Affinity electrophoresis used for determination of binding constants for antibody-antigen reactions.

The use of affinity electrophoresis in agarose gels for determination of binding constants for the interaction of antigens with monoclonal antibodies is exemplified for monoclonal anti-human serum albumin and anti-alpha 1-fetoprotein antibodies. The calculated binding constants are verified by independent binding assays. The electrophoretic separation of antigen-antibody complexes of different stoichiometry is also demonstrated. Thus, affinity electrophoresis represents an alternative method for both qualitative and quantitative assessment of antigen-antibody interactions.     

用硫氰酸盐洗脱法直接测定噬菌体抗体的相对亲和力

抗体与相应抗原的结合可以被硫氰酸盐洗脱而解离,抗体的亲和力越高则解离所需要的硫氰酸盐浓度就越大,这一原理在传统的免疫学实验中被用来测定单克隆抗体或多克隆抗体的相对亲和力。如果证明该原理同样适用于噬菌体抗体库技术,则可以建立一种直接测定噬菌体抗体相对亲和力的简便方法。首先将噬菌体抗体与工作浓度的硫氰酸盐共孵育,以证明这一过程并不影响其后的ELISA反应,然后参照硫氰酸盐洗脱法测定完整抗体分子和Fab段相对亲和力的方法,在ELISA实验中以酶标抗M13为二抗检测了5个单克隆噬菌体抗体的相对亲和力,并与相对应的可溶性Fab段的相对亲和力进行了比较。被测抗体中包括3个克隆的抗角蛋白抗体和2个克隆的抗乙型肝炎表面抗原抗体。结果发现,用硫氰酸盐洗脱法测定5个噬菌体抗体所得到的亲和力排序与测定其相应可溶性Fab段所得结果一致,表明硫氰酸盐洗脱法可作为一种简便快速的方法用来直接测定噬菌体抗体的相对亲和力。     

Determination of the affinity constants of recombinant human galectin-1 and -3 for simple saccharides by capillary affinophoresis

The affinity constants of recombinant human galectin-1 and galectin-3 for sugars were determined by capillary affinophoresis. The monoliganded affinophore contains p-aminophenyl-beta-lactoside as an affinity ligand in the matrix of succinylglutathione and has three negative charges. An analysis of the mobility change of the lectins caused by the affinophore and its inhibition by neutral sugars allowed, for the first time, a determination of the affinity constants between the binding sites of the lectins and sugars. The relative magnitude of the affinity constants for each of the sugars in terms of dissociation constants found to be consistent with previously reported data on the concentrations of sugars that caused a 50% inhibition (I50) in the binding assay of the lectin to oligosaccharide-immobilized agarose beads but the absolute values of the dissociation constants were considerably smaller than the I50 values. Capillary affinophoresis indicated microheterogeneity of the lectin preparations and enabled the separate analysis of the affinity of each component simultaneously showing the advantage in using a separation method for analysis of bioaffinity.     

Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding in solution

Affinity characterization by mass spectrometry (AC–MS) is a novel LC–MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC–MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC–MS was likewise extended to a larger set of soluble protein–ligand systems. It was established as a valuable resource for counter screen and structure–activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.     

Determination of antibody affinity by using antigenic and antibody immunosorbents

To determine the affinity of the active centers of antibodies, cellulose immunosorbents for antibodies and antigens have been used. The fixation of serum proteins on the sorbent, the interaction of fixed antibodies with a monovalent antigen and the graphic analysis of the results thus obtained allows one to assess not only the concentration of the effective active centers on the sorbent, but also all known characteristics of antibody affinity: the average association constant K0, the common association constant Kt, the geometric association constant Kg, the average association constants which determine the affinity of different antibody groups. The use of antigenic immunosorbent permits one to determine the value of the average internal association constant K0. The determination of antibody affinity in hyperimmune antiplague sera by means of immunosorbents and red blood cells coated with capsular antigen has resulted in obtaining similar values of affinity indices.     

Purification and specificity of antibodies to inosine 5'-monophosphate

Antibodies to inosine 5'-monophosphate elicited in rabbits by immunization with a conjugate of IMP (oxidized with periodate) and bovine serum albumin have been purified by affinity chromatography. By the use of two affinity columns, Sepharose-IMP and Sepharose-oligo(I), the antibodies have been fractionated into three fractions. By gel diffusion, the three fractions were found to react with the conjugates of bovine serum albumin and IMP, GMP and AMP respectively. The association constants for the binding of the Fab fragments purified on the Sepharose-oligo(I) column and several haptens have been deduced from fluorescence experiments. It is shown that the base and the phosphate group play an important part in the binding of IMP to Fab fragments. No reaction has been found between the antibodies and poly(I).poly(C) by gel diffusion. However, the antibodies interact with poly(I).poly(C) since they decrease the thermal stability of poly(I).poly(C).     

