Production of a monoclonal antibody that recognizes the lipopolysaccharide of a Campylobacter-like organism.

A monoclonal antibody was produced to a Campylobacter-like organism (RMIT 32A) which was isolated from the terminal ileum of a pig with proliferative enteritis. Isotyping of the antibody revealed that it was an IgG2a with kappa light chains. Immunoblots using the antibody against proteinase-K-treated whole cell lysates of RMIT 32A, a selection of Campylobacter species and other enteric bacteria showed that the antibody was specific for RMIT 32A and was directed against the lipopolysaccharide. This antibody can be used for the specific detection of RMIT 32A.     

Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan

Monoclonal antibodies were raised against proteoglycan core protein isolated after chondroitinase ABC digestion of human articular cartilage proteoglycan monomer. Characterization of one of the monoclonal antibodies (1/20/5-D-4) indicated that it specifically recognized an antigenic determinant in the polysaccharide structure of both corneal and skeletal keratan sulfate. Enzyme immunoassay analyses indicated that the mouse monoclonal IgG1 recognized keratan sulfate in native proteoglycan aggregate and proteoglycan monomer preparations isolated from hyaline cartilages of a wide variety of animal species (human, monkey, cow, sheep, chicken, and shark cartilage). The 1/20/5-D-4 monoclonal antibody did not recognize antigenic determinants on proteoglycan isolated from Swarm rat chondrosarcoma. This finding is consistent with several biochemical analyses showing the absence of keratan sulfate in proteoglycan synthesised by this tissue. A variety of substructures isolated after selective cleavage of bovine nasal cartilage proteoglycan (Heineg?rd, D., and Axelsson, J. (1977) J. Biol. Chem. 252, 1971-1979) were used as competing antigens in radioimmunoassays to characterize the specificity of the 1/20/5-D-4 immunoglobulin. Substructures derived from the keratan sulfate attachment region of the proteoglycan (keratan sulfate peptides) showed the strongest inhibition. Both corneal and skeletal keratan sulfate peptides as competing antigens in radioimmunoassays showed similar inhibition when compared on the basis of their glucosamine content. Therefore, the 1/20/5-D-4 monoclonal antibody appears to recognize a common determinant in their polysaccharide moieties. Chemical desulfation of the keratan sulfate reduced the antigenicity of the glycosaminoglycan. The antibody did not recognize determinants present in dermatan sulfate, heparin, heparin sulfate, or hyaluronic acid.     

An indel within the C8 alpha subunit of human complement C8 mediates intracellular binding of C8 gamma and formation of C8 alpha-gamma

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex, or MAC. It is an oligomeric protein composed of three subunits (C8alpha, C8beta, C8gamma) that are products of different genes. In C8 from serum, these are arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. In this study, the site on C8alpha that mediates intracellular binding of C8gamma to form C8alpha-gamma was identified. From a comparative analysis of indels (insertions/deletions) in C8alpha and its structural homologues C8beta, C6, C7, and C9, it was determined that C8alpha contains a unique insertion (residues 159-175), which includes Cys(164) that forms the disulfide bond to C8gamma. Incorporation of this sequence into C8beta and coexpression of the resulting construct (iC8beta) with C8gamma produced iC8beta-gamma, an atypical disulfide-linked dimer. In related experiments, C8gamma was shown to bind noncovalently to mutant forms of C8alpha and iC8beta in which Cys(164)-->Gly(164) substitutions were made. In addition, C8gamma bound specifically to an immobilized synthetic peptide containing the mutant indel sequence. Together, these results indicate (a) intracellular binding of C8gamma to C8alpha is mediated principally by residues contained within the C8alpha indel, (b) binding is not strictly dependent on Cys(164), and (c) C8gamma must contain a complementary binding site for the C8alpha indel.     

A Trypanosoma cruzi monoclonal antibody that recognizes a superficial tubulin-like antigen

A monoclonal antibody (MAB 10), obtained from mice infected with Trypanosoma cruzi, was found to recognize a superficial antigen in living or fixed parasites. It reacted more strongly with T. cruzi than with related parasites such as T. brucei and Leishmania. In immunoblots it recognized a single trypanosoma polypeptide and also brain tubulin, both of which had the same electrophoretic mobility. Further analysis suggested that the alpha-tubulin subunit contained the epitope recognized by MAB 10. These results suggest that a surface tubulin-like protein is present is T. cruzi.     

