Blockade of NK cell lysis is a property of monoclonal antibodies that bind to distinct regions of T-200

The previously described NK inhibitory monoclonal antibody 13.1 is shown to immunoprecipitate a series of high m.w. glycoproteins homologous with the murine T-200/Ly-5 molecules. Not all antibodies to the human T-200 molecule, however, have an inhibitory effect on NK cell function. A comparison is made between two noninhibitory anti-T-200 antibodies, 13.5 and 13.6, and two inhibitory anti-T-200 antibodies, 13.1 and 13.3. All antibodies are of the IgG1 subclass. Sequential immunoprecipitation experiments show that these antibodies react with the same set of molecules. The differences in NK-blocking activity could not be explained by the amount of antibody bound per cell in NK-enriched populations, nor by the avidity with which they bound. It is shown by competitive radiobinding assays that the 13.1 and 13.3 antibodies define a region, termed region A, distinct from that defined by the nonblocking antibodies 13.5 and 13.6, termed region B. Region B is shown to reside between the membrane and region A. These findings show that the inhibition of NK lysis by anti-T-200 antibodies is a function of the site on that molecule to which these antibodies bind. This may also explain the ability of antibodies to the A region of T-200 to block selectively the lysis of myeloid and erythroid tumor targets, with no effect on the lysis of T lymphoma targets.     

Immunomodulation of carcinogens-induced steroids-dependent human diseases

The experimental and clinical data about antibodies against environmental chemical carcinogens and endogenous steroids are represented. The conception of immunomodulation of carcinogens- and steroids-dependent human diseases is proposed. It is postulated that antibodies to polycyclic aromatic hydrocarbons and heterocyclic amines in cooperation with antibodies to cholesterol, sex hormones, mineralo- and glucocorticoids stimulate or inhibit cancer, malformation, cardiovascular and autoimmune diseases depending on their personal combination. It is recommended to use immunoassay of these antibodies for the human diseases prediction. The alternative approaches for prevention using the probiotics transformed by anti-carcinogen antibodies are substantiated.     

The problem of prozone in serum antibody titration and its mathematical interpretation

The question of the mechanism of "prozone" creation was considered from the point of view of the concentrations of free and semi-blocked bivalent antibodies in the mixture of these antibodies with monovalent antigen. Using the so called "coordinates of dilution", suggested by the author earlier, it was possible to calculate the relationships between the concentrations of either free or semi-blocked bivalent antibodies and the dilution of the antigen-antibody mixture. It was shown that dilution of antigen-antibody mixture leads to an increase of the concentration of free bivalent antibodies and simultaneous sharp decrease of the concentration of semi-blocked antibodies. It is suggested, that such a relationship is quite enough for the creation of prozone effect in reactions, when only bivalent antibodies are active and semi-blocked antibodies compete with free antibodies, providing inhibition of the reaction.     

Preparation and application of antibodies to phosphoamino acid sequences

In this review, we describe methods to generate and characterize sequence-specific phosphoamino acid antibodies. Several of the early contributions regarding the utility of such antibodies are summarized. Three antiphosphopeptide antibodies derived from sequences of the Bcr protein are described. They are anti-Bcr pSer-354, anti-Bcr pTyr-328, and anti-Bcr pTyr-360. These anti-Bcr phosphopeptide antibodies are directed toward phosphorylated sequences encoded by the first exon of the BCR gene, which is the critical portion of the Bcr sequence present in the Bcr-Abl oncoprotein. Using these antibodies, we established/confirmed the in vivo phosphorylation of Ser-354, Tyr-328, and Tyr-360 in Bcr and Bcr-Abl proteins. The cross-reactivity of these antibodies, which is a common problem with antipeptide antibodies, was also investigated and discussed.     

