Modification of a disulfide in the hinge region of rabbit IgG and study of the interaction of polyvalent ferritin antigen, protein A, and anti-IgG]

Since immunoglobulins are used in a vast variety of immunoassays, the problem of obtaining antibodies with enhanced antigen-binding activity is of great importance. In order to discriminate between putative approaches to activating antibody modification, some functional characteristics of rabbit IgG modified at the hinge disulfide by three reagents: iodoacetamide, N-ethylmaleimide, and 2.2'-dipyridyl disulfide, have been studied. As can be judged from gel-permeation chromatography data, the molecular sizes of modified rabbit IgG were slightly increased in comparison with the native protein. Using enzyme immunoassay, it was shown that modification by each of the above reagents results in the same degree of activation of the antibody binding to the protein polyvalent antigen-human ferritin, due to the increase in segmental flexibility, i.e., Fab motion around the Fo fragment of IgG. The type of concentration dependencies of antigen binding suggest that another determinant stimulating the antigen binding in addition to the increase in segmental flexibility, can be attributed to intra- or interdomain flexibility of domains constituting the Fab fragments. Using protein A and anti-IgG as conformational probes for the antibody Fo fragment, the conformation and conformational dynamics of both CD2 domain epitopes and the switch region between the CD2 and CH3 domains have been shown to be essentially unaffected by modification.     

Site of binding of IgG2b and IgG2a by mouse macrophage Fc receptors by using cyanogen bromide fragments

Cyanogen bromide fragments of murine IgG2b and IgG2a immunoglobulins were used to localize the sequences that are bound by specific IgG2b and IgG2a Fc receptors on murine macrophages. One fragment from the CH2 domain of IgG2b bound to the gamma 2b Fc receptor. Two fragments from IgG2a--one one from the CH2 domain, differing by only four amino acids from the homologous IgG2b fragment, and the other from the CH3 domain--specifically bound to the gamma 2a Fc receptor. In both a rosetting assay and a radioactive binding assay, these two fragments from IgG2a competed with intact IgG2a: however, they did not compete with each other. Rather, binding of the fragment from the CH3 domain of IgG2a augmented the binding of the fragment from the CH2 domain of IgG2a but not that of the homologous fragment from IgG2b. The binding of both IgG2a fragments was abolished by trypsin treatment of macrophages. These data suggest that 1) a sequence in the CH2 domain of IgG2b is sufficient for binding to the gamma 2b Fc receptor, 2) sequences from both the CH2 and CH3 domains of IgG2a bind to the gamma 2a Fc receptor, and 3) the binding of sequences from the CH3 domain of IgG2a may induce a conformational change in the gamma 2a Fc receptor that leads to enhanced binding of sequences from the CH2 domain.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

Anti-Ro autoantibody with cross-reactive binding to the heavy chain of immunoglobulin G.

Autoantibodies directed at the intracellular Ro ribonucleoprotein complex are found in the serum of patients with systemic lupus erythematosus (SLE) and related autoimmune diseases. The antigenic stimulus for the induction of these autoantibodies is unknown, although we have previously demonstrated that the Ro protein and immunoglobulin G (IgG) share immunologic determinants bound by anti-Ro antibodies. The present study further defines the fine specificity of this cross-reactive binding. Using both patient autoanti-Ro antibodies and antigen-induced rabbit anti-Ro serum, the binding specificity for IgG was located to the heavy chains of IgG outside the Fc domain. F(ab')2 fragments of IgG were observed to inhibit specific Ro binding by either human or antigen-induced rabbit sera, while Fc fragments of IgG failed to inhibit Ro binding. Anti-Ro sera were found to bind the heavy chains of IgG in immunoblots, and the antibodies eluted from these heavy chains were capable of immunoprecipitating the Ro particle from human cell extracts. Not all patient sera with anti-Ro antibodies possessed IgG binding antibodies. Studies of cyanogen bromide digestion fragments of IgG implicate the hinge region of IgG as the region cross-reactive with the Ro protein. The nature of this cross-reactivity may be important in understanding the induction and/or perpetuation of the anti-Ro response in patients with autoimmune disease.     

