Monoclonal antibodies as probes for Aedes aegypti trypsin

Two murine monoclonal cell lines secreting antibodies against mosquito trypsin were produced by the hybridoma technique. Selected clones of each line were expanded, injected into mice or rats and the immunoglobulins were purified from ascites fluid. Clones of one cell-line (Mab 1–7) recognized only the major 30 kD trypsin from imaginal midguts on SDS-western blots, while trypsin from larval midguts. bovine trypsin and trypsin from other mosquito species were not recognized. This antibody did not distinguish trypsin from 19 strains within the same species, collected around the world. The second line (Mab 11–12) exhibited crossreactivities with other species when applied to a native blot. Analysis of the digestive cycle with these antibodies revealed an increase in immunoreactivity after blood-feeding with both immunoblots and radioimmunoassays, confirming earlier reports based on enzymatic assays.     

Radioimmunoassay of the link proteins associated with bovine nasal cartilage proteoglycan.

Link proteins from bovine nasal cartilage have been purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Baker, J.R., and Caterson, B. (1979) J. Biol. Chem. 254, 2387-2393) and used to raise antisera in rabbits. A sensitive radioimmunoassay procedure utilizing binding of 125I-labeled antigen . antibody complexes to Protein A of Staphylococcus aureus has served to demonstrate the specificity of the antisera for the link proteins. The lack of reactivity with proteoglycan fractions indicates that link proteins and proteoglycan do not share antigenic determinants. This result is in accord with published cyanogen bromide peptide cleavage data (Baker, J.R., and Caterson B. (1977) Biochem. Biophys. Res. Commun. 77, 1-10) which showed proteoglycan and link protein to be structurally dissimilar. The radioimmunoassay procedure has been used to quantitate small amounts of link protein which remain associated with proteoglycan after purification by equilibrium density gradient centrifugation in 4 M guanidine HCl and by gel chromatography in sodium dodecyl sulfate.     

Immunological studies of bovine nasal cartilage proteoglycan "link proteins".

Bovine nasal cartilage proteoglycan aggregates are dissociated and separated by density gradient centrifugation in 4 M guanidine into proteoglycan subunit (PGS) and glycoprotein link (GPL) fractions, the latter containing hyaluronic acid and "link proteins" responsible for aggregate formation. It was previously concluded on the basis of immunodiffusion studies that GPL has two antigenic components, one in common with PGS and one specific for the link proteins. However, in the present study it was found that antisera to PGS, which should lack link proteins, reacted with both "subunit" and "link" components of GPL, and antisera to fragments of PGS derived from the hyaluronic acid-binding portion of the molecule reacted preferentially with the link component. Reduction and alkylation of GPL led to modification of the reactions of both anti-GPL and anti-PGS sera with its link component. These immunodiffusion results indicate that the proteoglycan subunit and the link proteins are immunologically related and suggest that the link proteins may be identical with and derived from the hyaluronic acid binding portion of the proteoglycan subunit.     

Monoclonal antibodies as probes of epithelial membrane polarization

Monoclonal antibodies directed against antigens in the apical plasma membrane of the toad kidney epithelial cell line A6 were produced to probe the phenomena that underlie the genesis and maintenance of epithelial polarity. Two of these antibodies, 17D7 and 18C3, were selected for detailed study here. 17D7 is directed against a 23-kD peptide found on both the apical and basolateral surfaces of the A6 epithelium whereas 18C3 recognizes a lipid localized to the apical membrane only. This novel observation of an apically localized epithelial lipid species indicates the existence of a specific sorting and insertion process for this, and perhaps other, epithelial plasma membrane lipids. The antibody-antigen complexes formed by both these monoclonal antibodies are rapidly internalized by the A6 cells, but only the 18C3-antigen complex is recycled to the plasma membrane. In contrast to the apical localization of the free antigen, however, the 18C3-antigen complex is recycled to both the apical and basolateral surface of the epithelium, which indicates that monoclonal antibody binding interferes in some way with the normal sorting process for this apical lipid antigen.     

Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin.

