The kinetics of hemolytic plaque formation. II. Inhibition of plaques by hapten.

We present a mathematical theory of hapten inhibition of hemolytic plaque formation. The treatment is based upon the mathematical model for plaque growth presented by DeLisi &; Bell (1974). The lymphocyte under consideration is embedded in an infinite three-dimensional medium, and is secreting antibodies isotropically at a constant rate. As the antibodies diffuse from the source they can bind reversibly to hapten, and in the most general case reversibly to red blood cell (RBC) epitope. The model leads to a non-linear diffusion equation coupled to a set of first order differential equations. The system must, in general, be solved numerically. However, in many cases of experimental interest simplifications arise which permit closed form solutions to be obtained. In this paper we have developed solutions for three special cases.In the first example antibodies can bind only univalently to RBCs, as would be expected if the epitopes are sparsely distributed. In this case reaction between antibody site and RBC epitope is rapid ( ⪆ 1 sec) and reversible and local equilibrium is assumed. This leads to a “pure” diffusion equation in the free antibody concentration, but with a reduced diffusion coefficient.In another example univalent attachment of an antibody site to a RBC epitope is followed by a rapid irreversible intramolecular reaction. This might be expected for example if the epitope density is large. An exact solution to the resulting diffusion equation was also found in this case. In order to assess an intermediate situation, we also solved the equations for a model in which intramolecular reaction is slow and irreversible.The theory predicts that the type of information one can obtain from inhibition experiments depends critically upon the preparation of the RBC. If the cell is sufficiently haptenated so that rapid irreversible multivalent attachment is favorable, a differential plot of the inhibition curve will reflect the affinity distribution of antibody sites for free hapten. If only univalent attachment with RBCs is possible, so that antibody sites bind to RBC hapten in the same way they bind to free hapten, then a differential plot of the inhibition curve will reflect the secretion rate distribution.     

Relationships between liquid- and solid-phase antibody association characteristics: implications for the use of competitive ELISA techniques to map the spatial location of idiotopes

A liquid-phase assay system based on small-zone size-exclusion chromatography was used to examine the binding of a monoclonal anti-idiotopic antibody, F6, to its idiotope on the murine plasmacytoma IgA, TEPC-15. Chromatographic behavior revealed a strong association between T-15 and F6, which was previously characterized by solid-phase immunoassay as recognizing a nonbinding site epitope of the T-15. This chromatographic pattern suggests that the inability of the hapten phosphorylcholine to inhibit the anti-idiotope:idiotope relationship in solid-phase immunoassay might be equally explained by the low affinity of the hapten relative to the high affinity of the anti-idiotope antibody. Bivalent interactions between solid-phase IgA and liquid-phase IgG should enhance the binding of the anti-idiotope to an extent that would prevent the hapten from dissociating the complex. Under these solid-phase assay conditions, observation of hapten inhibition may, in some cases, indicate site specificity, but absence of inhibition cannot be interpreted. Computer simulations of solid-phase hapten inhibition scenarios were used to evaluate the qualitative nature of binding inhibition profiles expected under various conditions of liquid- and solid-phase reactant affinities and concentrations. The apparently unusual inhibition curves previously observed in the T-15:anti-T-15 studies in which the degree of binding inhibition by hapten appeared to be independent of anti-idiotope concentration may be predictable in cases of excess solid-phase epitope; the plateau inhibition value is a function of relative affinity constants and concentrations of solid-phase and inhibitor components. The results additionally suggest that the affinity of a liquid-phase antibody may modulate the effective concentration of solid-phase epitope.     

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.     

Evidence for conformationally different states of interleukin‐10: binding of a neutralizing antibody enhances accessibility of a hidden epitope

We present the mapping of two anti‐human interleukin‐10 (hIL‐10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL‐10‐derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L ‐amino acids. The epitope‐derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody–hIL‐10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X‐ray structure of IL‐10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL‐10 can adopt a conformational state other than that observed in the crystal structure. Copyright © 1999 John Wiley & Sons, Ltd.     

Interaction of rabbit IgG with anti-IgG: localization of epitopes and absence of immunoreactivity of the pFc'-fragment]

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.     

