Splenic B cells and antigen-specific B cells process anti-Ig in a similar manner

B lymphocytes can process and present antigen to T cells. However, the fate of native antigen after its binding to specific B cells, i.e., the intracellular events involved in the processing and recycling of the antigenic fragments to the cell surface for antigen presentation, are not well understood. In the present study, we demonstrate that murine B cells degrade anti-Ig molecules bound to their surface and release acid soluble fragments into the supernatant. We also demonstrate that the kinetics of this process are identical for anti-mu, anti-delta, and anti-light chain antibodies, indicating that both surface IgM and surface IgD are equally effective in binding antigen and directing its processing. We also describe the effects of azide, chloroquine, and irradiation on this process. To extend these studies to the processing of specifically bound antigen, we demonstrate that highly purified trinitrophenyl antigen-binding cells degrade anti-Ig molecules with the same kinetics as unpurified splenic B cells. Thus, this purified population provides a suitable model system for the analysis of antigen degradation by antigen-specific cells.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Xenoantisera against a murine T cell tumor product cross-react with immunoglobulin Fab determinants

Some murine monoclonal T lymphoma cells express a surface component that reacts with chicken antisera produced against the Fab fragment of normal mouse IgG. In the present study, we use a solid phase immunoadsorbent consisting of affinity-purified chicken anti-Fab coupled to Sepharose to isolate a product produced by the in vitro T cell line, WEHI-7.1. The affinity-purified T cell surface molecule (IgT) migrated on SDS-PAGE as a single band of approximately 65,000 daltons. The object of these studies was to produce xenoantisera against the purified T cell product cross-reactive with Ig determinants and to characterize the antisera. Rabbits immunized with this purified molecule produced antibodies that reacted with Fab fragments of polyclonal mouse IgG and with the myeloma proteins MOPC-104E and MOPC-41, as detected by enzyme-linked immunosorbent assay (ELISA). This binding was eliminated by adsorption of the antisera with normal polyclonal IgG; however, adsorption with fetuin did not significantly affect the reactivity of the antisera. Radioimmune precipitation assays revealed that the rabbit anti-IgT bound to normal murine spleen and thymus cells; this reactivity was abrogated by adsorption with insolubilized polyclonal IgG. Competition radioimmunoassays demonstrated that detergent extracts of the thymus and the spleen contained material that inhibited the precipitation of MOPC-41; nonlymphoid cells lacked such material. The rabbit anti-IgT serum blocked the binding of antigen by normal T cells; adsorption of the antiserum with polyclonal IgG-Sepharose abrogated this blocking capacity. A solid phase immunoadsorbent prepared from the IgG fraction of the rabbit anti-IgT isolated a single component from formic acid-solubilized mouse thymus. This molecule had an approximate mass of 65,000 to 70,000 daltons. The anti-IgT serum isolated surface IgM and IgD from lactoperoxidase-catalyzed radioiodinated B cells. The anti-IgT serum detected IgM and IgG in mouse serum with the use of immunoelectrophoresis. The anti-IgT immunoadsorbent isolated several components from normal mouse serum, that, when analyzed by SDS-PAGE under reducing conditions, revealed bands corresponding to mu-, gamma-, and light chains as well as components that migrated between mu- and gamma-chains, and another component with an approximate mass of 45,000 daltons. Our results with antibodies to a purified T cell product indicate that a surface component of normal T cells and certain monoclonal T cell tumor lines is serologically related to the Fab fragment of serum Ig and is implicated in the binding of antigen.     

