A common idiotype expressed on a murine anti-Sm monoclonal antibody and antibodies in SLE sera.

A rabbit anti-idiotypic antiserum made against a murine monoclonal anti-Sm autoantibody (Y2) was used in a solid-phase radioimmunoassay to investigate idiotypic cross-reactivity among anti-Sm antibodies present in sera from patients with systemic lupus erythematosus. Sera from 25 of 51 SLE patients (49%) containing anti-Sm antibodies were positive for this Y2 idiotype compared to only one of 22 normal human sera. Nine of 28 SLE patients (32%) whose sera were anti-Sm negative were also positive for the Y2 idiotype in low titre. Binding was not due to rheumatoid factor-like activity but was specific for the Y2 determinant and could be eliminated by absorption with Y2 monoclonal antibodies. The anti-idiotypic antibody blocked the ability of 12 of 25 anti-Sm positive lupus sera to bind Sm. Conversely, Sm antigen inhibited the binding of anti-idiotypic antibody in nine of 12 lupus sera.     

Human EBV-transformed lymphocytes of patients with Schistosoma japonicum infection secrete idiotypically related immunoregulatory antibodies.

Lymphocytes derived from the peripheral blood of individuals infected with Schistosoma japonica were transformed in vitro with Ebstein-Barr virus (EBV). Serological characterization of antibody molecules revealed both antigen reactive (idiotypic) and anti-idiotypic transformants. One idiotypic EBV transformant, LO2C2, describes a major cross-reactive idiotype associated with anti-antigen binding molecules. Other antibody populations expressing idiotypic cross-reactivity were derived from separate individuals showing shared idiotypy in S. japonicum field study populations in the Republic of Philippines. Both idiotypic and anti-idiotypic molecules suppressed parasite antigen-driven blastogenesis of heterologous human peripheral blood lymphocytes. The data show a serologically related immunoregulatory immune network in patients in the Republic of the Philippines which is serologically distinct from idiotypy expressed in other selected S. japonicum endemic areas in the Far East.     

Demonstration of idiotypes expressed on basophil-bound IgE antibodies by using anti-idiotype-induced histamine release in grass pollen-allergic patients.

This study was designed to analyse further the idiotypic cross-reactivity between anti-Lol p I murine monoclonal antibodies of IgG isotype and basophil-bound human IgE antibodies from grass pollen-sensitive patients. It was also designed to determine the expression frequency of the idiotypes present on cell-bound IgE. Rabbit anti-idiotypic antisera were produced against idiotypes of three anti-Lol p I monoclonal antibodies (290A-167, 539A-6 and 348A-6) of different specificities. Basophils from 19 patients reacting to Lol p I allergen, as shown by positive skin test reactions and by the presence of serum-specific IgE antibodies (measured by RAST), were challenged with these rabbit anti-idiotypic antibodies and the histamine released was measured. Our data indicate that IgE-borne idiotypes were expressed as follows: (i) co-expression of the three idiotypes in 15% of patients; (ii) co-expression of two idiotypes in 21% of patients; and (iii) expression of a unique idiotype (290A-167) in 42% of patients. Among the three idiotypes, 290A-167 was shown to be a public idiotype since it was expressed in 80% of patients. Fab fragments of anti-idiotypic antibodies could inhibit anti-idiotype-induced histamine release, but optimal conditions varied from one patient to another.     

Oligoclonal immunoglobulins in subacute sclerosing panencephalitis and multiple sclerosis: a study of idiotypic determinants.

Studies have been made of the idiotypic determinants of subacute sclerosing panencephalitis (SSPE) antibodies using rabbit antisera to serum and spinal fluid fractions.Evidence is presented indicating that serum and cerebrospinal fluid (CSF) anti-measles antibodies, as judged by their idiotypes, differ in their relative concentrations in the two compartments. The results indicate that some of these antibody subpopulations originate within the CNS, while others are made largely or entirely outside. In addition to strong idiotypic specificity, a limited cross-idiotypic specificity relating antibodies from three out of fourteen SSPE patients has been identified. In the course of these studies, measles virus was found to agglutinate red cells coated with antibody fraction to high titres. This system has proved useful in demonstrating the competition between anti-idiotypic antibody and antigen for the combining sites of the measles antibody. Two anti-idiotypic antisera have also been obtained against the spinal fluid IgG of multiple sclerosis (MS) patients. The possible use of these marker reagnets as well as related methodologies in the search for the antigens involved in MS bands is discussed.     