Conformational changes in an epitope localized to the NH2-terminal region of protein C. Evidence for interaction of protein C domains

Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability to bind antigen in the presence of either CaCl2 or excess EDTA. Three stable clones were obtained which produced antibodies that bound to protein C only in the presence of EDTA. All three antibodies bound to the light chain of protein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized by a KD of 1.4 nM. In competition studies, it was found that the relative affinity of the antibody for protein C was 20-40-fold higher than for prothrombin, fragment 1 of prothrombin, or factor X. In contrast, 7D7B10 was unable to bind to factor IX or bovine protein C. The effect of varying Ca2+ concentration on the interaction of the antibody with protein C was complex. Low concentrations of Ca2+ enhanced the formation of the protein C-antibody complex with half-maximal effect occurring at approximately 60 microM metal ion. However, higher concentrations of Ca2+ completely inhibited 7D7B10 binding to protein C with a K0.5 of 1.1 mM. Furthermore, millimolar concentrations of Mn2+, Ba2+, or Mg2+ also completely abolished antibody binding to protein C. The location of the epitope was delineated by immunoblotting and peptide studies and found to be present in the NH2-terminal 15 residues of protein C. Although residues corresponding to positions 10-13 of human protein C were necessary for maximal binding of the antibody, they were not sufficient. No evidence could be found for involvement of the epitope in metal binding per se. Therefore, the effect of Ca2+ on antibody binding is thought to be due to metal-dependent conformational changes in protein C. It seems likely that Ca2+ occupation of a high affinity site, shown by others to be located in the epidermal growth factor-like domain, causes a conformational change in the NH2-terminal region of protein C which is favorable for antibody interaction, whereas Ca2+ binding to the low affinity site(s), known to be present in the gamma-carboxyglutamic acid domain, causes an unfavorable conformational change.     

Heymann antigen GP330 demonstrates affinity for fibronectin, laminin, and type I collagen and mediates rat proximal tubule epithelial cell adherence to such matrices in vitro.

We have utilized monoclonal antibodies directed against glycoproteins on the surface of proximal tubule epithelial cells (PTEC) to study their interaction with matrix components. PTEC exposed to monoclonal antibodies directed against a 330-kDa cell surface glycoprotein exhibited a significant epitope-specific inhibition of attachment and proliferation on type I collagen-, fibronectin-, laminin-, and gelatin-coated tissue culture surfaces. This effect was not due to antibody toxicity since such cells did not exhibit metabolic dysfunction in suspension cultures and the inhibition could be reversed upon removal of the antibody from the cell surface. Furthermore, detergent-solubilized gp330 demonstrated specific affinity for fibronectin, laminin, and type I collagen which was not inhibited by Arg-Gly-Asp-containing peptides. A monoclonal antibody directed against the receptor epitope was capable of promoting PTEC adherence and growth when such an antibody was immobilized on cell culture dishes. Although gp330 acted as a receptor for matrix proteins in primary cultures of freshly isolated PTEC, this effect was not demonstrable in established cultures. These results suggest that freshly isolated PTEC depend on gp330 for their attachment to matrix molecules while in vitro-adapted PTEC rely on other receptors activated by culture conditions. The affinity of gp330 for matrix molecules may be of pathogenic relevance in the persistence of gp330-containing immune complexes formed in the glomerular capillary wall in experimental membranous nephropathy (Heymann nephritis).     

Dialdehyde derivatives of purine mononucleotides: substrate properties and affinity modification of myosin ATPase

It was demonstrated that the dialdehyde derivative of ATP is a good substrate for Ca-ATPase of heavy meromyosin (Km = (1.2-1.4) X 10(-4) M; V = VATP). At the same time, this compound can induce irreversible inhibition of the enzyme. Since oxo-ATP is rapidly hydrolyzed by myosin to form oxo-ADP, this inhibition is the result of the enzyme interaction with oxo-ADP. It was found that the kinetics of heavy meromyosin inhibition by oxo-ADP are typical of affinity modification; in this case ATP fully protects heavy meromyosin from the activity loss. Similar results on the irreversible inhibition of the ATPase activity under the action of oxo-ADP were obtained in the presence of myosin, heavy meromyosin, subfragment I and natural actomyosin and in the absence of bivalent cations, thus suggesting the modification of the active center of myosin ATPase.     

Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody

X-ray structure analysis of 4 antibody Fab fragments, each in complex with human granulocyte macrophage colony stimulating factor (GM-CSF), was performed to investigate the changes at the protein-protein binding interface during the course of in vitro affinity maturation by phage display selection. The parental antibody MOR03929 was compared to its derivatives MOR04252 (CDR-H2 optimized), MOR04302 (CDR-L3 optimized) and MOR04357 (CDR-H2 and CDR-L3 optimized). All antibodies bind to a conformational epitope that can be divided into 3 sub-epitopes. Specifically, MOR04357 binds to a region close to the GM-CSF N-terminus (residues 11–24), a short second sub-epitope (residues 83–89) and a third at the C-terminus (residues 112–123). Modifications introduced during affinity maturation in CDR-H2 and CDR-L3 led to the establishment of additional hydrogen bonds and van der Waals contacts, respectively, providing a rationale for the observed improvement in binding affinity and neutralization potency. Once GM-CSF is complexed to the antibodies, modeling predicts a sterical clash with GM-CSF binding to GM-CSF receptor α and β chain. This predicted mutually exclusive binding was confirmed by a GM-CSF receptor α chain ligand binding inhibition assay. Finally, high throughput sequencing of clones obtained after affinity maturation phage display pannings revealed highly selected consensus sequences for CDR-H2 as well for CDR-L3, which are in accordance with the sequence of the highest affinity antibody MOR04357. The resolved crystal structures highlight the criticality of these strongly selected residues for high affinity interaction with GM-CSF.     

New approach in evaluating affinity of bivalent antibodies by the method of surface plasmon resonance. Theory

Theoretical aspects of the affinity evaluation for the interaction between bivalent receptors (or antibodies) and corresponding ligands (or antigens) are considered. It was shown that the ligand presence in the solution at the stage when the receptor dissociation occurs leads to the increase of the affinity evaluation accuracy. We demonstrated that the analysis of the dissociative curve of the receptor from the chip is not necessary for affinity determination; the analysis of associative curve is sufficient for this purpose. We also suggested a new approach for evaluating the affinity of bivalent receptors (or antibodies) when these reagents are present in the studied solution and the correspondent ligand (or antigen) is immobilized on the chip.     

Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins.

The mechanisms of neural crest cell interaction with laminin were explored using a quantitative cell attachment assay. With increasing substratum concentrations, an increasing percentage of neural crest cells adhere to laminin. Cell adhesion at all substratum concentrations was inhibited by the CSAT antibody, which recognizes the chick beta 1 subunit of integrin, suggesting that beta 1-integrins mediate neural crest cell interactions with laminin. The HNK-1 antibody, which recognizes a carbohydrate epitope, inhibited neural crest cell attachment to laminin at low coating concentrations (greater than 1 microgram ml-1; Low-LM), but not at high coating concentration of laminin (10 micrograms ml-1; High-LM). Attachment to Low-LM occurred in the absence of divalent cations, whereas attachment to High-LM required greater than 0.1 mM Ca2+ or Mn2+. Neural crest cell adherence to the E8 fragment of laminin, derived from its long arm, was similar to that on intact laminin at high and low coating concentrations, suggesting that this fragment contains the neural crest cell binding site(s). The HNK-1 antibody recognizes a protein of 165,000 Mr which is also found in immunoprecipitates using antibodies against the beta 1 subunit of integrin and is likely to be an integrin alpha subunit or an integrin-associated protein. Our results suggest that the HNK-1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated form of beta 1-integrin, which recognizes laminin in the apparent absence of divalent cations. We conclude that neural crest cells have at least two functionally independent means of attachment to laminin which are revealed at different substratum concentrations and/or conformations of laminin.     

