The mitochondrial genome of the S-type male-sterile cytoplasm of maize contains two linear episomes, S1 (6397 base pairs) and S2 (5453 base pairs). The S2 episome contains two large unidentified open reading frames, URF1 (3512 base pairs) and URF2 (1017 base pairs). We have demonstrated that a polypeptide with an apparent molecular mass of 130 kDa is the gene product of URF1. This polypeptide was first detected in Coomassie blue-stained protein gels of cms-S (where cms = cytoplasmic male sterile) but not in those of cms-T, cms-C, or normal mitochondrial proteins. The protein product of a translational fusion containing the 5' end of Escherichia coli lacZ and an internal segment from URF1 of S2 was recognized by antisera raised against the 130-kDa variant polypeptide. The mitochondria of fertile F(1) hybrids of cms-S x Ky21 (the male parent carrying nuclear fertility restoration genes) contain as much of the 130-kDa protein as is found in cms-S mitochondria of sterile plants. Spontaneous fertile cytoplasmic revertants from cms-S in a WF9 nuclear background also synthesized the 130-kDa polypeptide. Therefore, the mere presence or absence of the URF1 gene product of S2 does not determine the fertility status of maize plants, because male sterile and male fertile (nuclear restored and revertant) plants can contain equivalent amounts of the 130-kDa polypeptide.

Initial acceptance of Cibacron Blue 3G-A based matrices has made dye-ligand affinity chromatography an attractive proposition. This prompted the synthesis and search for new dye structures. A systematic library of 96 affinity resins was generated using novel analogs of Cibacron Blue 3G-A and also by varying spacer lengths for immobilization. The library was tested in a batch binding and elution mode using seven different proteins--four Aspergillus enzymes namely, NADP-glutamate dehydrogenase, laccase, glutamine synthetase and arginase, bovine pancreatic trypsin and the two serum proteins human serum albumin and immunoglobulin G. Unique binding patterns were observed for each of them indicating that the library displayed discriminatory interactions. The significance of spacer length in the interaction with proteins was discernable. Trypsin interacted best with affinity resins that had no spacer. It was possible to resolve IgG and HSA from a mixture using a combination of resins. There was a good spread of HSA binding capacity in the 96 affinity resins. While some showed better HSA binding capacity than the commercial CB3GA-based matrix, a few with lower capacity were also observed. Subsequent to an initial screen, one affinity resin (CR-017) could be used to enrich Aspergillus terreus NADP-GDH from crude cell extracts. The efficacy of this dye-affinity resin was rationalized by characterizing NADP-GDH inhibition kinetics with the corresponding free dye ligand. In the sum, the library provides a set of dye-ligand affinity matrices with a potential for use in high throughput screening for protein purification.

Successful embryo implantation and pregnancy in mammals depends on the establishment of immune tolerance between the maternal immune system and fetal cells. Monoclonal nonspecific suppressor factor beta (MNSFbeta), a cytokine produced by suppressor T cells in various tissues, possesses an antigen-nonspecific immune-suppressive function, and may be involved in the regulation of the uterine immune response during embryo implantation. In this study, anti-MNSFbeta IgG administered directly into the uterine lumen, significantly inhibited mouse embryo implantation in a dose-dependent manner in vivo, and this effect was reversed by co-administration of recombinant MNSFbeta. The effects of anti-MNSFbeta IgG on the gene pattern profiles in mouse uterine tissues were examined by cDNA microarray and several changes were confirmed by real-time PCR. Anti-MNSFbeta IgG caused up-regulation (> or = 2-fold) of 71 known genes and 17 unknown genes, and decreased expression (> or = 2-fold) of 74 known genes and 43 unknown genes, including several genes previously associated with embryo implantation or fetal development. Most of the known genes are involved in immune regulation, cell cycle/proliferation, cell differentiation/apoptosis, and lipid/glucose metabolism. These results demonstrate that MNSFbeta plays critical roles during the early pregnancy via multiple pathways.

