Distinct reactivities of four monoclonal antibodies with human interleukin 2 receptor

Two new murine monoclonal IgG1 antibodies, H-31 and H-A26, were characterized in comparison with two previously obtained monoclonal antibodies against human interleukin 2 (IL-2) receptor (IL-2 R), anti-Tac and HIEI. In immunofluorescence assays with various human hematopoietic cells, H-31 and H-A26 antibodies both reacted with only IL-2 R-positive cells, and they precipitated IL-2 R molecules, glycoproteins with molecular weights of 60K and 53K daltons (gp60/gp53), from human T-cell leukemia virus type I (HTLV-I)-carrying MT-2 cells, as demonstrated by sequential immunoprecipitation after absorption of IL-2 R with anti-Tac. Antibody-binding competition assays showed that H-31 and anti-Tac, and H-A26 and HIEI, respectively, competed reciprocally in binding to the cells, and that anti-Tac also inhibited the binding of HIEI but not vice versa. H-31, like anti-Tac, strongly inhibited the IL-2-dependent proliferation of normal activated T-cells, absorption of IL-2 and direct binding of IL-2 to the cells, while H-A26, like HIEI, inhibited those processes only weakly. The spectra of reactivities of these antibodies with various simian cell lines derived by HTLV-I infection were different, as revealed by immunofluorescence studies. Human IL-2 R was shown to express a unique antigenic determinant, detected with HIEI, that was not detectable in IL-2 R molecules of Old and New World monkeys, and also to express determinants common to simian IL-2 R molecules. These observations indicate that H-31 and H-A26 recognize human IL-2 R molecules and that the antigenic sites on the IL-2 R molecule defined by H-31, H-A26, anti-Tac, and HIEI are different.     

High- and low-affinity interleukin 2 receptors: distinctive effects of monoclonal antibodies

We previously established several mouse hybridoma cell lines producing monoclonal antibodies against the human interleukin 2 (IL 2) receptor molecule. As they bind to both high- and low-affinity IL 2 receptors, their effects on binding of 125I-labeled IL 2 to high- and low-affinity receptors were examined by Scatchard plot analysis. Two of these monoclonal antibodies, HIEI and H-47, reduced the IL 2 binding affinity of high-affinity receptors from a Kd of 14 to 20 pM to a Kd of 110 to 140 pM, but slightly raised that of low-affinity receptors. These two antibodies scarcely affected the numbers of high- and low-affinity receptors. On the other hand, H-31 completely blocked IL 2 binding to both high- and low-affinity receptors, and H-A26 slightly reduced the affinities of both high- and low-affinity receptors, from 17 pM to 28 pM and from 28 nM to 54 nM, respectively. H-48 had little affect on IL 2 binding to high- or low-affinity receptors. By use of these monoclonal antibodies, the inhibitory effect of IL 2 on growth of an HTLV-I-immortalized T cell line was demonstrated to be transmitted from high-affinity, but not low-affinity, receptors.     

A new monoclonal antibody recognizing an antigen of human lymphocytes similar or identical to Tac antigen

A new mouse monoclonal antibody (HIEI, IgG1 type) that reacts with a cell surface glycoprotein of human lymphocytes was isolated. Membrane immunofluorescence assay showed that HIEI, like the anti-Tac monoclonal antibody, reacted preferentially with activated normal human T-cells and adult T-cell leukemia (ATL) virus (ATLV)-carrying human T- and B-cell lines. However, an interesting difference between HIEI and anti-Tac antibody was that HIEI did not react with ATLV-transformed simian cell lines or those cultured with interleukin-2 (IL-2), whereas the anti-Tac antibody did. The immunoprecipitation assay showed that both HIEI and anti-Tac antibody precipitated a glycoprotein with a molecular weight of 60,000 daltons (gp60) from activated normal T-cells and ATLV-positive T- and B-cells, and also gp53 from MT-2 and MT-2-related T-cell lines transformed with ATLV in vitro by the MT-2 cocultivation method. HIEI inhibited the IL-2-dependent proliferation of normal T-cells, but its inhibitory effect was much weaker than that of the anti-Tac antibody. The anti-Tac antibody interfered with the binding of HIEI to target cells, but HIEI did not block binding of the anti-Tac antibody to the cells. These observations indicate that HIEI antibody recognizes a new antigenic determinant of the human Tac antigen.     

