Fusion of barnase to antiferritin antibody F11 VH domain results in a partially folded functionally active protein.

A chimeric protein, VH-barnase, was obtained by fusing the VH domain of anti-human ferritin monoclonal antibody F11 to barnase, a bacterial RNase from Bacillus amyloliquefaciens. After refolding from inclusion bodies, the fusion protein formed insoluble aggregates. Off-pathway aggregation was significantly reduced by adding either purified GroEL/GroES chaperones or arginine, with 10-12-fold increase in the yield of the soluble protein. The final protein conformation was identical by calorimetric criteria and CD and fluorescence spectroscopy to that obtained without additives, thus suggesting that VH-barnase structure does not depend on folding conditions. Folding of VH-barnase resulted in a single calorimetrically revealed folding unit, the so-called "calorimetric domain", with conformation consistent with a molten globule that possessed well-defined secondary structure and compact tertiary conformation with partial exposure of hydrophobic patches and low thermodynamic stability. The unique feature of VH-barnase is that, despite the partially unfolded conformation and coupling into a single "calorimetric domain", this immunofusion retained both the antigen-binding and RNase activities that belong to the two heterologous domains.

Folding and stability of chimeric immunofusion VL-barstar.

A chimeric protein VL-barstar that comprises the VL domain of anti-human ferritin monoclonal antibody F11 and barstar, the naturally occurring inhibitor of bacterial RNase barnase, has been constructed for study of structure-function characteristics of chimeric immunoglobulin fused proteins. Such chimeric constructs may be potentially employed for development of bivalent/bispecific antibodies on the basis of the high affinity interaction between barstar and barnase (the association constant is about 10(14) M(-1)). We have developed a protocol for VL-barstar expression in E. coli and purification and refolding from inclusion bodies that yields a homogeneous and soluble form of this protein. Differential scanning calorimetry in combination with fluorescence and CD spectroscopy revealed that the VL-barstar formed well-resolved ordered secondary and compact tertiary structures. However, partial loss of tertiary interactions resulted in low stability of the recombinant protein and the lack of functional activity of the two constituent modules. These conformational features suggest that the protein might be referred to the class of native molten globules, which comprises partially unfolded conformations stabilized under physiological conditions. Since individually expressed VL domain and barstar retain completely folded conformation and stable spatial structure, the incomplete folding of the chimeric protein may be attributed to interaction between heterologous domains, which appears at the folding stage preceding formation of a system of tertiary interactions in both structural modules. The results provide evidence for non-native interactions between heterologous modules that may occur in chimeric proteins composed of taxonomically distinct fusion partners.

Local conformational changes in ferritins with variable iron content.

Conformational changes were induced in human spleen ferritin by partial or complete removal of iron, and the immunoreactivity of the ferritin samples with variable iron content was analyzed. We established that a decrease in iron content resulted in bimodal changes in immunoreactivity of the epitopes recognized by the monoclonal antibodies G10 and F11. Immunoreactivity demonstrated a 3-6-fold decrease on lowering iron content from 800 to 40 atoms per protein molecule, followed by a sharp (4-14-fold) increase that was observed when low-iron ferritin was converted to iron-free apoferritin. These bimodal changes suggest the presence of more than two conformational states of ferritin with local alterations of the epitopes recognized by the monoclonal antibodies. The global conformation of ferritin, however, remained essentially unaltered, as demonstrated by ferritin interaction with polyclonal antibodies. Together, the results indicate that local conformational changes in the ferritin protein shell occur on progressive iron removal that results in low-iron and iron-free forms of ferritin. These changes are most clearly seen in apoferritin when compared to low-iron ferritin.

Thermodynamic stability and functional activity of tumor-associated antibodies.

