Non-inhibitory Antibodies Research Articles

Solid-phase monoclonal antibodies A novel method of directing the function of biologically active molecules by presenting a specific orientation

Monoclonal antibodies were produced against fibronectin and vitronectin, using the extracellular matrix from cultured bovine corneal endothelial cells as immunogen. Some antibodies inhibited the adhesion of BHK-21 cells to these molecules, in a standard assay with fibronectin or vitronectin coated on a plastic surface. By first coating the plastic surface with the monoclonal antibodies and subsequently binding vitronectin, the inhibitory effect was markedly increased. Divalent fibronectin required the presence of antibody in the medium as well as coated on the surface for maximal inhibition of cell adhesion. The inhibitory effect of surface-coated antibodies was stable in the presence of cells up to at least 24 h in vitro. Using this antibody coating method the surface could either be made compatible for cell adhesion (using non-inhibitory antibodies) or refractory. Assays performed by this method have considerable potential for quantifying particular functions of extracellular matrix molecules.

Noncoagulation Inhibitory Factor VIII Antibodies After Induction of Tolerance to Factor VIII in Hemophilia A Patients

We recently described tolerance induction with factor VIII/IX, cyclophosphamide, and high-dose intravenous IgG in hemophilia A or B patients with coagulation inhibitory antibodies. Circulating noninhibitory antibodies complexed with factor IX have been demonstrated in tolerant hemophilia B patients. Similar findings are now described in six tolerant hemophilia A patients. Complexes between factor VIII and the 'tolerant' antibody were demonstrated by subjecting plasma to gel filtration chromatography, void fractions containing factor VIII/vWF complexes being collected and adsorbed to protein A. Using 125I-labeled F(ab')2 fragments against IgG subclass and factor VIII antigen, complexes between an IgG4 antibody and factor VIII were found to adsorb to protein A. After infusion of factor VIII to tolerant patients, all factor VIII circulated in complex with IgG4 antibody. In three of the patients, the 'tolerant' antibodies inhibited an ELISA specific for factor VIII light chain but, unlike the pretolerant antibodies, did not bind radiolabeled factor VIII heavy chain. Although after induction of tolerance the patients still have circulating IgG4 antibodies against factor VIII, the antibodies differ in specificity, lack coagulation inhibitory activity, and do not enhance the rate of elimination of factor VIII.

Noncoagulation inhibitory factor VIII antibodies after induction of tolerance to factor VIII in hemophilia A patients

We recently described tolerance induction with factor VIII/IX, cyclophosphamide, and high-dose intravenous IgG in hemophilia A or B patients with coagulation inhibitory antibodies. Circulating noninhibitory antibodies complexed with factor IX have been demonstrated in tolerant hemophilia B patients. Similar findings are now described in six tolerant hemophilia A patients. Complexes between factor VIII and the ‘tolerant’ antibody were demonstrated by subjecting plasma to gel filtration chromatography, void fractions containing factor VIII/vWF complexes being collected and adsorbed to protein A. Using 125I-labeled F(ab')2 fragments against IgG subclass and factor VIII antigen, complexes between an IgG4 antibody and factor VIII were found to adsorb to protein A. After infusion of factor VIII to tolerant patients, all factor VIII circulated in complex with IgG4 antibody. In three of the patients, the ‘tolerant’ antibodies inhibited an ELISA specific for factor VIII light chain but, unlike the pretolerant antibodies, did not bind radiolabeled factor VIII heavy chain. Although after induction of tolerance the patients still have circulating IgG4 antibodies against factor VIII, the antibodies differ in specificity, lack coagulation inhibitory activity, and do not enhance the rate of elimination of factor VIII.

Direct inhibition of choline acetyltransferase activity by a monoclonal antibody raised against the plasma membrane of cholinergic nerve terminals

A monoclonal antibody raised against cholinergic synaptosomal plasma membranes isolated from Torpedo electric organ, inhibited completely amphiphilic and hydrophilic choline acetyltransferase (ChAT) activities extracted and separated by using Triton X-114 phase partition of synaptosomes. We tested whether ChAT inhibition was direct or not. We found that the antibody was inhibiting ChAT in preparations of very low purity as well as ChAT that was immunoprecipitated by using a non-inhibitory anti-ChAT polyclonal antibody. Also, inclusion of acetylcoenzyme A at 20 times its K m during incubation of ChAT and antibody, completely prevented ChAT inhibition. This same concentration of the ChAT substrate could significantly but not completely dissociate the complex enzyme-antibody. These results spoke in favour of a direct inhibition of ChAT; the antibody most probably binds to an epitope that may be located at or near the acetylcoenzyme A binding site. The inhibitory effect on hydrophilic and amphiphilic ChAT was dependent on the antibody concentration, but amphiphilic activity required higher concentrations to be affected to the same extent as hydrophilic activity. This was found not only with Torpedo, but also with rat and human ChAT activities. Thus, the antibody appears to be able to distinguish the two forms of ChAT activity.

Transfer of monoclonal antibodies into mammalian cells by electroporation

A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC 6.3.1.1) were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.