Mouse antibodies to the insulin receptor developing spontaneously as anti-idiotypes. I. Characterization of the antibodies

Mice immunized to ungulate insulins were found to develop antibodies of two specificities: insulin antibodies that were mostly IgG1 and IgG2 antibodies that acted both as anti-idiotypes to specific mouse insulin antibodies and as antibodies to the insulin receptor. There was a negative association between the presence of anti-idiotypic receptor antibodies and insulin antibodies bearing the specific idiotype; the specific idiotypic antibodies were confined to the early phase of the primary response while the anti-idiotypic receptor antibodies were detected only after the idiotypic antibodies had disappeared. To map the insulin epitope that triggered the specific idiotypic response, we chemically altered the insulin molecule so as to inhibit its interaction with the insulin receptor. The altered insulins triggered high titers of antibodies binding to antigenic determinants on native insulin, but no anti-idiotypic receptor antibodies. Thus, the epitope responsible for the specific idiotypic-anti-idiotypic network was probably the part of the insulin molecule whose conformation is recognized by the insulin receptor.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin is not detected, and free antibodies account for 68 to 98% of the original amount. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80-100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalency of serum antibodies, and monovalency of the toxin epitopes. A new model of the toxin-antibody interactions is proposed.     

Generation of recombinant antibodies and means for increasing their affinity

Highly specific interaction with foreign molecules is a unique feature of antibodies. Since 1975, when Keller and Milstein proposed the method of hybridoma technology and prepared mouse monoclonal antibodies, many antibodies specific to various antigens have been obtained. Recent development of methods for preparation of recombinant DNA libraries and in silico bioinformatics approaches for protein structure analysis makes possible antibody preparation using gene engineering approaches. The development of gene engineering methods allowed creating recombinant antibodies and improving characteristics of existing antibodies; this significantly extends the applicability of antibodies. By modifying biochemical and immunochemical properties of antibodies by changing their amino acid sequences it is possible to create antibodies with properties optimal for certain tasks. For example, application of recombinant technologies resulted in antibody preparation of high affinity significantly exceeding the initial affinity of natural antibodies. In this review we summarize information about the structure, modes of preparation, and application of recombinant antibodies and their fragments and also consider the main approaches used to increase antibody affinity.     

Early diagnosis of systemic lupus erythmatosus using ANN models of dsDNA binding antibody sequence data

In this paper a new method based on artificial neural networks (ANN), is introduced for identifying pathogenic antibodies in Systemic Lupus Erythmatosus (SLE). dsDNA binding antibodies have been implicated in the pathogenesis of this autoimmune disease. In order to identify these dsDNA binding antibodies, the protein sequences of 42 dsDNA binding and 608 non-dsDNA binding antibodies were extracted from Kabat database and encoded using a physicochemical property of their amino acids namely Hydrophilicity. Encoded antibodies were used as the training patterns of a general regression neural network (GRNN). Simulation results show that the accuracy of proposed method in recognizing dsDNA binding antibodies is 83.2%. We have also investigated the roles of the light and heavy chains of anti-dsDNA antibodies in binding to DNA. Simulation results concur with the published experimental findings that in binding to DNA, the heavy chain of anti-dsDNA is more important than their light chain.     

Single-step sandwich immunoassay of myoglobin with bifunctional monoclonal antibodies.

Two protocols for sandwich antigen-capture ELISA of human myoglobin were compared. In the first (routine) variant, 14D6 monoclonal antibodies conjugated to horseradish peroxidase were used as the secondary antibodies. Bifunctional antibodies specific for myoglobin/peroxidase were used as the secondary antibodies in the second variant. The myoglobin-binding site of the bifunctional antibodies was similar to that of the 14D6 antibodies, and the second antigen-binding site of the bifunctional antibodies was bound to horseradish peroxidase. When comparing standard calibration curves, the effective concentration of the bifunctional antibodies and that of antibodies conjugated to horseradish peroxidase were made equal. It is shown that the use of bispecific antibodies as the secondary antibodies does not improve the quality of the parameters tested, i.e., the sensitivity of the assay does not increase and the slope of the calibration curve remains constant.     