In this work a highly acetylated-ethylenediamine-Novarose (HA-EDA-Novarose) gel was synthesized and used as a new agarose-based salt-promoted adsorption chromatography (SPAC) matrix to effectively isolate serum immunoglobulins without the need of denaturing conditions. Samples of human serum in 0.5 M Na2SO4, 10 mM 3-(N-morpholino)-propane-sulfonic acid (MOPS), pH 7.6 were applied to a chromatographic column packed with the SPAC gel. Immunoglobulins (Igs) with affinity for the HA-EDA ligands were specifically adsorbed to the matrix, non-bound serum proteins were readily removed by washing the column with the same feed solution buffer. Bound Igs were effectively and very gently eluted by simply removing the salt from the feed solution buffer. The elution buffer consisted thus of only 10 mM MOPS, at pH 7.6 and no salt. The salt-dependent adsorption capacity of this system was estimated to be 7.3 mg/ml with protein recovery of about 93%. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis analysis, radial immunodiffusion and enzyme-linked immunosorbent assays showed that immunoglobulins G, M and A (IgG, IgM and IgA) were the main components present in the elution fraction. The new SPAC adsorbent was used to purify Igs from human serum and IgG and IgA from non-pure commercially available Igs preparations in a very gentle single step.

Ross River (RR) virus is a mosquito-borne alphavirus and one of the aetiological agents of epidemic polyarthritis in humans. Early detection of increased virus activity in mosquito populations enables public health authorities to implement measures to reduce the number of human infections during epidemics. However, current surveillance techniques require a minimum of four weeks for viruses to be isolated and identified.

This study was carried out to assess the use of enzyme immunoassays (EIA) as rapid alternatives to traditional cell culture techniques for detection of RR virus in mosquitoes.

Enzyme immunoassays and immunoperoxidase assays were developed using RR-specific monoclonal antibodies and compared to traditional methods for detection of RR virus in field-caught mosquito samples.

By inoculation of C6/36 cell cultures with mosquito homogenates and testing monolayers and culture supernatant by EIA, RR virus was detected and identified in all infected samples within 6 days.

The use of EIA provides a rapid, sensitive and specific alternative to traditional methods for the detection of RR virus in mosquito vectors.

A method suitable for the isolation of monoclonal antibodies (MAbs) on novel zirconia microspheres (20-30 microm) is described. Zirconia microspheres were generated by spray drying colloidal zirconia. Spray-dried zirconia microspheres were further classified and characterized by X-ray diffraction, BET porosimetry and scanning electron microscopy. Spray-dried zirconia microspheres were modified with ethylenediamine-N,N'-tetra(methylenephosphonic) acid (EDTPA) to create a cation-exchange chromatographic support. The chromatographic behavior of a semi-preparative column packed with EDTPA-modified zirconia microspheres was evaluated and implications for scale-up are provided. EDTPA-modified zirconia microspheres were further used to purify MAbs from cell culture supernatant. Analysis by enzyme linked immunosorbent assay and gel electrophoresis demonstrate that MAbs can be recovered from a cell culture supernatant at high yield (92-98%) and high purity (>95%) in a single chromatographic step.

We have probed the signaling characteristics of the macrophage low-density lipoprotein receptor-related protein (LRP) with monoclonal antibody 8G1, its Fab and F(ab')(2) fragments directed against the ligand binding heavy chain, and monoclonal antibody 5A6 directed against the membrane-spanning light chain of LRP. Ligation of LRP with 8G1, its Fab and F(ab')(2) fragments, or 5A6 increased intracellular Ca(2+) levels two- to threefold. Prior ligation of LRP with 8G1 did not affect the increase in [Ca(2+)](i) observed on subsequent ligation of LRP with lactoferrin, P. exotoxin A, or lipoprotein lipase. Binding to LRP by 8G1, its Fab and F(ab')(2) fragments, or 5A6 increased inositol 1,4,5-trisphosphate (IP(3)) levels by 50 to 100%. Incubation of macrophages with guanosine 5', 3'-O(thio)-triphosphate (GTP-gamma-S) before treatment with antibody potentiated and sustained the 8G1-induced increase in IP(3) levels. Treatment of macrophages with guanyl-5'-yl thiophosphate prior to GTP-gamma-S treatment abolished the GTP-gamma-S-potentiated increase in IP(3) levels in 8G1-treated macrophages. Antibody-induced increases in IP(3) and [Ca(2+)](i) in macrophages on ligation of LRP were pertussis toxin sensitive. Binding of 8G1 or its Fab or F(ab')(2) fragments to LRP stimulated macrophage protein kinase C (PKC) activity as evaluated by histone IIIs phosphorylation by about two- to sevenfold. Staurosporin inhibited the anti-LRP antibody-induced increase in PKC activity. Ligation of LRP with 8G1 increased cellular cAMP levels about twofold. Preincubation of macrophage with the LRP-binding protein receptor-associated protein suppressed the 8G1-induced increase in cAMP levels. Thus, binding of antibodies directed against either chain of LRP triggers complex signaling cascades.

