Antigen binding properties of highly purified bispecific antibodies.

A panel of three bispecific monoclonal antibodies (bsMAbs) binding to follitropin (FSH) and to beta-galactosidase have been prepared by fusion of hybridoma cell lines resistant to oubain and neomycin. One of these bispecific antibodies contains heavy chains of the same IgG subclass, and two are composed of heavy chains of different IgG subclasses. We have investigated methods for the purification of bispecific antibodies from hybrid hybridoma supernatant fluids grown in serum-free medium. Following ammonium sulfate precipitation, bispecific antibodies can be purified in a single step by mixed mode ion-exchange HPLC on Bakerbond Abx columns. In one case, three species were resolved by ion-exchange HPLC and functional analysis showed that two peaks contained parental antibodies, and the third contained the bispecific. Ion-exchange HPLC purification of serum-free preparations from two other hybrid hybridomas resolved seven protein-containing peaks, only one of which was active in a bispecific ELISA. The equilibrium affinity constants for each of the parental antibodies for both FSH and beta-galactosidase were determined and found to be similar to those of the purified bsMAbs. Further, the association of FSH to one binding site on a bispecific antibody was shown to have no effect on the equilibrium binding constant for beta-galactosidase binding to the other site. Our results suggest that bsMAbs can be readily purified from hybrid hybridomas by a simple and rapid method, and the binding of antigen to one binding site on a bsMAb is independent of antigen binding to the second site.

Sequences of variable regions of a monoclonal antibody specific to the thyroid hormone, triiodo-L-thyronine.

We have determined the nucleotide sequences of the variable regions of H and L chains of a monoclonal antibody 98QQ that interacts with the thyroid hormone triiodo-L- thyronine with high affinity. Analysis of the nucleotide sequence of the light chain V region of 98QQ revealed that the VL sequence is 99% identical to Balb/c germline Vk 21-E sequence. That is an interesting finding with this high affinity anti-T3 antibody, since occurence of predominantly germline variable region sequences is observed in some autoantibodies to self antigens but not usually in high affinity IgG antibodies. The sequence analysis also revealed that the heavy chain variable region sequence of 98QQ is similar to a V region of an anti-DNA antibody (MRL DNA 22). Thus the sequence analysis of our anti-T3 mAb 98QQ has revealed some features of autoantibodies to self antigens.

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages.

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.

Production and characterisation of monoclonal antibodies specific for staphylococcal enterotoxin B.

We have generated monoclonal antibodies (MABs) to staphylococcal enterotoxin B (SEB) in BALB/c mice. Five out of 20 clones which produce anti-SEB MABs have been characterised. Among them, three produce IgG1/kappa, one produces IgM/lambda, and one apparently produces both IgG1/lambda and IgM/lambda MABs. The anti-SEB titres of ascites fluids range from 3200 to greater than 819200 by ELISA. All of the MABs analysed thus far neutralise the mitogenic response of BALB/c splenocytes to a suboptimal dose of SEB. Also, the induction of suppressor cells by SEB in vitro is reversed by pre-incubating SEB with these MABs. Limited digestion with chymotrypsin, trypsin or Staphylococcus aureus V8 protease yields peptide fragments which have been tested by Western-blot analysis. MABs 1FD7 and 2GD9 are specific for the carboxy-terminal end of SEB, and have a similar, but not identical, binding epitope. MABs 2DA3 and 2HA10 bind to intact SEB but not to cleaved products, and are probably specific for antigenic determinants altered by the cleavage or by the denaturing conditions of the electrophoresis, or by both.

Differential protein synthesis by murine peritoneal macrophages elicited by various stimuli.

