鼻咽癌抗独特型单克隆抗体的制备和鉴定

用针对鼻咽癌癌细胞相关抗原的单克隆抗体Ｆｃ１（Ａｂ１）作免疫原，采用常规免疫和杂交瘤制备方法，获得了一株抗Ｆｃ１Ｖ区独特型的杂交瘤：２Ａ９。双夹心ＥＬＩＳＡ显示２Ａ９所分泌的抗体（Ａｂ２）对Ｆｃ１有效强的亲和力。ＥＬＩＳＡ结合抑制试验结果显示，Ａｂ２能抑制Ｆｃ１对鼻咽癌细胞ＣＮＥ１的反应。这就证明Ａｂ２作用于Ｆｃ１抗体的Ｖ区。用Ａｂ２与钥孔戚血蓝素（ＫＬＨ）交联物免疫小鼠产生的抗－抗独特型抗体（Ａｂ３）的血清，能与Ｆｃ１竞争ＣＮＥ１细胞株上的原始靶抗原。免疫组化证实，Ａｂ３与Ｆｃ１对鼻咽癌细胞有相同的反应，均为细胞膜染色。可以看出Ａｂ３与Ａｂ１（Ｆｃ１）具有相同的配位。以上结果表明：２Ａ９杂交瘤细胞所分泌的抗独特型抗体Ａｂ２是带有鼻咽癌相关抗原内影像的单克隆抗体。     

The immune response to anti-idiotype antibodies bearing an internal image epitope of tetanus toxin/toxoid. II. Comparison of the primary humoral immune response to xenogeneic Ab2 beta 1 and Ab2 beta 2 internal image anti-idiotype antibodies.

Mouse monoclonal (Ab1) anti-tetanus toxin/toxoid antibodies were used to raise Ab2 beta (tetanus toxin/toxoid internal image bearing) anti-idiotype antibodies in rabbits. Those rabbit serum antibodies (Ab2 beta) that did not bind to mouse serum proteins on an affinity column gave rise to an Ab3 anti-tetanus toxin/toxoid antibody response in mice. Rabbit serum antibodies that did bind to the affinity column, when eluted and used to inoculate mice also gave rise to an Ab3 anti-tetanus toxin/toxoid antibody response. It is suggested that one population of rabbit Ab2 beta anti-idiotype antibodies (unbound fraction) bears a partial or complete internal image of a tetanus epitope (Ab2 beta 1) while others (bound fraction) bear a complete or partial mirror image of a mouse immunoglobulin epitope as well (Ab2 beta 2).     

Analysis of anti-idiotypic antibodies against anti-microsomal antibodies in patients with thyroid autoimmunity.

Anti-microsomal antibody (AMA) activity was inhibited in 14 of 16 sera and in all 12 IgG preparations from patients with postpartum thyroiditis following incubation with F(ab')2 fragments from normal polyspecific immunoglobulin for therapeutic use (ivIg). Similar results were observed with sera from seven of seven patients with Graves' disease and five of six patients with autoimmune hypothyroidism. Results of these competitive binding assays and affinity chromatography of AMA IgG on Sepharose-bound F(ab'), fragments from ivIg indicated that AMA antibodies reacted with ivIg through idiotypic-anti-idiotypic interactions. Eight out of 10 IgG preparations from patients with autoimmune thyroid disease also showed inhibition of AMA activity when coincubated with autologous IgM at various IgG:IgM molar ratios. These observations suggest that ivIg can inhibit anti-microsomal antibodies through idiotype-anti-idiotype interactions and that such interactions occur with IgM anti-idiotype antibodies in vivo, providing evidence of a role for idiotypic network regulation in the control of thyroid autoimmunity.     