Immunocytochemistry of brain-reactive monoclonal antibodies in peripheral tissues

Among a total of 135 tissue-reactive monoclonal antibodies previously prepared, 81 were brain-selective and were classified into neuronal and non-neuronal categories. The neuronal antibodies were again subdivided into antineurofibrillar, antiperikaryonal-neurofibrillar, and antisynapse-associated groups. On the basis of morphologic, developmental, biochemical, and pathologic criteria, the antibodies in at least two of these groups were found to detect heterogeneous antigens (called "neurotypes") rather than different antigenic determinants in single antigens. On examining the distribution in peripheral organs of staining patterns of 11 antineuronal brain-reactive antibodies, we now confirm that these antibodies are, indeed, largely brain-specific. In general, non-neuronal elements in liver, lung, heart, thymus, intestine, adrenal, and spleen remained unstained. However, most of the antibodies stained peripheral neural elements. Occasional antibodies did stain selected, non-neuronal structures. Four out of five antineurofibrillar antibodies stained nerve fibers in adrenal medulla, intestine and thymus. All of three antiperikaryonal-neurofibrillar antibodies also stained nerve fibers in the adrenal medulla, but not in other organs. Two out of three antisynapse-associated antibodies stained what appear to be nerve contacts on adrenal medullary cells, but not on any other peripheral cells examined. The non-neuronal peripheral staining patterns were restricted to selective nuclear staining exhibited by two out of five antineurofibrillar antibodies and the staining of macrophage and selected cardiac muscle nuclei by two of three antisynapse-associated antibodies. However, one antineurofibrillar antibody also stained the cytoplasm of selected liver cells. Among non-neuronally reacting antibodies, two antibodies stained nuclei of all cells except neurons in brain as well as peripheral organs. An antibody staining the ciliary epithelium of choroid plexus also stained basal bodies of ciliated bronchial epithelium. The overall data suggest that the specificity of brain-reactive antibodies is high and that their cross-reactivity with epitopes in non-nervous tissue is rare. In these cases, the antibodies seem to provide specific reagents for these additional structures as well as for their specific brain antigens.     

A novel redox method for rapid production of functional bi-specific antibodies for use in early pilot studies

We demonstrate here a rapid alternative method for the production of functional bi-specific antibodies using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA). Following reduction of a mixture of two monoclonal antibodies with MESNA to break inter heavy chain bonds, this solution is dialysed under oxidising conditions and antibodies are allowed to reform. During this reaction a mixture of antibodies is formed, including parental antibodies and bi-specific antibody. Bi-specific antibodies are purified over two sequential affinity columns. Following purification, bi-specificity of antibodies is determined in enzyme-linked immunosorbent assays and by flow cytometry. Using this redox method we have been successful in producing hybrid and same-species bi-specific antibodies in a time frame of 6-10 working days, making this production method a time saving alternative to the time-consuming traditional heterohybridoma technology for the production of bi-specific antibodies for use in early pilot studies. The use of both rat and mouse IgG antibodies forming a rat/mouse bi-specific antibody as well as producing a pure mouse bi-specific antibody and a pure rat bi-specific antibody demonstrates the flexibility of this production method.     

Antibodies to the rhamnose determinants of the polysaccharide of streptococci group A in the sera of rheumatism patients and donors]

In the sera of patients with recurrent rheumocarditis, and especially in cases of primary rheumatism, the level of antibodies to group A streptococcal polysaccharide (A-PS) has been found, according to the results of the enzyme immunoassay, to be considerably higher than in the sera of healthy donors. The level of antibodies to rhamnose determinants (RD) of A-PS has been determined by the inhibition of the immunoenzyme reaction with A-PS under the influence of a variant of group A streptococcus and rhamnose disaccharides with the bonds alpha 1-2 and alpha 1-3. In patients with recurrent rheumocarditis the level of antibodies to A-PS has been shown to be considerably higher than in healthy donors having these antibodies. In acute primary rheumatism a high level of antibodies to A-PS has been detected only in a few cases, and at the same time the prevalence of antibodies to the specific RD of A-PS, bound with beta-N-acetylglucosamine, is observed. In the sera of patients with recurrent rheumocarditis and donors having a high content of antibodies to the rhamnose site of A-PS antibodies, seemingly active against at least two RD, have been detected. In acute primary rheumatism an insignificant amount of antibodies to the rhamnose site of A-PS may probably cause the autoimmune process accompanying rheumatism. This suggestion is substantiated by the previously established capacity of these antibodies for inducing the suppression of cytotoxic cell reactions to microbial antigens.     