Biosensor analysis of antigen-antibody interactions as a priority step in the generation of monoclonal bispecific antibodies

A biosensor system aimed at real-time measuring molecular interactions among label-free reactants has been used for a comparative analysis of the binding features (i.e., association-dissociation rates and affinity constants) as well as epitope mapping between bivalent monoclonal antibodies and the derived monovalent bispecific monoclonal antibody. The results show that observed different affinities between parental and derived bispecific antibodies concern the association rate constant, whereas the dissociation rate constants are unaltered. The apparent affinity-constant values determined by solid-phase radioimmunoassay yielded figures almost overlapping with those obtained with the biosensor instrument. The results of the present work indicate that the biosensor system has gained a key role not only as a tool for the study of antigen-antibody interactions, but also for setting up the reference parameters for the selection of the best candidates in the generation of bispecific monoclonal antibodies.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.     

Immunoglobulin G specifically binding plant N-glycans with high affinity could be generated in rabbits but not in mice

Xylosylated and core alpha1,3-fucosylated N-glycans from plants are immunogenic, and they play a still obscure role in allergy and in the field of plant-made protein pharmaceuticals. We immunized mice to generate monoclonal antibodies (mAbs) binding plant N-glycans specifically via the epitope containing either the xylose or the core alpha1,3-fucose residue. Splenocytes expressing N-glycan-specific antibodies derived from C57BL/6 mice previously immunized with plant glycoproteins were preselected by cell sorting to generate hybridoma lines producing specific antibodies. However, we obtained only mAbs unable to distinguish fucosylated from xylosylated N-glycans and reactive even with the pentasaccharide core Man3GlcNAc2. In contrast, immunization of rabbits yielded polyclonal sera selectively reactive with either fucosylated or xylosylated N-glycans. Purification of these sera using glyco-modified neoglycoproteins coupled to a chromatography matrix provided polyclonal sera suitable for affinity determination. Surface plasmon resonance measurements using sensor chips with immobilized glyco-modified transferrins revealed dissociation constants of around 10(-9) M. This unexpectedly high affinity of IgG antibodies toward carbohydrate epitopes has repercussions on our conception of the binding strength and significance of antiglycan IgE antibodies in allergy.     

Electrophoretic methods for studying protein-protein interactions.

Protein-protein interactions are involved in many biological processes ranging from DNA replication, to signal transduction, to metabolism control, to viral assembly. The understanding of those interactions would allow the effective design of new drugs and further manipulation of those interactions. Several useful analytical methods are available for the study of protein-protein binding, and among them, electrophoresis is commonly used. We describe two types of electrophoresis: gel electrophoresis and capillary electrophoresis. Gel electrophoresis is a well-established method used to study protein-protein interactions and includes overlay gel electrophoresis, charge shift method, band shift assay, countermigration electrophoresis, affinophoresis, affinity electrophoresis, rocket immunoelectrophoresis, and crossed immunoelectrophoresis. These techniques are briefly described along with their advantages and limitations. Capillary electrophoresis, on the other hand, is a relatively new method and affinity capillary electrophoresis has demonstrated its value in the measurement of binding constants, the estimation of kinetic rate constants, and the determination of stoichiometry of biomolecular interactions. It offers short analysis time, requires minute amounts of protein samples, usually involves no radiolabeled compounds, and, most importantly, is carried out in solution. We summarize the principles of affinity capillary electrophoresis for studying protein-protein interactions along with current limitations and describe in depth its application to the determination of stoichiometries of tight and weak binding protein-protein interactions. The protocol presented in the experimental section details the use of affinity capillary electrophoresis for the determination of stoichiometry of protein complexes.     

Determination of binding constants of antibodies fractionated according to their affinity to insulin

The number of populations of antibodies differing in their affinity for insulin was determined in guinea pig antiserum. The antibody fractions differing in their affinity were obtained by affinity chromatography on an antigenic sorbent. Elution with a stepwise pH gradient from 4 to 2 differing in the number and form of the steps resulted in 10 fractions of antibodies obtained from individual animal sera. The binding constants of fractionated antibodies were determined by a bioluminescent immunocofactor method. With a change in elution pH from 4 to 2, the equilibrious binding constant of antibodies increased from 10(6) to 5 X 10(9) M-1.     

Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.     

Traditional ELISA methods for antibody affinity determination fail to reveal the presence of low affinity antibodies in antisera: an alternative approach

Traditionally used methods of antibody affinity determination either by ELISA or by the surface plasmon resonance technique do not allow detection of the presence of low‐affinity antibodies in samples of high‐affinity antibodies. In this paper we demonstrate the possibility to reveal their presence and to determine the affinities of both categories of antibodies as well as the ratio of their concentrations. This is especially important since by using traditional methods for antibody affinity evaluation the admixture of low‐affinity antibodies in a sample diminishes the accuracy in determination of specific antibody affinity. In addition, the presence of an admixture of low‐affinity antibodies may be an important biological characteristic of the system under study; their revelation and the evaluation of their binding parameters may be valuable in many cases for obtaining a more complete characterization of the binding properties of the multiple antibodies generated in an immune response. Copyright © 2009 John Wiley & Sons, Ltd.     