Production and characterization of monoclonal anti-CD18 anti-idiotype antibodies

Three leukocyte adhesion receptors have been described which mediate intercellular binding of leukocytes: LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and p150/95 (CD11c/CD18). We have previously reported the production of several monoclonal antibodies against the common subunit of these receptors (CD18). We here describe the production of monoclonal anti-idiotype antibodies against one of the anti-CD 18 antibodies (H52) which has been shown to inhibit potently the function of leukocyte adhesion receptors. Three IgG1 and two IgM anti-idiotype antibodies were derived which recognized private idiotopes on the H52 molecule. Two of these antibodies blocked the binding of H52 to purified LFA-1 and to cell surface expressed antigen. One of the antibodies (AIM.6) was shown to be an internal image-type (Ab2β) antibody based on inhibition of its binding to H52 by purified LFA-1 and by its ability to induce Ab3 which recognize LFA-1 when used as immunogen. The AIM.6 Ab2β antibody was tested for recognition of leukocyte adhesion ligands in LFA-1-mediated leukocyte adhesion and activation assays. The AIM.6 antibody did not block intercellular adhesion of leukocytes or mitogen stimulation of T cells, functions which were completely inhibited by low concns of H52. AIM.6 Ab2β antibody bound to H52 very well at 0°C but bound very poorly or not at all at 37°C. Binding studies on a panel of anti-CD18 monoclonal antibodies showed that the idiotope defined by AIM.6 was unique to H52 and an antibody recognizing the same epitope on CD 18 (H5B9). This result showed that inhibitory anti-CD 18 monoclonal antibodies utilize at least two distinct paratopes in binding to CD18. The above results are in contrast to those obtained in other systems in which Ab2β antibodies against receptor-specific Ab1 antibodies recognize receptor ligands and are discussed in the context of ligand recognition by leukocyte adhesion receptors.     

Various expressions of a unique anti-human thyroglobulin antibody repertoire in normal state and autoimmune disease

Polyclonal anti-human thyroglobulin (hTgb) antibodies (Ab) were purified from sera of rabbits immunized with human thyroglobulin, normal humans and patients suffering from Graves' disease, Hashimoto's thyroiditis and thyroid carcinoma. The avidity of the various Ab preparations for hTgb ranged from 0.3 X 10(10) -2.2 X 10(10) M-1. By using well characterized mouse monoclonal antibodies (mAb) directed against hTgb, it was shown that the fine specificities of induced anti-hTgb Ab in rabbits, natural Ab in normal subjects and autoantibodies in diseased patients were similar; however, they differed from that of rabbit anti-bovine and anti-porcine thyroglobulin Ab which were able to inhibit the hTgb binding of only a few of the mAb. Anti-hTgb in rabbits and in patients with thyroid carcinoma varied from those in normal subjects only by uniformly elevated serum titers. In contrast, patients with Graves' disease and Hashimoto's thyroiditis showed an increased concentration essentially restricted to Ab reacting with few of the antigenic determinants recognized by the mAb. Our data suggest that the repertoire of anti-hTgb Ab is similar in mouse, rabbit and human. Furthermore, the finding of identical fine specificities for anti-hTgb Ab in normal and pathological conditions implies that autoantibodies are produced in normal subjects and held to a low level by regulatory processes which fail with respect to selected epitopes in autoimmune diseases.     

Specific detection of antibodies in cancer patients following immunotherapy with anti-idiotype

Assays were compared for specificity and sensitivity in detecting in cancer patients' sera antibodies (Ab) raised during the course of immunotherapy with goat anti-idiotypic antibodies (Ab2) bearing the internal image of a colon carcinoma-associated antigen defined by monoclonal antibody (MAb) CO17-1A (Ab1). The human Ab were tested for binding to tumor cells, isolated tumor antigen (Ag), and Ab2, and for the capacity to inhibit binding of Ab2 to Ab1. Chimeric (human/mouse) MAb CO17-1A was used as a positive control in all assays. Of the four different cell binding assays used, the mixed hemadsorption assay (MHA) showed the highest specificity and sensitivity. For detection of Ag-binding human Ab, the enzyme-linked immunosorbent assay (ELISA) with Ag as target and peroxidase (PO)-labeled anti-human IgG antibodies as tracer for detection of human Ab binding to the target, showed higher specificity and sensitivity as compared to radioimmunoassay (RIA). For detection of human Ab binding specifically to Ab2, three different ELISAs and three RIAs were used. Best results were obtained in the ELISA with anti-human IgG antibodies as target and biotinylated Ab2 as tracer for detection of human Ab binding to the target. Of four different inhibition assays used, the ELISA which measures inhibition of binding of biotinylated Ab2 to Ab1 by human Ab or chimeric antibody at 37 degrees C was the most sensitive and specific. These assays have general applicability for the characterization of human Ab responses in Ab2 vaccination approaches to various tumors and pathogens and therefore provide the basis for the establishment of a correlation between Ab responses and clinical outcome of the disease.     