Use of Inhibitory (Anti-Catalytic) Antibodies to study extracellular Proteolysis

The specific role of proteolytic enzymes in the degradation by live cells of fibrillar model matrices (fibrin, collagen) was studied using monoclonal and polyclonal inhibitory (anti-catalytic) antibodies. Dissolution of fibrin by plasminogen-supplemented human HT-1080 cells was blocked by (1) omission of plasminogen, (2) inhibitory anti-plasmin antibody, and (3) inhibitory anti-u-PA antibody but not by non-inhibitory control antibodies. Using a similar approach, it was shown that the dissolution of reconstituted type I collagen fibrils by trypsin-supplemented live human skin fibroblasts was blocked by inhibitory antibodies to fibroblast-type procollagenase but not by noninhibitory control antibodies. These findings permit us to deduce that, at least in culture, the dissolution of fibrin by plasminogen-supplemented HT-1080 cells was mediated by plasminogen-assisted proteolysis which entailed the extracellular conversion of plasminogen to plasmin by cell-derived u-PA, and that the dissolution of collagen fibrils by trypsin-supplemented skin fibroblasts was mediated by a collagenase-dependent pathway.

Soluble interleukin 2 receptors released from mitogen stimulated human peripheral blood lymphocytes bind interleukin 2 and inhibit IL2 dependent cell proliferation.

In this communication the binding characteristics and possible regulatory role of sIL2R were investigated. Soluble IL2R are released or secreted in high concentrations by phytohemagglutinin (PHA) stimulated human lymphoid cells. The addition of sIL2R, purified by gel filtration chromatography, to cultures of PHA stimulated lymphoblasts resulted in a dose-dependent inhibition of [3H]TdR incorporation that could be overcome by the addition of exogenous IL2. Scatchard analysis of IL2 binding demonstrated that the presence of sIL2R did not inhibit ligand interaction with the high affinity IL2R. Immunoprecipitation studies utilizing [125I]IL2 and the non-inhibitory anti-Tac protein antibody 7G7/B6 revealed that most of the 125I-labeled IL2 migrated with a protein of approximately 45-50 kDa on SDS/PAGE. Together, these results provide evidence that the sIL2R limits the availability of free IL2 to proliferating cells and down-regulates their response without directly affecting the number or function of the cell bound high affinity IL2R.

Structure-function relationships for the interleukin 2 receptor: location of ligand and antibody binding sites on the Tac receptor chain by mutational analysis.

The Tac protein plays a role in high- and low-affinity interleukin 2 (IL-2) receptors. A mutational survey of this molecule identified several small segments in which the binding of IL-2 was particularly sensitive to amino acid substitutions. Two of the segments (residues 1-6 and 35-43) located in the exon 2-encoded region of the molecule overlapped the apparent binding sites of three monoclonal antibodies (anti-Tac, GL439, and H31) that block high- and low-affinity Tac-IL-2 interactions, thus supporting the hypothesis that these segments of the protein are at or near sites of receptor-ligand contact. In contrast, the apparent binding sites of antibodies (Hiei and H47) that selectively inhibit high-affinity IL-2 binding were mapped to a distinct location (residues 158-160) within the region encoded by exon 4 of the Tac gene. Since high-affinity receptors consist of a heterodimer of Tac and a second ligand-binding protein (p70), this portion of the Tac molecule may be involved in the interaction between the two receptor subunits. As expected, the binding sites of noninhibitory antibodies (7G7/B6, residues 140-144; H48, residues 170-211) did not overlap those segments in which IL-2-binding mutants were observed. These results provide a preliminary correlation of structure and function for the Tac protein that should prove useful in evaluating detailed models of the IL-2-receptor complex.

Topographical separation of the catalytic sites of asparagine synthetase explored with monoclonal antibodies.

Monoclonal antibodies which inhibited the enzymatic activity of bovine pancreatic asparagine synthetase were mapped to two topographically separate regions of the enzyme surface using competitive binding assays. Three antibodies which all inhibited glutamine- and NH3-dependent synthesis of asparagine bound to a common antigenic region. A fourth monoclonal antibody which interfered with glutamine binding or cleavage but not with NH3-dependent synthesis of asparagine was mapped to a separate region of the enzyme surface. These findings suggest a topographical separation between the aspartyl-AMP and glutamine-binding sites of bovine pancreatic asparagine synthetase. Three noninhibitory antibodies exhibited conformation-dependent binding and were mapped to a third region of the enzyme. Binding assays were used to demonstrate extensive cross-reaction of these antibodies with asparagine synthetases isolated from bovine liver and sheep pancreas. Substantial cross-reactions were also seen with the enzyme isolated from rat liver or pancreas, a human tumor cell line, and a mouse tumor cell line. Of the four antibodies that inhibited glutamine- and NH3-dependent synthesis of asparagine from ruminant species, only one bound to and inhibited the enzyme from rat liver or mouse cells, which suggests significant structural differences between the ruminant and rodent enzymes.