Tumor-associated antibodies of human IgG1 subclass were eluted from cell-surface antigens of human carcinoma cells and studied by differential scanning calorimetry and binding to local conformational probes, protein A from Staphylococcus aureus and a monoclonal antibody targeted to the CH2 domain of the Fc fragment. At pH 2.0-7.0, we observed virtually identical enthalpies of thermal unfolding for IgG1 from normal human sera and tumor-associated IgG1. The exact values of calorimetric enthalpy (Delta h) at pH 7.0 were 6.1 and 6.2-6.3 cal/g for IgG1 from normal serum and IgG1 from carcinoma cells, respectively. The affinity constants of protein A binding to the CH2--CH3 domain interface demonstrated differences between serum IgG1 and tumor associated IgG1 that did not exceed 3-8-fold. The binding affinity toward the anti-CH2 monoclonal antibody determined for serum IgG1 and IgG1 from carcinoma cells differed not more than 2.5-fold. The thermodynamic parameters of IgG1 from carcinoma cells strongly suggest that protein conformational stability was essentially unaltered and that the Fc fragment of the tumor-derived IgG1 preserved its structural integrity.

Antigen-binding activity of monoclonal antibodies after incubation with organic solvents.

Effects of four organic solvents--methanol, trifluoroethanol, dimethylsulfoxide, and dimethylformamide (DMF)--on the ferritin-binding activity of three monoclonal mouse antibodies of IgG2a and IgG1 subclasses were studied. The ferritin-binding constants of monoclonal antibodies G10 and F11 (the IgG2a subclass) were increased 2-6-fold after incubation with DMF and removal of the organic solvent by gel filtration. The maximum effect on the F11 antibodies was found in the presence of 5-13% DMF and on the G10 antibodies at 11-40% DMF. The effect remained after the removal of DMF from the incubation medium, and this suggests that the incubation with DMF resulted in irreversible conformational changes of the antibodies and in production of active conformers of the G10 and F11 antibodies. These conformations occurred within 15-60 min. The long-term stability and the fluorescence of the antibodies exposed to DMF suggest that the conformational changes were not global, but involved small and relatively independent structural elements of the antibodies, either of hypervariable CDR loops in variable domains or of the hinge region of the antibodies. The affinity of the C5 antibodies of the mouse IgG1 subclass was decreased after incubation with DMF. The activation was a solvent-specific effect because incubation of the G10 antibodies with methanol and dimethylsulfoxide decreased the affinity for the antigen, and incubation with trifluoroethanol virtually did not affect it. Relatively small changes in the antigen-binding activity of the antibodies were found even after the incubation with 5% organic solvent.

Expression, refolding, and ferritin-binding activity of the isolated VL-domain of monoclonal antibody F11.

Expression of the VL-domain of mouse monoclonal antibody F11 to human spleen ferritin in Escherichia coli cells is associated with the formation of insoluble protein aggregates (inclusion bodies). The aggregates were solubilized in the presence of guanidine hydrochloride and the recombinant VL-domain was purified by immobilized metal affinity chromatography (IMAC). Subsequent renaturation results in approximately 99% pure preparation with high yield. The VL-domain forms dimers at concentrations from 1 to 10 mg/ml. Monomeric form is detected only at protein concentrations below 0.5 mg/ml. Functional activity of the VL-domain was verified by two variants of ELISA. The affinity of the VL-domain ((0.2-1.2). 108 M(-1)) is similar to the affinity of the full-length parental antibody F11 because when the immobilized VL-domain was used, the binding constant of ferritin to the VL-domain was only 4-6-fold lower than that in the case of F11 antibody. In another ELISA system with immobilized ferritin, affinity was decreased 30-fold. The VL-domain of antibody F11 is the first example of the recombinant variable domain of the immunoglobulin light chain that preserves the antigen-binding activity in the absence of the partner VH-domain. The data indicate that the recombinant VL-domain can be used in construction of chimeric immunotoxins and other antigen-binding proteins in immunotherapy and in studies of correlations between folding, stability, and activity of immunoglobulins.

Antiferritin single-chain Fv fragment is a functional protein with properties of a partially structured state: comparison with the completely folded V(L) domain.