Protein synthetic patterns of murine peritoneal macrophages were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) of 35S methionine-labeled proteins. While the protein synthetic patterns exhibited by resident, inflammatory, and activated macrophages had numerous common features that distinguished them from the other normal non-macrophage cell types examined, unique proteins also characterized each macrophage population from the others. The accumulation by resident macrophages of proteins 23, 25, and 37 distinguished them from elicited cells, as did the former's more abundant synthesis of proteins 54 and 52. The protein synthetic patterns of inflammatory thioglycollate- and proteose peptone-elicited macrophages were strikingly similar, save for the former's greater levels of accumulation of proteins 14 and 28, and the latter's more pronounced expression of p23.5. Peritoneal macrophages elicited by treatment with heat-killed Propionibacterium acnes, the live, attenuated Mycobacterium bovis strain BCG, Listeria monocytogenes, and the protozoan flagellate Trypanosoma rhodesiense, all exhibited tumoricidal activity in 16-h or 72-h functional assays. They shared a common protein synthetic profile that differentiated them from the synthetic patterns characteristic of the non-tumoricidal resident and inflammatory macrophages. These tumoricidal macrophages were unique in synthesizing a protein(s) of approximate molecular weight 26,000 daltons. A time-course study employing P. acnes-activated peritoneal macrophages indicated that p26 accumulation decayed with tumoricidal capacity as a function of time in culture, although no direct correlation between lytic activity and p26 expression could be definitively established. Peritoneal macrophages elicited with proteose peptone were not directly tumoricidal but were rendered so upon in vitro treatment with nanogram amounts of bacterial lipopolysaccharide. The accumulation of low levels of p26 by the newly explanted proteose peptone-elicited macrophages suggests the possibility that this protein characterizes macrophage populations primed as well as triggered for tumoricidal activity.

LPS regulation of specific protein synthesis in murine peritoneal macrophages.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) analysis of biosynthetically labeled proteins of murine peritoneal macrophages elicited by inflammatory and activating stimuli indicated that the accumulation of a small number of cell-associated proteins was altered after in vitro treatment with bacterial lipopolysaccharide (LPS). Both increases and decreases in the accumulation of specific proteins were observed after LPS stimulation. Proteins of approximately 87, 43, 37, 30, and 28 Kd were similarly regulated by LPS in proteose peptone-, P. acnes-, and M. bovis BCG-elicited macrophages. Thioglycollate-elicited and resident peritoneal macrophages showed very few changes in the pattern of proteins synthesized after LPS treatment. Many of the proteins whose accumulation was increased by LPS in the elicited macrophages (proteins of approximately 87, 52, 43, 37, and 28 Kd) were already synthesized at high levels in resident macrophages. LPS stimulation also altered the accumulation of many of the same proteins in bone marrow-derived macrophages, indicating the lack of T lymphocyte influence on the LPS-induced changes in macrophages. LPS stimulation of highly purified B cells caused changes in the accumulation of several proteins of 70 and 78 Kd, which were different from those regulated by LPS in peritoneal macrophages.

Thymocyte differentiation activity from the cloned monocyte/macrophage cell line RAW 264.7. Alterations in the expression of immature thymocyte surface antigens.

The cloned monocyte/macrophage cell line RAW 264.7 was previously shown to produce thymocyte mitogenic and co-mitogenic activity that eluted from a Sephadex G-75 column not only at approximately 16,000 daltons, the m.w. described for interleukin 1 (IL 1), but also at 30,000 to 40,000 daltons. The studies reported here indicate that the 30,000 to 40,000 dalton molecule has thymic differentiating activity. Thymocytes from A/J mice were fractionated on discontinuous BSA gradients, which yielded populations of cells enriched for immature and mature cells. The cells found at the interface between 35 and 29% BSA (band 1 cells), which are the most immature, were cultured for 48 hr with highly purified IL 1, with the 30,000 to 40,000 dalton form of thymocyte co-mitogenic activity obtained after Sephadex G-75 chromatography and chromatofocusing chromatography, or with media alone. The surface antigens TL-3, H-2Kk, Thy-1.2, Lyt-1, and Lyt-2 were examined by immunofluorescence. It was found that the highly purified 30,000 to 40,000 dalton species of co-mitogenic activity induced a significant increase in the content of surface H-2Kk, a decrease in TL-3, and a very small decrease in Thy-1.2 on the cell surface, whereas IL 1 was not capable of inducing a change in these surface antigens. There was no change in Lyt-1 on the surface of band 1 thymocytes after incubation with either IL 1 or the 30,000 to 40,000 dalton species. The 30,000 to 40,000 dalton species caused a significant decrease in the percentage of cells staining positive for Lyt-2, whereas IL 1 caused a smaller but significant decrease in Lyt-2. These changes in the surface markers TL-3, H-2Kk, and Thy-1.2 are consistent with changes that occur during thymocyte differentiation. It was also observed that the proliferative response to the 30,000 to 40,000 dalton form and IL 1 increased with increasing functional maturity of each band of thymocytes when used in the thymocyte mitogenic assay. However, only the 30,000 to 40,000 dalton form was capable of inducing a proliferative response in the immature band 1 thymocytes in the thymocyte co-mitogenic assay. These results indicate that the RAW 264.7 cells produce a factor that has, in addition to thymocyte co-mitogenic activity, thymocyte differentiation activity, and this factor is distinct from IL 1.