Two dual-specific (anti-IgG and anti-dsDNA) monoclonal autoantibodies derived from the NZB/NZW F1 recognize an epitope in the hinge region

The anti-IgG properties of two dual-specific (anti-dsDNA and anti-IgG) monoclonal NZB/NZW F1-derived autoantibodies, BV 17–45 and BV 16–13, were studied to resolve the location and possible commonality of the IgG epitope. To determine if BV 17–45 and BV 16–13 recognized the same IgG epitope, the relative temperature sensitivity of the conformational IgG epitopes were evaluated using the conformational sensitive immunoassay. Comparison of the temperature sensitivity of the conformational immunoglobulin epitopes over a temperature range of 25–100°C suggested that the epitope recognized by BV 17–45 was the same as the IgG epitope recognized by BV 16–13. Further studies with papain- and pepsin-generated F(ab′)₂, Fab, and Fc fragments of BV 17–45 and BV 16–13 revealed that the dual-specific autoantibodies BV 17–45 and BV 16–13 both bound an epitope in the hinge region of the IgG molecule. The potential correlation between these studies and the pathogenic nature of dual-specific autoantibodies is discussed.     

Two dual-specific (anti-IgG and anti-dsDNA) monoclonal autoantibodies derived from the NZB/NZW F1 recognize an epitope in the hinge region

The anti-IgG properties of two dual-specific (anti-dsDNA and anti-IgG) monoclonal NZB/NZW F1-derived autoantibodies, BV 17–45 and BV 16–13, were studied to resolve the location and possible commonality of the IgG epitope. To determine if BV 17–45 and BV 16–13 recognized the same IgG epitope, the relative temperature sensitivity of the conformational IgG epitopes were evaluated using the conformational sensitive immunoassay. Comparison of the temperature sensitivity of the conformational immunoglobulin epitopes over a temperature range of 25–100°C suggested that the epitope recognized by BV 17–45 was the same as the IgG epitope recognized by BV 16–13. Further studies with papain- and pepsin-generated F(ab′)₂, Fab, and Fc fragments of BV 17–45 and BV 16–13 revealed that the dual-specific autoantibodies BV 17–45 and BV 16–13 both bound an epitope in the hinge region of the IgG molecule. The potential correlation between these studies and the pathogenic nature of dual-specific autoantibodies is discussed.     

Studies on the Fc gamma receptor of the murine macrophage-like cell line P388D1. II. Binding of human IgG subclass proteins and their proteolytic fragments.

Binding studies of human IgG proteins to murine P388D1 cells indicated that they bind to an apparently homogeneous Fc receptor population. The association constant was 0.89 x 10(6)M-1 at 22 degrees C and was comparable to the binding affinities of homologous murine IgG2a and IgG2b. The number of receptor sites was found to be approximately 6 x 10(5)/cell. Fc gamma 1 and Fc gamma 3 fragments bound with an affinity comparable to that of the parent proteins. The P388D1 receptors could discriminate between the human IgG subclasses; the relative cytophilic activity was IgG3 greater than IgG1 greater than IgG4 and IgG2 was devoid of binding activity. Fragments corresponding to the C gamma 2 and C gamma 3 domains of human IgG1 were both unable to bind to the P388D1 receptors either alone or in equimolar combination. This suggests that the cytophilic site may be formed cooperatively by interaction between the two domains. The integrity of the hinge region appeared to be essential for full expression of cytophilic activity since reduction of the hinge-region disulfides in both human IgG1 and its Fc fragment markedly decreased their binding affinity. In addition, a mutant IgG1 molecule lacking the hinge region was significantly less cytophilic than its normal counterpart.     