Differential scanning calorimetry and spectroscopic probes were applied to study folding and stability of the single-chain Fv fragment (scFv) of the anti-human ferritin antibody F11 and its isolated variable light-chain (V(L)) domain. The scFv fragment followed variable heavy-chain domain (V(H))-linker-V(L) orientation and contained (Gly(4)Ser)(3) linker peptide. The two proteins were produced in Escherichia coli and refolded from denaturant-solubilized inclusion bodies. The isolated V(L) domain demonstrated a typical immunoglobulin fold with well-defined secondary and tertiary structure and was capable of binding human ferritin with K(a) = 1.8 x 10(7) M(-)(1), approximately (1)/(30) of the affinity of the parent F11 antibody. Involvement of this V(L) domain into the two-domain scFv fragment yielded a distorted secondary and significantly destabilized tertiary structure in which neither of the two constituent domains attained complete folding. The thermal unfolding enthalpy of scFv F11 at pH 7.0 was as low as 5. 0 J.g(-)(1) versus 16.3 J.g(-)(1) obtained for the V(L) domain and 24.7 J.g(-)(1) for the parent F11 antibody (mouse IgG2a subclass). Intrinsic fluorescence and near-ultraviolet circular dichroic (CD) spectra, and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonate, confirmed partial loss of tertiary interactions in scFv. The spectroscopic and calorimetric properties of scFv F11 under physiological conditions are consistent with a model of a partially structured state with a distorted beta-sheet as a secondary structure and partial loss of tertiary interactions, which closely resembles the alternatively folded A-state adopted by an immunoglobulin at pH 2-3 [Buchner, J., Renner, M., Lilie, H., Hinz, H.-J., Jaenicke, R., Kiefhaber, T., and Rudolph, R. (1991) Biochemistry 30, 6922-6929]. However, scFv F11 demonstrated only an approximately 4-fold decrease in the antigen-binding affinity (K(a) = 1.3 x 10(8) M(-)(1)) versus the parent F11 antibody. The scFv fragment F11 provides the first description of a functional protein trapped under physiological conditions in a partially structured state. This state is either close to the native one in the antigen-binding affinity or, alternatively, initial weak binding of the antigenic epitope induces folding of scFv F11 into a more structured conformation that generates relatively high affinity.

Two high-affinity monoclonal IgG2a antibodies with differing thermodynamic stability demonstrate distinct antigen-induced changes in protein A-binding affinity.

Two IgG2a monoclonal antibodies (G10 and F11) are described which have similar affinity for human spleen ferritin and identical protein A-binding affinity. The two mAbs display changes in protein A-binding affinity following binding of the antigen to its specific recognition site in the variable domains. However, while antigen-induced conformational changes in G10 enhance its affinity to protein A, interaction of F11 with ferritin results in a significant decrease in protein A-binding affinity. In contrast to the IgG2a antibodies, using a mouse IgG1 antiferritin antibody (C5) high-affinity binding of the antigen does not change an inherently low ability to bind protein A. Differential scanning calorimetry revealed that the enthalpy and Gibb's free energy of thermal unfolding for G10 was 19% and 23% higher, respectively, than the corresponding parameters for F11. The lower structural energetics of F11 are associated with the absence of a calorimetrically revealed folding unit, which may be responsible for interactions between the antigen-binding site and the protein A-binding site. This study provides the first demonstration that functionally significant interactions between two recognition sites in antibodies of the same subclass can be modulated by subclass-independent structural variations associated with different thermodynamic stability.

Distinct stability of recombinant L and H subunits of human ferritin: calorimetric and ANS binding studies.

Thermodynamic and pH stability of recombinant human L- and H-ferritins were probed by differential scanning calorimetry and 8-anilino-1-naphthalenesulfonate (ANS) binding in the pH range 2-7. At pH 2.0-2.8 they were dissociated into subunit monomers and in this pH interval the H-subunit displayed a single calorimetrically-revealed domain with properties of a molten globule-like state: low enthalpy (6.3-8.0 J/g or 169-172 kJ/mol) and Tm of thermal unfolding (approximately 50 degrees C), a wide transition range (approximately 20 degrees C) and high ANS binding. In contrast, at pH 2 the L-ferritin subunit showed two calorimetric domains with Tm of 35 and 40 degrees C with similar unfolding enthalpies and with moderate extent of interactions, as indicated by the ratio of calorimetric enthalpy (293.9 kJ/mol) and van't Hoff enthalpy (174.2 kJ/mol) for the thermal transition. A pH increase from 2.0 to 2.8 determined the coupling of the two domains into a single cooperative folding unit and drastic increase of the transition temperature (from 37 to 80 degrees C). The contacts between the two domains in the L-subunit appeared to contribute to about 30% of the total stabilization free energy. The unfolding enthalpies, heat capacity changes and pronounced ANS binding of the L-subunit at pH 2.0-2.8 indicated that part of the structure lacked 'meltable' tertiary interactions. The results indicate that H- and L-subunits are stabilized by largely different intra-chain interactions with a critical contribution to L-subunit stability of embedded salt bridge(s) absent in the H-subunit.