Spatially distinct allodeterminants of the H-2Kk molecule as detected by monoclonal anti-H-2 antibodies

In an attempt to delineate spatial relationships between various allodeterminants of cell surface MHC antigens, competitive binding studies were performed using 4 different monoclonal antibodies, each of which reacted with the H-2Kk antigen but with different serologic specificities. Competition was studied by examining the effect of unlabeled antibodies on the binding of each 125I-labeled antibody to spleen cells of the H-2a haplotype. Mutual inhibition was observed between 2 of the antibodies, and a 3rd antibody of lower affinity was inhibited by the first 2 antibodies but did not itself inhibit the binding of these antibodies. The 4th antibody did not block the binding of the other 3 labeled antibodies, and binding of this 4th labeled antibody was only partially inhibitable by the other 3 antibodies. These results indicate the presence of at least 2 spatially distinct allodeterminants on H-2Kk molecules expressed on the cell surface.     

Monoclonal antibody (H107) inhibiting IgE binding to Fc epsilon R(+) human lymphocytes

A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.     

Monoclonal antibodies to a nuclear protein (PCNA/cyclin) associated with DNA replication

Two hybridomas producing monoclonal antibodies to proliferating cell nuclear antigen. (PNCA)/cyclin were generated from spleen cells of BALB/c mice immunized with purified PCNA from rabbit thymus. The specificity of the monoclonal antibodies (19A2 and 19F4) was established by showing that they reacted in enzyme-linked immunosorbent assay (ELISA) with purified PCNA. Furthermore, they reacted in one-dimensional (ID) gel immunoblots with a 36 kD polypeptide which also reacted with human autoantibodies from lupus patients. Both monoclonals also recognized the nuclear polypeptide cyclin in two-dimensional (2D) gel immunoblots of HeLa cell proteins. Epitopes recognized by 19A2 and 19F4 were analysed by competitive inhibition test using a modified ELISA. The data suggested that the epitopes were closely related, but not identical. The data with human auto-antibodies were more difficult to interpret, although it suggested that some human anti-PCNA may share epitopes with 19A2 and 19F4, but in addition recognize different epitopes on the PCNA molecule.     

Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Development of rat anti-mouse interleukin 3 monoclonal antibodies which neutralize bioactivity in vitro

Several rat anti-mouse interleukin 3 (IL-3) monoclonal antibodies have been developed which inhibit the biologic activity of mouse IL-3. These antibodies were produced in rats immunized with preparations of purified, recombinant mouse IL-3, obtained from transiently transfected COS7 cell supernatant. Hybridomas secreting anti-IL-3 were selected initially either on the basis of their giving a positive signal in an indirect enzyme-linked immunosorbent assay, or for their ability to inhibit the proliferation of the IL-3-dependent mouse mast cell line, MC/9. Neutralizing rat monoclonal IgG1, IgG2a, and IgG2b antibodies have been identified; these also block IL-3-induced proliferation of the NFS-60 and IC2 cell lines. These antibodies also blocked the IL-3-induced proliferation of mouse bone marrow-derived colony-forming units-culture suggesting that the same epitopes on IL-3 influence receptor recognition for both the proliferation of factor-dependent cell lines as well as normal bone marrow cells. Fab fragments produced from certain of the IgG2a-neutralizing antibodies blocked as well as the parent IgG. Antibody cross-blocking studies identified one neutralizing antibody apparently recognizing an epitope that was spatially distinct from those recognized by the other blocking antibodies tested. The development of these neutralizing rat monoclonal antibodies to mouse IL-3 should facilitate further investigation on the role of this factor in hemopoietic regulation.     