Effect of epitope position on neutralization by anti-human immunodeficiency virus monoclonal antibody 2F5

The membrane-proximal region of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein (TM) is critical for envelope (Env)-mediated membrane fusion and contains the target for broadly reactive neutralizing antibody 2F5. It has been proposed that 2F5 neutralization might involve interaction of its long, hydrophobic, complementarity-determining region (CDR) H3, with adjacent viral membrane. Using Moloney murine leukemia virus (MLV) as a tool, we examined the effect of epitope position on 2F5 neutralization. When the 2F5 epitope was inserted in the proline-rich region of MLV Env surface protein (SU), 2F5 blocked cell fusion and virus infection, whereas MLV with a hemagglutinin (HA) epitope at the same position was not neutralized by anti-HA, even though the antibodies bound their respective Envs on the surface of infected cells and viruses equally well. When the 2F5 epitope was inserted in the MLV Env TM at a position comparable to its natural position in HIV-1 TM, 2F5 antibody blocked Env-mediated cell fusion. Epitope position had subtle effects on neutralization by 2F5: the antibody concentration for 50% inhibition of cell fusion was more than 10-fold lower when the 2F5 epitope was in SU than in TM, and inhibition was less complete at high concentrations of antibody; we discuss possible explanations for these effects of epitope position. Since membrane proximity was not required for neutralization by 2F5 antibody, we speculate that the CDR H3 of 2F5 contributes to neutralization by destabilizing an adjacent protein rather than by inserting into an adjacent membrane.     

Diversification of specificity after maturation of the antibody response to the HIV gp41 epitope ELDKWA

During maturing antibody responses the increase in affinity for target antigens is achieved by genetic diversification of antibody genes followed by selection for improved binding. The effect this process has on the specificity of antibody for variants of the antigen is not well-defined, despite the potential role of antibody diversification in generating enhanced protection against pathogen escape mutants, or novel specificities after vaccination. To investigate this, a library of single amino-acid substitution epitope variants has been screened with serum obtained at different time-points after immunization of mice with the HIV gp41 peptide epitope ELDKWA. The serum IgG response is shown to mature and increase affinity for ELDKWA, and the titre and affinity of IgG against most epitope variants tested increases. Furthermore there is a bias towards high affinity serum IgG binding to variant epitopes with conservative substitutions, although underlying this trend there is also significant binding to many epitopes with non-conservative substitutions. Thus, maturation of the antibody response to a single epitope results in a broadening of the high-affinity response toward variant epitopes. This implies that many pathogen epitope escape variants that could manifest as single amino-acid substitutions would not emerge by escaping immune surveillance.     

Neutralization Profiles of Primary Human Immunodeficiency Virus Type 1 Isolates in the Context of Coreceptor Usage

Most strains of human immunodeficiency virus type 1 (HIV-1) which have only been carried in vitro in peripheral blood mononuclear cells (primary isolates) can be neutralized by antibodies, but their sensitivity to neutralization varies considerably. To study the parameters that contribute to the differential neutralization sensitivity of primary HIV-1 isolates, we developed a neutralization assay with a panel of genetically engineered cell lines (GHOST cells) that express CD4, one of eight chemokine receptors which function as HIV-1 coreceptors, and a Tat-dependent green fluorescent protein reporter cassette which permits the evaluation and quantitation of HIV-1 infection by flow cytometry. All 21 primary isolates from several clades could grow in the various GHOST cell lines, and their use of one or more coreceptors could easily be defined by flow cytometric analysis. Ten of these primary isolates, three that were CXCR4 (X4)-tropic, three that were CCR5 (R5)-tropic, and four that were dual- or polytropic were chosen for study of their sensitivity to neutralization by human monoclonal and polyclonal antibodies. Viruses from the X4-tropic category of viruses were first tested since they have generally been considered to be particularly neutralization sensitive. It was found that the X4-tropic virus group contained both neutralization-sensitive and neutralization-resistant viruses. Similar results were obtained with R5-tropic viruses and with dual- or polytropic viruses. Within each category of viruses, neutralization sensitivity and resistance could be observed. Therefore, sensitivity to neutralization appears to be the consequence of factors that influence the antibody-virus interaction and its sequelae rather than coreceptor usage. Neutralization of various viruses by the V3-specific monoclonal antibody, 447-52D, was shown to be dependent not only on the presence of the relevant epitope but also on its presentation. An epitope within the envelope of a particular virus is not sufficient to render a virus sensitive to neutralization by an antibody that recognizes that epitope. Moreover, conformation-dependent factors may overcome the need for absolute fidelity in the match between an antibody and its core epitope, permitting sufficient affinity between the viral envelope protein and the antibody to neutralize the virus. The studies indicate that the neutralization sensitivity of HIV-1 primary isolates is a consequence of the complex interaction between virus, antibody, and target cell.     

