Determination of antibody affinity by ELISA. Theory

New approaches for the measurement of antibody affinity by ELISA are suggested and considered theoretically. It was shown that not only more precise and more convenient in comparison to that suggested earlier, but also more informative graphical representation of the experimental data in the appropriate coordinate could be used for evaluation of antibody affinity. The following cases were considered: (i) determination of antibody affinity for one kind of univalent antibodies, (ii) determination of antibody affinity for one kind of bivalent antibodies, (iii) determination of antibody affinity for two kinds of univalent antibodies, which are in a mixture, and (iv) determination of antibody affinity for two kinds of bivalent antibodies, which are in a mixture. Advantages and disadvantages of the different approaches are discussed.     

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.     

Determination of antibody affinity using ELISA

Some new approaches for the determination of antibody affinity were proposed. It was pointed out that the proposed methods are more simple, convenient, precise, and informative than that of Friguet et al. (1985). The approach that allows determination of two-valence antibodies affinity was also proposed. The example of two monovalent antibodies presented in the examined mixture was considered. It allows to estimate the affinity of both kinds of antibodies as well as to determine their concentration relations in the mixture.     

A new method for the evaluation of antibody affinity based on serial dilutions of studied antibody-antigen mixture

A new method for the evaluation of the affinity of bivalent antibodies were suggested. This method is based on the previously published by the author the idea of using so-called coordinates of dilutions. It was shown that the suggested method allows to evaluate the affinity of antibodies with high accuracy using this simple approach. It is supposed that at some conditions the suggested method could have substantial advantages in comparison to the traditional methods. This method allows to analyze situations when antibodies are already in a mixture with antigen, for example in the bloodstream in the case of infections or autoimmune diseases. The method provides useful approach for the evaluation not only antibody affinity, but also the concentration of circulated antigen.     

Avidity of bivalent antibodies. Problems of its experimental determination and theoretical evaluation

Some problems of experimental determination or theoretical evaluation of antibody avidity are considered. It was shown that in order to determine the fraction of nonoccupied antibodies in their mixture with the excess of the corresponding antigen which is required to estimate avidity the methods should be used which are more sensitive than ELISA. The available methods did not allow determining the avidity of bivalent antibodies because of many reasons. However, in the recent years new methods were suggested that make it possible to evaluate the avidity of bivalent antibodies and that of the receptors which consist of two binding sites connected by a flexible linker of the known length. Thus, there are all possibilities now for determining the avidity of bivalent antibodies in experiments or by theoretical methods.     

Analysis of antigen-specific antibodies and their isotypes in experimental malaria.

BACKGROUND: Measuring antibody production in response to antigen exposure or vaccination is key to disease prevention and treatment. Our understanding of the mechanisms involved in the antibody response is limited by a lack of sensitive analysis methods. We address this limitation using multiplexed microsphere arrays for the semi -quantitative analysis of antibody production in response to malaria infection. METHODS: We used microspheres as solid supports on which to capture and analyze circulating antibodies. Antigen immobilized on beads captured antigen-specific antibodies for semi- quantitative analysis using fluorescent secondary antibodies. Anti-immunoglobulin antibodies on beads captured specific antibody isotypes for affinity estimation using fluorescent antigen. RESULTS: Antigen-mediated capture of plasma antibodies enables determination of antigen-specific antibody "titer," a semi-quantitative parameter describing a convolution of antibody abundance and avidity, as well as parameters describing numbers of antibodies bound/bead at saturation and the plasma concentration-dependent approach to saturation. Results were identical in single-plex and multiplex assays, and in qualitative agreement with similar parameters derived from ELISA-based assays. Isotype-specific antibody-mediated capture of plasma antibodies allowed the estimation of the affinity of antibody for antigen. CONCLUSION: Analysis of antibody responses using microspheres and flow cytometry offer significant advantages in speed, sample size, and quantification over standard ELISA-based titer methods.     

