Polyreactive immunoglobulins recognize hydrophobic parts of proteins

It was shown that polyreactive immunoglobulins (PRIG), which are capable to interact non-specifically with various antigens, could differ from so called natural antibodies, which have also the capacity to react non-specifically with various antigens, including self-antigens. The main differences are that in contrast to the natural antibodies, which mainly interact, probably, with hydrophilic epitopes, PRIG recognise and bond preferentially to hydrophobic epitopes. We can consider this binding as a new type of interaction between immunoglobulins and antigens.     

Dynamics of the interaction of polyreactive immunoglobulins with immobilized antigens]

The kinetic of polyreactive immunoglobulins (PRIG) and immobilized antigen interaction was examined at different temperatures. It was shown that this process can be described by the so-called "competitive" model, and the relatively simple method for the rate constant determination for this process was developed. According to the "competitive" model PRIG molecule could be either in "active" or in "inactive" state and dynamic equilibrium exist between "active" and "inactive" molecules which strongly depend on incubation temperature. Only "active" PRIG can interact with antigens, and this is the reason of strong temperature dependence of PRIG-antigen interaction. The data also show that the mechanism of PRIG-antigen interaction differ from that of antibody-antigen interaction.     

Lipid-protein interactions and conformation of proteinaceous macromolecules

Analysis of the data as to the mechanism of specific antibody transformation into polyreactive immunoglobulins (PRIG) shows that for this transformation it is necessary and sufficiently to deprive antibodies of lipids, which are in norm tightly bound to the antibodies. Removal of these lipids by any methods (by treatment of antibodies with chaotropic ions, low/high pH, reactive oxygen species and lipases) leads to the loose by antibodies of their specificity and acquiring the ability to react with various non-related antigens, i.e. to their conversion into PRIG. Mathematical modeling of the PRIG--antigen interaction and values of thermodynamic characteristics of this process shows that antigen-binding domains of PRIG are in semi-melted state, thanks to what they can fit their structure to be complementary to structurally different antigens. Thus, we conclude that lipids bound to the so-called "hydrophobic pockets" of immunoglobulins (Ig) can stabilize the conformation of Ig and increase their rigidity, and removal of these lipids induce flexibility of Ig domains, responsible for interaction with antigens. It was presumed that lipids could exert the similar function of conformation stabilization not only in the case of antibodies, but also combining with some other proteins, for example, enzymes. Their removal could lead to the changing of protein conformation and loosing its biological activity. In this case the function of lipid removing and protein inactivation could exert cellular reactive oxygen species and cellular lipases and lipoxygenases.     

Specific and nonspecific interactions of antibodies and immunoglobulins with antigens and the methods of analysis of these interactions

The problem of specific and nonspecific interaction of serum immunoglobulins and antigens was considered. It was shown that high-sensitive methods allow to reveal low-affinity non-specific interaction of immunoglobulins and antigens. If the concentration of the specific antibodies in a studied sample of serum is low, the non-specific interaction of serum immunoglobulins may exceed substantially the effect of specific reaction. In this case the obtained results could be misinterpreted. In this connection the conclusion has been done that in such a case it is necessary to take into account the capability of serum immunoglobulins to interact non-specifically with antigens and to discriminate between specific and non-specific interaction. The methods of the diminishing the non-specific interaction are suggested.     

Mechanisms of interaction of polyreactive immunoglobulins and protein antigens

New features of interaction between polyreactive immunoglobulins (PRIG) and protein antigens were considered. It was shown that unlike specific antibodies, recognizing mainly hydrophilic epitops of proteins and interacting against them with high affinity according to the mechanisms "lock-and-key" and/or "induced fit", PRIG recognized and nonspecifically bound to hydrophobic patches of protein antigens. On this reason it is possible to prevent or markedly diminish PRIG-antigen interaction using the reagents that have high affinity to hydrophobic regions of proteins and therefore are capable to block these regions. The obtained data are in a good agreement with the former data concerning the kinetic and thermodynamics characteristics of PRIG-antigen interaction described by us earlier.     