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...     

Antiplatelet antibodies in cases of Glanzmann's thrombasthenia with and without a history of multiple platelet transfusion

Antiplatelet antibodies are known to be present in a wide spectrum of patients, which include chronic Idiopathic Thrombocytopenic Purpura (ITP), infections, etc., including Glanzmann''s thrombasthenia (GT) patients who receive multiple platelet transfusions. The presence of natural antibodies to platelet receptors is not studied in cases of GT. We studied the antiplatelet antibodies in 23 patients with GT, 15 of which had received multiple transfusions and eight that had not received transfusions, along with 50 cases of chronic ITP. The prevalence and specificity of platelet-bound antibodies were detected by inhibition assays using O-group platelets on flow cytometry. The mean antiplatelet antibodies in 15 patients of GT who had not received transfusions and eight patients with multiple transfusions was 8427 + 2131.88 and 9038 + 2856 antibodies/platelet, respectively, while in case of the 50 ITP patients studied, it was 22166 + 5616 antibodies/platelet (Normal Range 1500–3200 antibodies/platelet). We conclude that GT patients who have not received transfusions may develop antiplatelet antibodies to the missing/abnormal receptor. Whether this is due to a molecular mimicry or due to some other mechanism needs to be explored.     

Successful vaccine-induced seroconversion by single-dose immunization in the presence of measles virus-specific maternal antibodies

In humans, maternal antibodies inhibit successful immunization against measles, because they interfere with vaccine-induced seroconversion. We have investigated this problem using the cotton rat model (Sigmodon hispidus). As in humans, passively transferred antibodies inhibit the induction of measles virus (MV)-neutralizing antibodies and protection after immunization with MV. In contrast, a recombinant vesicular stomatitis virus (VSV) expressing the MV hemagglutinin (VSV-H) induces high titers of neutralizing antibodies to MV in the presence of MV-specific antibodies. The induction of neutralizing antibodies increased with increasing virus dose, and all doses gave good protection from subsequent challenge with MV. Induction of antibodies by VSV-H was observed in the presence of passively transferred human or cotton rat antibodies, which were used as the models of maternal antibodies. Because MV hemagglutinin is not a functional part of the VSV-H envelope, MV-specific antibodies only slightly inhibit VSV-H replication in vitro. This dissociation of function and antigenicity is probably key to the induction of a neutralizing antibody in the presence of a maternal antibody.     

Immunochemical properties of human low density lipoproteins as explored by monoclonal antibodies. Binding characteristics distinct from those of conventional serum antibodies

Spleen cells obtained from mice immunized with human plasma low-density lipoproteins (LDL) were fused with mouse myeloma cells. The resulting hybridoma cells secreting immunoglobulin specific for LDL were screened and scored by radioimmunoassay and cloned by multiple limiting dilutions. Immunochemical properties of the monoclonal antibodies were compared with convential mouse serum antibodies. It was found that conventional antibodies precipitated LDL and bound more than 95% of 125I-labeled LDL and the maximal binding was independent of temperature. The monoclonal antibodies were incapable of precipitating LDL and bound a maximum of only 20% of the total 125I-labeled LDL. The maximal binding between monoclonal antibodies and LDL was extremely temperature-dependent. An optimal degree of binding was observed at 4 degrees C, whereas binding at 37 degrees C was only 30% of that achieved at 4 degrees C. Although the binding at 37 degrees C was low, the maximal binding could be re-established following a subsequent incubation at 4 degrees C, suggesting that the antigenic structure of LDL is reversibly modulated at temperatures between 4 and 37 degrees C. Since the orientation of apolipoprotein B in LDL is known to be dynamic at different temperatures, this result suggests that monoclonal antibodies, but not conventional antibodies, are capable of detecting subtle conformational changes in LDL. In addition, we have determined the binding affinity of LDL to monoclonal antibodies and to conventional antibodies. Only monoclonal antibodies showed a linear Scatchard plot, suggesting that the binding was to a single site with a single affinity. The monoclonal antibodies also possessed high specificity and failed to react with porcine LDL, while serum antibodies could recognize both human and porcine LDL.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin was not detected, and free antibodies accounted for 68 to 98% of the original amount of the antibodies. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80–100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalence of serum antibodies, and monovalence of the toxin epitopes. A new model of the toxin-antibody interaction is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 235–242.Original Russian Text Copyright © 2005 by Titova, Sviridov.     