Modulation of immune response to Lol p I by pretreatment with anti-idiotypic antibody is not restricted to the idiotypic expression.

To study the role of anti-idiotypic antibodies in the regulation of the immune response to Lol p I (the major allergenic component of rye grass pollen), we have recently generated a panel of three MoAbs directed against distinct epitopes of Lolp I and an anti-idiotypic MoAb directed against the idiotype borne by one of the anti-Lol p I MoAbs (290A-167). The effects of pretreatment with this anti-idiotypic MoAb in BALB/c mice before immunization with the antigen have been examined. The anti-idiotypic MoAb or unrelated MoAb were given weekly for 8 weeks intraperitoneally. Mice then received the antigen (2 micrograms) adsorbed with alum (2 mg) at weeks 9, 11 and 13. Serum anti-Lol p I antibodies (IgG or IgE) and specific idiotypic responses were measured. Anti-Lol p I IgG antibodies could be detected before immunization with Lol p I only in mice pretreated with anti-idiotypic MoAb. Immunization with Lol p I induced an anti-Lol p I IgG response in both groups, but this response was higher in mice that received anti-idiotypic MoAb. Similar profiles were seen for specific IgE antibodies and idiotypic responses. Surprisingly, idiotypes borne by other anti-Lol p I MoAbs (539A-6 and 348A-6) had also been enhanced after pretreatment with the anti-290A-167 MoAb. These observations suggested that the pretreatment with this anti-idiotypic MoAb modulates not only the expression of the respective idiotype, but also affects other idiotype responses.     

Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5 antigen of porcine reproductive and respiratory syndrome virus

Syngeneic monoclonal anti-idiotypic antibodies (Mab2s) were generated against idiotypic antibodies to membrane glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) using the sequential immunization method. Six of 12 Mab2s possessed potential internal image characteristics by recognizing a common idiotype on murine and swine anti-GP5 antibodies. Further serological characterization demonstrated that one of the Mab2 (Mab2-5G2) represents internal image anti-idiotope which mimicked the GP5 antigen that inhibited the interaction between idiotypic anti-GP5 antibodies and GP5 antigen, its reaction with the idiotypic anti-GP5 antibody was inhibited by GP5 antigen and detected the common Id on anti-PRRSV antibodies from pigs that were experimentally infected with PRRSV. In addition, Mab2-5G2 identified a soluble protein on MA-104 and porcine alveolar macrophages. These results indicate that Mab2-5G2 may be a useful candidate as an alternative PRRSV serodiagnostic reagent and a useful probe to study PRRSV-cell interaction.     

Preparation and characterization of a monoclonal anti-T helper factor antibody.

Sprague-Dawley rats were immunized by injection of antigen B-specific T helper factor THF) eluted from Sepharose-antigen D adsorbents. Rat spleen cells from animals immunized with THF were fused with a BALB/c tumour cell (P3x63-Ag8.653) to prepare monoclonal anti-THF antibodies. The hybrids produced were screened for anti-THF antibodies by an enzyme-linked immunoassay (ELISA), and we shall describe the characteristics of one of the hybrid (hybridoma 6-2.2 anti-THF) antibodies produced. (i) The monoclonal hybrid 6-2.2 anti-THF antibody blocks water-soluble timothy extract-induced proliferation of timothy-specific T helper cells when these cells were preincubated with an excess of the anti-THF hybrid 6-2.2 antibody; (ii) incubation of timothy-specific T helper or T suppressor cells with an optimal dose of anti-THF 6-2.2 antibody induces significant levels of [3H]-thymidine incorporation in the absence of antigen; (iii) it binds specifically to the idiotypic determinant expressed on THFk, THFd, TSFk, and antigen B-specific IgE in an ELISA; and (iv) it has no effect upon spleen cells from mice primed with ovalbumin or Ascaris suum antigens. In addition, the monoclonal anti-THF 6-2.2 antibody cultured with normal spleen cells in mini-Marbrook chambers induced significant levels of antigen B-specific T suppressor cells. These studies indicate that the monoclonal anti-THF 6-2.2 antibody has anti-idiotypic antibody properties.     