Immunopotentiating properties of a multispecific α-anti-idiotype antibody

Multispecificity is not a well-understood property of some antibodies. Different functions have been attributed to multispecific natural antibodies, commonly associated with the neutralization and clearance of antigens. Much less is known about the role of antibodies like these, based on their idiotypic connectivity. B7Y33 is a chimeric IgG1 version of a polyreactive α anti-idiotype antibody that is able to interact with different immunoglobulin and non-immunoglobulin antigens. Here we report the capacity of this antibody to enhance the immunogenicity of several autologous IgMs in adjuvant-free conditions. Our results suggest that the formation of immune complexes seems to be necessary, but not sufficient, to this activity. The potential involvement of the interaction of B7Y33 with the FcγRIIb is discussed.     

The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies

Polyreactive antibodies bind to a variety of structurally unrelated antigens. The function of these antibodies, however, has remained an enigma, and because of their low binding affinity their biological relevance has been questioned. Using a panel of monoclonal polyreactive antibodies, we showed that these antibodies can bind to both Gram-negative and Gram-positive bacteria and acting through the classical complement pathway can inhibit bacterial growth by lysis, generate anaphylatoxin C5a, enhance phagocytosis, and neutralize the functional activity of endotoxin. Polyreactive antibody-enriched, but not polyreactive antibody-reduced, IgM prepared from normal human serum displays antibacterial activity similar to that of monoclonal polyreactive IgM. We conclude that polyreactive antibodies are a major contributor to the broad antibacterial activity of the natural antibody repertoire.     

Immunopotentiating properties of a multispecific α-anti-idiotype antibody

Multispecificity is not a well-understood property of some antibodies. Different functions have been attributed to multispecific natural antibodies, commonly associated with the neutralization and clearance of antigens. Much less is known about the role of antibodies like these, based on their idiotypic connectivity. B7Y33 is a chimeric IgG1 version of a polyreactive α anti-idiotype antibody that is able to interact with different immunoglobulin and non-immunoglobulin antigens. Here we report the capacity of this antibody to enhance the immunogenicity of several autologous IgMs in adjuvant-free conditions. Our results suggest that the formation of immune complexes seems to be necessary, but not sufficient, to this activity. The potential involvement of the interaction of B7Y33 with the FcγRIIb is discussed.     

Activation of "silent" antibodies and their interaction with antigens

Animal and human blood serum contains great amount of blocked (or "silent") immunoglobulins which, being activated by heating to 60 degrees C, pH decrease to 2.0-2.5 or treatment with 5M KSCN acquire a capacity to interact with different antigens. This interaction may be equally prevented or weakened by both identical and serologically non-related antigen, i.e. activated immunoglobulins are polyspecific. Polyspecific immunoglobulins show less affinity in comparison with monospecific antibodies, their interaction with antigens depends considerably on temperature.     

Determination of the concentration of ligand-specific molecules, found in mixtures with ligand-nonspecific molecules

A new method, which allows determination of the concentration of ligand-specific molecules in a mixture of these molecules with biochemically similar but ligand-unspecific molecules, is suggested. The method is based on a partial exhaustion of the mixture on a column with immobilized ligand and determination of the part of ligand-specific molecules presented in exhausted mixture. The concentration of monoclonal antibodies specific to bovine serum albumin in a commercial "Sigma" preparation and concentration of polyreactive immunoglobulins in a commercial "Sigma" preparation of bovine immunoglobulins were determined by suggested method.     

Complement-binding and immuno-modulating properties of polyreactive immunoglobulins

New data concerning biological properties of polyreactive immunoglobulins (PRIG) were obtained as a result of treatment of mouse serum immunoglobulins by 4 M KSCN and are presented in the paper. In particular, the capacity of PRIG to bind C1q, the subunit of the first component of complement was studied. It was shown that PRIG's binding capacity to C1q is similar to that of intact immunoglobulins. Intravenous administration of PRIG into mice together with either sheep red blood cells or heat-inactivated staphylococcal bacteria did not affect the immune response to these antigens. Meanwhile, the same administration of PRIG together with the purified protein derivate of tuberculin resulted in 10-fold increase of mouse antibody response to PPD. These results demonstrate that PRIG can have some immuno-modulating properties concerning low-immunogenic antigens.     

Suppression of growth of guinea pig line-10 hepatocarcinoma. I. Effect of simultaneous administration of tumor cells and antibodies from normal rabbits.