Characterization of a monoclonal antibody B1 that recognizes phosphorylated Ser-158 in the activation peptide region of human coagulation factor IX

Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as gamma-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent K(d) value of 5 nm in the presence of Ca(2+) (EC(50) = 0.58 mm). B1 bound to the isolated AP of FIX and retained the Ca(2+) dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca(2+). These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca(2+)-bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158.     

A monoclonal antibody that recognizes Golgi-associated protein of cultured fibroblast cells

We have obtained a hybridoma clone, JLJ5a, which secretes monospecific antibody directed against a 110-kdalton protein of gerbil fibroma cells, Rat-1 fibroblasts, and L6 myoblasts. It appears to be localized in the Golgi apparatus by the following criteria: (a) In double- staining experiments the localization of the 110-kdalton protein by the JLJ5a monoclonal antibody was coincident with the reaction products of thiamine pyrophosphatase (one of the enzyme markers of the Golgi apparatus; Novikoff and Goldfischer, 1961, Proc. Natl. Acad. Sci. U.S.A. 47:802-810) in the same cells. (b) The staining pattern of the JLJ5a monoclonal antibody became fragmented and dispersed into vacuoles after pretreatment of the cells with Colcemid or monensin.     

Studies of the developing chick retina using monoclonal antibody 8A2 that recognizes a novel set of gangliosides

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9-O-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab D1.1 which recognizes 9-O-acetyl GD3.     

MPM-12: a monoclonal antibody that predominantly stains mitotic cells and recognizes a protein kinase.

The monoclonal antibody MPM-12, raised by using partially purified extract of mitotic HeLa cells as the immunogen, preferentially stains the cytoplasm of mitotic cells by indirect immunofluorescence without exhibiting any species specificity. On immunoblots, MPM-12 recognizes three bands, of 155, 88, and 68 kDa, in mitotic HeLa cell extract but only the 68-kDa band in interphase cell extract. The 68-kDa band seems to be associated with chromatin while the other two are not. All three MPM-12 reactive peptides are phosphorylated, and the phosphorylation seems to be required for MPM-12 reactivity. The MPM-12 immunocomplexes exhibit autophosphorylating and histone H1 kinase activity.     

A monoclonal antibody that recognizes a basolateral membrane protein in A6 epithelial cells

The A6 cell line is a model for tight epithelia and studies of epithelial polarity. Monoclonal antibodies (MAbs) were produced by immunization of mice with intact A6 cells and fusion of spleen cells to generate hybridomas. Hybridoma supernatants were screened by ELISA to select MAbs binding to the apical membrane of confluent A6 cells. Localization of MAb binding was examined by indirect immunofluorescence using cross sections of A6 monolayers grown on collagen coated filters. One MAb, designated 13F12, was positive by apical surface ELISA but localized specifically to the basolateral membrane of cross sections of A6 monolayers on filters. Immunofluorescence labeling of confluent A6 cells grown on glass cover slips revealed that MAb 13F12 does not bind to the apical membrane, but binds to basolateral determinants in the regions of domes, where it appears able to penetrate cellular junctions. Subconfluent A6 cells express the antigen all over the cell surface. Cells approaching confluency express the antigen on the apical membrane of some cells but not others, and as the cells reach confluency, the antigen disappears from the apical surface, and the cells become fully polarized. A6 cells at confluency on glass cover slips are equally polarized as cells grown on filters with respect to this antigen. The antigen has been identified by immunoprecipitation as a 22 kDa protein. High concentrations of MAb 13F12 did not inhibit cell plating, indicating that the antigenic site is not directly involved in cell adhesion to the substrate. MAb 13F12 should prove to be a useful tool to study many aspects of epithelial polarity, including the signals involved in sorting of proteins to specific membrane domains.     

A novel monoclonal antibody that recognizes apical membrane of frog taste cells

We established a hybridoma clone 1N1 that produced a monoclonal antibody to stain the apical portion of frog taste cells, by directly immunizing taste discs of the bullfrog (Rana catesbeiana) without any dispersion procedure of the taste organ. The antibody stained discrete regions on the surface of the taste discs, but did not stain the epithelium sheet of the tongue devoid of taste discs. The antibody stained approximately 93% of the taste discs tested (172/184) derived from nine frogs, showing that distribution of the antigen was common to most of the taste discs. The following observations strongly suggested that the antibody recognized a certain antigen on the apical membrane of the taste cells. (i) The antibody selectively stained cross points of intermucus areas on the surface of the taste disc. Neither the mucus cells nor the wing cells that mainly cover the surface were stained with the antibody. (ii) Dispersed taste cells were prepared by calcium ion chelating and subsequently by collagenase treatment to avoid digestion of the antigen. The antibody stained the apical end of the taste cells.      