Thermodynamic stability of immunoglobulins and allosteric interactions with ferritin and protein A: distinct properties of the two antibodies of IgG2a subclass.

The two anti-ferritin monoclonal antibodies of mouse IgG2a subclass, G10 and F11, are described that have similar affinity to human spleen ferritin and identical protein A-binding affinity. Antigen binding was shown to change significantly the protein A-binding parameters of the IgG2a antibodies. Antigen-induced conformational changes result in enhanced protein A-binding affinity of the G10 antibody while reduced affinity of the F11 antibody. Antigen binding does not change inherently low affinity of the anti-ferritin IgG1 antibody C5 to protein A. Differential scanning calorimetry revealed that the enthalpy and Gibbs free energy of denaturation for G10 was respectively by 19 and 29% higher than the corresponding parameters for F11. The lower structural energetics of F11 is associated with the lack of a calorimetrically revealed folding unit that may be responsible for distinct interaction between the antigen-binding and protein A-binding sites. This work provides experimental demonstration of the fact that functionally significant interactions between the two spatially remote recognition sites in antibodies of the same heavy chain isotype can be modulated by relatively small structural variations that also result in different thermodynamic stability.

Antiferritin single-chain antibody: a functional protein with incomplete folding?

The pET(scF11) plasmid was constructed comprising the gene of a single-chain antibody against human ferritin. This plasmid encodes the leader peptide pelB followed by the heavy chain variable V(H) domain, (Gly4Ser)3 linker peptide, and light chain variable V(L) domain. The correctly processed scF11 antibody was expressed in Escherichia coli as an insoluble protein without the leader peptide. Purified soluble scF11 was obtained after solubilization in 6 M GdnHCl followed by a sequential dialysis against decreasing urea concentrations and ion-exchange chromatography. ScF11 demonstrated only a approximately 8-fold decrease in the affinity (Ka = 5.1 x 10(8) M(-1) in RIA and 1.8 x 10(8) M(-1) in ELISA) vs. the parent IgG2a/kappa monoclonal antibody F11. The emission maximum of intrinsic fluorescence strongly suggests a compact conformation with tryptophanyl fluorophores buried in the protein interior, consistent with the functionality of the protein. However, scF11 demonstrated (i) the lack of denaturant-induced fluorescence 'dequenching' effect characteristic of the completely folded parent antibody, and (ii) prominent binding, under physiological conditions, of a hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS) recognizing partially structured states of a protein. These findings are indicative of an incomplete tertiary fold that gives ANS access to the protein hydrophobic core. This work provides the first indication that the functional single-chain antibody scF11 displays some properties of a partially structured state and therefore may possess incomplete folding.

Immunoreagents based on polymer dispersions for immunochemical assays.

Immunoreagents based on polymer dispersions consisting of unimodal polyacrolein (PAL) microspheres with diameters in the range 0.3-2.0 microns have been prepared and evaluated by various immunoassay techniques such as immunoradiometric assay of ferritin and microtitre particle agglutination and immunofiltration dot assay of group-specific polysaccharide of S. pyogenes (A-PS) in comparison with conventional carriers and methods. The antibodies were covalently or indirectly bound to the PAL. The coupled antibodies to ferritin retained a high average affinity (Ka = 4.5 x 10(9) M-1). In comparison with microcrystalline cellulose-based immunosorbent, more than an order-of-magnitude lower amount of PAL-IgG was necessary for the analysis of ferritin. Use of PAL-IgG gave a higher sensitivity of assay with a detection limit of 0.7 x 10(-13) M l-1 and a wider concentration range of antigen detection (about four orders of magnitude) without manifestation of the high-dose hook effect. Particle agglutination assay of A-PS in microtitre plate was shown to be a simple, demonstrative and highly sensitive one-step analytical method with a detection limit of 0.05 ng A-PS/ml or 10(4) cells/ml. The sensitivity of immunofiltration assay using both enzyme and latex markers was shown to be approximately the same (50 ng A-PS/ml) and the duration of the assay was 3-5 min. No cross-reaction of latex conjugates with non-A Streptococcus cell lysates were observed.