A method suitable for the isolation of monoclonal antibodies (Mabs) is described. The protocol utilizes a zirconia based column modified with ethylenediamine-N,N'-tetra(methylenephosphonic) acid to create a novel cation-exchange chromatographic support. Initial experiments using a linear salt gradient demonstrate the ability of this support to efficiently separate Mab from transferrin and bovine serum albumin in a model matrix. Results of the purification of Mab from an actual cell culture supernatant over a range in protein concentrations are also shown. Analyses by enzyme-linked immunosorbent assay and gel electrophoresis demonstrate that Mabs can be recovered from a cell culture supernatant at high yield (92-98%) and high purity (> 95%) in a single chromatographic step.

Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 = 6 +/- 4 microM). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and IV can be isolated, these two isoforms do not show a calcium-dependent interaction. These results suggest that the self-association of synaptotagmins with different calcium binding features may create a variety of calcium sensors characterized by distinct calcium sensitivities. This combinatorial hypothesis predicts that the probability of a single SSV exocytic event is determined, in addition to the gating properties of the presynaptic calcium channels, by the repertoire and relative abundance of distinct synaptotagmin isoforms present on the SSV surface.

Kinetoplastid RNA editing consists of the addition or deletion of uridines at specific sites within mitochondrial mRNAs. This unusual RNA processing event is catalyzed by a ribonucleoprotein (RNP) complex that includes editing site-specific endoribonuclease, RNA ligase and terminal uridylnucleotidyl transferase (Tutase) among its essential enzymatic activities. To identify the components of this RNP, monoclonal antibodies were raised against partially purified editing complexes. One antibody reacts with a mitochondrially located 45 kDa polypeptide (p45) which contains a conserved repetitive amino acid domain. p45 co-purifies with RNA ligase and Tutase in a large ( approximately 700 kDa) RNP, and anti-p45 antibody inhibits in vitro RNA editing. Thus, p45 is the first kinetoplastid RNA-editing-associated protein (REAP-1) that has been cloned and identified as a protein component of a functional editing complex.

The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.

The murine monoclonal antibody A7 (MAb A7) is reactive against most human gastric cancer cell lines. Using a nude mouse peritoneal dissemination model of human gastric cancer, we investigated targeted chemotherapy using a conjugate of neocarzinostatin (NCS) with MAb A7 (A7-NCS). After demonstrating cytotoxicity of the complex against the human gastric cancer cell line MKN45 in vitro, we intraperitoneally injected A7-NCS, NCS or saline into nude mice bearing peritoneally disseminated human gastric cancer. A7-NCS inhibited peritoneal dissemination significantly more effectively than NCS. MAb A7 may prove to be an effective carrier for antineoplastic drugs in patients with peritoneal dissemination of gastric cancer.

Human choriogonadotropin (hCG), according to its three dimensional structure as determined by X-ray diffraction, has three beta-hairpin loops each in the alpha and beta subunit designated as alpha 1, alpha 2 alpha 3 and beta 1 beta 2 and beta 3, respectively. Since similar beta-hairpin loops in NGF and TNF beta have been implicated in their direct interaction with the receptor, it prompted the present investigation to determine the role of such loops in receptor binding and post-receptor signaling events in hCG. Based on the three dimensional structure of hCG, radical mutations were introduced in the alpha loops by replacing hydrophobic alpha 18Phe and alpha 74Phe by hydrophilic Thr residues in the alpha 1 and alpha 3 loops, respectively, and positively charged alpha 45Lys by negatively charged Asp in the helical segment in the alpha 2 loop. The beta loops were mutated by replacement of the beta 1, beta 2 and beta 3 sequences with the corresponding hFSH sequences. These replacements included beta 22Gly, beta 24Pro and beta 25Val with Glu, Arg and Phe in beta 1, 45Leu Gln Gly Val Leu Pro Ala Leu Pro53 with Tyr Lys Asn Pro Ala Arg Pro Leu Ile in beta 2 and 73Pro Arg Gly with Ala His His in the beta 3 loop. Six mutants, hCG alpha 1 beta, hCG alpha 2 beta and hCG alpha 3 beta and hCG alpha beta 1, hCG alpha beta 2 and h CG alpha beta 3, were obtained by co-infection of the insect High-Five cells with baculovirus containing mutant alpha or beta cDNAs and that containing complimentary wild type beta or alpha cDNAs. The mutants were almost completely secreted in the culture medium and were over expressed at levels ranging between 4.5 to 29 micrograms/ml indicating that mutations had no effect on the secretion or subunit assembly of hCG. In order to remove any contaminating beta-subunit, the culture medium was passed through a column of an hCG beta-specific monoclonal antibody, B158. The receptor binding activity of the mutant hCG alpha 1 beta, in which alpha 18Phe was replaced with Thr, increased almost 200% relative to rehCG. Similarly, increase in the cAMP and progesterone stimulation by the mutant ranged between 150 to 200%. This increase is believed to be due to a short range conformational change in the mutant as a result of the mutation rather than direct involvement of alpha 18Phe in the receptor binding. The evidence in support of this was derived from the fact that the affinity or interaction between the two subunits was impaired as indicated by the first order rate constant of hCG alpha 1 beta (km = 4.1 x 10(-2) min-1) at pH 3.0 at 23 degrees C which is one order of magnitude greater relative to rehCG (kw = 4.6 x 10(-3) min-1). All other mutations had no effect on the receptor binding or signal transduction of hCG indicating that, unlike NGF or TNF beta, beta-hairpin loops in hCG were not directly involved in receptor binding or post-receptor signaling events. However, since the mutation in the alpha 1 loop affects the receptor binding site, its presence in the vicinity of the alpha 1 loop is highly likely.

