Mouse Polyclonal Antiserum Research Articles

CDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica.

A gamma gt11 cDNA library was constructed from poly(U)-Sepharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-125I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degrees C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

Identification of a human protein homologous to the mouse Lyb-2 B cell differentiation antigen and sequence of the corresponding cDNA.

We have isolated and sequenced cDNA clones encoding the human homolog of the mouse Lyb-2 B cell differentiation Ag. Previous data suggest that Lyb-2 might represent a growth factor or lymphokine receptor. Human Lyb-2 mRNA is expressed in normal human tonsils and bone marrow cells, in the pre-B cell line REH, in three Burkitt lymphoma cell lines, and in some EBV-transformed B cell lines, but not in antibody-secreting myeloma cell lines, T cell lines, or a promyelocytic leukemia cell line. These data indicate that expression of human Lyb-2 is restricted to B lineage cells and turned off in antibody-secreting plasma cells. A polyclonal mouse antiserum was raised against human Lyb-2 and immunoprecipitates a Mr 42,000 protein from REH, Raji, and Daudi cells and from mouse L(tk) cells transfected with the human Lyb-2 cDNA in an expression vector. The human Lyb-2 protein is related to both the asialoglycoprotein receptor and CD23, the B cell-specific FcR for IgE. These data demonstrate that human B cells express a previously undescribed cell surface protein that is homologous to mouse Lyb-2 and has a similar pattern of expression during B cell development.

Cytotoxicity of glucose oxidase conjugated with antibodies to target cells: killing efficiency depends on the conjugate internalization

The cytotoxic action of glucose oxidase conjugated with antibodies against the target cells has been examined in a culture of human endothelial cells. Internalizable (anti-endothelial, MoaB E25) and non-internalizable (anti-fibronectin, MoAb FN) monoclonal antibodies were employed as vectors. Anti-endothelial monoclonal antibody E78 (whether it can be internalized by endothelial cells is unclear) and polyclonal mouse antiserum to the human endothelium were also used. The conjugates were prepared by oxidation of the enzyme carbohydrate moiety with periodate. Free conjugates display similar enzyme activity in glucose solution. In contrast to glucose oxidase, conjugated with no-immune IgG, antibody-conjugated glucose oxidase binds specifically to target cells. The efficiency of targeting was different for various conjugates. Targeting via the anti-fibronectin antibody and anti-endothelial antiserum provided maximal quantitative binding of glucose oxidase to endothelial cells, while the conjugates with MoAb E25 and MoAb E78 monoclonal antibodies provided less effective binding. In the presence of glucose, targeted glucose oxidase generated H 2O 2. Hydrogen peroxide is relatively stable in buffer, but rapidly decays in the culture medium supplemented with 20% human serum. Though the quantitative binding of MoAb E25-conjugated glucose oxidase was minimal comparing to other conjugates, targeting via MoAb E25 produced the maximal cytotoxic effect as well as targeting via polyclonal antiserum. The killing efficiencies of MoAb FN-conjugated and MoAb E78-conjugated glucose oxidase were about 30-fold lower. The high efficiency of the MoAb E25-conjugated enzyme may be due to its internalization by target cells. Internalization can lead to unaccessibility of generated H 2O 2 for extracellular scavengers and pH optimization for glucose oxidase activity, which provides valuable advantages for the cytotoxicity of the conjugate. Thus, cytotoxicity of antibody-conjugated glucose oxidase depends not only on the efficiency of specific binding to the target cell, but also on the fate of cell-bound conjugate. Cytotoxicity is extremely effective in case of ‘internalizable’ conjugate and drastically less effective in case of ‘non-internalizable’ conjugate.

Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells.

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.

A major antigen on the outer envelope of a human oral spirochete, Treponema denticola.

Human oral spirochetes are prominent inhabitants of subgingival plaque in patients with periodontal disease. By immunoelectron microscopy using protein A-gold complexes and either polyclonal mouse antiserum against the 53-kDa antigen or 53-kDa-antigen-specific monoclonal antibody, a major polypeptide antigen, with a molecular weight of 53,000 (molecular size, 53 kilodaltons [kDa]), of a human oral spirochete, Treponema denticola ATCC 33520, was found to localize on the surface of the outer envelope.

Demonstration of Membrane Association and Surface Location of Mycoplasma pneumoniae Antigens Using Monoclonal Antibodies

We examined 10 monoclonal antibodies (mAbs) directed against Mycoplasma pneumoniae proteins of 200, 170, 67, 46 and 42 kDa, and one mAb directed against a glycolipid component. The membrane association of the antigens reacting with our mAbs was investigated, in particular by phase-fractionation involving use of the detergent Triton X-114. The 170 kDa protein was shown to be membrane-associated, and surface exposure of this antigen was demonstrated by its disappearance from SDS-PAGE patterns after treatment of intact mycoplasmas with proteolytic enzymes. Cross-reactions with protein antigens of Mycoplasma genitalium were also shown. A mAb directed against a component of a lipid extract, prepared by the method used for preparation of the antigen used in the complement fixation (CF) test for serological diagnosis of M. pneumoniae infection, reacted with one major and a few minor bands in thin-layer chromatography (TLC) of the crude extract. The glycolipid character of this major antigen was demonstrated by treatment of the extract with sodium periodate, and by development of the TLC with orcinol/ferric chloride. These reactive bands were the same as those detected by the use of polyclonal mouse antiserum and a human convalescent serum, a result showing that the CF antigen contains a glycolipid moiety reacting with our mAb. The surface exposure of this antigen was demonstrated by binding of mAbs to intact cells.

