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.

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.

[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".

[Interaction of rabbit IgG with anti-IgG: localization of epitopes and absence of immunoreactivity of the pFc'-fragment]. / Vzaimodeistvie IgG krolika s anti-IgG: lokalizatsiia épitopov i otsutstvie immunoreaktivnosti pFc'-fragmenta.

To localize essential epitopes of rabbit IgG, a series of proteolytic IgG fragments obtained by papain (Fab, Fc) or pepsin (pFc', F(ab')2) proteolysis have been prepared and their interaction with sheep antibodies against rabbit IgG has been studied. The data obtained suggest that essential immunoreactive epitopes of rabbit IgG are located in the CH2 domain and hinge region. This finding is in line with the results obtained by computing the antigenic sites of immunoglobulins. However, the deviation from the computed antigenic structure was deduced from the complete lack of immunoreactivity of the pFc fragment, it being a dimer of the terminal CH3 domain of the Fc fragment. The hinge region comparable in size with the dimensions of the epitope reveals high affinity binding to anti-IgG, thus testifying to the localization of the expressed epitope or its essential part in the hinge region. Proteolytic cleavage of this region leads to a significant decrease in the binding of the IgG fragment to anti-IgG. In addition to the CH2 domain and hinge region, a relatively low interaction of the antigen-binding antibody fragments with anti-IgG was found.

[Study of rabbit IgG, modified with carboxycarbonic anhydride of diethylenetriaminopentacetic acid]. / Issledovanie krolich'ego IgG, modifitsirovannogo karboksikarbonovym angidridom diétilentriaminpentauksusnoi kisloty.

The affinity-purified by chromatography on immobilized antigen rabbit IgG was modified with mixed carboxycarbonic anhydride of DTPA which markedly alters the interaction of charged residues in the protein molecule. To study the correlation between the antigen binding activity and the conformational mobility of IgG, the reactivity of modified IgG towards conformational probes targeted at variable and constant IgG domains, was investigated. The antibody against CH2 domains of IgG, staphylococcal protein A and protein antigen ferritin were used as conformational probes. It was found that modification of IgG amino groups entails the global increase in conformational mobility involving the Fab fragments, CH2 and, probably, the CH3 domains of the Fc portion of IgG. Taking advantage of Fab fragments modification it was shown that two processes contribute to the global increase in the conformational mobility of IgG. These processes are: i) stimulation of segmental flexibility and, ii) increase in the mobility within the Fv domains of the Fab fragments.

[Oxidative iodination of rabbit IgG: localization of markers in an Fc-fragment and effects of modification]. / Okislitel'noe iodirovanie IgG krolika: lokalizatsiia metki v Fc-fragmente i éffekty modifikatsii.

A comparative study of rabbit IgG, both native and modified ones, designed to assess the functional activity of these proteins under oxidative iodination conditions has been carried out. Polyclonal IgG, its antigen-specific fraction and Iodogen as an oxidant were used. Polyclonal antibodies directed against the CH2 domain of IgG, protein A targeted at the CH-2-CH3 domain interface and ferritin testing the conformation of the antigen-binding Fv fragment, were applied as conformational probes for assessing the changes in the IgG conformation. By taking advantage of pepsin proteolysis of [125I]-IgG, from 80% to 92% of the label was found to be localized within the CH3 domain, thus implying the domain-selective nature of iodination, when the degree of modification was below 0.1 atom of iodine per IgG molecule. Yet, when the three above-mentioned conformational probes were used, considerable alterations in the conformation of not only the CH2 domain and CH2-CH3 domain interface, but in the Fv domain being a part of the Fab fragment, were observed. By using competitive enzyme immunoassay for the straightforward comparative evaluation of functional properties of "cold" (native) and 125I-modified IgG, the deleterious effect of the oxidant (Iodogen) rather than iodine atom substitution at the phenolic ring of Tyr residues was shown to be the major determinant of alterations in the IgG molecule.