Human chorionic gonadotropin (hCG) has four N-glycosyl chains, two in each subunit. Several analogs lacking one or more specific N-linked carbohydrate chains have been purified from insect cells by immunoaffinity chromatography on a monoclonal antibody, B17, column Traces of the hCG beta mutant present, if any, were removed by a second immunoaffinity chromatography on a column of hCG beta specific monoclonal antibody, B158. N-glycosylation was inhibited by the replacement of either Asn or Thr to Gln in the consensus sequence. -Asn x Ser/Thr-, for N-glycosylation. All analogs were overexpressed in High-Five insect cells with the expression levels ranging between 1.5 to 15 micrograms/ml and were found homogeneous by SDS-PAGE under nonreducing and reducing conditions. Their molecular sizes ranged between 34k to 44k. The receptor binding affinity of all the analogs was unaltered as determined by radio receptor assay using rat ovarian membranes. The availability of these analogs should facilitate studies on the effect of a specific carbohydrate chain on the conformation and in vivo properties of hCG.

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG2a and had kappa light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G1, G2 and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characterize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis. The nuclear protein(s) detected by Th-10a monoclonal antibody was mostly 95 kDa and also 83 kDa polypeptide. The results also indicated that the 95 kDa nuclear protein was phosphorylated in vivo.

We investigated the difference in natural resistance to Legionella pneumophila infection between aged (18-20-month-old) and young (3-month-old) mice of ddY strain. Aged mice were more susceptible to the bacterial infection than young mice; 50% lethal doses of L. pneumophila for aged and young mice were 2.2 x 10(7) and 8.5 x 10(7) colony forming units (CFU), respectively, after intraperitoneal injection of the bacteria. The bacterial burden in the livers was larger in aged than young mice after a challenge with a sublethal dose of L. pneumophila. However, peritoneal macrophages of aged mice paradoxically had a greater capacity to kill intracellular L. pneumophila than those of young mice. Interferon-gamma (IFN-gamma) production from naive spleen cells was compared after an in vitro stimulation with formalin-killed L. pneumophila. Spleen cells of aged mice produced significantly less IFN-gamma than those of young mice. When anti-murine IFN-gamma monoclonal antibody was administered before the bacterial infection, the subsequent bacterial burden in the livers significantly increased in young but not in aged mice. These data suggest that, in aged mice, IFN-gamma production is depressed at an early phase of L. pneumophila infection and it renders aged mice more susceptible to the infection.

Protein A Superose was employed to separate affinity-purified anticarbohydrate antibodies according to immunoglobulin G (IgG) subclass. Separation was achieved with a novel buffer system (disodium phosphate-sodium acetate-sodium chloride-glycine), which allowed the generation of a linear pH gradient from pH 8 to 3. Protein A-bound anti-carbohydrate antibodies were eluted as three peaks, two of them mainly containing IgG2 and one consisting of highly enriched IgG1. The enriched antibody preparations retained their functional activity. This separation procedure can be considered as an alternative to the preparation of IgG subclasses with subclass-specific monoclonal antibodies and could be employed whenever contamination with immune complexes has to be avoided.