Detection of conformational changes in human chorionic gonadotropin upon binding to rat gonadal receptors

After binding to rat testicular or ovarian luteinizing hormone (LH) receptors, human chorionic gonadotropin (hCG) and mammalian LH can be detected with monoclonal antibodies directed against a conserved epitope on the beta subunit of the hormones. Two such anti-hCG/anti-LH monoclonal antibodies, known as B105 and B110, compete with one another for binding to this epitope region on free and receptor-bound hormone. By comparing the affinities of B105 and B110 for these two forms of hCG, we have detected apparent changes in the structure of the hormone which develop subsequent to receptor binding. Whereas the affinity of B105 for receptor-bound hCG is approximately 10-fold lower than that for free hCG, the affinity of B110 for receptor-bound hCG is nearly 20-fold greater than that for free hCG. Both B105.hCG and B110.hCG complexes bind to the receptor; however, they have approximately 25 and 50% lower affinity than hCG. Thus, although B110 binds better to the form of hCG which is bound to receptors, binding of B110 to hCG does not appear to induce a conformational change in the hormone which facilitates hormone-receptor binding. Consequently, both B105 and B110 partially inhibit binding of hCG to its receptors. Fab fragments of B105 and B110 are as effective as intact B105 and B110 in inhibiting the binding of labeled B105 and B110 to hCG-receptor complexes, suggesting that circular complexes which might be formed by the interaction of divalent antibody, two molecules of hCG, and two membrane-bound receptors or one divalent receptor are not contributing to the affinity of the antibodies for receptor-bound hCG. Alternatively, formation of circular complexes can explain an increase in apparent affinity of B105 for ovine or bovine LH-receptor complexes. Data obtained with B105 suggest either that the structure of the epitope is altered following binding or that a portion of the epitope is partially obscured when hCG binds to the receptor. In contrast, the data obtained using B110 are not explained by models in which steric factors reduce the affinity of the antibody for the hormone-receptor complex. Therefore, as a minimal explanation for these observations, we postulate that the conformation of the B105/B110 epitope region is altered following binding of the hormone to receptors. The nature of the conformational change and its relationship to LH/hCG action is unknown.     

Qualitative analysis of a mathematical model of a open enzyme reaction

A general case of the set of two differential equations, describing an open reaction v1 leads to S v reversible E P v2 leads to, has been considered. The requirements to the character of the functions v1([S]), v2([P]) and v([S], [P]) were formulated for the case of existence and absence of alternative steady states and sustained oscillations. The formulae were derived to determine the slope of the unstable portion of the quasi-steady state characteristic. The generalized model of Monod, Wyman and Changeux has been considered as an example of v([S], [P]). It has been shown that with monotonically decreasing v1 and monotonically increasing v2, the alternative steady states and oscillations are possible only in the presence of substrate inhibition or product activation. However, under the joint action of substrate inhibition and product activation, the system will exhibit bistability rather than an oscillatory behavior. In the case of an irreversible two-substrate reaction which can be described by a similar mathematical model, inhibition by the first and second substrate is equivalent to substrate inhibition and product activation.     

Structural consequences of target epitope-directed functional alteration of an antibody. The case of anti-hen lysozyme antibody,HyHEL-10

Decreased affinity of an antibody for a mutated epitope in an antigen can be enhanced and reversed by mutations in certain antibody residues. Here we describe the crystal structures of (a) the complex between a naturally mutated proteinaceous antigen and an antibody that was mutated and selected in vitro, and (b) the complex between the normal antigen and the mutated antibody. The mutated and selected antibody recognizes essentially the same epitope as in the wild-type antibody, indicating successful target site-directed functional alteration of the antibody. In comparing the structure of the mutated antigen-mutant antibody complex with the previously established structure of the wild-type antigen-wild-type antibody complex, we found that the enhanced affinity of the mutated antibody for the mutant antigen originated not from improvements in local complementarity around the mutated sites but from subtle and critical structural changes in nonmutated sites, including an increase in variable domain interactions. Our findings indicate that only a few mutations in the antigen-binding region of an antibody can lead to some structural changes in its paratopes, emphasizing the critical roles of the plasticity of loops in the complementarity-determining region and also the importance of the plasticity of the interaction between the variable regions of immunoglobulin heavy and light chains in determining the specificity of an antibody.     