Failure of the usual anti-Ig antibody method for quantitative measurement of low affinity antibodies

The usual anti-Ig antibody method, consisting of the precipitation of soluble antigen-antibody complexes by heterologous anti-Ig antibody, was applied for quantitative estimation of guinea pig IgG2 anti-ovalbumin and anti-2,4-dinitrophenyl (DNP) antibodies by measuring the maximum amounts of antibody-bound antigens. However, the amounts of antibodies estimated were less than those obtained by other methods: the precipitin reaction, the precipitation of antigen-antibody complexes with 50% saturated ammonium sulfate, and equilibrium dialysis. In particular, the anti-Ig antibody method greatly underestimated the amount of anti-DNP antibody with low affinity for epsilon-DNP-L-lysine. Thus, it was concluded that partial dissociation of the antigen-antibody complexes occurring upon precipitation with anti-Ig antibody made the anti-Ig antibody method unsuitable for quantitative determination of antibodies.     

Antibody affinity measurements

The use of antibodies in immunoaffinity separations represents one of the most specific methods for purifying substances of biological interest. Since the binding affinity of antibody greatly influences its behavior in such separations, it is often important to know the value of the antibody affinity expressed as an equilibrium constant K. The present review discusses the equations used in the quantitative analysis of antigen/antibody interactions and describes currently used experimental methods for measuring K values. Advantages and shortcomings of the solution phase and solid phase approaches used for measuring antibody affinity are discussed.     

A Radioimmunoassay-Based Method for Measuring the True Affinity of a Monoclonal Antibody with Trace Amounts of Radioactive Antigen: Illustration with the Products of a Cell-Free Protein Synthesis System

This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.     

重组人骨形态发生蛋白-2抗体的制备及纯化

骨形态发生蛋白-2是骨科研究领域的重要生物蛋白。用重组人骨形态发生蛋白-2(rhBMP₂)与BSA连接成复合物作为免疫原,免疫家兔制备出多克隆抗血清。分别用免疫沉淀法和Sepharose4B-BSA反向免疫亲和吸附法去除抗血清中载体BSA抗体。用盐析法和蛋白层析法纯化出抗rhBMP₂抗体。研究发现,家兔产生高效价抗体的最佳时间为14-16周,从rhBMP₂抗体的特异性、相对纯度及回收率综合分析,免疫沉淀法去除BSA抗体优于反相亲和吸附法。     

Feedback by early and late primary antisera on the primary and secondary adoptive immune responses of mice to burro erythrocytes

Experiments on antibody feedback inhibition of the immune response have confirmed that control is more effective against a primary response than against a secondary response. The cells producing antibodies in primary and secondary responses are different both in terms of number of IgFC and amount of antibody produced by individual IgFC (plaque size). Late primary anti-burro RBC sera (greater than 200 days), despite low titers, are, on a volume for volume basis, feedback inhibitors at least as good as early (8-12 days) primary antisera on primary responses but are more effective in suppressing secondary responses (B memory cells). Late primary antisera, due to the process of affinity maturation, have a high affinity for antigen. The suppressive effect of early and late antisera is equally removable by absorption with burro erythrocytes: a result which it is thought, decreases the likelihood of feedback by anti-idiotype being involved in the observed suppression. It is suggested that feedback antibody acts (a) in competition with receptors, inter alia removing antigen into immunologically irrelevant pathways, (b) by a process involving the linking of antigen to Fc receptors, and (c) as a blocking antibody coating B cells (Bm) or APC which are already binding epitopes, thus preventing their cooperation with specific helper or other accessory cells.     

Determination of the binding constants of immune serum antibodies to human IgG

The possibility of using two variants of the enzyme-linked fluorescent cofactor immunoassay for the determination of antibody binding constants has been demonstrated. The determination of binding constants for antibodies isolated by affinity chromatography techniques has been carried out. These techniques permit the isolation of fractions, differing in their affinity by 5-10 times, from the whole population of antibodies.     

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.     

Selecting and screening recombinant antibody libraries

During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity. Innovative strategies enable directed evolution of binding sites with ultra-high affinity, high stability and increased potency, sometimes to a level that cannot be achieved by immunization. Automation of the technology is making it possible to identify hundreds of different antibody leads to a single therapeutic target. With the first antibody of this new generation, adalimumab (Humira, a human IgG1 specific for human tumor necrosis factor (TNF)), already approved for therapy and with many more in clinical trials, these recombinant antibody technologies will provide a solid basis for the discovery of antibody-based biopharmaceuticals, diagnostics and research reagents for decades to come.     

Characterization of the binding epitope of the monoclonal antibody DMAb-1 to ganglioside GM2.

Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.     

Prediction of affinity and kinetics in biomolecular interactions by affinity chromatography

Computer simulation of affinity chromatography is a valuable tool for accurate prediction of column performance. In our study affinity pairs based on lectin and antibody interactions with carbohydrates have been used as model systems. In this well-characterized system we have demonstrated the usefulness of the simulation approach for determination of affinity and kinetics. These properties are typically difficult to obtain for many weakly interacting molecular species (i.e., when dissociation constants (K(D)) are greater than 10(-5) M). The influence of affinity and kinetics on peak broadening in affinity chromatography has also been investigated.     

MALDI/MS-based epitope mapping of antigens bound to immobilized antibodies

Proteolytic digestion of proteins bound to immobilized antibodies, combined with matrix assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound peptides, can be a powerful technique for epitope determination. Binding of the protein to the antibody is done while the protein is in its native, folded state. A purified protein is not required for this procedure, because only proteins containing the antigenic determinant will bind to the antibody in the initial step. The method makes use of the resistance of the antibody to enzymatic digestion. Enzymatic cleavage products of the antigenic protein not containing the epitope are washed off the beads, leaving the epitope-containing fragments affinity bound to the immobilized antibody. Dissociation of the antigen-antibody complex prior to mass spectrometric analysis is unnecessary because the affinity-bound peptides are released by the MALDI matrix crystallization process, although the antibody remains covalently attached to the sepharose beads. This epitope-mapping protocol has been used in the determination of both continuous and discontinuous epitopes on both glycosylated and unglycosylated proteins.     

一种构建改形单域抗体的方法

为验证一种构建改形单域抗体的实用新方法,与以往方法不同的是,该方法不需要对抗体进行空间结构模拟,以确定人源抗体的FRs接受序列及在人源FRs接受序列中哪些氨基酸残基需要突变,并且该方法将抗体的改形与亲和力成熟于同一过程完成,利用该方法构建了改形抗CD28重链单域抗体,根据一种鼠源抗CD28重链单域抗体的氨基酸序列,于GenBank中查得两条与之最同源的人源抗体序列,利用其中一条的FRs作为改形抗体的主框架进行改形构建,将鼠源抗体的CDR区插入到人源FR区后,对人源FR区的一些氨基酸残基进行替换突变,替换的氨基酸残基数及替换原则主要是根据对查到的人源抗体序列,鼠源抗体序列,以及这些序列与Kabat分类中的种属序列进行的比较,为了增加改形抗体基因的多样性,对要被替换的氨基酸残基在基因合成中采用简并的方式,使要被替换的氨基酸残基和替换的氨基酸残基都有机会出现,二者出现的几率各为50%,同时,在将大小不同的合成核苷酸片段采用重叠PCR扩增以获得完整改形抗体基因时,采用高Mg^2 浓度下和使用TaqDNA聚合酶,以进一步随机引入突变,利用重叠PCR产物构建了一个噬菌体抗体库,经过3轮淘选后,获得了几个具有较高免疫学活性的改形抗体,对其中的两个抗体进行了进一步研究,将两个抗体的基因在大肠杆菌BL21（DE3）中表达,复性后的表达蛋白仍具有较高的免疫学活性,结果表明该方法是有效可行的。     

Determination of the affinity of high- and low-affinity antibodies, which are in a mixture, using ELISA and the method of non-linear regression

It was shown that application of the method of non-linear regression for the solution of the equation, which relates the fraction of free antibodies in a mixture and antigen concentrations, allows to determine the affinity constants for two antibodies in a mixture. Such method is easier and more accurate than the suggested by us earlier method, which use the numerical solution of the appropriate four equations, that describe the relations between the experimental data obtained by ELISA, competing antigen concentration, and values of antibody affinity. In addition, the proposed method allows using much less quantity of experimental measurements without diminishing of the accuracy for the affinity constants evaluations.     

单克隆抗体生产的新时代

由fd噬菌体与质粒重组载体噬菌粒(phagemid)构建大容量,高效筛选的表面表达抗体基因片段文库,经突变、模仿体内B细胞亲和力成熟过程(affinity maturation)以免疫亲和层析筛选高亲和力特异抗体片段.取代单克隆抗体,应用于免疫分析诊断.