Activation of human B cells and inhibition of their terminal differentiation by monoclonal anti-mu antibodies

Anti-mu antibody preparations have been found to exert both positive and negative effects on B cell activation and differentiation. To explore these paradoxical influences of IgM cross-linkage on human B cells, three gamma 1 kappa murine monoclonal antibodies specific for human mu-chains (DA4.4, AB6.4, 145.8) were examined for their comparative effects on activation of B cells and inhibition of terminal plasma cell differentiation. All three antibodies appeared equally efficient in immunoprecipitation of surface IgM molecules; however, fluorescence-activated cell sorter analysis revealed that the DA4.4 and AB6.4 antibodies saturated the B cell surface IgM at slightly lower concentrations than did the 145.8 antibody. When the affinity-purified antibodies were added in varying concentrations to cultures of small resting B cells, all three antibodies induced B cell enlargement and DNA synthesis, but with varying degrees of efficiency (DA4.4 greater than AB6.4 much greater than 145.8). In striking contrast, large B cells isolated either by FACS or density gradient separation were unresponsive. The anti-mu-induced proliferative response of small B cells required relatively high B cell densities, but not T cells or the Fc portion of the antibody molecules. The maximal proliferative response was obtained during the third day of culture, and the response curve suggested that anti-mu induced only one round of B cell replication. All three antibodies were capable of completely inhibiting T cell factor-induced differentiation of large B cells into IgM plasma cells; both F(ab')2 fragments and intact anti-mu antibodies were effective in final concentrations as low as 1 microgram/ml. Significant suppression of IgG and IgA plasma cell differentiation was also achieved, but required higher concentrations of the anti-mu antibodies. For each antibody, there was a close correlation between the efficiency of inducing small B cell proliferation and of inhibiting large B cell differentiation into plasma cells. The results show that the B cell response to cross-linkage of cell surface IgM varies according to the differentiation stage. We postulate that the mature resting B cell represents the only stage in the life history of the B cell during which surface Ig cross-linkage leads to a positive signal, negative signals being the rule at other stages in B cell replication and differentiation.     

Structure and biologic functions of human IgD. XI. Identification and ontogeny of a rat lymphocyte immunoglobulin having antigenic cross-reactivity with human IgD.

Chicken anti-human IgD antiserum (anti-delta) has demonstrated an antigenically cross-reactive homologue on rat lymphocytes. IgD and IgM are the only cell surface immunoglobulins detectable by the lactoperoxidase radiolabeling technique employed. The results indicate that, although rat surface IgD is antigenically distinct from rat IgM, the respective H chains co-electrophorese in 10% polyacrylamide-SDS gels. Rat delta-chain has an apparent m.w. of 73,000 daltons and exhibits a minor 65,000 dalton component which probably represents a partially degraded delta-chain. The ontogenic emergence of rat IgD occurs approximately 3.5 weeks after birth whereas IgM, in contrast, is apparent by 6 days of age. Thus, as in the human, IgM develops before IgD. IgD receptors are undetectable in the thymus but are present in increasing levels in spleen, blood, lymph nodes, and Peyer's patches.     

IgM binding protein expressed by activated B cells

We have identified an IgM binding protein, a single chain polypeptide of Mr 60,000, that is expressed on the surface of B lymphocytes within 18 hr following their activation with phorbol myristate acetate. The IgM binding protein was also detected on fresh leukemic B cells from individuals with chronic lymphocytic leukemia, and the level of its expression was increased after phorbol myristate acetate activation. Resting and phorbol myristate acetate-activated T cells, monocytes, and polymorphonuclear leucocytes did not express detectable amounts of the IgM binding protein. The 60-kDa protein on activated human B cells could bind secreted IgM molecules of both mouse and human origin, as well as endogenous membrane-bound IgM molecules following their cross-linkage with anti-mu antibodies. The binding of soluble IgM molecules to the surface of activated B cells was also demonstrated by indirect immunofluorescence analysis.     