A highly selected panel of anti-CD4 antibodies fails to induce anti-idiotypic antisera mediating human immunodeficiency virus neutralization.

Anti-CD4 antibodies directed to the N terminus of CD4 can inhibit human immunodeficiency virus (HIV) infection. Therefore, it has been proposed that some of these reagents may contain idiotypic determinants which conformationally model the binding site expressed on gp120. In this report, we have selected a panel of anti-CD4 monoclonal antibodies as idiotypic mimics of gp120 by employing cross-blocking techniques, and CD4 epitope mapping using site-directed mutagenesis. These studies suggest that only 4 out of the original panel of 12 would be expected to represent suitable candidates for modelling the gp120 binding site. Nevertheless, anti-idiotypic antisera raised against these antibodies failed to inhibit gp120 binding to CD4. This negative result may reflect the incomplete modelling of the virus binding site by anti-CD4, or the lack of internal image antibody in the anti-idiotypic preparations. Alternatively, the binding site on gp120 may not be accessible to antibody neutralization, excluding the possibility of an idiotypic vaccine to HIV based on anti-CD4 antibody as surrogate antigen.     

A study of idiotypic suppression in adult rabbits immunized with Salmonella abortus-equi.

It is possible to induce idiotypic suppression in adult rabbits immunized with Salmonella abortus-equi (S.a.e.). Ten months after priming we injected the rabbit with anti-idiotypic serum prepared against its own antibodies to S.a.e. and, 3 weeks later, gave it a booster injection of bacteria. A new anti-idiotypic serum was preparedwith the serum to S.a.e. collected after this boost and was used for the following isiotypic suppression attempt made 10 months after the first one. Using this procedurewe succeeded in two successive idiotypic suppression attempts in the same rabbit. Inthe three attempts we carried out, idiotypic suppression was totally effective, i.e. idiotypes detected by the serum used for the suppression totally disappeared after thesuppression, and the suppression lasted during the life of the rabbits (maximum 10 months). This observation is consistent with a suppression resulti-g from an interaction of anti-idiotypic antibodies with the complementary receptors at the surface of memory cells. This suppression was without effect on antibody to S.a.e. titre and on IgG concentration. Idiotypes detected by the anti-idiotypic serum prepared withthe serum to S.a.e. collected after the suppression. These idiotypes were different from those detected by the anti-idiotypic serum used for the suppression. This observation confirms that idiotypic recognition is confined to a limited number of clonal products, despite the fact that a very heterogeneous antibody population was used forthe anti-idiotypic immunization. Thus we did not observe the appearance of new idiotypes produced previously silent cell clones. All the different idiotypes we detected during the successive idiotypic suppression attempts carried determinants shich remained peculiar to each individual rabbit.     

Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach

We report a novel competitive type immunoassay for oestradiol based on the idiotypic anti-idiotypic approach. This has been achieved by the production of an anti-idiotypic antibody (anti-Id) which is directed against the oestradiol binding site of the primary idiotypic antibody (Ab1). In this format the primary Ab1 was captured onto the surface of microtitre wells and oestradiol standards or serum samples were then allowed to compete with europium labelled anti-Id for the binding sites of Ab1. Fluorescence was proportional to the concentration of oestradiol over the range 0-8 ng/ml. The sensitivity of the assay was 80 +/- 20 pg/ml, whilst the intra-assay variation ranged from 3 to 10%, and the inter-assay variation from 7.3 to 15%. The results obtained by the fluorescence immunoassay correlated well with those obtained by an extraction radioimmunoassay using tritiated antigen and dextran-coated charcoal for separation of bound and free ligand (n = 60, r = 0.98). The idiotypic anti-idiotypic approach in hapten immunoassays enables antibodies to be labelled instead of haptens, and thus permits the development of robust and sensitive immunoassays.     

Preparation and characterization of the anti-idiotypic properties of rabbit anti-timothy antigen B helper factor and anti-mouse timothy IgE antisera.

with AgB-primed B or T cells and injected into syngeneic X-irradiated recipients. Anti-THF and anti-Eid purified by an AgB-specific T suppressor factor (TSF) affi-gel adsorbent retain their ability to specifically initiate [3H]-thymidine incorporation of AgB-primed T cells. The data indicate that both anti-THF and anti-Eid recognize unique determinants present on AgB-specific T-helper, T-suppressor and B cells, and suggest that the receptors on AgB-specific T and B cells share cross-reactive idiotypic determinants.     

