexose in the `hinge' region than in the Fc fragment. The initial cleavage of susceptible IgG molecules into Fab and Fc fragments by papain under the conditions given by Porter (1959) had reached completion after digestion for 2hr., though bovine IgG was digested somewhat more slowly than human or rabbit IgG. The release of `hinge' peptides from human and rabbit IgG had also reached completion by 2hr., but was slower from bovine IgG and continued for several hours longer. Since bovine IgG molecules contained on the average a greater amount of hexose in the `hinge' region, carbohydrate on this part of the γ-chain may influence not only the initial rate of enzymic hydrolysis into Fab and Fc fragments, but also, and to a greater extent, the rate of further limited hydrolysis of the N-terminal regions of the Fc fragment. The presence of carbohydrate in the `hinge' region does not appear to account for the resistance of some IgG molecules to papain digestion and of some Fc fragments to N-terminal degradation.     

Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain

IgG antibodies (Abs) and fragments of IgG Abs are becoming major biotherapeutics to treat an assortment of human diseases. Commonly prepared fragments of IgGs include Fc, Fab, and F(ab')2 fragments, all of which can be made using the sulfhydryl protease papain, although prolonged digestion times and/or excessive amounts of papain typically result in further cleavage of the Fc domain into smaller fragments. During our attempts to use papain to isolate Fc fragments from different IgG monoclonal Abs, it was observed that prior removal of Fc glycans resulted in a faster rate of papain-mediated degradation of the Fc domain. Subsequent time-course experiments comparing glycosylated and deglycosylated versions of IgG antibodies showed that the majority of molecules in a deglycosylated IgG sample were converted into Fab, Fc, and smaller Fc fragments in less than one hour, whereas the original glycosylated IgG required more than two hours to convert into a comparable amount of Fab and Fc fragments. Furthermore, whereas papain digestion converted almost all of a deglycosylated Fc fragment into smaller fragments of approximately 10 and approximately 12 kDa within 4 h, more than 40% of a glycosylated Fc fragment remained intact even after 24 h of digestion. These results indicate that the presence of CH(2) domain glycans in either IgGs or purified Fc fragments increases resistance to papain digestion. Increased sensitivity of non-glycosylated Fc domains to papain is consistent with the Fc domains lacking a defined structure, as exemplified by their inability to bind Fcgamma receptors, since misfolded proteins are often degraded by proteases because of increased accessibility of their proteolytic cleavage sites. Based on these observations it is possible to use papain sensitivity as a means of assessing proper Fc structure of IgG molecules.     

Human B-cell differentiation by Fc fragment of IgG. I. Fc fragment from human IgG induces plasma cell generation but cannot induce lymphocyte proliferation

Circulating mononuclear cells (MNC) from normal donors were examined for lymphocyte proliferation and plasma cell differentiation following stimulation by Fc and Fab fragments or by intact IgG. Lymphocyte differentiation and DNA synthesis were examined as a function of culture duration and concentration of Fc, Fab fragments, and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin and with specific antisera (anti-mu, -gamma, -alpha, -delta, -kappa, and -lambda chains). DNA synthesis of mononuclear cells cultures was analyzed by measuring [3H]thymidine incorporation. The results indicated that only the Fc fragments are able to induce the differentiation of B cells. The polyclonal plasma cell response to Fc fragments was dose dependent, peaked on the sixth day of culture, and was isotypically diverse (IgM greater than IgA greater than IgG). This activity requires the presence of T helper cells and monocytes. In contrast, the Fc fragments were unable to induce a proliferative response.     

Alternative mechanism of protein A-immunoglobulin interaction the VH-associated reactivity of a monoclonal human IgM

The immunoglobulin site(s) that mediates the alternative mechanism of interaction between immunoglobulins and staphylococcal protein A (SpA) was studied by using a monoclonal human IgM. Several IgM fragments were tested for their inhibitory effect in a competitive binding assay of 125I-IgM to SpA. Only those fragments containing Fab mu pieces showed some inhibitory activity. The reactivity of the Fab mu region was retained in some of its subfragments, such as Fv or the VH domain, unlike isolated light chains or VL domains. Furthermore, antibodies specific for the VH domain completely inhibited the SpA-IgM interaction. These results indicate that the alternative SpA-binding site of IgM is located in VH regions.     

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments.

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.     

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.     

Immunoelectron microscopic localization of idiotypes and allotypes on immunoglobulin molecules

We have developed a novel approach to the analysis of antigenic (allotypic and idiotypic) determinants on intact immunoglobulin molecules. Immune complexes composed of IgG in combination with anti-idiotype or anti-allotype antibody were "visualized" by transmission electron microscopy. Individual Fab fragments of anti-idiotype or anti-allotype antibody, when bound to the IgG, altered the "Y" configuration in a reproducible and interpretable manner. Anti-idiotype antibody (either as Fab or IgG) bound to the terminus of the presumed V region of the IgG molecule, thus extending the apparent length of the Fab arms. Analysis of a rabbit VH framework allotype (a1) revealed that the determinant(s) is (are) located on the lateral portion of the V region of IgG. Binding of the anti-a1 Fab fragments was always at approximately right angles to the axis of the Fab arms of IgG. Fab antibody to the rabbit kappa light chain (b4) allotype bound to the lateral portion of the terminal half of the IgG Fab arms. This technique should be of value in localizing less well defined immunoglobulin determinants.