Monoclonal antibodies were raised that specifically recognize the NH2-terminal neoepitope sequence present in link protein cleavage products derived from stromelysin-degraded proteoglycan aggregate. Competitive enzyme-linked immunosorbent assay, using synthetic peptides as inhibitors, showed that one of these antibodies (CH-3) required, for antibody recognition, the free NH2-terminal amino acid isoleucine (residue 17 of the intact protein) in the sequence NH2-IQAENG at the stromelysin cleavage site of link protein 3. Human proteoglycan aggregate was digested with recombinant human stromelysin, bovine chymotrypsin, bovine trypsin, and porcine elastase, and their respective link protein degradation products were tested for immunoreactivity with antibody CH-3. Only stromelysin- and chymotrypsin-generated link protein 3 were recognized by antibody CH-3. Both of these enzymes generate link protein NH2 termini with the sequence 17IQAENG. . .; hence these studies indicated that monoclonal antibody CH-3 recognized this neoepitope sequence in only specific proteolytically modified link protein molecules. Since the occurrence of link protein 3 increases with aging, the incidence of CH-3 epitope in proteoglycans isolated from human knee articular cartilage of individuals of different ages was investigated. The prevalence of CH-3 epitope was found to be highest in newborn and adolescent articular cartilage samples. However, little CH-3 epitope was detected in older adult cartilage, although considerably more link protein 3 was present in these samples. These results suggest that additional proteolytic agents are responsible for the increased occurrence of link protein degradation products with aging.     

Monoclonal antibodies as probes for plasma membrane domains in the exocrine pancreas

Monoclonal antibodies (mAb) were generated as probes for the plasma membrane domains of pancreatic acinar cells. Primary monolayer cultures of mouse pancreatic acinar cells, which have an expanded apical surface relative to normal pancreas, were used to immunize rats. With conventional immunization and fusion protocols, 3% of the hybridomas were positive against the acinar lumen by indirect immunofluorescence of mouse pancreas cryosections. Culturing of spleen cells from an immunized rat on the apical surface of acinar cell monolayer cultures before fusion with the myeloma (an in vitro boost) doubled the percentage of hybridomas producing apical membrane-specific mAb. Monoclonal antibodies were characterized by immunofluorescence, ultrastructural immunoperoxidase cytochemistry, immunoprecipitation, and immunoblotting. One antibody, acinar-1 (IgG2a), labeled the apical membranes of pancreatic acinar cells, hepatocytes, salivary and lacrimal gland acinar cells, and the brush border of small intestine enterocytes. This mAb precipitated and blotted a protein of 94 KD. Acinar-2 (IgM) also labeled pancreatic acinar cell apical membranes but did not label other tissues and did not precipitate or blot. Acinar-3 labeled pancreatic acinar cell lateral membranes. Duct-1 (IgM) labeled pancreatic duct apical membrane and ducts in liver and salivary glands but did not precipitate or blot. These domain-specific mAb demonstrate that common antigenic determinants occur in the apical surfaces of several exocrine epithelia and may be important in secretion.     

Monoclonal antibodies to lipocortin-1 as probes for biological function.

We have developed two monoclonal antibodies to human lipocortin-1 (103 and 105) as reagents for quantitating the protein in biological systems and neutralizing its activity. Lipo 105 is a high affinity antibody that is functional in ELISA and Western blot formats. The antibody recognizes a site between amino acids 30 and 55 in the lipocortin-1 sequence and can be used on native or denatured protein. Lipo 103 is an antibody that neutralizes the phospholipase A2 inhibitory activity of lipocortin-1 by blocking binding of the protein to phospholipid surfaces. The antibody is specific for native human lipocortin-1. Lipo 103 was recently shown to block lipocortin-1-dependent differentiation of a squamous carcinoma cell line, demonstrating its usefulness as a probe for function.     

The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage.

A preparation containing the link proteins may be obtained from bovine nasal cartilage by extraction with 4 M guanidine hydrochloride and by equilibrium density gradient centrifugations of the extract as commonly employed in the isolation of proteoglycan monomers. In the present paper, protein-rich proteoglycans have been removed from such a preparation to give purified link proteins by chromatography on Sepharose CL-6B in 1% sodium dodecyl sulfate. The individual link proteins, which in order of increasing electrophoretic mobility are termed link proteins 1, 2, and 3, have been separated and isolated in a subsequent preparative gel electrophoresis step. The link proteins present in largest amount, link proteins 1 and 2, have essentially the same amino acid compositions, and following partial digestion with the V8 protease from Staphylococcus aureus and analytical electrophoresis in sodium dodecyl sulfate, their peptide patterns closely resemble each other. Therefore,it is probable that link proteins 1 and 2 are structurally similar. Link protein 1 contains more carbohydrate than link protein 2 (9.5% and 3.0%, respectively) and it is suggested that the major difference between them is in carbohydrate content.     