Structural basis of the interaction between IgG and Fcgamma receptors

The binding of multivalent antigen-antibody complexes to receptors for the Fc portion of IgG (FcgammaR) induces the clustering of the FcgammaR and triggers cell activation leading to defence reactions against pathogens. The Fc portion of IgG consists of two identical polypeptide chains which are related to each other by a 2-fold axis and are folded in two structural domains, the C(H)2 domain, near the flexible hinge region of the IgG molecule, and the C(H)3 domain. We studied the interaction in solution between the Fc fragment of mouse IgG2b and the extracellular region of mouse FcgammaRII. We find that one Fc molecule binds one FcgammaRII molecule only. Using NMR spectroscopy, we show that FcgammaRII binds to a negatively charged area of the C(H)2 domain, corresponding to the lower hinge region, and that the binding of FcgammaRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. We therefore propose a model that explains why IgG molecules are unable to trigger FcgammaR-mediated cellular responses spontaneously in the absence of crosslinking by multivalent antigens.     

Stabilisation of the Fc fragment of human IgG1 by engineered intradomain disulfide bonds

We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of T(m) of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the T(m) of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the T(m) of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule.     

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.     

Cleavage of IgGs by proteases associated with invasive diseases

The effective functioning of immunoglobulins and IgG mAbs in removing pathological cells requires that the antigen binding regions and the Fc (effector) domain act in concert. The hinge region that connects these domains itself presents motifs that engage Fc receptors on immune effector cells to achieve cell lysis. In addition, sequences in the lower hinge/CH2 and further down the CH2 region are involved in C1q binding and complement-mediated cell killing. Proteolytic enzymes of little relevance to human physiology were successfully used for decades to generate fragments of IgGs for reagent and therapeutic use. It was subsequently noted that tumor-related and microbial proteases also cleaved human IgG specifically in the hinge region. We have shown previously that the “nick” of just one of the lower hinge heavy chains of IgG unexpectedly prevented many effector functions without impacting antigen binding. Of interest, related single-cleaved IgG breakdown products were detected in breast carcinoma extracts. This suggested a pathway by which tumors might avoid host immune surveillance under a cloak of proteolytically-generated, dysfunctional antibodies that block competent IgG binding. The host immune system cannot be blind to this pathway since there exists a widespread, low-titer incidence of anti-hinge (cleavage-site) antibodies in the healthy population. The prevalence of anti-hinge reactivity may reflect an ongoing immune recognition of normal IgG catabolism. Tumor growth and bacterial infections potentially generate hostile proteolytic environments that may pose harsh challenges to host immunity. Recent findings involving physiologically-relevant proteases suggest that the potential loss of key effector functions of host IgGs may result from subtle and limited proteolytic cleavage of IgGs, and that such events may facilitate the incursion of invasive cells in local proteolytic settings.     

Monoclonal antibodies to the bovine immunoglobulin G2a allotypes A1 and A2.

Production of murine monoclonal antibodies (Mabs) to bovine IgG2a-A1 and A2 allotypes resulted in three Mabs being selected as anti-IgG2a-A1 and A2 reagents. Two Mabs recognize the A1 allotype and 1 recognizes the A2 allotype. Initial epitope mapping with the Mabs indicates that one of the A1 epitopes resides in the hinge region and the other epitope resides more toward the C-terminus of the immunoglobulin. The A2 epitope recognized by the A2 Mab does not appear to reside in the hinge region of the immunoglobulin but apparently resides more C-terminal.     

A monoclonal anti-IgE antibody against an epitope (amino acids 367-376) in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23)

We have produced three different mAb specific for human IgE-Fc. Their binding pattern to either heat-denatured IgE or a family of overlapping IgE-derived recombinant peptides and their ability to affect interaction of IgE with its low affinity receptor Fc epsilon R2/CD23 demonstrate that they recognize distinct epitopes on the IgE molecule. All three mAb were able to induce basophil degranulation as measured by the induction of histamine release. mAb 173 recognizes a thermolabile epitope in the CH4 domain. It does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 272 recognizes a thermostable epitope that maps to a sequence of 36 amino acids (AA) spanning part of the CH2 and CH3 domain and it does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 27 recognizes a thermolabile epitope located on a 10 AA stretch (AA 367-376) in the CH3 domain. This area contains one N-linked oligosaccharide (Asn-371), but the antibody is not directed against carbohydrate because it binds to Escherichia coli-derived IgE peptides. mAb 27 inhibits the binding of IgE to Fc epsilon R2/CD23 but is still capable of reacting with IgE already bound to Fc epsilon R2/CD23. These data suggest that upon binding to Fc epsilon R2/CD23, the IgE molecule engages one of two equivalent-binding sites close to the glycosylated area of the CH3 domain.     

