A. D. Dmitriev

Analysis of the binding of bispecific monoclonal antibodies with immobilized antigens (human IgG and horseradish peroxidase) using a resonant mirror biosensor

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.

Quantitative analysis of the products of IgG chain recombination in hybrid hybridomas based on affinity chromatography and radioimmunoassay.

On the model of a hybrid hybridoma (quadroma) to alpha-endorphin (END) and horseradish peroxidase (HRP), we have elaborated a general approach to analyse H and L chain interactions in hybrid hybridomas and to evaluate their efficiency as producers of bispecific antibodies (bAbs). This strategy is based on quantitative analysis of quadroma produced Abs by affinity chromatography and radioimmunoassay. First, Abs produced by quadroma cells in culture media (IgG pools from three quadroma clones) were fractioned with respect to specificity. Second, Ab concentrations in each fraction (bispecific, anti-END, anti-HRP and inactive) were measured by specific radioimmunoassays, using rabbit antiserum against mouse IgG and 125I-labelled affinity purified quadroma Abs. Then the experimentally obtained Ab distributions were compared with the predicted Ab distributions for different models of IgG chain recombination in quadroma cells (random H/L pairing, preferential homologous H/L association). As follows from these models, in a random H/L recombination the yield of bAbs in quadroma produced IgG cannot exceed 12.5%, and the ratio of bAbs and inactive Abs cannot exceed 0.5. In the analysed clones the yield of bAbs amounted to about 30% of total IgG, and the ratio of bAbs and inactive Abs was about 5-8, giving strong evidence for preferential homologous H/L association in these cells. The ratio of anti-HRP and anti-END Abs was about 10:1, suggesting unequal production of parental IgG chains in quadroma cells. The result of quantitative analysis of quadroma IgG was further supported by two-dimensional gel analysis of affinity-purified fractions of quadroma IgG and of two parental mAbs.

Construction of a quadroma to α-endorphin/horseradish peroxidase using an actinomycin D-resistant mouse myeloma cell line

Abstract A hybrid hybridoma (quadroma), secreting antibodies with double specificity to α-endorphin (α-EP) and horseradish peroxidase (HRP), has been produced. The bispecific antibodies constituted about 28–29% of all immunologically active IgG, produced by quadroma. The quadroma was isolated by fusion of two mouse hybridomas (anti-HRP and anti-α-EP) with distinct phenotypes: double mutant AMD R /HAT S , and wild type (AMD S /HAT R ). A novel strategy for the construction of a double-mutant was applied, based on the use of an actinomycin D-resistant (AMD R ) mouse myeloma for initiation of one of the parental hybridomas.

Effect of conditions of monoclonal antibody adsorption on antigen-binding activity

The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013–0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.

Analysis of Bispecific Monoclonal Antibody Binding to Immobilized Antigens Using an Optical Biosensor

The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (Kass) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The Kass of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (kdiss) for anti-HRP shoulder of Babs was 21 times higher than kdiss for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody–enzyme conjugates in the case of binding of bivalent Mabs.

The comparison of the ability of monoclonal antibodies directed to different proteins (human IgG, human myoglobin and HRP) and bispecific antibodies derived thereof to bind antigens immobilized on a surface of a solid phase

BACKGROUND Bindings of mouse monoclonal antibodies (mAbs) and affinity purified bispecific antibodies (bAbs), derived thereof, to antigens adsorbed on immunoplates have been compared, using ELISA and RIA methods. METHODS The analysed panel of antibodies included mAbs specific to human myoglobin (Mb), human IgG (hIgG) and horseradish peroxidase (HRP) and biologically produced bAbs with double specificity to Mb and HRP, and to hIgG and HRP. RESULTS The degree of difference between different mAbs and corresponding bAbs varied markedly from antibody to antibody, depending on whether the parental mAbs could bind immobilized antigens bivalently. The observed equilibrium binding constant (K(obs)) for anti-HRP mAbs was 21-38 times higher that of anti-HRP site of bAbs (anti-hIgG/HRP or anti-Mb/HRP, respectively), due to bivalent binding of mAbs. Anti-Mb mAbs also bound bivalently with immobilized Mb. On the contrary, anti-hIgG mAbs bound monovalently with immobilized hIgG in the same conditions. The avidity of anti-Mb/HRP bAbs increased, if both antigens were simultaneously adsorbed on a solid phase. CONCLUSIONS The obtained data indicate that the use of bAbs in heterogeneous immunoassays instead of traditional mAb-enzyme conjugates hardly can provide the significant gain in assay performance if parental mAbs bind bivalently.