A monoclonal antibody recognizes an O-acylated sialic acid in a human melanoma-associated ganglioside

Monoclonal antibody D1.1 originally prepared against the B49 cell line derived from a rat brain tumor was shown to react with a ganglioside present in fetal rat brain. We have found that this antigen is also present in human malignant melanoma tumors as well as many melanoma cell lines. The ganglioside from human melanoma cell lines migrates between GM1 and GM2 on one-dimensional thin layer chromatography. Analysis by two-dimensional thin layer chromatography with intermediate ammonia treatment suggests that the ganglioside contains one or more base-labile O-acyl esters. Mild base hydrolysis under conditions known to remove O-acyl esters results in complete loss of antigenic reactivity. Thus, the alkali-labile moiety is a critical component of the epitope recognized by the antibody. Analysis of the sialic acids of total gangliosides from [6-3H]glucosamine-labeled melanoma cells showed that approximately 10% of these molecules are O-acylated. Similar analysis of the purified ganglioside showed that greater than 30% of the sialic acids comigrated with authentic 9-O-acetyl-N-acetylneuraminic acid. The antibody did not cross-react with normal human skin melanocytes nor with any of a large number of normal human adult and fetal tissues. The antibody also did not react with numerous other malignant cell lines studied. These findings suggest that the antigenic epitope defined by antibody D1.1 contains an O-acylated sialic acid and may arise from aberrant O-acetylation occurring in human malignant melanoma cells.     

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.     

Experimental allergic encephalomyelitis: successful treatment in vivo with a monoclonal antibody that recognizes T helper cells

Monoclonal antibody W3/25, which recognizes rat T helper cells, was injected i.p. or i.v. into Lewis rats with clinical signs of experimental allergic encephalomyelitis (EAE). This treatment, which was given 12 or 13 days after inducing EAE by the injection of purified myelin basic protein, significantly affected the course of the disease in that treated animals recovered, on the average, in 2 days, whereas control animals exhibited signs of EAE for a minimum of 4 days. Two pan-T cell monoclonal antibodies given in similar dosage failed to affect the course of the disease.     

A monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor CD44.

A monoclonal antibody, AF3, was previously shown to specifically inhibit poliovirus binding to HeLa cells and to detect a 100-kDa glycoprotein only in cell lines and tissues permissive for poliovirus infection. These results suggested that the 100-kDa protein may be involved in the pathogenesis of poliomyelitis and the cellular function of the poliovirus receptor site. To study further the role of the 100-kDa protein in poliovirus attachment, immunoaffinity purification, amino acid sequencing, and cDNA cloning were undertaken. The results demonstrate that antibody AF3 reacts with the lymphocyte homing receptor CD44, a multifunctional cell surface glycoprotein involved in the homing of circulating lymphocytes to lymph nodes and the modulation of lymphocyte adhesion and activation. Antibody AF3 reacts with a subset of CD44 molecules (AF3CD44H), which appears to be a small fraction of the heterogeneously glycosylated CD44 molecules expressed on hematopoietic and nonhematopoietic cells. Anti-CD44 monoclonal antibodies, previously reported to induce CD44-mediated modulation of lymphocyte activation and adhesion, compete with 125I-AF3 in binding assays, demonstrating functional overlap among the epitopes. The anti-CD44 monoclonal antibody A3D8, which binds to a greater molecular weight range of CD44 than does AF3, inhibits poliovirus binding to a similar degree. CD44 does not act as a poliovirus receptor, since CD44-expressing mouse L-cell transformants did not bind poliovirus. The poliovirus receptor and AF3CD44H may be noncovalently associated, or they may interact through the cytoskeleton or signal transduction pathways.     

Characterization of a novel anti-peptide antibody that recognizes a specific conformation of the platelet-derived growth factor receptor.