Characterization of the IgG antibody response to timothy grass pollen antigens

The IgG and IgE antibody responses against timothy grass pollen antigen B (AgB) in several mouse strains were determined. Considerable variability in both responses was demonstrated, and (CBA/J X C57BL/6)F1 mice were selected for use in subsequent experiments. Anti-idiotypic-antibody-induced T suppressor cells almost completely suppressed AgB-specific IgE, but the IgG response was not altered. Further studies with photooxidized AgB (Ox-AgB) indicated that the IgG response was directed against an antigenic determinant expressed on both native antigen and Ox-AgB. Our data indicates that AgB possesses two distinct antigenic determinants, one that induces an IgE response, and one that induces an IgG response.     

Generation of the Single Chain Antibody Fragment Conserves the Idiotypic Profile of the Anti-CD30 Monoclonal Antibody HRS3

Recombinant single chain antibody fragments (scFv) derived by combining immunoglobulin VL and VH regions provide valuable antibody-like reagents. A number of them are shown to have retained the antigen specificity of the parental monoclonal antibody (MoAb). Little is known about the idiotypic profile of scFv fragments compared with that of the parental MoAb. To address this question we analysed the idiotypic profile of a scFv that was derived by phage-display techniques from the anti-CD30 MoAb HRS3.We assayed (i) binding of HRS3-scFv to recombinant CD30-Fc antigen and to four different anti-idiotypic MoAbs defining at least three different idiotopes on HRS3, and (ii) cross-competition with the parental MoAb HRS3 and the closely related anti-CD30 MoAb HRS4. The assays revealed that the HRS3-scFv fragment exhibits the same specificity for both CD30 antigen and the tested anti-idiotypic MoAbs compared with the parental MoAb demonstrating that the recombinant scFv fragment has retained the complete idiotope of the parental MoAb.     

Maintenance of antigen-specific immunological memory through variable regions of heavy and light chains of anti-idiotypic antibody

Immunological memory is characterized by a quick and enhanced immune response after re-exposure to the same antigen. To explain the mechanism involved in generation and maintenance of immunological memory, we had earlier proposed a hypothesis involving the relay of memory by idiotypic and anti-idiotypic B cells. The peptidomimic present in the hypervariable region of anti-idiotypic antibody was hypothesized to carry forward immunological memory. In the present work, we provide evidence supporting a role for the anti-idiotypic antibody in eliciting antigen-specific B-cell and T-cell responses. Employing the idiotypic monoclonal antibody (Ab(1)) specific for haemagglutinin (H) protein of rinderpest virus, Ab(2beta) was generated, which possesses an internal image of the H protein in the region between amino acids 527 and 556. We demonstrate that antigen-specific memory is perpetuated by immunization with Ab(2), as shown by maintenance of antigen-specific T-cell responses upon restimulation in vitro of Ab(2) immune splenocytes by antigen-presenting cells expressing H protein or pulsed with H-protein-derived peptides. We have also shown that boosting with antigen-specific anti-idiotypic B cells generates a memory response in antigen-primed mice. Evidence has been provided for the existence of an antigen-specific B-cell idiotypic network in the body that supports the perpetuation of immunological memory as proposed in the relay hypothesis.     

A human monoclonal IgG reactive with a private idiotope of a monoclonal IgM with autoantibody activity against myelin-associated glycoprotein.

One hundred and thirteen sera from patients with monoclonal IgG were tested for reactivity against a panel of 13 human monoclonal IgM having various autoantibody activities: 6 to myelin-associated glycoprotein (MAG), 2 to vimentin intermediate filament protein and 5 to red blood cell antigens [cold agglutinins with specificity directed to I antigen (3 cases), i antigen (1 case) or Pr antigen (1 case)]. One IgG was found to react with a monoclonal IgM with anti-MAG activity. This reactivity was characterized as idiotypic and directed against a private idiotope of the monoclonal IgM. This work provides further evidence for the existence of anti-idiotypic antibody activity of monoclonal Ig occurring in human B cell neoplasias.     