Age-related changes in the structure of proteoglycan link proteins present in normal human articular cartilage.

Reduced-minus-oxidized difference spectra were recorded on particle preparations of the cyanobacterium Anacystis nidulans. Physiological oxidation of anaerobic membranes was effected either by O2 or by light. In both cases the spectral changes observed in the 550-570nm region were essentially the same. The results were confirmed by dual-wavelength spectrophotometry. It is concluded that a membrane-bound cytochrome f-b complex participates in both respiratory and photosynthetic elevtron transport.     

Monoclonal antibodies as probes for functional domains in cAMP-dependent protein kinase II

The antigenic regions of the type II regulatory subunit of cAMP-dependent kinase from bovine heart have been correlated with the previously established domain structure of the molecule. Immunoblotting with both serum and monoclonal antibodies of fragments generated by limited proteolysis or chemical cleavage of the R-subunit established that the major antigenic sites were confined to the amino-terminal portion of the polypeptide chain (residues 1-145). Radioimmunoassays using two different antisera suggested that one or more of the high affinity serum antibody recognition sites were further restricted to residues 91-145. This amino-terminal portion of the R-subunit includes the hinge region which is particularly sensitive to proteolysis, allowing the R-subunit to be cleaved readily into a COOH-terminal domain which retains the cAMP-binding sites and an NH2-terminal fragment which appears to be the major site for interaction of the R-subunits in the native dimer. Monoclonal antibodies that recognized determinants on both sides of this hinge region were characterized and their specific recognition sites localized. Accessibility of antigenic sites in the holoenzyme in contrast to free R2 was compared. Although cAMP did tend to slightly increase the affinity of the holoenzyme for one of the monoclonal antibodies, all of the antigenic sites clearly were exposed and accessible in the holoenzyme. Furthermore, despite the presumed close proximity of antigenic sites to interaction sites between the R- and C-subunits, in no case did binding of antibody to the holoenzyme promote dissociation of the complex. The fact that the monoclonal antibodies would precipitate holoenzyme as well as free R2 was used to ascertain the importance of specific amino acid residues in the interaction of the R- and C-subunits. cAMP-binding domains were isolated following limited proteolysis with chymotrypsin and thermolysin. These fragments differed by only three amino acid residues at the NH2-terminal end. U of these fragments in conjunction with immunoadsorption established that the chymotryptic fragment, which contained the Asp-Arg-Arg preceding the site of autophosphorylation, was capable of forming a stable complex with the C-subunit. In contrast, the thermolytic fragment which differed by only those three residues no longer complexed with the C-subunit, indicating that the arginine residues not only contribute to the specificity of the phosphorylation site but also are an essential component for energetically stabilizing the holoenzyme complex.     

Monoclonal antibodies used as probes for the structural organization of the central region of fibronectin

Two monoclonal mouse antibodies against human plasma fibronectin were compared in their reactivity for proteolytic fragments of the antigen by enzyme immunoassay and immunoblotting. These antibodies were shown to react with two different structures within a short segment (about 30 kDa) located about one-third away from the C-terminus of the fibronectin chains.     

Monoclonal antibodies as probes of conformational changes in protein-engineered cytochrome c

Determination of the nature of the antigen-antibody complex has always been the ultimate goal of three-dimensional epitope mapping studies. Various strategies for epitope mapping have been employed which include comparative binding studies with peptide fragments of antigens, binding studies with evolutionarily related proteins, chemical modifications of epitopes, and protection of epitopes from chemical modification or proteolysis by antibody shielding. In this study we report the use of protein engineering to modify residues in horse cytochrome c that are in or near the epitopes of four monoclonal antibodies specific for this protein. The results demonstrate not only that site-specific changes in the antigen binding site dramatically affect antibody binding, but, more importantly, that some of the site-specific changes cause local and long-range perturbations in structure that are detected by monoclonal antibody binding at other surfaces of the antigen. These findings emphasize the role of native conformation in the stabilization of the interaction between protein antigens and high affinity monoclonal antibodies. Furthermore, the results demonstrate that monoclonal antibodies are more sensitive probes of changes in conformation brought about by protein engineering than low resolution spectroscopic methods such as circular dichroism, where similar spectra are observed for all the analogues. These findings suggest a role for monoclonal antibodies in detecting conformational changes invoked by nonconservative amino acid substitutions or substitutions of evolutionarily conserved residues in protein-engineered or recombinant proteins.     