Protection of mice against the lethal toxicity of a lipopolysaccharide (LPS) by immunization with anti-idiotype antibody to a monoclonal antibody to lipid A from Eikenella corrodens LPS.

We produced anti-idiotype antibodies to antibody to lipid A from Eikenella corrodens. The ALA-1 monoclonal antibody (immunoglobulin M [IgM] isotype), which had already been produced in our laboratory (T. Kato, I. Takazoe, and K. Okuda, Infect. Immun. 57:656-659, 1989), had reacted strongly with lipid A from E. corrodens, Escherichia coli, and Salmonella minnesota. Four anti-idiotype monoclonal antibodies to ALA-1 (Ab1), designated A2LA-1 (IgG1 isotype), A2LA-2 (IgG2a isotype), A2LA-3 (IgG2a isotype), and A2LA-4 (IgG3 isotype), which recognized the idiotype Ab1, were produced. A2LA-1, A2LA-2, and A2LA-3 were capable of over 61% inhibition of ALA-1 reactivity to E. coli J5 lipid A in an enzyme-linked immunosorbent assay system. The sera of mice and rabbits immunized with the anti-idiotype antibodies revealed that the internal image anti-idiotype antibody induced the production of IgG antibodies that cross-reacted with or bound to lipid A. These studies indicate that A2LA-1 and A2LA-2 contained an antigenic epitope that mimicked lipid A. Immunization of mice with A2LA-1 resulted in prevention of lethal toxicity from E. coli J5 lipopolysaccharide.     

Studies on thyroid cell surface antigens using cultured human thyroid cells.

Human thyroid cells in primary culture were used for studies of thyroid cell surface antibodies in patients with thyroid autoimmune disorders. Radioiodinated IgG preparations containing thyroid microsomal antibody (TMAb), thyroid stimulating antibody (TSAb) and/or thyroglobulin antibody (TgAb) were tested for binding to thyroid cells. Binding was observed with radioiodinated IgG from patients with Graves' disease, Hashimoto's thyroiditis and idiopathic myxoedema containing TMAb, irrespective of the presence of TSAb and TgAb, while negative results were obtained with normal IgG. A dose-dependent inhibition of binding to thyroid cells was produced by the addition of the corresponding unlabelled IgG preparations. Evidence for tissue specificity was provided by the absence of binding to human skin fibroblasts used as controls. Preabsorption with human thyroid microsomes completely abolished the binding to thyroid cells of a radioiodinated TMAb positive IgG preparation, while only incomplete removal of the reactivity to thyroid microsomes was produced by preabsorption with thyroid cells. These data suggest that some but not all microsomal antigenic determinants are expressed on the thyroid cell surface. Binding to thyroid cells was also observed with purified TgAb, indicating that thyroglobulin antigenic determinants are present on the surface of thyroid cells. No evidence of binding was obtained with a TSAb positive Graves' IgG preparation with undetectable TMAb and TgAb. Unlabelled IgG preparations containing TMAb from patients with either Hashimoto's thyroiditis or idiopathic myxoedema were shown to inhibit the binding to thyroid cells of radioiodinated TMAb positive Graves' IgG and vice versa. These data indicate that antibodies present in these thyroid autoimmune disorders share common thyroid cell surface antigens. However, the binding of radioiodinated IgG from a patient with idiopathic myxoedema was only partially inhibited by Graves' or Hashimoto's IgG, suggesting that some of the thyroid cell surface antibodies of idiopathic myxoedema may not be detectable in other thyroid autoimmune disorders.     

Anti-idiotypic immunization provides protection against lethal endotoxaemia in BALB/c mice.