Monoclonal antibodies to immunodeterminants of lipoteichoic acids.

Murine hybrid cell lines producing monoclonal antibodies directed against determinants present on lipoteichoic acids were generated. Hapten inhibition studies showed that one group of monoclonal antibodies was inhibited by deacylated cardiolipin, and the second group was inhibited by kojibiose. Thus, antibodies directed against the polyglycerophosphate chain, which is common to the lipoteichoic acids of many gram-positive species, and against the streptococcal group D antigen were obtained.

Production and characterization of three monoclonal antibodies to Candida albicans proteins.

Three monoclonal antibodies, designated A2C7, C2C7, and F19, were produced which recognize proteins from Candida albicans. All are of the immunoglobulin G1 heavy chain and kappa light chain class. A2C7 and C2C7 immunoprecipitated three proteins contained in a partially purified fraction (region A) of a mycelial cytoplasmic extract of C. albicans. The apparent molecular weights of these proteins are 120,000 (120K) to 135K, 44K to 52K, to 38K. Monoclonal antibody F19 was reactive with proteins of 42K, 43K, and 50K in immunoblotting experiments. F19 was also able to form a precipitin band in agarose gel with protein(s) contained in region A. Limited proteolytic digestion of the three proteins immunoprecipitated by A2C7 and C2C7 demonstrated that both monoclonal antibodies recognized the same three Candida proteins and that there exists a significant degree of relatedness in primary structure among the three proteins. Proteins with apparent molecular weights of 120K to 135K, 44K to 52K, and 35K to 38K that were immunoprecipitated by sera from two patients with invasive candidiasis and by the serum from a rabbit immunized against a 48K (44K to 52K) Candida protein were also analyzed by limited proteolysis. Patterns of peptide fragments generated by enzymatic digestion of these proteins showed that the proteins recognized by the monoclonal antibodies are the same proteins recognized by antibodies in the sera of patients during an invasive Candida infection and by antibodies in the serum of the immune rabbit.

Identification and molecular weight characterization of antigens from Candida albicans that are recognized by human sera.

Antigenic components in the cytoplasmic extract of Candida albicans were examined after fractionation by concanavalin A-Sepharose and DEAE-Sephacel ion-exchange chromatography. Fractions from the DEAE column were tested by fused rocket immunoelectrophoresis for their reactivity with antibodies in the sera of 20 patients with disseminated candidiasis. Three groups of fractions (regions A, B, and C) from the DEAE column were defined by their reactivity with these sera. Immunoblot analysis with 20 human sera identified 18 antigenic components in regions A, B, and C. Region A contained nine antigens, region B contained four antigens, and region C contained five antigens. Region A contained an antigen with an apparent molecular weight of 48,000 that was recognized by 7 of 10 sera from patients with disseminated candidiasis. Immunoprecipitation experiments with labeled proteins from region A and 51 human sera also demonstrated the presence of a major antigen whose apparent molecular weight is 48,000 to 52,000. The 48- to 52-kilodalton protein is an abundant protein in region A and is the most frequently recognized protein by antibodies in the sera of patients with disseminated candidiasis. Patients with disseminated candidiasis had significantly higher levels of antibody (immunoglobulin G) (P less than 0.001) directed against the 48- to 52-kilodalton protein than did patients with noninvasive forms of candidiasis, patients with other fungal infections, or normal, healthy persons.

Identification of multiple-molecular-weight forms of thymocyte comitogenic activity from the monocyte/macrophage cell line RAW 264.7.