Interleukin 2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis and are inducible by lymphokine treatment of normal human lung macrophages, blood monocytes, and monocyte cell lines

Expression of receptors for IL 2 was believed initially to be restricted to T cells after their activation by IL 1 and antigen. However, recently IL 2 receptors (IL 2R) were demonstrated on activated B cells by using an anti-IL 2R monoclonal antibody (anti-Tac). In this study, we examined the capacity of cultured human alveolar macrophages, blood monocytes, and myelomonocytic (HL-60) or monoblast (U937) cell lines to bind three different anti-IL 2R monoclonal antibodies before or after stimulation with the monocyte-activating agents IFN-gamma, LPS, phorbol ester, or lymphokine-containing conditioned medium. For each of the four cell populations examined, resting unstimulated cells bound little or no anti-IL 2R antibody, as shown independently by quantitative cell binding assay and by immunoperoxidase labeling. By contrast, incubation with recombinant IFN-gamma, conditioned medium, or to a lesser extent, native or recombinant IL 2 itself, resulted in a significant enhancement of anti-IL 2 receptor monoclonal antibody binding by all four populations, whereas LPS, PMA, or IL 1 had no effect. In addition, membrane binding of anti-Tac antibody, similar to that seen after stimulation of normal lung macrophages with IFN-gamma, was detected by using macrophages obtained by bronchoalveolar lavage of five patients with active pulmonary sarcoidosis. These findings are consistent with the expression of a functional IL 2R on activated cells of the monocyte lineage, since anti-Tac binding to IFN-gamma-treated HL-60 cells was inhibited by addition of excess IL-2; specific binding of anti-IL 2 monoclonal antibodies was detected in the presence of exogenous IL 2; and a 50 to 55 kD molecule was immunoprecipitated from both activated lung macrophages and T lymphoblasts by using anti-Tac antibody. We conclude that human mononuclear phagocytes can be induced by lymphokines to express IL 2R, and that such IL 2R+ macrophages can be detected in vivo during inflammation.     

Preparation and characterization of monoclonal antibodies against native membrane-bound penicillin-binding protein 1B of Escherichia coli.

We prepared monoclonal antibodies against penicillin-binding protein 1B (PBP 1B) of Escherichia coli to study the membrane topology, spatial organization, and enzyme activities of this protein. The majority of the antibodies derived with PBP 1B as the immunogen reacted against the carboxy terminus. To obtain monoclonal antibodies recognizing other epitopes, we used PBP 1B lacking the immunodominant carboxy-terminal 65 amino acids as the immunogen. Eighteen monoclonal antibodies directed against membrane-bound PBP 1B were isolated and characterized. The epitopes recognized by those monoclonal antibodies were located with various truncated forms of PBP 1B. We could distinguish four different epitope areas located on different parts of the molecule. Interestingly, we could not isolate monoclonal antibodies against the amino terminus, although they were specifically selected for. This is attributed to its predicted extreme hydrophilicity and flexibility, which could make the amino terminus very sensitive to proteolytic degradation. All antibodies reacted against native PBP 1B in a dot-blot immunobinding assay. One monoclonal antibody also recognized PBP 1B in a completely sodium dodecyl sulfate-denatured form. This suggests that all the other monoclonal antibodies recognize conformational epitopes. These properties make the monoclonal antibodies suitable tools for further studies.     

Rat monoclonal antibodies to rabbit and human serum low-density lipoprotein.