Antibodies against urinary light chain idiotypes as agents for detection and destruction of human neoplastic B lymphocytes

Patients with B cell neoplasms frequently have low levels of tumor-related light chains in their urine; these light chains can be isolated with the use of relatively simple methods and then used to raise antibodies to the idiotypic determinants. In this study, anti-light chain idiotypes were raised against monoclonal light chains from the urine of four patients with chronic lymphocytic leukemia. The antibodies reacted specifically with the tumor cells of the homologous patient, assessed by immunofluorescence, and can therefore be used for tumor cell detection. In one case for which serum idiotypic IgM was available, the anti-light chain idiotype was shown to bind whole idiotypic IgM, and such binding could be inhibited by idiotypic IgM or idiotypic light chains, which demonstrates recognition of similar antigenic determinants. The binding of antibody to tumor cells was also totally inhibited by idiotypic IgM. The analysis of separated sera from the four patients for free light chains demonstrated only low levels (3.0 to 8.6 micrograms/ml of serum with a mean of 5.8), which suggests that light chain is rapidly cleared and therefore does not present a major barrier to antibody attack. It should be feasible to use such antibodies for both analysis and therapy of B cell neoplasms.     

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.     

Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation

B cell hybridomas with Ia and IgM molecules on the cell membrane were treated with either purified goat anti-mouse mu antibody (anti-mu) or monoclonal rat anti-mouse IgM antibody (anti-IgM). The spontaneous uptake of [3H] thymidine by these cells was markedly inhibited by both reagents. These hybrid cells could be induced to differentiate into IgM-secreting cells in the presence of these reagents at high frequency. Furthermore, the induction of IgM secretion by B cell hybridomas treated with these antibodies was completely T cell independent, and cell division was not required for the differentiative response to anti-mu. In addition, F(ab')2 fragments of anti-mu showed more effects on proliferation and differentiation of these cells than intact anti-mu. Interestingly, TH2.54, a subline of B cell hybridomas, could generate IgG2a production as well as IgM when incubated with anti-mu. These findings suggest very strongly that the interaction of either goat anti-mu or monoclonal rat anti-IgM with surface IgM molecules on the cell membrane of the B cell hybridomas inhibits in vitro spontaneous proliferation, and results in providing signals for differentiation into Ig-secreting cells without T cell factors.     

Molecular basis of a unique tumor antigen of radiation leukemia virus-induced leukemia B6RV2: its relation to MuLV gp70 of xenotropic class

Hybridomas secreting monoclonal antibodies that reacted with the B6 radiation leukemia virus (RadLV)-induced leukemia B6RV2 were produced by fusion of BALB/c NS-1 myeloma cells with spleen cells from (BALB/c X B6)F1 mice immunized with B6RV2. By direct and absorption analyses with 28 B6 and BALB/c leukemias, the monoclonal antibodies NU7-4 and NU7-99 were shown to react only with B6RV2, indicating that they recognized an individually distinct antigen on B6RV2 that was identified previously with conventional (BALB/c X B6)F1 anti-B6RV2 serum. Another monoclonal antibody, NU1-132, showed relatively restricted reactivity with B6 RadLV leukemias. These three monoclonal antibodies all precipitated material of approximately 80,000 daltons, which is the same size as that precipitated by anti-xenotropic MuLV gp70 serum. Sequential immunoprecipitation analysis revealed that the molecules precipitated by NU7-4 were not removed by pretreatment of NU7-99 or NU1-132 and that the molecules precipitated by NU7-99 were not removed by NU7-4 or NU1-132. The molecules precipitated by NU1-132 were partially removed by pretreatment with NU7-4, but not with NU7-99. The molecules precipitated by these three monoclonal antibodies were removed by pretreatment with anti-xenotropic gp70. These results suggested heterogeneity of the xenotropic MuLV gp70-related molecules expressed on B6RV2 and a possible relation between serologically defined unique tumor antigens and gp70-related molecules.     