A baculovirus polyhedral envelope-associated protein: Genetic location, nucleotide sequence, and immunocytochemical characterization

Using a polyclonal mouse antiserum produced against purified virions of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV), two immunoreactive λgtII clones were identified which contained nonoverlapping insert DNAs which mapped to a single open reading frame (ORF) in the HindIII-M fragment. Analysis of nucleotide sequence data indicates that this ORF encodes a protein with a MW of 32.4 kDa. A trpE-p32 gene fusion containing the entire p32 ORF was constructed, and the fusion protein was purified and used to immunize rabbits. Western blot analysis and immunofluorescence studies using the anti-TrpE-p32 antiserum detected a polyhedra-derived virus (PDV)-associated protein of 32 kDa at 24 hr postinfection (hr p.i.). The protein was observed in the cytoplasm nuccleus at 24 hr p.i. and became concentrated in the cytoplasm late in infection. Western blot analysis and immunofluorescent microscopy of polyhedra solubilized under various conditions indicated that p32 is associated with the polyhedral envelope. The predicted amino acid sequence for p32 showed 58% amino acid identity with the predicted amino acid sequence for an ORF (ORF 3) in a similar region of the genome of the MNPV of Autographa californica (AcMNPV). The solubility properties of the p32 protein and reciprocal immunoblotting experiments indicate the OpMNPV p32 gene encodes a protein which is homologous to the polyhedral envelope-associated phosphoprotein of AcMNPV, pp34, recently reported by M. A. Whitt and J. S. Manning [(1988) Virology 163, 33–42].

Production and characterization of monoclonal antibodies against Staphylococcus aureus α-toxin

L. Blomqvist and AM. Sjögren. Production and characterization of monoclonal antibodies against Staphylococcus aureus α-toxin. Toxicon 26, 265 – 273, 1988. — Murine monoclonal antibodies against staphylococcal α-toxin were produced using a well-characterized α-toxin fragment preparation as immunizing agent. Three monoclonal antibodies were selected for anti-α-toxin ativity in an ELISA using α-toxin as antigen. The monoclonal antibodies (MAbs) belonged to different immunoglobulin classes/subclasses and showed different abilities to neutralize the hemolytic, cell-membrane-damaging, dermonecrotizing and lethal action of α-toxin. One MAb was superior to mouse polyclonal antiserum in all test systems except for hemolysis, whereas another MAb neutralized essentially as the polyclonal serum. The third MAb did not neutralize the hemolytic or dermonecrotic effect but still inhibited the lethal and membrane-damaging effect of α-toxin. These results indicate that the three MAbs recognize different epitopes on the toxin molecule and that different biological activities might correspond to these epitopes.

Neuron localization and neuroblastoma cell expression of brain-derived growth factor

Bovine brain-derived growth factor (BDGF) is a approximately 16-17 kD polypeptide mitogen with a broad spectrum of cell specificity. Using a highly specific mouse polyclonal anti-BDGF antiserum for indirect immunoperoxidase and immunofluorescent stainings, BDGF was found to be specifically localized in the neurons of bovine brain cortex. The indirect immunofluorescent staining was blocked by the presence of excess purified BDGF. Human neuroblastoma cells showed cytoplasmic staining with anti-BDGF antiserum. The cell lysates of neuroblastoma cells elicited a BDGF-like activity which could be completely inhibited by preincubation with anti-BDGF antiserum.

Mouse antisera specific for desmosomal adhesion molecules of suprabasal skin cells, meninges, and meningioma.

Mouse polyclonal antisera were raised to the Mr 130,000 and Mr 115,000 cell surface glycoproteins, desmocollins, of desmosomes from bovine nasal epithelium. Immunoblotting confirmed that the antisera were specific for the desmocollins. An immunofluorescence study showed that the antisera distinguished between the basal and suprabasal layers of bovine and human epidermis. The antibodies reacted with cultured keratinocytes only after calcium-induced stratification. In epidermis, therefore, there appears to be a difference between the desmocollins of basal and suprabasal cells that may be important in relation to epidermal differentiation. Previous work has shown that polyclonal antisera raised in other animals (guinea pigs and rabbits) against desmocollins, as well as against other desmosomal components, react with all desmosome-containing epithelia. In contrast, an immunofluorescence survey of bovine, rat, and human tissues showed that the present mouse antisera stained only suprabasal skin cells and the arachnoid layer of the meninges, demonstrating that these have common determinants that distinguished their desmocollins from those of all other tissues. The antibodies also stained 11 of 12 meningiomas and, therefore, may be useful as a marker not only for the diagnosis of these tumors but also for investigation of their histogenesis.