A total of 16 hybrid myeloma clones secreting monoclonal antibodies (McAb) to rabbit or human serum low-density lipoprotein (LDL) were derived from the fusion of spleen cells from LOU or DA rats immunized with rabbit or human LDL and the rat myeloma lines Y3 Ag1.2.3 or YB2/0. Anti-(rabbit LDL) McAb showed limited reactivity with LDL from human, rhesus-monkey, rat and mouse serum. Six out of seven anti-(human LDL) McAb reacted with rhesus-monkey LDL, and only one showed partial cross-reaction with rabbit LDL. Binding-competition experiments indicated that the epitopes recognized by the anti-(rabbit LDL) IgG could be grouped into two major clusters: McAb in the first cluster reacted either with apo-(lipoprotein B-100) (apoB-100) and apo-(lipoprotein B-74) (apoB-74) or with apoB-100 but not with apo-(lipoprotein B-48) (apoB-48), the lower-Mr form of apoB of intestinal origin; the McAb in the second cluster all reacted with apoB-48 in addition to apoB-100 or apoB-100 and apoB-74. The six anti-(human LDL) IgG bound to separate epitopes on LDL. Further data on the epitope specificity of these McAb were obtained by antibody blotting after partial proteolysis of apoB-100 with trypsin or staphylococcal V8 proteinase, and the data confirmed the results obtained with the binding-competition experiments. One McAb to rabbit LDL inhibited the binding of LDL to the fibroblast LDL receptor (50% inhibition at a McAb/LDL molar ratio of 10). A similar result was produced by two other McAb at higher concentrations of antibody.     

Monoclonal antibody-defined antigens of human prostate cancer cell line PC3

Summary Over 600 hybridomas were derived from the immunization of mice with live cells and aqueous extracts of the human prostatic carcinoma cell line PC3. A total of 26 hybridomas with restricted reactivities were selected, subcloned and antibodies tested on a variety of tumor and normal cells. Seven monoclonal antibodies showed reactivity for prostate cancer and other tumor cell lines, including breast carcinomas. Three of the antibodies obtained after immunization with live cells reacted with live cells only and three of the four antibodies obtained after immunization with cell extract reacted with cell extracts and spent culture media. The fourth antibody in the latter group was reactive only in the immunoperoxidase staining assay. Antibody PrS5 recognized a 90,000 molecular weight molecule from ¹²⁵I-surface-labeled cells in immunoprecipitation analysis. Antibodies PrE3 and PrD8 detected a nonacid glycolipid pentasaccharide from PC3 cells and meconium, and a glycoprotein of 115,000 molecular weight from ¹²⁵I-surface-labeled red blood cells. The similar patterns of reactivity in RIAs and antigen analysis suggest that antibodies PrE3 and PrD8 recognize the same molecule. The results emphasize the usefulness of immunohistochemistry in the testing of monoclonal antibodies and the impact of the form in which the antigen is presented on the resultant antibody specificity     

Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the binding of the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies

A systematic approach for the determination of epitope specificities of monoclonal antibodies to a complex antigen system is described. After initial screening to identify antigen-binding monoclonal antibodies, one or more of the clones are isolated by limiting dilution cloning, grown in ascites, and the resulting antibodies secreted into the ascitic fluid are affinity purified on Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice were immunized with either purified carcinoembryonic antigen (CEA) or the CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned, resulting in 33 different clones. The antibodies produced by the different cloned hybrids and the remaining uncloned hybrids recognized a total of five different epitopes on CEA. All of the epitopes reside on the protein moiety of the molecule as determined by antibody binding to deglycosylated CEA. The monoclonal antibodies with five different epitope specificities were reacted with tissue sections of normal and cancerous tissues and with peripheral blood smears. Each of the five monoclonal antibodies reacted with tissue sections from colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted with normal liver tissue. Granulocytes in peripheral blood smears bound three antibodies strongly and one antibody weakly, and one antibody was not bound. One monoclonal antibody that reacted with normal liver tissue was not bound by granulocytes. The ability of these five monoclonal antibodies to differentially detect three different CEA-related antigens in normal and malignant tissues may have clinical utility.     

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K^k. In the absence of exogenous IL-2 the clones require the presence of Ia⁺, Thy-1⁻ accessory cells and of Thy-1⁺, Lyt-1⁺2⁻ cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A_αA_β and/or E_αE_β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2⁺ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E_αE_β when B10.A(4R) spleen cells, which do not express E_αE_β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K^d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A_αA_β and E_αE_β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.     

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.     

Production and characterization of monoclonal antibodies to rat liver arginase

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.     

