Optimization of methods of immobilization of anti-immunoglobulins. Equilibrium parameters of the interaction of immobilized antibodies with an antigen

Methods for immobilization of anti-immunoglobulins on insoluble supports were optimized, and the interaction of immunoadsorbents obtained with [125I]-labeled rabbit IgG was investigated. It was shown that this interaction can be adequately described by a rather simple equilibrium model which reflects the interaction of a monovalent antigen with two independent types of binding sites. Within the framework of this model the association constants as well as the concentrations of high affinity binding sites which influence the capacity and efficiency of the separation system were determined. Optimization of the immobilization methods implicated a study on the role of certain functional groups of the antibody involved in the formation of covalent bonds, on the effect of the spacer arm length on the properties of immobilized antibody as well as on the role of the degree of immobilization. It was found that immunoadsorbents obtained after antibody immobilization via lysine or tyrosine residues on matrices with a specific spacer group are the optimal ones.     

Identification of a protein cross-reacting with anti-phosphotyrosine antibodies in yeast insoluble cytoplasmic matrices

Immunoblot analysis with anti-phosphotyrosine antibodies of total extracts from exponentially growing yeast cells reveals a unique cross-reactive polypeptide of about 75 Kd (p75). The specificity of the immunodecorations has been checked by experiments of competition with phosphoaminoacids. A common behaviour has been observed for the 75 kd band and the 170 kd band corresponding to the platelet-derived growth factor receptor from Swiss 3T3 cells, which it has been known to be autophosphorylated on tyrosine upon ligand binding and used as a control throughout this work. We have found that p75 is associated to detergent insoluble cytoplasmic matrices. The stability of p75 detection by antibodies following treatments that specifically hydrolyze phosphohistidine and its susceptibility to potato acid phosphatase treatment provide further evidences that the epitope recognized by these antibodies in the yeast p75 polypeptide is indeed phosphotyrosine.     

Effect of succinylation of antibodies on their conformation and interaction with the antigen

Using succinic anhydride, a succinylated derivative of anti-urease IgG having 49 ± 6% modification was prepared and its physicochemical and immunological properties were studied. IgG undergoes substantial changes in its native conformation on succinylation, which was mainly attributed to electrostatic destabilization of the native protein conformation. The modified IgG exhibited a decrease in its cross-reactivity with urease. This decrease is attributed to the conformational change in IgG upon succinylation and/or is due to the disruption of the lysine residues in the antigen-binding site of IgG upon succinylation, which may be involved in binding the antigen. IgG was able to bind to the specific antigen although its conformation was partially modified. Therefore, partial modification of the conformation of the antigen-binding site of IgG is permissible in order to bind to the antigen. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 12, pp. 1642–1647.     

Peptide ligands that bind IgM antibodies and block interaction with antigen.

We have selected a peptide-display phage library on IgM Abs and identified a panel of phage-expressing peptides that bind to IgM Abs in general, but not to Abs of other classes. A synthetic peptide corresponding to one of the displayed peptide sequences also binds to IgM Abs. The peptides bind to both soluble pentameric Abs and to monomeric cell-surface IgM. The phage-displayed and synthetic peptides inhibit the binding of IgM Abs to Ag. These peptides may create confounding artifacts when IgM Abs are used for epitope mapping studies. Nonetheless, the peptides may have both experimental and therapeutic utility.     

Optimization of process parameters for the continuous production of alcohol with a cell immobilized bioreactor

The optimum values of substrate concentration, pH and temperature for higher yields of alcohol in cell immobilized bioreactor using alginate entrapped cells of yeasts for continuous fermentation of alcohol were obtained employing full factorial search. The results indicate that the yield of alcohol is predominantly influenced by the substrate concentration and temperature, both individually and in combination. The pH, on the other hand, has no significant influence. The path of steepest ascent method has been used to optimise the alcohol yield. A best alcohol yield has been obtained with 23.5% substrate concentration, 30°C temperature and at pH 5.0.     

Analysis of some "insoluble" problems of determining the binding parameters of ligand-receptor interaction and methods of their solving

Some problems of the estimation of the parameters of ligand-receptor interaction (affinity, rate constants, valency, etc.) were considered. It was demonstrated that not only the Scatchard plot but also Klotz plot could be used for determining the parameters of ligand-receptor interaction for two types of binding sites of different affinity. A new approach and new coordinate systems for the estimation of the parameters of ligand-receptor interaction were suggested. It was shown that for the estimation of the affinity of putative monovalent antibodies by ELISA various equations, which are more precise and convenient than the Friguet et al. equations, could be obtained by the transformation of mass action law equation. The problem solution for the estimation by ELISA the affinity of two types of bivalent antibodies with different affinity and their concentrations for the case of the mixture of these antibodies was also suggested. The application of the proposed coordinate of dilution allows to solve the problem of determination of the parameters of ligand-receptor interaction (including antigen-antibody system) for the pre-existing ligand-receptor mixture without their preliminary separation and purification. This approach is especially important for the cases when the receptor is not stable enough to be isolated in the intact form from this mixture. It was shown that the well-known phenomenon of the prozone often observed under the titration of serum antibodies by the method of agglutination may get a mathematical explanation. Analytical solution of the problem of determining the velocity constant and the amount of the end product of the first order irreversible and reversible reaction kinetics was suggested, despite the fact that the process is described by the system of irrational equations. Mehods of asymptotic solution of transcendental irrational equations which describe the dynamics of reactions which mechanisms are subject to the so-called heterogeneous, successive, or competitive models have been considered. These methods permit the finding of the reaction rate constants and the amount of the end product, if the kinetics of the transformation of either initial, or end product of the reaction is known.     