Anti-Idiotypic Antibodies against the Receptor Antibodies in Myasthenia Gravis

Anti-idiotypic antibodies were prepared against purified acetylcholine receptor antibodies from two patients with myasthenia gravis. The purified idiotypes did not crow-react. Reaction with idiotypic from other patients were found in 8% and 37%, respectively, which suggest that shared idiotypic specificities occur. The anti-idiotypic gm fractions had no receptor-like activity and did not bind cholinergic ligands. Receptor antibodies from two mothers and their newborn children with neonatel myasthenia gravis showed marked differences in the reactions with an anti-idiotypic antibody. This suggests that not passive transfer of maternal antibodies but a transient synthesis of a receptor antibody with a different specificity is an important factor in the pathogenesis of neonatal myasthenia gravis.     

Recombinant idiotypic TCRbeta chain immunization in mice generates antigen specific T cell response

Vaccination remains the most cost-effective means of preventing infectious diseases. Success of vaccination depends on generation of effective memory response. Understanding the mechanism of generation and maintenance of immunological memory would help in the design of rational vaccines. T lymphocytes play a central role in the generation of protective immune response against many microbial infections. A hypothesis known as relay hypothesis was earlier proposed, which explains the maintenance of immunological memory through interaction of idiotypic and anti-idiotypic lymphocytes. In the present study, we have shown that immunization with a model antigen, chicken ovalbumin specific T cell receptor beta chain (idiotypic TCR) generates TCR specific antibody and anti-idiotypic T cell responses as well as ovalbumin specific T cell response. We further show that boosting of ovalbumin primed mice with ovalbumin specific idiotypic TCRbeta DNA or TCRbeta protein gives memory response for ovalbumin. This study provides experimental evidence for perpetuation of immunological memory through idiotypic network interactions.     

Production and isolation of anti-idiotypic B cells

Summary Techniques are described for the production of anti-idiotypic B cells by immunization with idiotypic antibodies complexed to rheumatoid factor. Screening for anti-idiotypic antibody production and immunoaffinity isolation of the B cells, actively producing the anti-idiotype antibody, by idiotypic antibody-coated magnetic beads is described. This methodology can be used to isolate anti-idiotypic B cells for immune regulation studies or to provide fusion partners for the production of monoclonal anti-idiotypic antibodies.     

Terasaki-ELISA for murine IgE-antibodies. I. Quality of the detecting antibody: production and specificity testing of antisera specific for IgE

In order to develop an ELISA for the quantitative determination of murine IgE, antisera specific for murine IgE were prepared in the goat and rabbit. As immunogen, monoclonal IgE antibody mixtures of several allotypically different hybridomas were used. Before use, these antibodies were purified employing procedures that allow maximum recovery of binding activity. The goat and rabbit mouse epsilon chain-specific antisera were adsorbed on normal mouse serum. The purified antisera were found to be free of allotypic activity. However, immunoadsorption on NMS could not always remove contaminating anti-idiotypic antibodies. Repeated adsorptions with monoclonal antibodies of different isotypes carrying a similar idiotype were necessary to remove all detectable anti-idiotypic activity. Only after these precautions were the antisera suitable for detecting IgE molecules on nitrocellulose blots as well as for quantitating circulating IgE antibodies (ELISA) and IgE-secreting cells (plaque assay and reverse ELISA plaque assay). The purity and reactivity of several commercially available anti-IgE preparations were tested in similar types of specificity assays. Since the specificity of antibodies used in ELISA determines the monospecificity of the assay, retesting for contaminating cross-reactivities in commercial preparations was shown to be necessary.     

Sharing of idiotypic specificities between different antibody populations from an individual rabbit.

Rabbits hyperimmunized with tobacco mosaic virus synthesize very heterogeneous antibodies. Despite this, specific anti-idiotypic sera recognizing a large part (70%) of these antibodies can be raised in rabbits matched for allotypic specificities a1, a2, a3, b4, b5, b6, c7, and b9. Different rabbits synthesize antibodies with different idiotypic specificities. However, in the serum of a single rabbit antibody fractions of different isoelectric pH share some idiotypic specificities. The results show that, at least in certain cases, antibodies against one antigen are not simply a random collection of immunoglobulin which happen to fit with this antigen, but that some definite relationship exists between the products of different clones which have been activated by antigen. These findings are discussed in the light of network concepts of the immune system.