The cloned monocyte/macrophage cell line RAW 264.7 was investigated for interleukin 1 (IL-1) production. Of the inducers tested, bacterial lipopolysaccharide was found to be the most effective. The cyclic nucleotide analogs 8-BrcAMP and 8-BrcGMP were also tested, with only 8-BrcGMP being capable of inducing a small amount of IL-1 activity. Gel filtration studies revealed thymocyte mitogenic and comitogenic activity in three molecular-weight peaks: greater than 70,000, 30,000 to 40,000, and 12,000 to 18,000 Da. The multiple-molecular-weight forms were present when samples were prepared under serum-free conditions and also when samples were prepared and chromatographed in high ionic strength NaCl or under disulfide reducing conditions. Molecular charge heterogeneity was observed when proteins were chromatographed using column chromatofocusing (PBE 94). The intermediate-molecular-weight form eluted from the column over a pH range of 5.0 to 5.4; while the low-molecular-weight form eluted at three separate pH's: greater than or equal to 7.4 (unbound material), 5.2, and 4.8. The low-molecular-weight and intermediate-molecular-weight forms exhibited different dose-response curves when assayed under conditions used by other investigators (1 X 10(7) cells/ml; phytohemagglutinin, 1 microgram/ml), but very similar dose-response curves when assayed under conditions used by our laboratory (2 X 10(6) cells/ml; concanavalin A, 0.25 microgram/ml) in a thymocyte comitogen assay. The possible relationship of these multiple-molecular-weight species of thymocyte comitogenic activity from RAW 264.7 to other biological activities from cloned and noncloned cellular sources is discussed.

Immunocytochemical evidence for 3',5'-cGMP and 3',5'-cGMP-dependent protein kinase involvement in lymphocyte proliferation.

In preliminary experiments cyclic nucleotides and cyclic nucleotide-dependent protein kinase subunits were localized in murine splenocytes using immunofluorescence and immunoperoxidase techniques. Cyclic nucleotides, presumably protein bound, and protein kinases (PK) were found in both cytoplasm and nucleus. Following mitogen stimulation the localizations did not change. In experiments reported here using the peroxidase-antiperoxidase technique not all cells in the population were stained with antisera against the various antigens. At early times (5-60 min) following stimulation with lipopolysaccharide (LPS) or concanavalin A (ConA) the fraction of cells staining positively for cGMP and cGMP PK increased relative to non-stimulated cells. Radioimmunoassay measurements showed elevated intracellular concentrations of cGMP beginning at 15 min following mitogenic stimulation. The data presented is consistent with a role for cGMP and cGMP PK in lymphocyte activation.

Structure and developmental expression of the chick alpha-actin gene.

Recombinant DNA clones containing chick alpha-actin mRNA sequence have been isolated and used as probes to analyze the structure and developmental expression of the chick alpha-actin gene. The full length, 2000 nucleotide alpha-actin mRNA is detected in poly(A) RNA at early and late stages of in vivo leg muscle development. As expected, the alpha-actin mRNA is present at very low levels at early myogenic stages but is a high abundance species in terminally differentiated muscle. However, most of the alpha-actin mRNA from fused leg muscle is shorter than 2000 nucleotides, and occurs in relatively discrete size classes. An alpha-actin-like mRNA can be detected in poly(A) RNA from early embryonic brain, indicating that transcription of the alpha-actin gene may not be strictly muscle-specific at all stages of development. We have identified at least 3, very short (< 100 base pairs) intervening sequences in the alpha-actin gene which was isolated from a chick genomic library. The structure of the chick alpha-actin gene differs, therefore, from the structures of actin genes from yeast and Drosophila, both of which contain a single, relatively long, intervening sequence.

Some Physical Characteristics of the Enzymes of l-Tryptophan Biosynthesis in Higher Plants.

Anthranilate synthetase, phosphoribosyltransferase, phosphoribosyl anthranilate isomerase, and indoleglycerol phosphate synthetase were examined in partially purified extracts of the monocotyledon, Zea mays and the dicotyledon, Pisum sativum. The plant extracts were chromatographed on DEAE-cellulose and Sephadex G150. The molecular weights of the enzymes were determined and found to be similar to those observed for many bacteria. None of the plant tryptophan enzyme activities was aggregated in vitro as is also the case with most bacteria. This is in contrast with the complex aggregation patterns observed in other eucaryotic organisms that have been examined (fungi and Euglena gracilis). The tryptophan enzymes from peas and corn were generally similar but some differences in stability were observed.