S1, a heterophile antigen present on human sarcoma cell lines in culture, has been previously defined by this laboratory [1,2]. This antigen is also present in guinea-pig kidney. Purification of the antigen to homogeneity has now been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose, sephadex, high pressure liquid chromotography and affinity chromotography. S1 is a monomeric protein of 70,000 Da, as indicated by the presence of a single band on SDS-PAGE. Amino acid analysis demonstrates the prevalence of glycine, lysine and glutamic acid. Aspartic acid was found to be the N-terminal residue with further sequence of glycine-valine-alanine-glutamic acid (gly-val-ala-glut).

Systemically injected anti-acetylcholinesterase antibodies in rats cause selective lesions of preganglionic sympathetic neurons. Adult rats were examined up to four months after a single i.v. injection of murine monoclonal acetylcholinesterase antibodies or normal immunoglobulin G (1.5 mg). Within 4 h, antibody-treated rats developed ptosis, a sign of sympathetic dysfunction that was never reversed. Persistent pupillary constriction reflected preserved and unopposed parasympathetic function. Weight gain was depressed, but locomotor activity, excitability, and sensorimotor responses were normal, and gross neuromuscular performance was near normal. These findings were supported by biochemical evidence for selective sympathetic damage. Acetylcholinesterase activity was reduced for the whole period of observation in sympathetic ganglia and adrenal glands but fell only transiently in muscle and serum. At all times, choline acetyltransferase activity (a marker of presynaptic terminals) was unaffected in muscle but grossly depleted in ganglia. Light and electron microscopy showed that preganglionic sympathetic terminals of superior cervical ganglia were severely damaged while parasympathetic ganglia were less affected and motor endplates of skeletal muscle were apparently spared. Immunocytochemistry revealed punctate deposits of murine immunoglobulin G and complement component C3 in ganglionic neuropil 12 h after antibody injection. This finding was consistent with complement-mediated lysis of preganglionic terminals. Morphometric analysis of preganglionic neurons in the intermediolateral nucleus of the spinal cord showed progressive loss of cholinergic perikarya over several months. We conclude that antibody-induced destruction of ganglionic terminals leads to death of preganglionic sympathetic neurons and, hence, permanent dysautonomia.

To determine if systemically administered antibodies could reach antigenic targets and cause immunologic lesions in brains of newborn rats, murine monoclonal antibodies against rat acetylcholinesterase were injected i.p. on the first postnatal day. As early as 24 h after injection, antibodies were detected immunocytochemically in brain parenchyma, along with punctate debris that showed intense cholinesterase activity. Total acetylcholinesterase activity in the brain dropped by 30%, and 10S activity was almost undetectable at day 3, implying true enzyme loss since the antibodies did not directly impair catalytic function. At day 7, 10S acetylcholinesterase began to recover but the activity remained only half that of controls. At day 12, total acetylcholinesterase activity was still reduced (30% in whole brain, 40% in cerebral cortex), consistent with lasting damage to cholinesterase-expressing cortical neurons. This conclusion was confirmed by histochemical experiments showing a nearly complete disappearance of acetylcholinesterase fiber-staining in cerebral cortex and basal ganglia at days 4 and 8, with residual deficits at day 12. Choline acetyltransferase activity decreased in the cerebral cortex, implying a loss of cholinergic terminals, but specifically immunoreactive perikarya remained abundant in the basal forebrain. Immunocytochemistry showed no obvious changes in three non-cholinergic markers: tyrosine hydroxylase, tryptophan hydroxylase, and glutamic acid decarboxylase. Overall, it appeared that acetylcholinesterase antibodies induced widespread but reversible damage of cholinergic fibers and terminals, while sparing cholinergic cell bodies and many other neural systems.

How to obtain concentrated monoclonal antibodies from culture supernatant is of great importance to many scientists today. In this report, a simple, single-step technique is described to concentrate monoclonal antibodies directly from hybridoma-culture supernatant. The contaminants can be removed from the supernatant by using an ultrafiltration unit which allows macromolecules of less than 100,000 MW to pass through leaving behind monoclonal antibodies which is purified and concentrated more than 25-fold.