Production of bifunctional monoclonal antibodies to human IgG and horseradish peroxidase and their utilization for testing anti-HIV antibodies

Bispecific antibodies are purified from ascitic fluid by successive affinity chromatography on HRP-Sepharose and IgG-Sepharose. The ability of the affinity purified bispecific antibodies to bind human IgG and horseradish peroxidase is demonstrated in the immunoenzyme assay (identification of anti-HIV antibodies.Bispecific antibodies are purified from ascitic fluid by successive affinity chromatography on HRP-Sepharose and IgG-Sepharose. The ability of the affinity purified bispecific antibodies to bind human IgG and horseradish peroxidase is demonstrated in the immunoenzyme assay (identification of anti-HIV antibodies.

Monoclonal antibodies requiring coating buffer with low pH for efficient antigen capture in sandwich ELISA: the rarities or practically important phenomena?

This article reexamines some opinions concerning pH requirements for optimal immobilization of monoclonal antibodies (mAbs) by passive adsorption in antigen capture ELISA. It was discovered that substitution of “classical” sodium phosphate (pH 7.5) and carbonate (pH 9.5) coating solutions by acid (pH 2.8) buffers maximized antigen capture 4 out of 10 different tested anti-HBsAg mAbs, resulting in a 1.5–2.5 increase of binding curve coefficients. By measuring both mAbs amounts and functionality, the enhancement effect was attributed to the better preservation of solid phase antibodies activity.

Analysis of Serotonin Transporter in Human Platelets by Immunoblotting Using Site-Specific Antibodies

We have produced a panel of site-specific antibodies recognizing different regions of the human serotonin transporter (SERT). This panel included: 1) monoclonal antibodies 23C5 (mAbs 23C5) to the C-terminal region (amino acid residues 597-630); 2) polyclonal antibodies (pAbs) to the N-terminal region (amino acid residues 69-83); 3) pAbs to the region (amino acid residues 86-100) in the beginning of the first transmembrane domain (TMD). The antibodies were produced using recombinant proteins and synthetic peptides (containing certain sequences of SERT) as antigens. These antibodies were purified by affinity chromatography, conjugated to horseradish peroxidase (HRP), and used for immunoblotting analysis of SERT in extracts of human platelets. Sodium dodecyl sulfate extracts were prepared under conditions preventing non-specific proteolytic degradation of the proteins. In platelet extracts, all antibodies were able to detect the 67 kD protein, apparently corresponding to full-length SERT molecule (its theoretical mass is about 70 kD). These antibodies also detected several polypeptides of smaller size (56, 37, 35, 32, 22, and 14 kD), apparently corresponding to N-terminal, C-terminal, and non-terminal SERT fragments. Specificity of immunostaining was confirmed by preincubation of HRP-labeled anti-SERT antibodies with excess of corresponding antigen, which resulted in disappearance of protein band staining. It is suggested that SERT undergoes a programmed proteolytic cleavage (processing) resulting in formation of several SERT-derived polypeptides of smaller size. It is possible that one of the cleaved SERT species is required for serotonin transport activity. Possible sites for specific proteolysis may be located in the region near TMD1 and in the intracellular loop between TMD4 and TMD5.

[Monoclonal antibodies to alpha-endorphin effective in immunohistochemistry and immunoblotting].

: Four mouse monoclonal antibodies (E11, A8, F8, H5) to alpha-endorphin have been produced. The antibodies bind 12.5, 20.6, 9.6, 6.6% of 125I-beta-endorphin and 35.5, 15.1, 12.8, 12.2% of 125I-gamma-endorphin; the binding of 125I-alpha-endorphin being taken for 100%. The binding of antibodies E11, A8, F8 and H5 to 125I-alpha-endorphin was 50% inhibited by unlabeled ligand in concentrations 5, 50, 30 and 35 nM respectively. Using tissue sections of rat pituitary it was shown that antibody E11 can be used for the localization of endorphin producing cells by immunofluorescence. The antibodies F8 and H5 effectively detected endorphin precursor proopiomelanocortin by immunoblotting.