Antigenic properties of keratan sulfate: influence of antigen structure, monoclonal antibodies, and antibody valency.

The influence of (a) antigen structure, (b) type of monoclonal antibody, and (c) antibody bivalency on the immunochemical detection and quantification of keratan sulfate (KS) from aggrecan has been studied. Apparent KS epitope levels were determined by immunoglobulin G (IgG)-enzyme-linked immunosorbent assay (ELISA) in preparations of human aggrecan and in a defined series of lower molecular weight proteoglycan preparations generated by proteolytic and alkali treatment of aggrecan. Gel filtration chromatography showed KS epitope to be preferentially detected in the higher molecular weight fragments of the preparations. In single KS chains the epitope was detected in the chains of higher M(r). The ability of the proteoglycan to inhibit in the IgG-ELISA decreased with a reduction in proteoglycan fragment size, ranging between 6- and 260-fold, depending on the antibody used. This was considered to be a cooperative binding effect. With most antibodies, the sensitivity of the IgG-ELISA (represented by the steepness of the inhibition slope) was also reduced with smaller inhibitor sizes. The lowest limit of detectability (the amount of KS required to generate 20% inhibition) varied by up to 60-fold depending on the antibody used. The use of monovalent Fab fragments instead of the whole IgG anti-KS antibody in the ELISA showed that the bivalency of the antibody also affected the quantitation of the assay. In the Fab-ELISA the assay was found to have an increased detectability (by 9.5-fold with aggrecan as the inhibitor), and the proteoglycan fragments and aggrecan all generated parallel inhibition curves. Although the Fab-ELISA was somewhat influenced by the structural presentation of the KS, this was not apparent for small fragments and single chains. Thus the effects of cooperative binding and antibody valency could be overcome and quantitative data could be obtained for all samples, using papain-digested samples and the Fab-ELISA. Application of this assay to analysis of body fluids showed the KS-containing fragments in synovial fluid, serum, and urine were of different sizes and could be quantified.     

Analysis of binding curves in multivalent antigen-heterogeneous antibody systems

The binding of antibodies to N-valent antigens can be utilized to gain information on the antibody affinity distribution, under the assumption, that the formation of each bond occurs as an independent event. The analysis of the most widely used plots of binding data (double reciprocal, Scatchard, Sips and the so-called “avidity” plot) leads to expressions which correlate asymptotical features of the binding curves to the antibody site concentration to the antigen valence and to the affinity moments <K^?1>, <K> and <K²>. Only in the “avidity” plot is the shape of the curve independent of the valence of the antigen, depending merely on the antibody affinity distribution.     

Characteristics of affinity modification of myosin ATPase under the action of monoaldehyde derivatives of ADP]

It was shown that the highly purified monoaldehyde derivative of ADP obtained by partial reduction of the dialdehyde derivative of ADP causes strong irreversible inhibition of the Ca-ATPase activity of myosin subfragment I, the inhibiting effect being of the affinity modification type. The addition to the reaction medium of Mg2+ (but not Ca2+) during the subfragment I interaction with the inhibitor fully prevents the inhibiting effect at all substrates used (Ca-, Mg- or K, EDTA-ATPases). Contrariwise, the subfragment I modified in the absence of Mg2+ exhibits the same degree of inhibition for all the three types of the ATPase activity. An unexpected result that was previously unobserved for other affinity modifiers of myosin ATPase is the maintenance of activity in 50% of active centers, when "two-head" forms of the enzyme (the myosin proper and heavy meromyosin, HMM) are modified. Noteworthy that the affinity modification reaction is characterized by the same values of inhibition constants as in the case of myosin subfragment I (Ki = 3.3-3.5 X 10(-4) M; ki = 0.03-0.04 min-1). This finding provides additional evidence in favour of functional asymmetry of myosin heads in the myosin molecule which seems to be due to the screening of the active center of one head by the other one.