The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated. MN12 was freeze-dried in different formulations [without lyoprotectant or in the presence of sucrose, dextran, or hydroxypropyl-beta-cyclodextrin (HP beta CD)] and under varying conditions (with or without secondary drying). Subsequently, the monoclonal antibody was stored for 18 or 32 days at various temperatures (4, 37, or 56 degrees C). For comparison, solutions of MN12 were stored under the same conditions. Regardless of the lyoprotectant used, precipitation and a concomitant reduction of the antigen-binding capacity by about 10% were observed upon reconstitution of lyophilized MN12. HP beta CD proved to be the most effective stabilizer to prevent degradation of lyophilized MN12 during storage. Compared with MN12 solutions, HP beta CD-containing lyophilized MN12 cakes were more resistant to heat-induced charge alterations and loss of antigen-binding capacity.

Mice are highly resistant to Legionella pneumophila infection. To study the natural resistance, we used A/J and C57BL/6 mice which have macrophages permissive and non-permissive for the intracellular growth of L. pneumophila, respectively. The LD50 for A/J and C57BL/6 were 2.7 x 10(7) and 7.2 x 10(7) CFU, respectively, indicating that the difference in macrophage ability to regulate the bacterial growth had some effect on susceptibility to L. pneumophila. There was no difference between both strains in elimination of the bacteria from the blood stream within 5 h after infection. When mice were challenged intravenously with a sublethal dose (4 x 10(6) CFU), the bacterial burden in the liver at day 1 was significantly higher in A/J than in C57BL/6. The bacteria, thereafter, were eliminated rapidly from the liver at a similar rate in both strains. Elimination of the bacteria from the spleen and lungs was also delayed in A/J as compared to C57BL/6. Naive spleen cells of both strains in vitro could produce a large amount of interferon-gamma (IFN-gamma) one day after they were stimulated with formalin-killed L. pneumophila. When anti-murine IFN-gamma monoclonal antibody was administered, the bacterial burden in liver, spleen and lungs significantly increased in A/J, and also in C57BL/6 to some extent. We suggest that the innate macrophages' ability to regulate the intracellular bacterial growth and the endogenous IFN-gamma produced in a very early phase play a critical role in murine natural resistance against L. pneumophila infection.

1. The tubulovesicles of hog and rabbit gastric parietal cells were immunopurified from microsomes using monoclonal antibodies against the (H+, K+)-ATPase. 2. The best yields of immunoprecipitation were obtained with an ATPase/mAb molar ratio of 0.3: the immunoprecipitate contained 79 and 90% of the hog and rabbit microsomal PNPPase activity respectively and K(+)-stimulated ATPase specific activity was 221 +/- 29 mumoles Pi per hr and per mg of membrane protein. 3. The immunoprecipitate contained vesicles that were 85% cytoplasmic-side out, like tubulovesicles in vivo, demonstrating that the epitopes were cytoplasmic. 4. The alpha-beta protomer of (H+, K+)-ATPase accounted for 80 +/- 12% of the immunopurified proteins. 5. The major other proteins ran at 80, 75, 69, 57, 47, 44, 39, 34 and 32 kDa on the SDS-PAGE. 6. Comparative analysis between sucrose-gradient purified fractions and immunopurified tubulovesicles demonstrated that carbonic anhydrase and actin were contaminants and that the 53 kDa and presumably the 50 kDa bands of the gradient fraction were alpha and beta subunits of F1 ATPase.

Immunoenzymometric assays (IEMAs) for human insulin and intact proinsulin were developed using the amplification system developed by Johannsson et al. (Clin. Chim. Acta 148 (1985) 119-124) for the detection of the enzyme alkaline phosphatase. The detection limit of the assays was 0.8 pmol/l for proinsulin and 0.8 pmol/l for insulin whereas it was 1.8 pmol/l and 2.3 pmol/l respectively for the homologous immunoradiometric assays (IRMA). These assays are superior to immunoradiometric assays in terms of sensitivity, shelf-life of the labelled antibody and suitability for automation.

