The generation of monoclonal anti-idiotype antibodies to human B cell-derived leukemias and lymphomas

We developed murine anti-idiotype monoclonal antibodies for each of four patients with B cell-derived leukemias and lymphomas. Idiotypic immunoglobulin was isolated from mouse X human tumor-cell hybridomas or from patients' serum and was used to immunize mice for the development of murine anti-idiotype monoclonal antibodies. Each patient's anti-idiotype antibodies demonstrated reactivity restricted to the immunizing immunoglobulin, thereby limiting their therapeutic utility to a single individual. In addition, we isolated isotype switch variants of hybridomas producing monoclonal anti-idiotypic antibody. The restricted specificity of these antibodies was found to be of value for the analysis of the extent of malignant B cell infiltration in a variety of tissues from several patients. Large populations of idiotype-bearing cells were detectable in biopsy specimens from patients K.T. and L.H. In contrast, although bone marrow specimens from patient G.D. were apparently devoid of morphologically abnormal cells, a small, highly fluorescent population of cells was demonstrable underscoring the potential utility of these antibodies for posttreatment evaluation as well as for therapy. In a fourth patient, H.M., anti-idiotype antibodies developed against the circulating macroglobulin isolated from his plasma failed to react with either his circulating or bone marrow hairy cell leukemia cells. However, examination of an enlarged inguinal lymph node revealed the presence of a large number of idiotype-bearing cells. Thus, the presence of two distinct malignant B cell clones were discovered in this individual through the use of anti-idiotype monoclonal antibodies. Anti-idiotype antibodies, therefore, represent a highly specific tool for the evaluation and potential therapy of B cell malignancies in individual patients.     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997     

Mapping the idiotopes of a monoclonal anti-myoglobin antibody with syngeneic monoclonal anti-idiotypic antibodies: detection of a common idiotope

A panel of syngeneic monoclonal anti-idiotypic antibodies was prepared by immunizing A.SW mice with keyhole limpet hemocyanin-coupled A.SW monoclonal anti-myoglobin (HAL 19, IgG1) and screening the cloned hybridomas for production of IgG2 binding to idiotype but not to certain other anti-myoglobin antibodies of the same subclass in an ELISA. With these antibodies, we identified three nonoverlapping idiotopes, based on three clusters of monoclonal anti-idiotopes that mutually inhibit within each cluster, but not between clusters (Cluster I: S2, S6, S8; Cluster II: S5, S7; Cluster III: S9). Only Cluster II antibodies block the binding of myoglobin to HAL 19 and so identify a binding site-related idiotope(s). Binding of both Cluster II monoclonals (S5 and S7) to Hal 19 is inhibited by a rabbit anti-idiotype that we previously reported detects a common cross-reactive anti-myoglobin idiotope in immune sera. However, only one of these, S7, and not S5, identifies an idiotope that is present on 20 to 30% of A.SW anti-myoglobin antibodies in immune sera and ascites. The panel of syngeneic monoclonal anti-idiotype antibodies also detects new idiotopes not detected by the rabbit anti-idiotype. The development of a panel of syngeneic monoclonal anti-idiotypic antibodies to different clusters of idiotopes on the same antibody molecule, including one that identifies a major common idiotope in immune sera, should allow the analysis of possible idiotype network regulation in vivo and in vitro in a completely syngeneic system.     

Anti-idiotypic antibodies in a patient with monoclonal rheumatoid factor after pneumococcal bacteremia

A 51-yr-old Japanese female patient with monoclonal IgM gammopathy with rheumatoid factor activity was admitted because of pneumococcal bacteremia. About 2 wk after admission, her rheumatoid factor activity became undetectable by RAHA test and radioimmunoassay, subsequent to the initial marked elevation. The suppressive capacity of the patient's IgG fraction on the rheumatoid activity of her monoclonal IgM on January 11 was determined. The IgG fraction obtained on February 22 blocked the binding of the rheumatoid factor to rabbit IgG. The suppressive activity in the IgG fraction of February 22 was shown to be localized within the F(ab')2 fragment. Furthermore, the specificity of the suppressive serum factor was shown by the inability to block the binding of SRBC coupled with diazotized phosphorylcholine to anti-pneumococcal antibody. Thus, the marked reduction of rheumatoid factor activity was considered to result from anti-idiotypic antibody transiently appearing in her serum after pneumococcal bacteremia.     