Against lipid A (the conserved moiety of lipopolysaccharides from Gram-negative bacteria) neutralizing IgM monoclonal antibodies (mAb) 8-2 and 26-20 anti-idiotypic (Ab2) mAb were produced: Ab2 mAb KM-04 (IgG1) against mAb 8-2, and Ab2 mAb PW-1 (IgG2a) and PW-2 (IgG1) against mAb 26-20. The binding of Ab2 mAb KM-04 to 8-2 (Ab1) was strongly inhibited by a lipopolysaccharide (LPS) extract from either Salmonella minnesota R595 (Re LPS) or Escherichia coli J5 (Rc LPS), whereas the binding of Ab2 mAb PW-1 and PW-2 to 26-20 (Ab1) was only marginally inhibited by both Re LPS and Rc LPS. The results indicated that Ab2 mAb KM-04 recognizes a lipid A-binding site related idiotope on mAb 8-2 and therefore KM-04 might bear the internal image of a neutralization determining epitope of lipid A. Consequently Ab2 KM-04 might induce antibodies to lipid A. Indeed anti-idiotypic immunization of syngeneic BALB/c mice with Ab2 mAb KM-04 resulted in development of lipid A-binding anti-anti-idiotypic (Ab3) antibodies in serum. Similar immunizations with Ab2 mAb PW-1 and PW-2 were unsuccessful. However, induction of lipid A-binding Ab3 by mAb KM-04 proved to be genetically restricted to BALB/c mice. DBA/2 mice, Swiss mice and rabbits did not develop lipid A-binding antibodies upon immunization with mAb KM-04. In protection experiments, it was shown that BALB/c mice vaccinated with mAb KM-04 showed significantly enhanced survival from challenge with either rough (Re) LPS from Salmonella minnesota or smooth LPS from E. coli 0111:B4 when compared to BALB/c mice immunized with a non-relevant Ab2 mAb. The results suggest that mAb KM-04 constitutes a non-internal image vaccine to the lethal effect of lipid A in BALB/c mice. Furthermore an Ab3 mAb was prepared against Ab2 mAb KM-04 that showed reactivity with Re LPS. This Ab3 mAb, designated LE-21 (IgG2a) protected mice against an otherwise lethal challenge of Re LPS.     

Vaccination with syngeneic monoclonal anti-idiotype protects against a tumour challenge.

Rat X rat hybridomas secreting monoclonal anti-idiotypic antibodies have been prepared from Hooded rats immunized with two tumour-reactive, syngeneic monoclonal antibodies 11/160 and M10/76 (specific, respectively, for the Hooded rat sarcomata HSN and MC24). The hybridomas were selected on the basis that the secreted antibodies competed with antigen for binding to the immunizing idiotype. One monoclonal anti-idiotype (HIM/1/230, gamma 2a isotype) that recognizes an antigen-binding site idiotope of antibody 11/160 has been found to substitute for antigen. Hooded rats vaccinated by three challenges with HIM/1/230 produce serum Ab3 that is indistinguishable in antigen specificity from the 11/160 Ab1, and show reduced tumour take following an i.v. challenge with 10(6) HSN cells. The response to vaccination with anti-idiotype was both qualitatively and quantitatively dependent on the mode of immunization. High titre 11/160-like Ab3 was generated only when the vaccine contained Freund's adjuvant, whereas resistance to tumour challenge was found only in animals vaccinated with anti-idiotype in the absence of adjuvant.     

带有HBsAg表位内影像的单克隆抗独特型抗体的建立和鉴定

本文采用鼠单克隆抗-HBs(Ab_1)作抗原免疫同系鼠,取免疫鼠的脾细胞与SP_2/0骨髓瘤细胞融合,经过反复筛选、克隆化获得两株(B_3,C_2)持续分泌单克隆的抗独特型抗体其(Ab_2)。对其中之一B_3作了深入鉴定。B_3的Ig类型为IgG,亚类为IgG_2a。核型分析结果染色体数为106条。通过ELISA抑制试验对B_3进行鉴定,发现B_3一方面能与纯化HBsAg发生阳性的竞争性抑制,另一方面又能与不同来源的抗-HBs发生结合,出现阳性的中和抑制。无论竞争性抑制还是中和抑制,都呈现剂量依赖关系。将B_3Ig免疫同系鼠,经一次免疫后就能诱导出具有抗-HBs活性的抗-抗-独特型抗体(Ab_3)。以上结果表明:B_3株杂交瘤细胞所分泌的单克隆抗独特型抗体是带有HBsAg表位内影像的单克隆抗体。并对B_3Ig的可能应用意义进行了讨论。     

Comparative study on IgG and IgA antibodies against human thyroid and eye-muscle antigens in Graves' ophthalmopathy.

Circulating IgG and IgA anti-thyroid and anti-eye muscle antibodies were investigated in 87 patients with Graves' disease (60 cases with ophthalmopathy). The ELISA method was used. Both IgG and IgA antibodies were demonstrated against human thyroid and eye-muscle membrane or cytosol antigens. Anti-eye-muscle antibodies of the IgA type were observed more frequently than those of the IgG type (25 cases vs. 18 were demonstrated with membrane antigens and 37 cases vs. 23 with cytosol antigens). The respective distributions for thyroid antigens the cytosol fraction were 55 cases vs. 13 and 18 cases vs. 36. A significant difference was observed in the anti-thyroid IgG levels and the anti-eye-muscle membrane or cytosol levels between the patients with Graves' disease and those in control group (P less than 0.001). The difference in the IgA antibody to thyroid and eye-muscle antigens was significant between the patients with and without ophthalmopathy (P less than 0.002). The strong correlation between the levels of IgA antibodies to thyroid and those to the eye-muscle cytosol fractions might be connected with the theory of the common aetiology of the thyroid and eye diseases in Graves' ophthalmopathy (P less than 0.001). Circulating IgA anti-human thyroid and eye-muscle antibodies seemed to have a diagnostic relevance in the development of ophthalmopathy in Graves' ophthalmopathy.     

The production and characterisation of a chimaeric human IgE antibody, recognising the major mite allergen Der p 1, and its chimaeric human IgG1 anti-idiotype.

BACKGROUND: Two mouse monoclonal antibodies have been described, namely: mAb 2C7 (IgG2bkappa), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1kappa), which is an anti-idiotypic antibody raised against mAb 2C7. Given its broad IgE specificity, anti-idiotype mAb 2G10 could potentially have immunomodulatory applications. For example, a chimaeric human IgG version of mAb 2G10 could prove to be a useful molecule for binding to mast cell and basophil FcepsilonRI bound IgE, and in doing so co-ligating FcepsilonRI with FcgammaRIIB, which has been reported to have downregulatory effects. AIMS: To produce a chimaeric human IgE version of mAb 2C7 (mAb 2C7huE) and a chimaeric human IgG1 version of its anti-idiotype mAb 2G10 (mAb 2G10huG1). METHODS: The Vkappa and VH regions of mAb 2C7 and its anti-idiotype mAb 2G10 were engineered into human constant regions of the IgE and IgG1 isotypes, respectively. RESULTS: The production of chimaeric mAb 2C7huE and its anti-idiotype mAb 2G10huG1 confirmed that the respective mouse antibody V regions were successfully engineered into human constant regions and still retained the specificity of the original murine V regions. CONCLUSION: The newly constructed chimaeric antibodies will be useful to investigate the downregulation of IgE mediated hypersensitivity by the crosslinking of FcepsilonRI with FcgammaRIIB.     

The common idiotypic specificity of anti-a2 allotype antibodies: contribution of H and L chains to idiotype expression

Previously an idiotypic specificity common to anti-a2 allotype Ab from 15 rabbits was detected and characterized. The contribution of H and L chains to the expression of this common idiotypic specificity was determined. Two types of recombinant IgG were prepared: (1) H chains from anti-a2 Ab with L chains from normal a3b4 IgG. and (2) L chains from anti-a2 Ab with H chains from normal a3b4 IgG. By inhibition of binding radioimmunoassay, recombinant IgG having either H or L chains from anti-a2 did not inhibit the binding between anti-a2 Ab and anti-idiotype Ab. By direct binding radioimmunoassay, recombinant IgG of H chains from anti-a2 Ab with L chains from a3b4 IgG reacted with anti-idiotype Ab whereas recombinant IgG with H chains from normal a3b4 IgG and L chains from anti-a2 Ab did not react. These results indicate that the common idiotypic specificity of anti-a2 Ab is determined primarily by the H chain; however, the full expression of the common idiotypic specificity requires the interaction of both H and L chains from anti-a2 Ab.     

The immune response to anti-idiotype antibodies bearing an internal image epitope of tetanus toxin/toxoid. I. Induction of the humoral immune response

Primary immune responses to tetanus toxoid (TT) and primary and secondary immune responses to a rabbit TT internal image bearing anti-idiotype antibody (Ab2 beta 1) inoculated in Freund's complete adjuvant (FCA), saline (SAL) or syntex adjuvant formulation vehicle (SAF) via the intraperitoneal or subcutaneous route, were examined in mice. High anti-TT antibody (Ab3) titres are reported although the titre and persistence of the antibody response varied according to the adjuvant used in the priming and challenge inocula of Ab2 beta 1. Mouse Ab3 antibodies were elicited in mice inoculated with rabbit Ab2 beta 1 antibodies which in turn were elicited by an inoculum of mouse monoclonal anti-TT Ab1 antibody. Ab3 was shown to be identical to Ab1 by immunoblot analysis. Primary and secondary immune responses elicited by rabbit Ab2 beta 1 antibody protected mice against a lethal dose of tetanus toxin.     

Development and characterization of an anti-idiotype antibody to the capsular polysaccharide of Neisseria meningitidis serogroup C.

A monoclonal anti-idiotypic antibody (Ab2) whose antibody combining site contained a surrogate image of the meningococcal group C capsular polysaccharide was developed. To accomplish this, a monoclonal antibody against the group C capsular polysaccharide was developed by the fusion of splenocytes from mice immunized with Neisseria meningitidis group C strain MP13 with Sp2/0-Ag14 plasmacytoma cells. Monoclonal antibody 1E4, an immunoglobulin M isotype, demonstrated binding to the serogroup C polysaccharide in enzyme-linked immunosorbent assay (ELISA). Monoclonal antibody 1E4 reacted with 30 of 30 group C strains and 1 of 36 group B strains in immunodot assay, slide agglutination, inhibition ELISA, and bactericidal assay. This monoclonal antibody was selected as idiotype (Ab1) for the development of hybridomas producing an anti-idiotype antibody. One of the hybridomas developed, designated 6F9, was capable of over 70% inhibition of 1E4 in binding in the meningococcal C polysaccharide-specific ELISA. Studies with convalescent human serum demonstrated 100% inhibition of a serogroup C-specific ELISA with 200 micrograms of 6F9 per ml and 50% inhibition of this ELISA was achieved with 50 micrograms of 6F9 per ml. Monoclonal anti-idiotype antibodies (Ab3) with specificities similar to Ab1, 1E4 were generated from BALB/c mice immunized with the Ab2 (6F9). Immunization of rabbits with 6F9 resulted in an immunoglobulin G response which was significantly greater than that of control to a titer of 1:160. These studies indicate that monoclonal 6F9 contained a surrogate image on the combining antibody site which mimicked meningococcal C polysaccharide. This surrogate image is capable of evoking antibodies to the meningococcal C polysaccharide in syngenic and xenogenic species.     

An anti-idiotype antibody which mimics the inner-core region of lipopolysaccharide protects mice against a lethal challenge with endotoxin.

Recently, we described the generation and characterization of an Armenian hamster Ab2 beta anti-idiotype monoclonal antibody (MAb4G2) specific for the binding site of a mouse monoclonal antibody, MAbY1-4A6, directed against the conserved 2-keto-3-deoxyoctulosonate (Kdo)-containing inner-core region of lipopolysaccharide (LPS) (S. K. Field, M. Pollack, and D. C. Morrison, Microb. Pathog. 15:103-120, 1993). In that study, mice and hamster immunized with MAb4G2 generated serum immunoglobulin G and M (IgG and IgM) antibodies which cross-react with Salmonella minnesota R595-chemotype rough mutant LPS (Re-LPS). In this report, we demonstrate that in C3Heb/FeJ mice, MAb4G2 elicits an immune response which is characterized by specific binding of antibody to Re-LPS, as assessed by enzyme-linked immunosorbent assay. The practical use of MAb4G2 as a potentially effective therapeutic agent against gram-negative bacterial sepsis is suggested by the demonstration that immunization of these mice with MAb4G2 results in significant protection of D-galactosamine-sensitized animals against an otherwise lethal dose of Re-LPS. Assessment of the temporal changes in Re-LPS-specific serum antibody titers from mice immunized with MAb4G2 or Re-LPS over a 40-day period indicates that immunization with Re-LPS elicits significantly higher titers of serum IgM antibodies compared with those in animals immunized with MAb4G2. Conversely, two immunizations with MAb4G2 result in an up to 10-fold increase in anti-Re-LPS-specific IgG serum antibody titers relative to those obtained in mice immunized with Re-LPS. Nineteen days after the secondary boost with MAb4G2, anti-Re-LPS-specific IgG serum antibody titers were significantly higher (three- to fourfold) compared with those in Re-LPS-treated animals. Initial immunization with the anti-idiotype antibody primes animals for enhanced secondary responses to Re-LPS, as assessed by the titers of anti-Re-LPS-specific IgG profiles. These data suggest the potential utility of MAb4G2 as a candidate vaccine against the lethal properties of gram-negative bacterial LPS.     

IgA class and subclass thyroid auto-antibodies in Graves' disease and Hashimoto's thyroiditis

The reported prevalence of IgA class thyroid antibodies in Hashimoto's thyroiditis is variable and the IgA subclass distribution in unknown, despite recent reports of IgG subclass restriction in the thyroid auto-antibody response. Using an ELISA, IgA class antibodies were found against thyroglobulin (Tg) and microsomes (Mic) in 40-52% of patients with Graves' disease and Hashimoto's thyroiditis, and, against thyroglobulin, they were detected in the absence of IgG antibodies in 10% of the cases. Both IgA1 and IgA2 subclasses were detected in all patients with IgA class antibodies, although a significantly higher proportion of IgA2 relative to IgA1 was found in microsomal compared with thyroglobulin antibodies. In view of the high turnover rate and unique complement-fixing properties of IgA2 antibodies, this class of thyroid auto-antibody may play an important role in determining the response in thyroid auto-immunity.     

The IgG subclass distribution of thyroid autoantibodies

There have been several conflicting reports concerning the subclass distribution of thyroid autoantibodies. We have therefore reinvestigated this with both a radioimmunoassay and an ELISA based on the use of a variety of monoclonal antibodies. Our results demonstrate the presence of subclasses IgG1, IgG2, IgG3 and IgG4 in both thyroglobulin and microsomal autoantibodies from patients with either Graves' disease or Hashimoto's thyroiditis. However, in many patients there is over-representation of the IgG4 subclass. We also found marked differences in the binding characteristics of monoclonal antibodies directed against the same subclasses, underlining the need for appropriate selection of such monoclonal reagents in any assay.     

Immunogenetic analysis of variable regions encoding AB1 and gamma-type AB2 antibodies from the NeuGc-containing ganglioside family

The variable regions from P3, a murine monoclonal antibody (MAb) against NeuGc-containing gangliosides, and two anti-idiotype MAbs directed to P3 MAb were cloned and sequenced. Comparisons with previously reported sequences showed that P3 is a germline antibody encoded by genes from the V(H)Q52 and V(kappa)19 families. Analysis of nucleotides at the heavy chain CDR3 (H-CDR3) showed the presence of an extensive 3' N region that contains almost 50% of the nucleotides of this CDR. In addition, amino acid sequence analysis of the H-CDRs of this MAb revealed the presence of three arginines, two of which are present in the H-CDR3, that could be involved in the interaction of P3 MAb with its electronegative epitope on gangliosides. Anti-idiotype 1E10, which seems to define a "regulatory" idiotope on P3 MAb (it induces Id+ Ab3), represents a germline Ab2 that belongs to the V(H)J558 and V(kappa)10 gene families. By contrary, the anti-idiotype 3B11 is an extensively mutated antibody that belongs to the V(H)3660 and V(kappa)4/5 gene families, defining a "private" idiotope on P3 MAb. Even when different V genes contribute to the variable regions of 1E10 and 3B11 MAbs, they share an acidic motif E/D-D-Y/D-Y-D in H-CDR3, suggesting that both Ab2s recognize paratope positive residues on the Ab1. Therefore, complementary electrostatic interactions involving H-CDR3 from both Ab1 and Ab2, might provide a clue to understand the molecular basis for the generation of gamma-type anti-idiotype antibodies to V regions recognizing glycolylated ganglioside antigens.