A decline in cell surface gamma-glutamyl transpeptidase specific activity was previously observed to be concomitant with C6 glial cell proliferation. To elucidate the underlying factor(s) mediating gamma-glutamyl transpeptidase down-regulation, the effects of C6 cell density and culture conditions on cell surface transpeptidase activity levels were investigated. After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities. The lower-density cultures showed an induction within 24 h of plating. As the cultures proliferated, the specific transpeptidase activities declined to a common low level at post-confluency. The gamma-glutamyl transpeptidase down-regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation. Induction and down-regulation of gamma-glutamyl transpeptidase activity at low cell densities were not a result of trypsinization. Supplementation of low-density cultures with conditioned medium, use of matrix-coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity. Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that down-regulation was not due to allosteric factors or an alteration in enzyme character. A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that gamma-glutamyl transpeptidase down-regulation is related to the concentration of enzyme at the cell surface. Immunocytochemical localization of gamma-glutamyl transpeptidase demonstrated that gamma-glutamyl transpeptidase antigen levels decrease as C6 cell density increases. These results led us to propose that cell-cell contact stimulates the disappearance of gamma-glutamyl transpeptidase from the surface of cultured C6 glial cells.

The selective retention of proteins on matrix-linked histidine has been shown to depend on chromatographic conditions: pH, temperature and ionic strength. An extension of this study to separate mouse monoclonal antibodies on histidyl-Sepharose is presented here; the roles of different functional groups such as imidazole, primary amine and carboxyl groups are elucidated by using histamine-Sepharose and histidine linked via the carboxyl group of the alpha-amino acid. We separated two monoclonal antibodies, immunoglobulin G1 (IgG1) from a culture supernatant and IgG2b from ascites fluid precipitated with 50% ammonium sulphate. The pseudoselective retention of monoclonal IgG1 on the three different matrices and IgG2b on histidyl-aminohexyl-Sepharose was achieved at pH 7.4. The purity of the final monoclonal antibody preparation determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions proved the separation of the monoclonal antibodies (IgG1, IgG2b) from other contaminating proteins such as albumin and transferrin. Quantitation of the mouse monoclonal antibodies was carried out using enzyme-linked immunosorbent assay.

We describe the production and functional characterization of 2 monocytic-cell-lineage-specific immunotoxins constructed with saporin emitoxin (SAP) from Saponaria officinalis. Interest in the production of these immunotoxins, of possible clinical relevance, has been raised by the availability of 2 MAbs of high specificity for circulating monocytes and M5b ANLL, thus envisaging their potential use in bone-marrow purging. SAP emitoxin was selected on the basis of the low cytotoxicity in unconjugated form, as opposed to highly specific cytotoxicity and favourable pharmacokinetical properties in the conjugated form. SPDP conjugation produced immunotoxins which retained serological specificity and protein-synthesis-inhibitory activity. The 2 immunotoxins did not interfere with bone-marrow progenitor-cell growth in a CFU-GM colony assay. On the contrary, they were capable of killing monocytic cells selectively, as demonstrated in phenotypical and functional assays. Thus these 2 novel immunotoxins appear to be promising reagents in purging autologous bone marrow prior to transplantation in patients suffering from monocytic leukaemia.

The neuronal specific protein NP185, found associated with brain clathrin-coated vesicles, formed a complex with unphosphorylated, but not with phosphorylated, clathrin light chains. The NP185-clathrin light chain complex was associated with casein kinase II activity, which, in the presence of polylysine, phosphorylated clathrin light chain b but not the NP185. The dissociation of this complex with 50% ethylene glycol pH 11.5 suggests that NP185 binds to hydrophobic domains of clathrin light chains. When NP185 molecules were retained by monoclonal antibody-linked Sepharose beads, they bound synaptic vesicles, decoated vesicles and synaptosomal plasma membrane. Immunohistochemistry on mouse cerebellar tissue sections using 8G8, a monoclonal antibody raised against NP185, showed neuronal specific labeling closely following synaptic distribution. In immunoblots, NP185 shares similar epitopes to those detected in another assembly polypeptide, AP-180, an indication that both proteins are identical. It appears that NP185 plays a specific role in nerve ending functions through its ability to induce clathrin to polymerize into cages, its interaction with synaptic vesicles, with the plasma membrane and with clathrin coat components.

The monoclonal antibody (mAb) 2G8 (subclass IgG2a) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys flesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. No crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.1.8) from human serum. Binding of mAb 2G8 to the globular G2 form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen-tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme-linked immunosorbent assay and Western blot showed that heat-denatured, diisopropylfluorophosphate-treated, CNBr- and trypsin-digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N-glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N-linked carbohydrates.