From gel filtration to biosensor technology: the development of chromatography for the characterization of protein interactions

The objective of this review is to summarize the development of chromatographic techniques for the determination of reaction stoichiometries and equilibrium constants for solute interactions of biological importance. Gel chromatography is shown to offer a convenient means of characterizing solute self-association as well as solute-ligand interactions. Affinity chromatography is an even more versatile method of characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reactants. Adoption of different experimental strategies such as column chromatography, simple partition equilibrium experiments and biosensor technology has created a situation wherein affinity chromatography affords a means of characterizing the whole range of reaction affinities-from relatively weak interactions (binding constants less that 10(3)M (-1)) to tight interactions with binding constants greater than 10(9)M (-1). In addition to its established prowess as a means of solute separation and purification, chromatography thus also possesses considerable potential for investigation of the functional roles of the purified reactants-an endeavour that requires characterization as well as identification of the interactions responsible for a physiological phenomenon.     

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.     

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.     

The tandem beta-zipper model defines high affinity fibronectin-binding repeats within Staphylococcus aureus FnBPA

Binding of the fibronectin-binding protein FnBPA from Staphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.     

Structural analysis of the epitopes recognized by monoclonal antibodies to angiotensin II

Six clones were obtained that secrete anti-angiotensin II antibodies after somatic cell fusions between splenocytes of immunized BALB/c or outbred OF1 mice and NS-1 myeloma cells. The dissociation constants for angiotensin II ranged from 0.3 to 2.9 nM. A panel of 20 structural analogs of the hormone were used as probes to analyze the specificity of binding. From the binding studies and the putative three-dimensional structures of the tested peptides, three families of antibodies could be distinguished that recognized overlapping epitopes; the conservation of the native conformation of the angiotensin II molecule in the analogs appeared essential for the preservation of a high affinity to the antibodies. With one antibody, the affinities of the angiotensin II analogs have been correlated with their intrinsic biologic activities (as measured by in vivo pressor tests), and not with their binding affinity to the membrane receptor. These results are interpreted as mimicry, by the antibody binding site, of the active conformation of the receptor site.     

Qualitative aspects of thyroglobulin antibodies in Hashimoto's thyroiditis and Graves' disease

We have tried to characterize the thyroglobulin antibodies (TgAb) involved in Hashimoto''s thyroiditis and Graves'' disease by studying their affinity constants and binding capacities. Two populations of antibodies were found in half of the patients with either disease, TgAb₁ with a high affinity (K₁) but a low binding capacity (B₁) and TgAb₂ with a lower affinity (K₂) but a much higher binding capacity (B₂). The mean affinity constants and binding capacities were similar in the two diseases. In the other half of the patients only one population of antibody was present, with a low affinity constant (K_t) and a very high binding capacity (B_t), thus comparable to TgAb₂. The mean K_t and B_t were similar in the two diseases. From these results it would appear that circulating thyroglobulin antibodies in Hashimoto''s thyroiditis and Graves'' disease are similar in their affinity constants and binding capacities, so that these characteristics do not reflect the different pathogenesis of each condition.     

Analysis of hemoglobin oxygenation from combined equilibrium and kinetic data. Is quaternary enhancement necessary?

An experimental approach based on four independent techniques, in which kinetic and equilibrium measurements of subunit assembly reactions are combined with concentration-dependent oxygen-binding curves, has previously been used to resolve parameters of the linkage system for human hemoglobin over a wide range of conditions [(G.K. Ackers and H.R. Halvorson, Proc. Natl. Acad. Sci. U.S.A. 71 (1974) 4312; F.C. Mills et al., Biochemistry 15 (1976) 1093; M.L. Johnson et al., Biochemistry 15 (1976) 5363). Throughout this extensive body of results it has been found that the affinity for binding oxygen to tetramers at the fourth step exceeds the mean affinity of dissociated dimers. The existence of this "quaternary enhancement" effect has recently been questioned by Gibson and Edelstein (J. Biol. Chem. 262 (1987) 516) and by Philo and Lary (J. Biol. Chem. 265 (1990) 139) on the basis of kinetically derived oxygen-binding constants that do not exhibit quaternary enhancement. These authors have also suggested that quaternary enhancement might not be necessary to explain the oxygen-binding data mentioned above. In this study, we have explored the effect of constraining the numerical analysis of oxygen-binding data against the new kinetically derived binding constants. It is found that the sets of linkage constants which are compatible with both the oxygen-binding data and the new kinetically derived dimer binding constant require both quaternary enhancement and substantial dimer cooperativity. Increasing the dimer cooperativity to compensate completely for quaternary enhancement requires both dimeric and tetrameric binding constants that disagree with the kinetically derived values. Thus, the quaternary enhancement effect cannot be eliminated by readjustment of the remaining constants of the linkage system. Possible sources of the discrepancy between the kinetically derived binding constants and the otherwise self-consistent data from the other four techniques are discussed.