[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".

[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.

[Monoclonal antibodies to human spleen ferritin. I. Preparation and study of the interaction with isoferritin and apoferritin]. / Monoklonal'nye antitela k ferritinu selezenki cheloveka. I. Poluchenie i issledovanie vzaimodeistviia s izoferritinami i apoferritinom.

Three monoclonal antibodies to human spleen ferritin were produced and their interaction with soluble and immobilized ferritins studied. An immunoassay was developed to monitor the interaction of soluble biotinylated ferritin and the antibody with subsequent separation of the soluble complexes on streptavidin-cellulose. Analysis of immunoreactivities of a series of isoferritins (human liver, spleen, heart and equine spleen ferritins) revealed that all the three monoclonal antibodies bound to only human L-type ferritins (spleen and liver ferritins), suggesting a high species- and tissue specificity of these antibodies. The monoclonal antibodies were specifically directed against conformation-dependent antigenic determinants as could be evidenced from the lack of their binding to the subunits of the dissociated ferritin. The affinity of the monoclonal antibody for the ferritin deprived of iron (apoferritin) was higher than that for native ferritin due to the greater conformational flexibility of the apoferritin molecule. The latter property may underly a complete loss of immunoreactivity by the apoprotein adsorbed on the polystyrene surface as a result of conformational changes induced in the apoferritin molecule by adsorption. These findings provide additional support for recognition by all of the three antibodies of conformational antigenic epitopes in the ferritin molecule.

[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.