Functional activation of antibodies on modification with Pd(II) coproporphyrin I N-Hydroxysuccinimide ester.

Metalloporphyrin-antibody conjugates provide a significant advantage over other types of conjugates in biomedical use for phosphorescence immunoassay, targeted immunotherapy, and internal imaging of malignant tumors. Monoclonal HSF102 antibody of mouse IgG2a subclass and monospecific rabbit IgG, both antibodies directed to human spleen ferritin, were modified with the new reagent Pd(II) coproporphyrin I tetra-N-hydroxysuccinimide ester. Functional study of the conjugates obtained revealed a 7- and 1.4-fold increase in the antigen binding activity for monoclonal HSF102 and monospecific IgG antibodies, respectively. For rabbit monospecific IgG, a concomitant increase in binding to anti-rabbit IgG antibodies directed to the epitopes of the CH2 domain in the Fc fragment was observed. In all cases, the maximum functional activation was found after conjugating one mole porphyrin per mole antibody. These results suggest that functional activation of the conjugates might be due to an increase in conformational flexibility of an antibody molecule after the modification. This increase in flexibility involves the Fab fragments and a pair of the CH2 domains in the Fc fragment and might be due to a significant charge shift (minus 5 charge units per modified amino group) that occurs after conjugation of an antibody with tetracarboxylic porphyrin.

[Non-native conformational states of immunoglobulins: thermodynamic and functional analysis of rabbit IgG]. / Nenativnye konformatsionnye sostoianiia immunoglobulinov: termodinamicheskii i funktsional'nyi analiz IgG krolika.

By changing pH within the pH range 2-7, the processes of intramolecular rearrangement in the multidomain structure of rabbit IgG were induced which resulted in the formation of four IgG conformers - N, NA, I and NI, differing in the amount of structure in the CH2 domain and as well as in its interaction with the neighboring domains. The stability of the IgG conformers was studied by differential scanning calorimetry and expressed in terms of thermodynamic parameters, delta H, Tm, and delta G. The pH-dependent changes in thermal stability of IgG and the range of stability for the conformers were described by conformational (phase) diagrams. The native N-conformer exists at pH 7.0-5.5. At pH < 5.5 the formation of the NA conformer showing significant decooperation (lower extent of interaction) of the domains occurs. Decooperation permits the CH2 domain in the NA conformer to undergo conformational transitions independently of the rest of the molecule-the property which cannot be observed for the more cooperative N-conformer of IgG. The formation of the intermediate I-state occurs at pH < 3 as a consequence of unfolding of the tertiary structure of the CH2 domain, while its secondary structure and compact tertiary structure of the remainder of the IgG domains remain unchanged. Such partially structured and stable states of immunoglobulins have not yet been described. Reversibility of pH-induced transitions in IgG was studied using calorimetry and ligand-binding assays involving the C1q component of the complement, protein A, antigen and monospecific anti-CH2 domain antibodies as conformational probes reporting local conformational changes in the CH2 domain and its interdomain interfaces. Refolding of IgG from the I-state by increasing pH results in the formation of a functionally active N1 conformer differing from native IgG by a changed balance of interdomain interactions. In addition to previously reported data suggesting a greater extent of the CH2 and CH1 domain interactions in the NI conformer, this conformer was shown to possess a lower extent of interaction of the CH2 and CH3 domains. The changed stability and a balance of domain interactions in the NI conformer results in changes in some of the effector functions including enhanced two-site binding to protein A and antigen-dependent binding to C1q with a concomitant decrease in the affinity of protein A binding site and reduced antigen binding capacity of the high affinity IgG. Low affinity IgG does not change the antigen-binding parameters after refolding to the N1 conformer.

Modification of monoclonal and polyclonal IgG with palladium (II) coproporphyrin I: stimulatory and inhibitory functional effects induced by two different methods.