IgD-secreting murine plasmacytomas: identification and partial characterization of two IgD myeloma proteins

We have used an immunofluorescence inhibition assay to identify 2 BALB/c plasmacytomas, TEPC-1017 and TEPC-1033, that secrete large quantitites of IgD. Both TEPC-1017 and TEPC-1033 myeloma proteins bound to anti-kappa as well as hybridoma and heterologous anti-delta antibodies, but not to anti-mu, gamma, alpha, or lambda antibodies. Both myeloma proteins were purified by (NH4)2SO4 precipitation, ion exchange chromatography, gel filtration, and Staphylococcus aureus Protein A absorption. These IgD kappa myeloma proteins were used to prepare affinity purified rabbit antibodies to delta-chain and the TEPC-1017 and TEPC-1033 idiotypes. Native TEPC-1017 and TEPC-1033 both had mobilities between those of mouse IgA kappa dimers and trimers when analyzed by polyacrylamide gradient gel electrophoresis. Both IgD myeloma proteins broke down under mild reducing conditions into subunits with electrophoretic mobilities slightly slower than those of an IgA kappa monomer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced TEPC-1017 and TEPC-1033 demonstrated kappa-chains and heavy chains that co-migrated with alpha chain. These data suggested that secreted IgD contains 2 delta 2 kappa 2 subunits that are linked by an easily reducible disulfide bond. The kappa-chains of IgD secreted by TEPC-1017 and TEPC-1033 have apparent m.w. of approximately 63,000 daltons, whereas the apparent m.w. of intracytoplasmic delta-chain, intracytoplasmic delta-chain synthesized in the presence of tunicamycin, and the cellfree translation product of TEPC-1017 delta-chain mRNA are 54,000, 43,000, and 44,000 daltons, respectively. This is compatible with the interpretation that the delta-chain peptide has a leader sequence and is N-glycosylated during or shortly after peptide synthesis and is glycosylated further shortly before IgD secretion.     

In vitro and in vivo B lymphocyte-activating properties of monoclonal anti-delta antibodies. I. Determinants of B lymphocyte-activating properties

To appreciate better the mechanisms by which B lymphocytes are activated by anti-Ig antibodies, we characterized seven monoclonal mouse allo-antibodies to IgD of the a allotype for their isotypes, fine specificities, IgD-cross-linking abilities, avidities, and abilities to activate B cells in vitro and in vivo. Three of the monoclonal antibodies tested bound to the Fc fragment of IgD with relatively high avidity and were effective at cross-linking IgD, since they precipitated soluble IgD and rapidly capped B cell membrane IgD. These were the only antibodies tested that induced B cell DNA synthesis in vitro and were the most effective antibodies at inducing in vivo increases in B cell size and DNA synthesis and in vitro and in vivo increases in B cell surface Ia expression. Two antibodies bound to the Fd fragment of IgD with relatively high avidity but could not rapidly cap cell membrane IgD or precipitate soluble IgD even in the presence of 2% polyethylene glycol. These high-avidity, poorly cross-linking antibodies were unable to stimulate B cell DNA synthesis in vitro and were much less effective than the first group of anti-delta antibodies at stimulating in vivo increases in B cell DNA synthesis, size, or surface Ia expression or in vitro increases in surface Ia expression. One antibody, which bound to the Fc fragment of IgD with an intermediate avidity, was unable to rapidly cap B cell membrane IgD or precipitate soluble IgD in saline, but could precipitate soluble IgD in the presence of 2% polyethylene glycol. This antibody failed to induce B cell DNA synthesis in vitro and was as effective as the higher-avidity, poorly cross-linking antibodies at stimulating increases in B cell size, surface Ia expression, and DNA synthesis in vivo, and surface Ia expression in vitro. One antibody, which bound to the Fd fragment of IgD with low avidity and was unable to precipitate soluble IgD or to cap cell membrane IgD, had little ability to activate B cells by any of the parameters studied. Each of the monoclonal anti-delta antibodies, regardless of isotype or fine specificity, when bound to agarose to increase its ability to cross-link IgD, was mitogenic for B cells in vitro. None of the monoclonal antibodies to IgD of the a allotype stimulated B cells from b allotype mice to increase their size, surface Ia expression, or synthesis of DNA in vitro or in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice.