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.     

Analysis of the monocyte Fc receptors and antibody-mediated cellular interactions required for the induction of T cell proliferation by anti-T3 antibodies

The induction of human T cell proliferation by antibodies that cross-link T3 antigens is dependent on functional interactions of anti-T3 antibodies with monocyte Fc receptors. In this report, we used a panel of anti-T3 antibodies of differing heavy chain isotype and a variety of other monoclonal antibodies to analyze several features of the antibody-mediated interactions between T cells and monocytes that are required for mitogenesis. Whereas three IgG2a anti-T3 antibodies were mitogenic for cells from all individuals, IgM and IgG2b anti-T3 antibodies did not induce T cell proliferation in any donor and could block the proliferative responses induced by other mitogenic anti-T3 antibodies. Dose-response analyses with four IgG1 anti-T3 antibodies demonstrated donor heterogeneity as reported by other investigators. However, in contrast to these previous reports, high concentrations of IgG1 anti-T3 antibodies were found to be mitogenic for all donors, indicating that this heterogeneity is based on relative rather than absolute defects in low responder monocytes. Cell mixing experiments in which monocytes from two different low responder donors were co-cultured with T cells and IgG1 anti-T3 antibodies did not identify any complementary defects, suggesting that the low responder phenotype results from a relatively restricted polymorphism. To assess the nature of the signals required for inducing T cell proliferation, nonmitogenic anti-T3 antibodies were co-cultured with other pan-T cell antibodies having the IgG2a isotype. The combination of signals from T3 antigen cross-linkage and those independently generated by other IgG2a antibodies bound to monocyte Fc receptors did not induce T cell proliferation. Hence, it appears that the T3 antigen or closely associated structures must be clustered at the monocyte membrane for mitogenesis. Finally, in competitive inhibition experiments, the isotype specificity of monocyte Fc receptors involved in the induction of T cell proliferation was examined. Two distinct Fc receptor sites, one that binds murine IgG2a and IgG3 antibodies and a second that binds murine IgG1 antibodies, were identified. Murine IgM or IgG2b did not appear to bind either of these receptor sites. Taken together, these data indicate that human monocytes have two distinct Fc receptor sites, which must specifically and directly interact with T cell-bound anti-T3 antibodies for mitogenesis.     

Production of antibodies against rhodopsin after immunization with beta gamma-subunits of transducin: evidence for interaction of beta gamma-subunits of guanosine 5'-triphosphate binding proteins with receptor

The light-detecting system of retinal rod outer segments is regulated by a guanyl nucleotide binding (G) protein, transducin, which is composed of alpha-, beta-, and gamma-subunits. Transducin couples rhodopsin to the intracellular effector enzyme, a cGMP phosphodiesterase. The beta gamma complex (T beta gamma) is required for the alpha-subunit (T alpha) to interact effectively with the photon receptor rhodopsin. It is not clear, however, whether T beta gamma binds directly to rhodopsin or promotes T alpha binding to rhodopsin only by binding to T alpha. We have found that serum from rabbits immunized with T beta gamma contained a population of antibodies that were reactive against rhodopsin. These antibodies could be separated from T beta gamma antibodies by absorbing the latter on immobilized transducin. Binding of purified rhodopsin antibodies was inhibited by T beta gamma, suggesting that the rhodopsin antibodies and T beta gamma bound to the same site on rhodopsin. We propose that the rhodopsin antibodies act both as antiidiotypic antibodies against the idiotypic T beta gamma antibodies and as antibodies against rhodopsin. This hypothesis is consistent with the conclusion that T beta gamma interacts directly with the receptor. It is probable that in an analogous way, G beta gamma interacts directly with receptors of the adenylate cyclase system.