Suppression of growth of the line-10 hepatocarcinoma in strain-2 guinea pigs occurred when line-10 cells were injected intradermally together with sera or immunoglobulins derived from normal rabbits. A significant number of animals were resistant to subsequent rechallenge with tumor cells. This immunity was specific, depended on contact of immunoglobulins with tumor cells and on the concentration of immunoglobulins. Repeated injections acted as potent vaccines and resulted in the development of immunity in 84.6% of recipients. Fc receptors were not detected on line-10 cells. Antibodies reacting with line-10 cell unique antigens as well as with antigens common to line-10, line-1 and normal guinea pig spleen cells were found in NRS. Injection of line-10 cells together with rabbit immunoglobulins from which antibodies reacting with antigens derived from line-10 cells had been removed did not result in tumor suppression. The specific antigen(s) recognized by antibodies that suppressed growth of the line-10 tumor in vivo was not determined.     

Inhibition of normal mouse lymphocyte mitogen responses by xenogeneic or allogeneic antibodies to the MuLV glycoprotein gp71.

Xenogeneic and allogeneic antisera to the major envelope glycoprotein (gp71) of murine leukemia viruses (NyLV) inhibited the mitogenic response of normal mouse splenic lymphocytes to phytohemagglutinin (PHA) and lipopolysaccharide (LPS). This inhibition was specific for gp71 as demonstrated by the inability of xenogeneic antisera to other viral glycoproteins or structural proteins to inhibit and by the ability of purified antigens to block specifically the inhibitory effect. The ability of antisera to gp71 to inhibit LPS responses, however, is highly dependent on the strain and age of mouse spleen cells used and appears correlated with the expression of endogenous viruses. Moreover, the preferential inhibition of LPS responses suggests that this expression may be predominately B cell specific. The results suggest that the inhibitory effect is mediated via antibody binding to lymphocytes and that expression of viral envelope antigens on the cell surface which bind immunoglobulins can block or interfere with the binding or uptake of mitogens. A variety of natural mouse immune sera and "tumor" sera, having antibodies directed against gp71, can similarly inhibit mitogen responses; and this inhibition can be specifically blocked with MuLV or gp71.     

金抗体替代ELISA中的天然抗体用于溶菌酶检测

目的 酶联免疫吸附测定(ELISA)被广泛用于抗体或抗原的检测,并被视为临床实践中的金标准,可提供相对可靠、灵敏和特异的检测结果.ELISA的本质是抗原与相应抗体之间的特异性相互作用.然而,天然抗体固有的不稳定性是ELISA的一个难以克服的弱点,并可能导致检测结果的重现性差甚至错误的诊断结果.本课题组先前应用构象工程方...     

Mechanisms for increasing the activity of polyreactive immunoglobulins in vivo

The mechanisms of increasing of polyreactive immunoglobulin (PRIG) activity in vivo are suggested. It was shown that at some conditions, which can be observed in organism in sites of inflammation, activity of serum PRIG could be enhance considerably. This enhancing might be induced either by reactive oxygen species, mainly by hydroxyl radicals, or by lipases. The increasing of PRIG activity could be not only a result of preexisting PRIG unblocking, but also a result of transformation specific antibodies into PRIG, caused by peroxide degradation or lipolysis of lipids, tightly but noncovalently linked to antibodies, either by hydroxyl radicals or lipase. It was also suggested that most (if not all) specific antibodies consist of not only polypeptide chains, but also some lipids. Deprived of these lipids antibodies loose their specificity and transform into polyreactive immunoglobulins.     

Role of specific IgE antibodies in peroxidase (EPO) release from human eosinophils

After the demonstration of cytophilic IgE immunoglobulins (Ig) on human blood and lung eosinophils, their role in cell activation was studied by eosinophil peroxidase (EPO) assay. Hypodense human eosinophils from filariasis-infected patients were activated by anti-human Ig or various antigens. A selective release of EPO occurred after incubation with anti-human IgE, but not with anti-human IgG. The activation by antigens showed a strict antibody specificity of cytophilic IgE antibodies. The direct involvement of IgE antibodies in activation by the specific antigen was evidenced by inhibition experiments with aggregated human IgE myeloma protein. Circulating IgE antibodies exhibiting the same specificity and able to induce EPO release were detected in the sera from filariasis patients by a passive sensitization assay. Only the hypodense eosinophils were able to release EPO after IgE-dependent activation both in the direct assay and in the passive sensitization test, confirming the functional heterogeneity of human eosinophils. These results suggest that the interaction between IgE antibodies and human eosinophils can play a role both in protective immunity and pathology by releasing active pharmacologic mediators.     