During the course of B lymphocyte development, newly emerging surface Ig+ B cells pass through a stage when Ag-Ag receptor interactions lead not to immune responsiveness but to a state of functional tolerance. We have explored the molecular basis of antigenic nonresponsiveness and tolerance susceptibility using tolerance-susceptible surface Ig+ splenic B lymphocytes from neonatal mice and anti-mu chain antibodies as a polyclonal ligand. In this population of cells, surface IgM is uncoupled from the inositol phospholipid (PI)-hydrolysis pathway at a point proximal to the receptor; anti-mu antibodies did not stimulate inositol phosphate generation despite the fact that PI-hydrolysis was observed after treatment with A1F4, implicating the existence of a functional G protein and phospholipase C. Further evidence for a difference early in the signal transduction pathway stems from the finding that anti-mu stimulation does not induce the expression of two immediate/early PKC-linked genes egr-1 and c-fos. This appears to be the primary signaling difference between the mature and immature B cells from the neonatal mouse splenic population, as these cells undergo a G0-G1 cell cycle phase transition when surface IgM is bypassed using phorbol diester and calcium ionophore. Interestingly, despite undetectable levels of PI-hydrolysis, we observed equivalent receptor-mediated changes in intracellular calcium when comparing the immature and mature populations. These results indicate incomplete coupling of surface IgM to the signal transduction machinery operative in mature, immunocompetent B cells and suggests a molecular mechanism accounting for the differential processing of surface IgM signals into activation vs tolerogenic responses observed in these two stages of B cell development.     

Mouse monoclonal antibodies to chicken VH idiotypic determinants reactivity with B and T cells

We have prepared mouse monoclonal antibodies against idiotypic (Id) determinants on chicken antibodies to N-acetylglucosamine (NAGA) and p-aminobenzoic acid (PABA) made by inbred line EL 6(3) birds. The monoclonal anti-NAGA Id antibody, termed CId-1, reacted with affinity purified antibodies to NAGA, but not with antibodies specific for PABA, arsanilic acid (Ars), phosphorylcholine (PC), or with normal chicken IgG and IgM. The monoclonal anti-PABA ID antibody, termed CId-2, reacted with anti-PABA antibodies and to a lesser extent with anti-Ars antibodies, but not with anti-NAGA, anti-PC, and normal IgG and IgM. The Id determinants were found among antibodies to NAGA and PABA made by outbred and inbred lines of White Leghorn chickens. The binding of the CId-1 and CId-2 antibodies to intact homologous anti-NAGA and anti-PABA antibodies, respectively, was not hapten-inhibitable in either case. Both anti-Id antibodies reacted specifically with isolated homologous heavy chains, suggesting VH Id specificities. The monoclonal CId-1 and CId-2 antibodies were reactive by immunofluorescence with approximately 0.9 and 0.2%, respectively, of the circulating lymphocytes and with approximately 0.4 and 0.15 of plasma cells. CId-1+ and CId-2+ bursal cells were first detected on the 16th and 14th days of incubation, respectively; both reached maximal frequencies by the 17th day of incubation. The CId-2 antibody reacted exclusively with immunoglobulin-positive cells. The CId-1 antibody also reacted with a subpopulation (0.4%) of immunoglobulin-negative lymphocytes from normal and agammaglobulinemic chickens, and thus would appear to recognize an idiotypic determinant expressed by certain clones of B and T cells.     

Cross-reactivity of rat, mouse, and human IgD.