Idiotype vaccines against human T cell acute lymphoblastic leukemia. I. Generation and characterization of biologically active monoclonal anti-idiotopes

A murine monoclonal anti-tumor antibody termed SN2 (Ab1), isotype IgG1-kappa, that defines a unique human T cell leukemia-associated cell-surface glycoprotein, gp37 (m.w. 37,000), was used to generate monoclonal anti-idiotype antibodies (Ab2) in syngeneic BALB/c mice. The Ab2 were screened on the basis of their binding to the F(ab')2 fragments of SN2 and not to the F(ab')2 of pooled normal BALB/c mice sera IgG1 or to an unrelated BALB/c monoclonal antibody of the same isotype. Fifteen Ab2, obtained from two fusions, were specific for the SN2 idiotope and not against isotype or allotype determinants. To find out whether these Ab2 are directed against the paratope of SN2, the binding of radiolabeled SN2 to leukemic MOLT-4 and JM cells which contain gp37 as a surface constituent was studied in the presence of these anti-idiotopes. Clone 4EA2 inhibited the binding 100% at a concentration of 50 ng and 4DC6 inhibited 90% at a concentration of 250 ng. A third clone 4DD6 gave about 50% inhibition. Similar was the inhibition of SN2 binding to insolubilized MOLT-4 antigen or cell membrane preparation. The binding of SN2 (Ab1) to 4EA2 and 4DC6 was also inhibited by semipurified preparation of gp37 antigen. These results demonstrate that at least two of the anti-idiotope antibodies are binding either at or near the binding site idiotope of SN2. Next, the purified Ab2 was used to immunize syngeneic mice to induce antibody binding to MOLT-4 cells or gp37. Sera from mice immunized with 4EA2 and 4DC6 coupled to keyhole limpet hemocyanin contained antibodies which bind to semipurified gp37 antigen and MOLT-4 cells. Immune sera inhibited the binding of iodinated Ab2 and Ab1 indicating that an anti-anti-idiotopic antibody (Ab3) in mice shares idiotopes with Ab1 (SN2). Also, the binding of iodinated Ab2 to Ab1 was inhibited by rabbit antisera specific for gp37. Collectively, these data suggest that anti-idiotype antibodies 4EA2 and 4DC6 may be useful in the generation of idiotype vaccines against human T cell leukemia.     

Chicken developmental antigens: analysis of erythroid populations with monoclonal antibodies

Fusions were performed between the mouse PAI myeloma cell line and spleen cells from Balb/c mice immunized with intact erythrocytes from 1-day Cornell K-strain White Leghorn chickens. Following single cell cloning, four hybridoma clones were found to secrete erythroid specific monoclonal antibodies. Based on its pattern of reactivity, the antibody (IgG2a, kappa) secreted by clone 10C6 detects a specific avian oncodevelopmental antigen associated with the hematopoietic system: chicken fetal antigen (CFA). Two other clones, designated as 3F12 and 4C2, produced antibodies (IgM, kappa) that recognize another avian developmental antigen: chicken adult antigen (CAA). A fourth clone, 9F9, produced an antibody (IgM, kappa) that reacts with all peripheral erythrocytes from both Japanese quail and chicken regardless of age. Clone 10C6 antibody apparently detects an erythrocyte specific (ES) determinant of CFA associated with determinant #8 while antibodies of clones 3F12 and 4C2 recognize a chicken specific determinant of CAA. Analysis by complement mediated microcytotoxicity indicated that the epitopes detected by 10C6 vs 3F12 and 4C2 antibodies were expressed on erythrocytes in a reciprocal fashion during development. Furthermore, strain variations in the incidence of erythrocytes carrying these epitopes were observed. The usefulness of these monoclonal antibodies for the study of erythroid populations is discussed.     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

Mitogen-like monoclonal anti-actin antibodies

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.     