Three mouse monoclonal antibodies (Mab), RIV6, MN12, and WT31, were purified from cell culture supernatants containing foetal bovine serum (FBS) by two-step purification protocols, involving protein A affinity and ion exchange chromatography. Provided that the purification conditions were adapted to the physico-chemical properties of the individual Mab, clinical grade products could be obtained. The residual levels of bovine IgG originating from FBS were below 1% on a protein basis. Endotoxin levels were below 1 ng/ml. The contents of other serum proteins, DNA, and protein A were below or near the detection limits. The final products met the requirements for therapeutic Mab. Special attention was paid to the behaviour of foetal bovine IgG in the different purification steps. Large variations in the IgG contents of different batches of FBS were observed. However, the properties of the IgG fractions of the batches were very similar. A major IgG fraction with a low affinity for protein A and with components with relatively acidic isoelectric points (pIs) was distinguished from a minor fraction exhibiting a high affinity for protein A and a more diverse pI pattern. The impact of these findings on the purification strategy used for the Mab is discussed.

Estradiol (E2) induces an increase in the peptide elongation rate of isolated uterine ribosomes assayed in a cell-free protein synthesis system. An inhibitory factor, extracted from ribosomes of E2-deprived rats, was found to inhibit the peptide elongation reaction by acting on certain tRNAs to render them incapable of binding to aminoacyl-tRNA synthetases, thus reducing the availability of specific aminoacylated tRNAs required for the sequential translation of the codons in mRNA. The uterine ribosome-associated tRNA inactivator (RATI) has been partially purified and monoclonal antibodies (MABs) to RATI have been prepared. Specificity of the MABs for RATI was indicated by the inactivation of RATI in vitro by the anti-RATI MABs. RATI selectively inactivates deacylated, but not acylated, tRNAs and the inactivation does not appear to involve nuclease cleavage of the tRNA. Within 1 h after E2 treatment 50% of both RATI activity and immunoreactivity were lost from the uterine ribosome extracts, suggesting that E2 regulation of tRNA reutilization may occur through dissociation of RATI from the ribosomal site of tRNA deacylation or alteration in the structure of RATI resulting in inactivation both biologically and immunologically. We propose that RATI may function as an E2-regulatable 'switch' mechanism which inactivates, delays or defers the aminoacylation of certain tRNAs in the absence of E2 and which participates in the regulation of protein synthesis at the translational level by creating rate-limiting levels of certain tRNAs in the E2-deprived uterus.

The polyphosphoinositides, PIP and PIP2, have been proposed to regulate actin polymerization in vivo because they dissociate actin/gelsolin complexes in vitro. We tested this hypothesis by comparing the ability of EGF and bradykinin to affect PI metabolism and the actin cytoskeleton in A431 cells. EGF, but not bradykinin, was found to induce ruffling and dissociation of actin/gelsolin complexes in these cells. However, both EGF and bradykinin stimulated the accumulation of inositol phosphates in [3H]inositol-labeled cells indicating that stimulation of PI turnover is not sufficient for the induction of changes in actin/gelsolin complex levels. EGF stimulated a twofold increase in the level of PIP in A431 cells. Other phosphoinositide levels were not markedly altered. Treatment of the cells with cholera toxin abrogated the EGF-induced rise in PIP levels without altering the dissociation of actin from gelsolin. These data indicate that increases in PIP and/or PIP2 are not necessary for dissociation of actin/gelsolin complexes. Overall, these experiments suggest that in A431 cells, the effects of EGF on the actin cytoskeleton are unlikely to be mediated through changes in PIP or PIP2 levels.

The epitope specificity of two monoclonal antibodies against the S1 subunit (A4, A12) and one MAb against the S3 subunit (B9) of pertussis toxin, all protective in the mouse aerosol model of B. pertussis infection, but with different effects in assays of toxin-neutralizing activity, was examined in competitive binding enzyme immunoassays using biotinylated anti-pertussis toxin monoclonal antibodies or biotinylated goat anti-pertussis toxin polyclonal antibody after preincubation with unlabelled antibody. Biotinylated A4 was blocked by A4, A12, and B9; A12 was blocked by A4, A12, and B9. In contrast, biotinylated B9 was blocked by B9 and A4, but not by A12. All three monoclonal antibodies successfully blocked the anti-pertussis toxin polyclonal antibody; a mixture of the three anti-pertussis toxin monoclonal antibodies was more effective than any monoclonal antibody alone P less than or equal to 0.01). These data suggest that these three anti-pertussis toxin monoclonal antibodies recognize separate, but closely linked epitopes on pertussis toxin, and that epitopes on the S1 subunit and B-oligomer may induce protective immunity.