Two site-specific anti-peptide antibodies (AbP1 and AbP2) were raised against the platelet-derived growth factor (PDGF) receptor. These two sites correspond to amino acid residues 977 through 988 (peptide 1) and 932 through 947 (peptide 2) of the murine PDGF receptor. Both antibodies recognized human and murine PDGF receptors in immunoprecipitation and immunoblotting analyses. None of the antibodies was directed to phosphotyrosine. One of the antibodies (AbP2) showed unusual antigen recognition specificity. This antibody specifically recognized the tyrosine-phosphorylated PDGF receptor and not the unphosphorylated native receptor, suggesting that recognition by this antibody requires a specific conformation that is induced by PDGF-stimulated autophosphorylation.     

Characterization of human sperm surface antigens with monoclonal antibodies

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.     

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.     

Characteristics of a monoclonal antibody (WT-31) that recognizes a common epitope on the human T cell receptor for antigen

We describe a monoclonal antibody, WT-31, that reacted with all human T lymphocytes. Electrophoretic analysis of the material reacting with WT-31 revealed that it precipitated predominantly an 80-kD disulfide-linked heterodimer from the cell surface-labeled T leukemic cell line HPB-ALL. This heterodimer was identical to the one precipitated with a recently described monoclonal reagent, T40/25, which recognizes a clonotypic structure on HPB-ALL. The target antigen of WT-31 comodulated with T3 after incubation of T cells with excess anti-T3 antibody, indicating that the WT-31 target antigen is associated with T3. We also found that anti-T3 reagents, but not the clonotypic reagent T40/25, blocked binding of FITC-labeled WT-31 to HPB-ALL cells. This indicates that the T cell receptor epitope recognized by WT-31 is located close to the epitopes recognized by the anti-T3 reagents anti-Leu-4 and SPV-T3b but distal from the clonotypic T40/25 epitope. Functional studies showed that WT-31 reacts similar to anti-T3 antibodies. It is mitogenic for resting T cells, blocks cytolysis mediated by alloantigen-specific CTL clones, and induces antigen-nonspecific cytolysis by CTL clones against Daudi target cells. WT-31 did not inhibit the formation of conjugates, but it blocked cytolysis just before or during the Ca2++-dependent programming for lysis. We conclude that WT-31 is an antibody that recognizes a common determinant on the T cell receptor for antigen. The present results support the notion that the two chains of the T cell receptor (alpha and beta) form a functional protein ensemble with the three invariable T3 polypeptide chains (T3-gamma-, delta-, epsilon).     

A monoclonal antibody that recognizes mammalian cortical granules and a 32-kilodalton protein in mouse eggs

The fertilization-induced exocytosis of egg cortical granules (CGs) is responsible for a block to polyspermy, crucial to the viability of many species. The contents of mammalian CGs have been an elusive target for analysis because of picogram quantities of CG proteins. By using media enriched in secreted CG contents from calcium ionophore-induced eggs as an immunogen, a monoclonal antibody was raised that immunolocalized to structures in the mouse egg cortex with all the hallmarks of CGs. These structures were the correct size, absent from the region over the metaphase II spindle, and greatly reduced after fertilization. Double-labeling experiments confirmed that the antibody recognized the same population of CGs as those recognized by Lens culinaris agglutinin. On Western blots, the antibody primarily recognized a 32-kDa protein (and secondarily one at approximately 25 kDa) in mouse eggs. Analysis of biotin-labeled secreted proteins from activated eggs confirmed that CGs release only a small number of major proteins (45, 34, 32, 28, and approximately 20 kDa by SDS-PAGE). We therefore propose that the 32-kDa protein identified by this antibody is likely to correspond to the 32-kDa protein released from activated eggs and that it may be involved in the block to polyspermy. These methods should make it possible to generate additional antibodies to study the structure of CG components as well as their roles in the polyspermy block and CG biogenesis.     

A unique monoclonal antibody that recognizes mature p17 of HIV-1 but not its precursor.

The entire and partial gag regions of human immunodeficiency virus type 1 (HIV-1) were overproduced in Escherichia coli and used for epitope mapping of antibodies against p17. We found that a mouse monoclonal antibody to p17, V17 recognizes the mature p17 but not the unprocessed Gag proteins containing the entire p17 moiety. Further analysis revealed that V17 recognizes the C-terminal 12-amino-acid region of p17 having free C-terminus. This monoclonal antibody may be useful for monitoring the maturation of virus particles.