Idiotypic analysis of myeloma proteins: anti-DNA activity of monoclonal immunoglobulins bearing an SLE idiotype is more common in IgG than IgM antibodies

The anti-idiotype 3I which recognizes a determinant on kappa-chains of anti-DNA antibodies in SLE patients also recognizes a determinant on kappa-chains of 82/706 myeloma proteins tested. Twenty-nine of these 82 proteins bind to double-stranded DNA, including two monoclonal IgM, one monoclonal IgA, and 26 monoclonal IgG proteins. DNA binding is much more frequent in the IgG than in the IgM myeloma proteins (p less than 0.001), and is also associated with cationic antibody charge. Two-dimensional gel electrophoresis reveals markedly increased charge heterogeneity of both heavy and light chains of the monoclonal IgG as compared with the monoclonal IgM proteins. We postulate that the increased charge heterogeneity of the IgG-associated 3I-reactive kappa-light chains may reflect somatic mutation, and that DNA specificity within the 3I idiotype system arises by somatic mutation of germ-line genes found in normal individuals. DNA binding may be associated with those mutations that give rise to a cationic immunoglobulin charge.     

Secretions of anti-myelin-associated glycoprotein antibodies by B cells from patients with neuropathy and nonmalignant monoclonal gammopathy

Four patients with peripheral neuropathy and nonmalignant monoclonal gammopathy with anti-myelin-associated glycoprotein (MAG) antibodies were studied to determine whether secretion of anti-MAG IgM antibodies by B cells was autonomous, or whether the monoclonal B cells were responsive to T cells. Secretion of anti-MAG IgM by isolated B cells was stimulated by the addition of increasing numbers of pokeweed mitogen (PWM)-activated autologous OKT4+ helper T cells in all four patients. Secretion of anti-MAG IgM by peripheral blood lymphocytes was dependent on the ratio of OKT4+ T helper cells to OKT8+ T suppressor/cytotoxic cells. In three patients with an OKT4+ to OKT8+ T-cell ratio of 2:1, PWM activation stimulated secretion of anti-MAG IgM; in one patient with an OKT4+ to OKT8+ ratio of 1:2, activation by PWM suppressed anti-MAG IgM secretion. These studies suggest that the monoclonal B cells that secrete anti-MAG IgM are responsive to regulatory T cells.     

Detection of cross-reactive determinants shared by human monoclonal IgM reacting with myelin-associated glycoprotein

Monoclonal IgM from patients with peripheral neuropathy frequently have anti-myelin-associated glycoprotein (MAG) activity. We investigated the idiotypes of 10 monoclonal anti-MAG by using rabbit polyclonal antisera. Three groups of anti-idiotypic antisera could be distinguished. Four sera reacted only with the immunizing protein. Two sera reacted with a single other anti-MAG IgM in addition to the immunizing one. Immunoenzymatic studies showed that these two couples of anti-MAG IgM reacted identically with 100% cross-inhibition, indicating that the whole set of idiotypes identified by the rabbit antiserum was present on both IgM antibodies. The four other anti-idiotypic sera reacted with the homologous IgM, as well as with most of the other anti-MAG IgM. In contrast to the previous antisera the binding of these four sera to the homologous IgM could not be inhibited by other cross-reacting anti-MAG IgM. However, when a heterologous IgM was used for coating, these antisera with one exception showed complete cross-inhibition. The absence of inhibition by purified MAG of the patient of the anti-idiotypic antisera sera suggests that these antibodies are most likely to be directed against framework determinants rather than against combining site epitopes.     

Idiotypic analysis of a monoclonal anti-Sm antibody

Among murine models of autoimmunity, MRL mice are unique in their expression of antibodies to the nuclear antigen Sm. To assess genetic mechanisms in the control of this response, the idiotypes borne by a monoclonal anti-Sm antibody of MRL-Ipr/Ipr origin were investigated. Rabbit antisera were prepared against Y2, a hybridoma product with anti-Sm activity, and were rendered specific for idiotype by extensive absorption with normal globulins from BALB/c mice. In assays of idiotype by an inhibition ELISA, Y2 was shown to share idiotypes with Y12, another monoclonal anti-Sm derived from the same fusion as Y2; other monoclonal autoantibodies of MRL origin but different antigenic specificity failed to display idiotype activity in this assay. The presence of other anti-idiotypic specificities was revealed by absorption and elution of the anti-idiotype from an MRL globulin column; sera from both anti-Sm-positive and negative mice demonstrated these idiotypes. These results suggest that the predominant specificities detected by the anti-idiotype were unique to the monoclonal antibodies of the same animal, although there was also activity to idiotypes not related to anti-Sm binding molecules.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.     