A Plasmodium falciparum antigen containing clusters of asparagine residues.

A genomic DNA fragment that encodes a Plasmodium falciparum antigen has been isolated by using human antibodies eluted from the membrane of infected erythrocytes. The antigen has a very unusual primary structure; it is exceptionally rich in asparagine residues, many of which are distributed in clusters (2-15 residues) along the polypeptide chain. Unlike many P. falciparum antigens, this protein lacks tandemly repeated sequences. The antigen is distinct from Pf 155, a merozoite-derived antigen deposited in the membrane of infected erythrocytes, but contains epitopes that crossreact with anti-Pf 155 antibodies. Antisera prepared in mice against the asparagine-rich protein react with late-stage parasites in indirect immunofluorescence. In an in vitro merozoite reinvasion assay, the IgG fraction of a mouse polyclonal antiserum, as well as a mouse monoclonal antibody, gave significant inhibition. Three polypeptides (Mr 36,000, 30,000, and 15,000) were recognized by these antibodies on immunoblots of P. falciparum extracts.

The synergistic neutralization of Newcastle disease virus by two monoclonal antibodies to its haemagglutinin-neuraminidase protein.

Monoclonal antibodies (MAbs) to 2 epitopes on the haemagglutinin-neuraminidase (HN) protein of the Ulster strain of Newcastle disease virus neutralized synergistically: MAbs to HN-1 and HN-2 neutralized 1.2 and 1.7 log10 infectious units (i.u.) of virus when single as compared to 4.1 when combined. Although MAb to HN-1 but not to HN-2 inhibited haemagglutination they both neutralized more virus on desialyzed cells compared to normal cells and were considered to have interfered with viral attachments in a cooperative manner when combined. A third MAb to fusion (F) protein reduced infectivity by five log10 i.u. HN-1, HN-2 and F were not the only immunodominant epitopes because mutants, which simultaneously lacked all 3 epitopes, were indistinguishable from wild type when neutralized by polyclonal mouse, rabbit or chicken antiserum to whole virus.

A monoclonal antibody equivalent to anti-rat neural antigen-1 as a marker for schwann cells

The monoclonal antibody 217c, raised by Peng et al. [(1982) Science, Wash. 215, 1102–1104 in mice against the rat glioma cell line C6, can be used as a marker for normal Schwann cells. In mixed cultures of Schwann cells and fibroblasts from neonatal rat sciatic nerve, this monoclonal antibody, detected by indirect immunofiuorescence, bound to the surface of cells with the same elongated morphology as those that express a previously described surface antigen, rat neural antigen-1 (Ran-1), defined by polyclonal mouse antisera. In these experiments Ran-1 and the antigenic determinant recognized by monoclonal 217c were both found on normal rat Schwann cells and on the rat glial tumor cell lines C6, 33B and 21A and the pheochromocytoma PC 12. Neither ami-Ran-1 nor the monoclonal antibody bound to neurons, fibroblasts or glial cells in newborn rat cerebellum cultures, the rat muscle cell line L6, the transformed rat fibroblast cell line Rat 1, the rat brain tumor cell line B28 or the mouse Schwannoma cell line TR6B. Thus the monoclonal 217c behaved as if it were detecting Ran-1 by binding to normal rat Schwann cells and to those tumor cells that have this antigen. Our data show that this monoclonal antibody is a reliable and convenient marker for rat Schwann cells in culture.

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients.

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.

Diurnal variation in asthma.

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.

Neutralization of Newcastle disease virus by monoclonal antibodies to the hemagglutinin-neuraminidase glycoprotein: requirement for antibodies to four sites for complete neutralization.

The neutralizing characteristics of monoclonal antibodies directed to four antigenic sites on the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus were determined. Neutralization by each antibody resulted in a persistent fraction of nonneutralized virus which varied from 1 to 17% depending on the hemagglutinin-neuraminidase site recognized, but not on the antibody. The addition of antibodies to all four sites on the hemagglutinin-neuraminidase glycoprotein was required to give a level of neutralization comparable with that obtained with polyclonal mouse antiserum. The high persistent fractions were not due to viral aggregates, a high level of variants in the virus stock, the use of insufficient antibody, low antibody avidity, or an effect peculiar to the use of the chicken cells as host. The addition of rabbit anti-mouse immunoglobulin to the persistent fraction left by any of the antibodies resulted in a further reduction in infectivity, often by as much as two logs. Thus, some viral particles are capable of binding antibody while retaining their infectivity. The implications of these findings to the mechanism of neutralization are discussed.