A hingeless Fc fusion system for site-specific cleavage by IdeS

Fusion of proteins to the Fc region of IgG is widely used to express cellular receptors and other extracellular proteins, but cleavage of the fusion partner is sometimes required for downstream applications. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a protease with exquisite specificity for human IgG, and it can also cleave Fc-fusion proteins at a single site in the N-terminal region of the CH2 domain. However, the site of IdeS cleavage results in the disulfide-linked hinge region partitioning with the released protein, complicating downstream usage of the cleaved product. To tailor the Fc fragment for release of partner proteins by IdeS treatment, we investigated the effect of deleting regions of IgG-derived sequence that are upstream of the cleavage site. Elimination of the IgG-derived hinge sequence along with several residues of the CH2 domain had negligible effects on expression and purity of the fusion protein, while retaining efficient processing by IdeS. An optimal Fc fragment comprising residues 235–447 of the human IgG1 heavy chain sufficed for efficient production of fusion proteins and minimized the amount of residual Ig-derived sequence on the cleavage product following IdeS treatment. Pairing of this truncated Fc fragment with IdeS cleavage enables highly specific cleavage of Fc-fusion proteins, thus eliminating the need to engineer extraneous cleavage sequences. This system should be helpful for producing Fc-fusion proteins requiring downstream cleavage, particularly those that are sensitive to internal miscleavage if treated with alternative proteases.     

Comparison of the Fc fragment from a human IgG1 and its CH2, pFc', and tFc' subfragments. A study using reductive methylation and 13C NMR

The Fc fragment of a human monoclonal IgG1 was compared with subfragments containing (a) the intact CH2 domain (CH2 fragment) or (b) the intact CH3 domain (pFc' and tFc' fragments). All fragments were reductively 13C-methylated and their resulting dimethyllysyl resonances characterized in 0.1 M KC1 as a function of pH by 13C NMR spectroscopy. Seven resonances were characterized for the 18 lysine residues of the Fc fragment, eight for the 12 lysines of the CH2 fragment, and five each for the 18 lysine residues of the Fc fragment, eight for the 12 lysines of the CH2 fragment, and five each for the 9 lysines of the pFc' and the 6 lysines of the tFc' fragments, respectively. The multiplicity of resonances indicates that the lysine residues in each fragment exist in a variety of microenvironments and that the fragments are all highly structured. The correspondence between 6 of the 12 or 13 perturbed lysine residues in the Fc fragment and the smaller subfragments indicates that the conformation of the CH2 and CH3 domains is largely unchanged in the smaller fragments. However, in addition to three lysines at the CH2-CH3 domain interface, whose environments were known to be disrupted in the smaller fragments, three or four lysine residues have somewhat different properties in the Fc fragment and in the subfragments, indicating that some local perturbations are induced in the domain structure in the subfragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential mapping of Fc gamma-binding and monoclonal antibody-reactive epitopes on gE, the Fc gamma-binding glycoprotein of herpes simplex virus type 1.