Monoclonal antibodies as molecular probes for cell wall antigens of the brown alga,Fucus

Monoclonal antibodies to cell wall carbohydrates were produced against carbohydrates extracted from the brown alga, Fucus distichus ssp. edentatus (de la Pyl.) Powell. Mouse spleen cells were immunized in vitro with alginate and fucans, and hybridoma cultures were screened by enzyme immunoassay. Most antibodies were immunoglobulin (Ig)M and one was IgA. Antigens were localized on methacrylate sections of Fucus tissues by indirect immunofluorescence. Each antibody labelled tissues with a distinctive distribution pattern in cell walls and extracellular matrix regions, demonstrating that each antibody was specific for a different extracellular epitope (i.e., antigenic determinant). Most antibodies also labelled intracellularly on at least one cell type. Punctate, fibrous or clumped labelling was characteristic of individual antibodies and provided information related to carbohydrate structure and solubility. These antibodies are molecular probes for small regions on cell wall polymers and should be valuable in studies of cell wall synthesis, secretion, assembly and modification as well as carbohydrate fine structure and function.Abbreviations EDTA ethylenediaminetetraacetic acid - EIA enzyme immunoassay - Ig immunoglobulin (IgG, IgM and IgA are immunoglobulin types)     

Monoclonal antibodies as probes of the antigenic structure of tobacco mosaic virus.

The antigenic structure of tobacco mosaic virus has been analysed by measuring the ability of nine monoclonal antibodies to distinguish between wild-type virus and 13 mutants showing single and double amino acid substitutions in the coat protein. Although the majority of antibodies detected those substitutions that were located at the outer surface of the virion, some of them also recognized conformation alterations induced by exchanges occurring deep inside the subunit. In the case of five mutants, the antibody reactivity was reduced compared with wild-type virus, while in the case of three others, it was significantly higher. Each monoclonal antibody possessed a unique discrimination pattern with respect to the different substitutions. The simultaneous presence of two exchanges led to the complete disappearance of any binding with six of the nine antibodies and to reduced binding with three others. The superior discriminatory capacity of monoclonal antibodies compared with polyclonal antisera was demonstrated by the fact that three exchanges not detected with antisera were found to alter the antigenicity when tested with monoclonal antibodies.     

Immunohistochemical localization of articular cartilage proteoglycan and link protein in situ using monoclonal antibodies and lectin-binding methods

Lectins have specificity for certain carbohydrate structures in macromolecules. Lectins are, therefore, useful histochemical tools for demonstrating the composition and localization of components of connective tissue matrices, such as articular cartilage. In order to assess the significance of observed lectin-binding patterns, experiments were performed in which monoclonal antibodies against chondroitin sulphate- and keratan sulphate-containing proteolgycans and link proteins were applied to sections of bovine articular cartilage after enzymatic digestion with chondroitinase ABC and keratanase. The following conclusions were made: (1) Binding of peanut agglutinin (PNA) in the interterritorial matrix predominantly indicates the presence of keratan sulphate, but may also detectO-linked oligosaccharides of proteoglycans. (2) In normal cartilage wheat germ agglutinin (WGA) binds nearly exclusively to keratan sulphate. In cartilage degraded with chondroitinase ABC and keratanase this lectin may also detect carbohydrates in link protein due to enhanced accessibility. Binding of WGA toO-linked oligosaccharides may eventually occur. (3) In enzymatically digested cartilage matrix, staining with soybean agglutinin (SBA) may be due to link protein, but not to chondroitin sulphate, because specific breakdown of the glycosaminoglycan chain is required for binding of SBA. (4)Ulex europaeus agglutinin I (UEA I) binding sites are only detectable in digested cartilage matrix.