Influence of a poly-ethylene glycol spacer on antigen capture by immobilized antibodies

The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency. In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E. coli O157:H7, and ovalbumin. The antigen capture efficiency was determined using a surface ELISA method. Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction. The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin. Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens. Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.     

Equilibrium and kinetic parameters for the interaction of a monoclonal antibody with liposomes bearing fluorescent haptens

We have obtained equilibrium and rate constants for the interaction of monoclonal IgG and its monovalent Fab fragment with a hapten (fluorescein) attached to the surface of a liposome. Binding was detected at nanomolar hapten concentrations by the quenching of the hapten's fluorescence on antibody binding. The binding parameters were computed from nonlinear least squares fits, using mass-action models. Crypticity of the hapten was observed and interpreted as an equilibrium between two states, extended and sequestered, the latter representing haptens associated with the membrane surface. Depending on the lipid composition of the liposomes, the fraction of sequestered hapten ranged from 0.25 to 0.975; transitions between the two states took place on the time scale of minutes. Fab interactions with extended hapten on the membrane were similar to interactions with water-soluble hapten. The ability of IgG to bind bivalently to membrane gave it an avidity two to six times the affinity for purely monovalent binding. However, the equilibrium constant for the monovalent-bivalent binding equilibrium was effectively four to five orders of magnitude less than that for the initial binding step. This probably reflects steric penalties for the simultaneous binding of two haptens on a membrane.     

Studies on the interaction of cholesterol with soluble and insoluble elastins

The exchange of ³H-cholesterol in an aqueous solution of taurodeoxycholate with insoluble elastin and kappa-elastin peptides has been quantitatively studied. At higher cholesterol concentrations, and in similar experimental conditions, 0.3 μg and 0.2 μg of cholesterol could be adsorbed, respectively, on to 1 mg insoluble elastin and 1 mg kappa-elastin coacervate. Therefore, the above amounts of cholesterol fixed per mg elastin represent minimal estimates. The replacement of Na⁺ ions by Ca²⁺ ions increased the cholesterol binding both of fibrous and of soluble elastins. The binding curves tend towards a limiting value in the presence of Na⁺, but no saturation effect was observed in the presence of Ca²⁺. The replacement of sodium ions by calcium ions in the taurodeoxycholate solutions also lowered the coacervation temperature of the kappa-elastin peptides. Fibrous elastin retained more cholesterol at 65°C than at 37°C, suggesting the importance of hydrophobic interactions in cholesterol elastin association. These results suggest that conformational changes could be induced in both soluble and insoluble forms of elastin by calcium ions increasing their affinity for cholesterol. This enhancement of cholesterol fixation by elastin in the presence of Ca²⁺ ions may well have a physiopathological importance. So does also the fact that elastin-peptides exhibit a higher affinity in the presence of Ca²⁺ for cholesterol than insoluble elastin, suggesting the possibility of increased cholsterol (and calcium) fixation as elastin is degraded in situ by elastases.     

Monoclonal anti-idiotopic antibodies against myasthenia-inducing anti-acetylcholine receptor monoclonal antibodies. Preponderance of nonparatope-directed antibodies affecting antigen binding

To analyze components of the idiotypic network in experimental autoimmune disease, we produced 17 isogeneic anti-idiotopic monoclonal antibodies (anti-Id) against two experimental autoimmune myasthenia gravis-producing anti-acetylcholine receptor (anti-AChR) monoclonal antibodies. We studied the binding of five of the anti-Id to the anti-AChR monoclonal antibodies bearing the complementary idiotopes (Id-mAb). They bound with Kd values ranging from 0.06 to 0.86 nM, values comparable to those of Id-mAb:AChR complexes (0.26 and 0.34 nM). All of the anti-Id tested moderately inhibited the binding of AChR to Id-mAb, whereas for each anti-Id, AChR either strongly inhibited anti-Id binding or had no effect on anti-Id binding. Hence, the inhibition of Id-mAb:AChR binding by anti-Id was not reciprocal with the inhibition of anti-Id:Id-mAb binding by AChR. For each anti-Id, the relative affinities of anti-Id and AChR for Id-mAb together with the lack of symmetry of inhibition by anti-Id compared to inhibition by AChR indicate that these two "ligands" are not competitive inhibitors. Consequently, anti-Id and AChR do not bind to overlapping sites on the Id-mAb, suggesting that the observed inhibition is mediated allosterically. This may be a common mechanism of anti-Id:Id binding, which would have important implications for the mechanism of anti-Id-induced suppression.     

Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen.

Five murine hybridoma lines that produce monoclonal antibodies against Epstein-Barr virus membrane antigen (MA) were established. Immunoprecipitation experiments demonstrated that three of the antibodies precipitated both the 236,000 (236K) MA and the 212K MA. The other two antibodies precipitated the 86K MA. Antibodies against the 236K-212K MA and the 86K MA mediated complement-dependent cytolysis of Epstein-Barr-virus-infected cells. The antibodies against the 86K MA neutralized both the B95-8 and P3HR-1 viruses.     

Determination of rate constants for the antigen-antibody reaction. Kinetic characteristics of interaction of soluble and corpuscular antigen with specific antibodies

Using the previously developed procedure, the rate and equilibrium constants for the interaction of antibodies with soluble or corpuscular antigens were determined. Cow milk casein was used as a soluble antigen, while heat-inactivated Staphylococcus aureus cells--as a corpuscular antigen. The values of kinetic constants for the above reactions are close to those for various antigen--antibody pairs obtained by other investigators.     

Chimeric and humanized antibodies with specificity for the CD33 antigen.

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.     

A comparison of methods for measuring intestinal antibodies against Vibrio cholerae in mice.

A re-evaluation was made of the efficiency with which some of the commonly used assays would detect intestinal antibodies. The data indicate that the most sensitive assays for the detection of intestinal antibodies are the baby mouse protection test and the radioimmunoassay. The reasons for the lack of sensitivity with other assay methods are discussed.     

Production of antibodies against the porcine luteinizing hormone by two immunization methods.

Two methods of rabbit immunization were applied: multisite intradermal injections of small doses and intramuscular injections of large hormone doses for obtaining antibodies against the porcine luteinizing hormone (LH). A high titre, specificity towards LH and affinity were obtained in rabbits immunized by both methods. In most animals the highest titre was noted 10 weeks after the first immunization.     

Monoclonal antibodies against the human lymphocyte differentiation antigen CD 76 bind to gangliosides.

Two monoclonal antibodies, HD 66 and CRIS-4, by which the new CD 76 B-cell-associated cluster was defined, bound to several gangliosides (sialic acid containing glycolipids) of different polarity. One of the gangliosides recognized by HD 66 could be identified as NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-beta 1-1'Cer. This antigen was enzymatically synthesized. Sialidase treatment of the ganglioside antigens abolished binding of HD 66 and CRIS-4.     

An antigen chimera of poliovirus induces antibodies against human papillomavirus type 16.

It has been established that the surface of poliovirus type 1 can be extensively modified to incorporate antigenic domains from other poliovirus serotypes and from unrelated viruses. The fact that the modified (chimeric) viruses exhibit dual antigenicity and immunogenicity led us to explore the possibility of using the Sabin vaccine strain of poliovirus type 1 as a vector for the presentation of antigenic domains from human papillomavirus type 16 (HPV-16), a virus associated with the development of cervical carcinoma. We report here the construction and characterization of a chimeric poliovirus containing a 16-residue sequence derived from the major capsid protein (L1) of HPV-16. This virus chimera stimulated the production in rabbits of antibodies which recognized the HPV-16-derived peptide and an L1 fusion protein synthesized in Escherichia coli and detected HPV-16 in human biopsy material by immunoperoxidase staining. The possibility that poliovirus-HPV chimeras could be used as vaccines against HPV-16 is discussed.     

Effect of various parameters on viability and growth of bacteria immobilized in sol–gel-derived silica matrices

Immobilized bacteria are being extensively used for metabolite production, biocatalysts, and biosensor construction. However, long-term viability and metabolic activity of entrapped bacteria is affected by several conditions such as their physiological state, the presence of high-osmolarity environments, porous structure and shrinkage of the matrix. The aim of this work was to evaluate the effect of various parameters on bacteria immobilized in sol–gel-derived silica matrices. With this purpose, we evaluated the stress of immobilization over bacteria cultures obtained from different growing states, the effect of cell density and bacteria capability to proliferate inside matrices. Best results to attain longer preservation times were obtained when we immobilized suspensions with an optimized bacterial number of 1 × 10⁷ cfu/gel in the presence of LB medium using aqueous silica precursors. Furthermore, the impact of osmotic stress with the subsequent intracellular trehalose accumulation and the addition of osmolites were investigated. Shorter preservation times were found for bacteria immobilized in the presence of osmolites while trehalose accumulation in stressed cells did not produce changes on entrapped bacteria viability. Finally, nutrient addition in silica matrices was studied indicating that the presence of a carbon source without the simultaneous addition of nitrogen was detrimental for immobilized E. coli. However, when both carbon and nitrogen sources were present, bacteria were able to survive longer periods of time.