Antibodies conjugated with porphyrins and metalloporphyrins have a great potential for applications in fluorescence or phosphorescence immunoassays as well as in photodynamic therapy, radioimaging and internal radiation therapy of cancer. Here we describe how the new preactivated metalloporphyrin, palladium (II) coproporphyrin I-tetra-N-hydroxysuccinimide ester, can be covalently attached to mouse monoclonal and rabbit anti-human ferritin antibodies. The advantages of the proposed reagent over the previously reported carboxylic porphyrins coupled through carbodiimide activation are indicated. Conformational changes in antibodies caused by each of the two methods were assessed from their binding to the antigen (a probe for the antibody Fv domain) and anti-IgG antibodies probing the global conformation of the CH2 domain in the Fc fragment. Porphyrin coupling through carbodiimide activation resulted in a decrease in both functional activities of modified antibodies even at low levels of modification. In contrast, when the N-hydroxysuccinimide (NHS) derivative of porphyrin was used, enhancement of the antigen-binding affinity of porphyrin-antibody conjugates occurred due to an increase in the conformational mobility (flexibility) of the modified antibodies. The stimulatory effect of conjugation was maximal when one porphyrin molecule was coupled to an antibody molecule. Coupling of NHS-activated porphyrin at pH 7.4, 7.8 and pH 8.5 suggested that the high efficiency of the reaction at pH 8.5 could be attributed predominantly to the formation of antibody aggregates, only 50% of which were covalently cross-linked. The lowest percentage of aggregates in porphyrin-antibody conjugates was found at pH 7.4 and a molar reagent-to-protein ratio in the 10:1-40:1 range. Thus, the use of the NHS-activated carboxylic porphyrin provides a mild, simple and convenient procedure for preparing antibody conjugates with enhanced antigen-binding affinity.

[Polyacrolein latex as a solid phase carrier for radioimmunoassay. Comparison with microcrystalline cellulose and polystyrene test tube surface]. / Poliakroleinovyi lateks v kachestve tverdofaznogo nositelia dlia immunoradiometricheskogo analiza. Sravnenie s mikrokristallicheskoi tselliulozoi i poverkhnost'iu polistirol'nykh probirok.

The immunosorbent obtained through a covalent immobilization of antibodies on a polyacrolein latex was studied in comparison with the sorbents based on microcrystalline cellulose and with the surface of polystyrene test tubes in a two-center radioimmunoassay for ferritin. It was shown that the polyacrolein latex provides a covalent binding of up to 75 mg protein per g dry polymer with an immobilization efficiency of 50-70% and a high effective association constant of immobilized antibodies (Ka = 4.5 x 10(9) M-1). The same parameters for the sorbent based on microcrystalline cellulose were 33 mg/g, 25-35%, and 4.3 x 10(8) M-1, respectively. In the radioimmunoassay for ferritin, the immunosorbents based on polyacrolein latex and cellulose ensured a nearly the same analytical sensitivity at the level of (0.7-0.9) x 10(-13) M ferritin and pointed to the possibility of eliminating the "hook-effect". Compared to the immunosorbent based on polystyrene test tubes, the advantages of the polyacrolein latex are the decrease in the minimum detectable concentration (increase in analytical sensitivity), the extension of the dynamical range of analysis, and the absence of the "hook-effect".

Thermodynamic and functional characterization of a stable IgG conformer obtained by renaturation from a partially structured low pH-induced state.

At pH2, rabbit IgG adopts a partially structured state that exhibits loss of thermal unfolding transition, tentatively assigned to the CH2 domain, whilst retaining a well-defined tertiary structure for the rest of the molecule and extensive secondary structure. Renaturation of IgG from this state yields a stable conformer that differs from native IgG by a lower degree of interaction between the CH2 and CH3 domains, and stronger interaction between the CH1 and CH2 domains, as judged by differential scanning calorimetry and probing the IgG conformation with specific ligands (C1q component of complement, protein A and monospecific antibodies to the CH2 domain and hinge region.

Large increase in thermal stability of the CH2 domain of rabbit IgG after acid treatment as evidenced by differential scanning calorimetry.