The entire 396 residue extracellular sequence of gE the HSV-1 Fc gamma-binding glycoprotein has been studied to determine epitopes binding to two mAb II-481 and 88S previously demonstrated to react with gE at or near the Fc gamma-binding regions. Overlapping 7-mers constructed from the established sequence were tested with mAb II-481 and 88S along with their Fab fragments. Control mAb of the same IgG 2b subclass as well as whole rabbit and human IgG and Fc were also tested for binding to overlapping linear sequences using the ELISA pin assay to map Fc gamma-binding regions. Six sequences PKTSWRRVS, GLYTLSV, QVASVVLVVQP, PAPPRSWP, CLYHPQLP, and ASTWTSRL were found that constituted major regions binding to the two different mAb of the same specificity. Glycine substitution for each residue within these sequences indicated that arginine 29, tryptophane 70, valine 144, valine 157, arginine 208, histidine 283, and arginine 305 constituted important portions of the II 481 mAb-reactive epitope. Many of the same regions along with one other, GPLHPSW, appeared to be involved in Fc gamma binding. Substitution of glycine for each residue indicated that histidine 67, tryptophane 70, valine 71, valine 157, valine 158, valine 160, valine 161, tryptophane 210, serine 279, cysteine 280, leucine 281, tyrosine 282, histidine 283, proline 284, glutamine 285, proline 287, tryptophane 302, and arginine 305 were important for Fc gamma-binding. Inhibition by gE peptides of rosetting of E sensitized with rabbit IgG antibody around HSV-1-infected cells, as well as inhibition of rosetting using F(ab)2 fragments of rabbit antibodies to these peptides was used to assay relative contributions of all seven regions to Fc gamma-binding activity. Our results provide a tentative map of mAb binding and Fc gamma-reactive sites on gE. mAb and Fc gamma binding of a limited number of individual antigenic amino acids widely distributed among the separate reactive regions suggest that many of the same separate residues contribute both to antigenicity as well as to Fc gamma-binding activity.     

Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.     

Development of a compact anti-BAFF antibody in Escherichia coli

Recombinant antibodies, especially single-chain antibody fragment (scFv), can be applied as detection reagents and even substitute for some reagents used in immunoassays. For scFv fragments, there is no such universal system available up to now. We have constructed vectors for the convenient, rapid expression of a novel compact antibody composed of anti-B-cell-activating factor of the TNF family (BAFF) scFv and the Fc portion (the hinge region, CH2, and CH3 domains) of the human IgG1 in Escherichia coli. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The scFv-Fc antibody was demonstrated to retain high binding affinity to antigen, including membrane-bound BAFF and soluble BAFF, and to possess some human IgG crystallizable fragment domain functions, such as human complement C1q and protein A binding. Both size-exclusion high-performance liquid chromatography column analysis and Western blotting of proteins subjected to nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested that scFv-Fc antibody is homodimeric with relative molecular mass of 110 kDa. These findings suggest that the compact antibody may be useful in diagnostic application for the prediction of BAFF relevant to autoimmune diseases in human.     

Location of antigenic epitopes on antibody molecules

Using X-ray crystallographic co-ordinates of immunoglobulins, surface regions accessible to a large spherical probe, comparable in size to an antibody domain, were computed. Locations of these exposed regions were compared with those of experimentally determined antigenic sites, i.e. idiotypic, allotypic and isotypic serological markers. In all cases, an excellent agreement was found. The most prominent computed epitopes correspond to convex parts of antibody surface made by reverse turn segments of the polypeptide chain. The computed epitopes occur in homologous positions in all the immunoglobulin domains, and most of the beta-sheet surfaces on the domains are poorly antigenic. The CH2 domain (Fc fragment) has many more antigenic sites than the Fab fragments (antigen-binding fragments). Variable domain epitopes (idiotypes) involve both hypervariable and framework residues, and only about 25% of the hypervariable residues are strongly antigenic. The results indicate that, in a vertebrate body, each antibody molecule may be recognized, and its concentration regulated, by at least 40 complementary anti-immunoglobulin antibodies; therefore, a possibility of an "immune network" with much higher connectivity than is generally assumed should be seriously contemplated.     

Identification and antigenic epitope mapping of immunodominant region amino residues 510 to 672 on the spike protein of the severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510-672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539-559), S1C4 (548-567), S1C7/8 (583-606), and S1C10/11 (607-630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome.