Production and characterization of monoclonal antibodies to Bacteroides gingivalis.

The rapid detection of Bacteroides gingivalis by immunological methods using monoclonal antibodies could greatly improve the diagnosis and prognosis of severe periodontal disease in adults. In this study, three distinct hybridomas were produced which secrete monoclonal antibodies to soluble and whole-cell antigen preparations of B. gingivalis. The BGII, V F9/2D hybridoma produced a murine antibody that can detect all of the B. gingivalis isolates studied while never cross-reacting with the other oral microbial antigens tested.

Importance of antibody isotype in monoclonal anti-idiotype therapy of a murine B cell lymphoma. A study of hybridoma class switch variants.

An initial panel of four syngeneic monoclonal antibodies directed against the idiotype of a murine B cell lymphoma was used to treat this tumor in vivo. The antibody in the panel of the IgG2a isotype was more effective in treatment than the other antibodies, which were of the IgG1 and IgG2b isotypes. To independently assess the role of antibody isotype in mediating antitumor effects, switch variant hybridoma families were isolated from the hybridomas secreting the less effective IgG1 and IgG2b antibodies. A family isolated from an IgG1-secreting parent consisted of IgG1-, IgG2b-, and IgG2a-secreting members, and an IgG2a variant was isolated from an IgG2b-secreting parent for another family. Antibody members of each family differed only in heavy chain composition and were the same with respect to their light chains and their affinity and specificity for idiotype. The IgG2a members of both families were superior to the other members in inhibiting tumor growth with an order of effectiveness of IgG2a greater than IgG1 greater than IgG2b. These in vivo results paralleled the abilities of these different isotype antibodies to mediate antibody-dependent cellular cytolysis in vitro. For the IgG2b----IgG2a family, in vivo treatment with the IgG2a member given i.p. after i.p. tumor challenge at one-tenth the dose of the IgG2b member was still superior to the latter. At one-hundredth the dose of the IgG2b, the IgG2a was still superior to the latter when the antibodies were given i.p. and tumors subcutaneously. These data and those showing that the clearance of these antibodies from the serum differed in only a relatively minor way indicate that the IgG2a antibodies in this system had greater antitumor effects primarily by virtue of their greater capacity for host effector interaction.

Use of JH4 joining segment gene by an anti-arsonate antibody that bears the major A-strain cross-reactive idiotype but displays diminished antigen binding.

One of the antibody families utilized by the A/J mouse in its response to p-azophenylarsonate (Ars) is characterized by the expression of the major anti-arsonate cross-reactive idiotype (CRI) of the A strain. This family has been termed the Ars-A family. A hybridoma antibody (HP 101F11 ) obtained after immunization of an A/J mouse with Ars was identified initially as displaying the CRI, but was subsequently found to bind antigen at a level much lower than most members of the Ars-A family. The results of binding studies suggested that HP 101F11 possesses reduced avidity for antigen. When isolated light and heavy chains were allowed to recombine with the heavy and light chains of a strongly antigen-binding, strongly CRI-positive antibody of the Ars-A family (HP 93G7 ), the low level of antigen binding by HP 101F11 was found to be due to a structurally variant heavy chain. Whereas antibodies of the Ars-A family with normal avidity for antigen had been shown to use the JH2 joining segment gene, amino acid sequence analysis of HP 101F11 revealed that this antibody has a JH segment with a sequence identical to that encoded by a portion of a different JH gene, JH4 . The implication that 101F11 uses the JH4 gene instead of JH2 was supported by the observation that the productively rearranged gene is associated with an Eco R1 restriction fragment 0.95 Kb smaller than the corresponding fragments of Ars-A hybridomas with normal avidity for antigen. The size difference of 0.95 Kb corresponds exactly to the known distance between the JH2 and JH4 genes in BALB/c germline DNA. In addition to the structural differences immediately attributable to the use of JH4 , HP 101F11 has shown an amino acid interchange in the DH segment, and a single amino acid deletion at the DH-JH boundary. These results show that variation among members of the Ars-A family in the DH and/or JH segments provides alternative structural forms of Ars-A antibodies upon which selective processes can operate during the course of an immune response.

Induction of a cross-reactive idiotype dextran-positive antibody response in two IgH-Cb mouse strains treated with anti-J558 cross-reactive idiotype antibodies.

The effect of IdX-specific rabbit and allogeneic antiidiotype antibodies (Ab2) was investigated in vivo in Igh-Cb mouse strains with respect to the induction of a cross-reactive idiotype (IdX)-positive anti-alpha (1-3) Dextran (Dex) response. These C.B20 and C57Bl/6 mice have an allotype-linked incapacity to respond with IdX-positive anti-alpha (1-3) Dex antibodies upon conventional immunization with Dex B1355. 7 d after the rabbit Ab2 injections, IdX-positive Ig (Ab3) and IdX-positive anti-alpha (1-3) Dex antibodies (Ab1') were detected in the sera of each tested mouse. The affinity-purified Ab1' were idiotypically indistinguishable from reference BALB/c IdX-positive myeloma proteins and BALB/c anti-alpha (1-3) Dex antibodies (Ab1) in a competitive inhibition radioimmunoassay, while Ab3 Ig appeared idiotypically deficient and did not bind to Dex. The response to the alpha (1-6) linkage of Dex was not affected in these mice. A large fraction of the Ab1' and Ab3 responses of both mouse strains were of the IgG1 class. The Ab1' antibodies differed from BALB/c Ab1 by lower relative binding to five of eight tested Dex, and by expressing the Igh4b allotype determinants on the IgG1 antibodies. This study identifies the products of a VHDex gene that appears to be under regulatory control in the Ighb mice. Its association with the b haplotype suggests that this gene may differ structurally from the BALB/c VHDex gene.

Idiotypic relatedness of human monoclonal IgG cryoglobulins.

The serological relatedness of the idiotypic (ID) determinants of one type lambda light chain dimer and fifteen monoclonal IgG cryoglobulins were assayed. Rabbits were immunized with 9/15 IgG cryoglobulins, and twenty-three antisera were obtained and absorbed to render them specific for the ID determinant of the immunizing IgG cryoglobulin. By use of haemagglutination-inhibition, cross-reactivity was detected among five cryoglobulins. This was localized to the Fab region of the IgG, was not related to identity of the variable region subgroups of the heavy and light chains of the cross-reactive cryoglobulins, and was not detected in eighteen non-cryoprecipitable IgG myeloma proteins. The serological relatedness of the ID determinants suggests that a subset of IgG cryoglobulins may possibly have similar variable region structures and/or antigenic specificities.

Functional analysis of GAT-specific T cell clones: H-2-restricted monoclonal T helper cells do not regulate expression of antibody isotypes.

We obtained T cell clones specific for poly (Glu60, Ala30, Tyr10) (GAT) from GAT-primed lymph node cells of BALB/c mice. The clones consisted of helper cells that were carrier-specific and H-2-restricted and required a hapten-carrier bridge to activate B cells. They may be considered TH1 cells. They could all induce antibody responses of various isotypes in adoptive transfer in vitro. Isotypic patterns of the responses obtained with the clones were identical to those obtained with polyclonal T cells and were not dependent on the number of T helper cells added to the culture. Therefore, our observations demonstrate that classic TH1 cells do not regulate isotype expression on secondary responses. The expression of idiotype-like determinants on the GAT-specific clones was assayed. A syngeneic anti-idiotypic serum (B658), prepared in BALB/c mice against BALB/c anti-GAT antibodies previously characterized, was shown to block specifically the helper activity of GAT-specific T cell lines from BALB/c mice. Under the same experimental conditions the activity of none of the BALB/c clones was affected by serum B658.

A monoclonal antiidiotypic antibody to MOPC 315 IgA inhibits the growth of MOPC 315 myeloma cells in vitro.

Spleen cells from BALB/c mice immunized with MOPC 315 IgA were fused with P3X63/Ag8 myeloma cells. Hybrid clones were screened for antibody production by a plate-binding radioimmunoassay in which MOPC 315 IgA was reacted with culture supernatants and 125I-protein A. One antibody-producing hybridoma clone (D10) was selected and injected i.p. into BALB/c mice. Ascitic fluid of tum or-bearing animals reacted specifically with MOPC 315 IgA and the reaction was inhibited by DNP- aminocaproic acid, indicating that the monoclonal antibody was directed against the hapten-binding site of MOPC 315 IgA. The monoclonal antiidiotypic antibody was of the complement (C)-binding IgG2a subclass and inhibited IgA production and growth of MOPC 315 cells in vitro in the presence of guinea pig C, as assessed by inhibition of formation of plaques and colonies by MOPC 315 cells in agar.