Characterization of the restricted component of Epstein-Barr virus early antigens as a cytoplasmic filamentous protein.

Four monoclonal antibodies produced against the restricted component of the Epstein-Barr virus (EBV) early antigen (EA-R) precipitated a polypeptide with an approximate molecular weight of 85,000. Three of these antibodies prepared against the native 85,000-molecular-weight protein (85K protein) reacted by immunofluorescence with acetone-fixed smears but not methanol-fixed smears of EBV-producing cells activated with tumor-promoting agent and sodium butyrate. The fourth monoclonal antibody which was produced against the denatured 85K protein reacted with both acetone-fixed cells and methanol-fixed cells. Blocking of direct immunofluorescence by the different monoclonal antibodies established that these monoclonal antibodies were directed against three different epitopes expressed on the 85K protein. The cytoplasmic staining pattern produced by each antibody was granular during the first 24 to 28 h after induction, developed into filamentous structures about 36 h after induction, and then began to aggregate after 48 h. Similar structures were observed in human placental cells transfected by EBV DNA and stained with three of the monoclonal antibodies. These results suggest that the EA-R polypeptide is assembled into filaments during the EBV lytic cycle. The significance of this in regards to replication has yet to be determined. Biochemical characterization of this major EA-R component did not reveal any major differences in this protein isolated from different cell lines.     

An approach to the study of structure-function relationships of MHC class I molecules: isolation and serologic characterization of H-2Kb somatic cell variants

Somatic cell variants expressing an altered antigenic form of the H-2Kb molecule were isolated for the purpose of performing structure-function analysis of a class I MHC molecule. Over 25 independently isolated variants were derived from an Abelson virus transformed pre- B cell line (R8) by mutagenesis with ethyl methane sulfonate or ethyl nitrosourea. Negative selection was performed by complement-dependent cytotoxicity with anti-H-2Kb monoclonal antibodies subsequently followed by positive selection to separate the H-2Kb surface negative variants from structural variants. Biochemical characterization of a random selection of three independent variants indicated that the variant H-2Kb molecule was present in normal amounts in lysates, and was unchanged in size. Cytofluorometric analysis with the use of a panel of seven monoclonal antibodies against H-2Kb indicated that all of the variants had lost one or more alloantigenic determinants (monoclonal antibody binding sites). For these variants, the pattern of monoclonal antibody loss of recognition suggested that antibody defined alloantigenic determinants appear to be discretely localized to a single domain, either the alpha 1 or the alpha 2 domain, of the H-2Kb molecule. In contrast, CTL recognition of the Kb molecule of these variants depends on involvement of both alpha 1 and alpha 2 domains as shown in the companion paper.     

Common Epitopes In the Circumsporozoite Proteins of Plasmodium Berghei and Plasmodium Gallinaceum Identified By Monoclonal Antibodies to the P. Gallinaceum Circumsporozoite Protein

ABSTRACT. Monoclonal antibodies that react with the circumsporozoite protein of the avian malaria Plasmodium gallinaceum sporozoites also reacted with circumsporozoite protein of the rodent malaria Plasmodium berghei. Two types of reactivity were identified: 1) two monoclonal antibodies reacted with P. berghei sporozoite protein by enzyme-linked immunosorbent assay, Western blot and indirect immunofluorescence antibody, 2) six other monoclonal antibodies reacted with P. berghei sporozoites by ELISA and Western blot only. We studied whether these differences could be explained by reactivity in enzyme-linked immunosorbent assay with different P. berghei circumsporozoite peptides. Although all P. gallinaceum monoclonal antibodies reacted with the P. berghei repeats, the first group reacted with a conserved peptide sequence, N1, whereas the second group did not. These results suggest that circumsporozoite proteins from P. gallinaceum and P. berghei share common epitopes. the biological significance of our finding is not yet clear. Indeed, the cross-reactive monoclonal antibodies giving a positive indirect immunofluorescence antibody with the P. berghei sporozoites only caused a borderline effect on the living P. berghei parasites in vitro as measured by inhibition of sporozoite infectivity.