Highly purified sheep anti-rat lymphocyte membrane IgD (mIgD) was used to detect cross-reactivity with the putative murine-delta chain on mouse lymphocytes. Cross-reactivity is demonstrated by indirect immunofluorescent staining and by immunoprecipitation of 125I-labeled lymphocyte membrane extracts followed by electrophoresis on 10% polyacrylamide gels. In addition, cross-reactivity of anti-rat-delta with human IgD is shown by gel diffusion analysis. The anti-rat-delta reagent stained both Ig5a+ and Ig5b+ lymphocytes. Preincubation of Ig5b+ (but not Ig5a+) cells with monoclonal allotype-specific antibodies (anti-Ig5b) under capping conditions caused inhibition of staining by the sheep anti-rat-delta reagent, indicating that it is the delta-chain that is recognized on mouse lymphocytes and that the anti-rat-delta reagents does not distinguish between mouse-delta allotypes. Furthermore, absorption of the sheep anti-rat-delta serum with purified human IgD reduced subsequent staining of mouse lymphocytes by approximately 50%; staining was not affected by absorption with human IgM. This xenogeneic anti-delta antiserum appears to detect determinants on the delta-heavy chain, which are shared by at least three species of mammals, suggesting that these determinants represent important molecular features conserved during evolution.     

Delineation and characterization of human B cell subpopulations at various stages of activation by using a B cell-specific monoclonal antibody

Human tonsillar B cells were separated into three distinct subpopulations, Ba-/IgD+, Ba+/IgD+, and Ba+/IgD-, by using a B cell-specific monoclonal antibody (anti-Ba) that recognizes only activated B cells, and anti-IgD antibody. Stimulation of Ba-/IgD+ cells with anti-mu plus PHA-conditioned culture supernatant (PHA-sup) or TPA induced Ba+/IgD+ cells, which reverted to Ba-/IgD+ phenotype in the absence of continuous stimulation. Further stimulation of Ba+/IgD+ cells with several B cell activators, such as TPA plus anti-mu or PWM plus T cells, resulted in the loss of IgD expression. Three-color FACS analysis showed that the expression of transferrin receptor (TFR) was at its maximum in Ba+/IgD- cells, and the intensity of this expression was proportional to that of Ba expression in Ba+/IgD+ cells. PHA-sup induced maximum proliferation in Ba+/IgD- cells, and the degree of response was a function of the intensity of Ba expression in Ba+/IgD+ cells. PHA-sup or purified BCDF (BSF-2) induced Ig secretion preferentially in Ba+/IgD- cells. Taken together, these results show that resting B cells (Ba-/IgD+) are activated into Ba+/IgD+ cells, and then into Ba+/IgD- cells, under mitogenic stimulation, and BCDF induces the final maturation of Ba+/IgD- cells into Ig-secreting cells. Ba+/IgD- cells, which maximally expressed TFR as well as Ba and displayed maximum proliferative response to PHA-sup, did not express any Tac antigen. On the other hand, in vitro activated B cells expressed Ba and TFR as well as Tac antigen.     

Cloning and expression of partial Japanese flounder (Paralichthys olivaceus) IgD

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7 d Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.     

In vitro immune response of human peripheral lymphocytes. VII. Effect of anti-mu and anti-delta antibodies on B colony formation and detection of abnormal B cells in patients with juvenile rheumatoid arthritis

Effect of anti-mu and anti-delta antibodies on PHA- and protein A-induced B colony formation was studied. Anti-mu antibody at any concentrations tested did not show inhibitory or enhancing effect on colony formation. On the other hand, anti-delta antibody enhanced both PHA- and protein A-induced colony formation. Optimum concentration of anti-delta antibody for maximum enhancement was 0.1 microgram/ml. and F(ab')2 fragment of anti-delta antibody also showed comparable enhancing effect. Simultaneous addition of IgD with anti-delta antibody abrogated anti-delta-induced enhancement, and anti-delta antibody did not show any facilitation of colony formation in IgM+ IgD- cell population. In marked contrast with normal B cells, anti-mu antibody showed a remarkable enhancing effect on protein A-induced colony formation of B cells from JRA patients. F(ab')2 fragment of anti-mu antibody also showed comparable enhancing effect. Anti-mu antibody did not show any enhancement of colony formation of B cells from several other autoimmune diseases. The result indicated the presence of abnormal B cells in JRA patients.