Structural similarities among enzyme pterin binding sites as demonstrated by a monoclonal anti-idiotypic antibody

BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.     

Direct visualization of IgM antibodies bound to tissue antigens using a monoclonal anti-type III collagen IgM as a model system

A mouse monoclonal IgM antibody directed against human Type III collagen was utilized to immunolocalize Type III collagen by transmission and scanning electron microscopy without the use of an electron-dense conjugate. Because bound IgM can be directly visualized, primary or secondary antibody conjugates, such as ferritin, HRP, colloidal gold, etc., are unnecessary in this method. Immunolocalization to Type III collagen in the matrix of human skin and to fibrils formed in vitro using only IgM antibody reveals uninterrupted IgM binding which exactly matches the banding period of the collagen fibrils. In contrast, colloidal gold-conjugated secondary antibody complexes directed against primary IgM binding sites reveal less precise labeling. The data suggest that direct visualization of primary monoclonal IgM antibodies may be useful in a wide variety of highly specific ultrastructural immunolocalization studies without requiring the use of electron-dense conjugates.     

Analysis of the anti-alpha 1 leads to 3 dextran response with monoclonal anti-idiotype antibodies

The antibody response to alpha 1 leads to 3 dextran (DEX) in BALB/c mice consists of a family of closely related yet highly heterogeneous molecules. Although these antibodies have been previously characterized both idiotypically and structurally, detailed analysis of responding clones has not been possible using conventional anti-idiotype antibodies. Monoclonal syngeneic and allogeneic anti-idiotype antibodies (MAIDs) specific for anti-DEX antibodies were used in this study to dissect the serum antibody response to DEX in BALB/c mice. The constructed MAIDs showed considerable heterogeneity by isoelectric focusing and by their binding characteristics to a series of DEX specific myeloma and hybridoma proteins. The predominant heavy chain isotype of these MAIDs was gamma 1. These antibodies were used to identify individual idiotypic structures (IdI) on J558, or M104E as well as cross-reactive determinants common to both (IdX). Although both IdX and IdI MAIDs were obtained, IdI specific antibodies were obtained more frequently. BALB/c mice immunized with DEX produced antibodies expressing both IdI but in highly variable amounts. A large percentage of, but not all DEX specific antibody, could be accounted for by IdX bearing antibodies. Suppression of adult and neonatal mice by IdI specific MAIDs was effective with precise elimination of only those clones expressing IdI determinants leaving the total lambda bearing anti-DEX response intact. Suppression of adults and neonates by an IdX specific MAID resulted in a temporary and partial suppression of the total lambda bearing anti-DEX response along with total suppression of the IdX portion of the response. Unlike other systems these monoclonal antibodies produce only suppression, and under a variety of conditions enhancement of anti-DEX responses has not been observed.     

Specific and shared idiotypes found on hybridoma anti-DNA autoantibodies derived from rheumatoid arthritis and systemic lupus erythematosus patients

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.     

Production and characterisation of monoclonal anti-idiotype antibody to Vibrio anguillarum

Seven monoclonal anti-idiotype antibodies (mab2) were raised against mouse monoclonal antibody (mab1) 4A6. Identification of subclass showed that 1H5, 1D1, 2B12 and 2F12 belonged to IgG2b, 2H12 and 1H12 to IgG2a and lE10 to IgG3. The titres of these mab2 ascitic fluids ranged from 1 x 10(-4)-1 x 10(-6). The capacity of the mab2 to inhibit the binding between the corresponding rabbit antiserum and Vibrio anguillarum was investigated with the competitive inhibition ELISA. The results showed that mab2 1D1, 1E10, 1H5 and 1H12 were able to inhibit this binding. Another experiment demonstrated that mab2 1D1, 1E10 and 1H5 might induce Balb/c mice to produce Ab3 and these Ab3 competed the same antigen epitopes with Ab1. These results indicate that mab2 1D1, 1E10 and 1H5 are likely to represent an internal image of V. anguillarum and may thus be described as Ab2-beta anti-idiotype antibodies. In protection experiments, Japanese flounders vaccinated with mab21D1, 1E10 and 1H5 showed significantly enhanced survival from challenge with V. anguillarum. Thus. mab21D1, 1E10 and 1H5 may have use as idiotype vaccines for fish in aquaculture.