Rabbit IgG after exposure to 0.05 M glycine-HCl, pH 2.0, and native IgG were compared by differential scanning calorimetry (DSC) at pH 3.5 and C1q binding studies at pH 7.8. For acid-treated IgG, a large increase (by approx. 12-15 degrees C) in thermal stability of the CH2 domain occurs and this domain no longer demonstrates a separate and thermodynamically independent unfolding at 56 degrees C seen for native IgG. The results suggest that stabilization of the CH2 domain in acid-treated IgG arises from stronger, relative to the native protein, interaction of the CH2 domain with adjacent and more stable IgG domain(s). Conformational differences of the two forms of IgG were confirmed at neutral pH by a 4-fold increase of C1q-binding affinity of acid-treated IgG.

[A stable conformer of IgG, prepared by an acidic influence: study by calorimetry, binding of the C1q complement component, and monospecific anti-IgG]. / Stabil'nyi konformer IgG, poluchennyi kislotnym vozdeistviem: issledovanie s pomoshch'iu kalorimetrii, sviazyvaniia C1q komponenta komplenta i monspetsificheskikh anti-IgG.

Thermal stability and functional activity of rabbit IgG in its native conformation and after incubation at pH 2.0 were studied using differential scanning calorimetry and binding of conformational probes, i.e., the C1q component of the complement and two monospecific anti-IgG antibodies. The latter reacted selectively with the "hinge" region joining the Fab and Fc fragments of IgG or with the CH2 domain in the Fc fragment. At pH 2.0 complete unfolding of rabbit IgG did not occur: the protein demonstrated the presence of secondary and compact tertiary structures but differed from the native conformation by decreased overall enthalpy and TM of thermal denaturation as well as by changed secondary structure parameters as could be evidenced from CD spectroscopy and scanning calorimetry data. Incubation at pH 2.0 followed by renaturation at neutral pH led to irreversible conformational changes in IgG. The most significant differences between the two IgG conformers were demonstrated by calorimetry at pH 3.5 which revealed that the acid-treated conformer differs from the native one by enhanced thermal stability of the CH2 domain. Using the combination of thermodynamic and functional studies, it was shown that the origin of stabilization was the increase in the extent of interaction between the CH2 domain in the Fc fragment and the CHI domain in the Fab fragment. This resulted in the increase of the functional link between the antigen-binding domain and the C1q binding site in the CH2 domain of the acid-induced IgG conformer. In parallel with the increase in stability of the CH2 domain, conformational changes in the "hinge" region were found, together with the absence of intrinsic conformational changes in the CH2 domain proper as could be judged from C1q and monospecific anti-IgG binding assays. The results obtained demonstrate one of possible mechanisms whereby functionally significant rearrangements in the IgG molecule can be induced by changes in the interactions between invariably folded domains rather than by intrinsic changes in the domain conformation.

[Monoclonal antibodies to human spleen ferritin. II. Localization of epitopes and quantitative parameters of antigen binding]. / Monkonal'nye antitela k ferritinu selezenki chekoveka. II. Lokalizatsiia épitopov i kolichestvennye parametry sviazyvaniia antigena.

The interaction of three monoclonal antibodies with human spleen ferritin has been studied. Using titration of various monoclonal antibody-containing media with the immobilized antigen, the specific content of active antibodies capable of binding to ferritin was determined, which was 22-27% for ascitic fluids, 35-50% for total mouse IgG and 88% for affinity-purified HSF102 antibody. Using [125I]ferritin and a novel computer-aided technique for determining the antigen-antibody binding, the affinity constants were obtained which ranged from 6.10(8) to 3.10(9) M-1. The monoclonal antibodies inhibited by 77-95% the binding of rabbit polyclonal antibodies to ferritin. This result is suggestive of a compact distribution on the ferritin surface of immunodominant epitopes forming clusters of closely related antigenic sites recognized by monoclonal and rabbit polyclonal antibodies. Competitive binding and additivity assays revealed that the epitope for the HSF102 antibody was sterically remote from the epitopes recognized by HSF101 and HSF103 antibodies to allow for noncompetitive binding of two different monoclonal antibodies. The competition between HSF101 and HSF103 antibodies pointed to the overlapping of their epitopes. It was found that no more than four [125I]IgG molecules could simultaneously be bound to one ferritin molecule which reflected the maximal valency of this antigen during its interaction with IgG antibodies.