Substances involved in the natural resistance of fish to infection–A review

Natural‘antibodies’are substances found in the blood of animals that have not been immunised against infective agents. However, exposure to these agents or to cross-reacting antigens may well have taken place. Fish contain naturally-occurring, relatively nonspecific, lectin-like proteins or glycoproteins, which are distinct from immunoglobulins, and which react with a wide variety of antigens and may confer some degree of immunity against natural infection. In most cases the cause of the antigenic stimulus is not obvious although the formation of these‘antibodies’may have been brought about by exposure to various micro-organisms. Many of these antibody-like molecules behave in a similar manner to immune antibodies or immunoglobulins and cross-react with specific carbohydrate moieties on the cell walls of bacteria, erythrocytes and certain other cellular antigens, due to the presence of similar antigenic determinants. It is difficult to ascribe an appropriate definition to the term‘natural antibody’. In fish, these‘antibodies’have been so designated on the basis of functional rather than structural criteria. Such naturally-occurring, low grade, antibody-like‘immune’substances include‘acute phase’proteins, lysozyme and chitinase, interferon, agglutinins, lysins, complement and properdin, precipitins, and non-immunoglobulin, lectin-like molecules. In addition to the above non-immunoglobulin materials, natural immunoglobulins identifiable as IgM have also been reported in fish. Furthermore, mucus contains many biochemical agents capable of reaction against infective organisms and thus providing the host with an immediate or a first line of defence mechanism. This review compiles some of the relevant information in the literature concerned with natural‘immune’substances, present in the serum and mucus of fish, involved in protection against pathogens. Wherever possible the basic physicochemical properties of these substances are indicated and their potential immunobiological functions discussed.     

研究报告：金抗体替代ELISA中的天然抗体用于溶菌酶检测

目的酶联免疫吸附测定(ELISA)被广泛用于抗体或抗原的检测,并被视为临床实践中的金标准,可提供相对可靠、灵敏和特异的检测结果。ELISA的本质是抗原与相应抗体之间的特异性相互作用。然而,天然抗体固有的不稳定性是ELISA的一个难以克服的弱点,并可能导致检测结果的重现性差甚至错误的诊断结果。本课题组先前应用构象工程方法开发了一种基于金纳米粒子的人工抗体(简称金抗体)。金抗体可以像天然抗体一样特异性地与抗原相互作用,并且具备远优于天然抗体的稳定性。出色的稳定性使金抗体可能成为天然抗体更好的替代物,用于ELISA中。方法经过必要的优化并与辣根过氧化物酶(HRP)耦联后,制得酶标金抗体10HRP-(Au-400P1),然后用酶标金抗体代替天然酶标抗体用于ELISA检测中。结果通过一系列的实验证明,抗溶菌酶金抗体可用于ELISA特异性检测1～16 mg/L范围内的鸡蛋清溶菌酶(HEWL)样品。结论金抗体可以替代天然抗体用于ELISA检测,并具有优于传统ELISA法的检测准确性和一致性。     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

A Cryptic Polyreactive Antibody Recognizes Distinct Clades of HIV-1 Glycoprotein 120 by an Identical Binding Mechanism

Polyreactive antibodies play an important role for neutralization of human immunodeficiency virus (HIV). In addition to intrinsic polyreactive antibodies, the immune system of healthy individuals contains antibodies with cryptic polyreactivity. These antibodies acquire promiscuous antigen binding potential post-translationally, after exposure to various redox-active substances such as reactive oxygen species, iron ions, and heme. Here, we characterized the interaction of a prototypic human antibody that acquires binding potential to glycoprotein (gp) 120 after exposure to heme. The kinetic and thermodynamic analyses of interaction of the polyreactive antibody with distinct clades of gp120 demonstrated that the antigen-binding promiscuity of the antibody compensates for the molecular heterogeneity of the target antigen. Thus, the polyreactive antibody recognized divergent gp120 clades with similar values of the binding kinetics and quantitatively identical changes in the activation thermodynamic parameters. Moreover, this antibody utilized the same type of noncovalent forces for formation of complexes with gp120. In contrast, HIV-1-neutralizing antibodies isolated from HIV-1-infected individuals, F425 B4a1 and b12, demonstrated different binding behavior upon interaction with distinct variants of gp120. This study contributes to a better understanding of the physiological role and binding mechanism of antibodies with cryptic polyreactivity. Moreover, this study might be of relevance for understanding the basic aspects of HIV-1 interaction with human antibodies.