Specific inhibition of ATP-ADP translocase in cardiac mitoplasts by antibodies against mitochondrial creatine kinase

Mitochondrial creatine kinase was purified from rat hearts and used to produce antibodies in chicken and rabbits. Antibodies were purified to a high degree of homogeneity by an affinity chromatography method. Chicken antibodies against mitochondrial creatine kinase inhibited this enzyme in rat-heart mitochondrial inner membrane and matrix preparation, and simultaneously blocked oxidative phosphorylation. Under these conditions respiratory chain activities remained unchanged, but adenine nucleotide translocase was inhibited. Removal of mitochondrial creatine kinase from the membrane by pretreatment with 0.15 M KCl and 20 mM ADP completely abolished the effect of antibodies against mitochondrial creatine kinase on oxidative phosphorylation. Noninhibitory antibodies from rabbit with high affinity to rat mitochondrial creatine kinase inhibited neither creatine kinase activity nor oxidative phosphorylation. These data show close and specific spatial arrangement of mitochondrial creatine kinase and adenine nucleotide translocase in mitochondria. It is supposed that there is a fixed orientation of these proteins in the cardiolipin domain in the membrane and that their interaction may occur by a frequent collision due to their lateral movement.

Adhesion of Tumor Cells to Hepatocytes: Different Mechanisms for Mammary Carcinoma Compared With Lymphosarcoma Cells

Mechanisms of adhesion between tumor cells and hepatocytes, which are likely to play a role in liver metastasis formation, were studied in vitro. TA3 mammary carcinoma and MB6A lymphosarcoma cells were added to rat hepatocytes that had been cultured for 24 hours. Adhesion was quantified by counting adherent cells seen in sections of pelleted, Epon-embedded culture fragments. Adhesion of TA3, but not of MB6A cells, was inhibited by antibodies prepared from an antiserum raised against sinusoidal face-enriched liver plasma membranes. Detergent-solubilized liver components, affinity purified on immobilized inhibitory antibodies, neutralized inhibition, whereas a subfraction separated from this material with the use of immobilized noninhibitory antiliver antibodies had no neutralizing activity. Adhesion of MB6A but not of TA3 cells was inhibited by the calcium ionophore A23187 and the local anesthetic procaine. The calmodulin inhibitor trifluoperazine inhibited adhesion of MB6A cells more strongly than that of TA3 cells. Finally, adhesion of TA3 cells was dependent on external calcium, whereas in the case of MB6A cells calcium could be replaced by magnesium. These observations suggested that adhesion of the two tumor cell types to hepatocytes involved distinct hepatocyte surface molecules and required distinct biochemical machinery.

The receptor-binding domain of human apolipoprotein E. Monoclonal antibody inhibition of binding.

To investigate the potential of monoclonal antibodies as probes to determine the receptor-binding domain of apolipoprotein E (apo-E), five apo-E antibodies were tested to see if any of them inhibited 125I-apo-E3 . dimyristoylphosphatidylcholine binding to apo-B,E receptors on cultured fibroblasts. Only one of the five antibodies, referred to as 1D7, was found to inhibit binding, blocking greater than 90% of the receptor-binding activity of apo-E3 dimyristoylphosphatidyl-choline. The 1D7 Fab fragments were also effective inhibitors. The 1D7 bound to a Mr = 22,000 NH2-terminal thrombolytic fragment of apo-E (residues 1-191) and to a 93-residue cyanogen bromide fragment of apo-E (residues 126-218). The four noninhibitory antibodies bound only to the NH2-terminal thrombolytic fragment. These results suggested that the 1D7 epitope is contained between residues 126 and 191, and that the epitopes of the other antibodies are not contained in this region. The use of synthetic apo-E fragments, which cover various lengths of the sequence from residues 129-169, and human apo-E variants with substitutions at residues 145, 146, or 158, narrowed the location of the 1D7 epitope to residues 139-169 and, most likely, to the immediate vicinity of residues 140-150. It is of interest that 1D7 was found to bind to the same region of apo-E that has been implicated as the receptor-binding domain in receptor-binding studies using human apo-E variants and apo-E3 fragments.

Single-step purification of epoxide hydrolase from rat liver microsomes using monoclonal-antibody chromatography

Monoclonal antibody to rat liver microsomal epoxide hydrolase has been obtained after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to CNBr-activated Sepharose 4B, to give a specific affinity column. Monoclonal-antibody affinity chromatography provided a rapid single-step method of purifying to homogeneity active epoxide hydrolase from crude solubilized microsomes. The techniques used offer an effective method for characterization of a non-inhibitory monoclonal antibody.

Immunoenzymology of acetylcholinesterase-IV. Studies on an enzyme-inhibiting antibody

Inhibitory antibody was obtained from the serum of a rabbit immunized with AChE. This antibody differed from previously described noninhibitory antibody only in its effect on the hydrolysis of ATCh by AChE. Both inhibitory and noninhibitory antibody-enzyme complexes showed increased stability to heat and demonstrated similar activity and kinetic parameters with other substrates. When in complex with the enzyme, both the whole inhibitory IgG molecule and F(ab′) 2 fragments inhibited hydrolysis of ATCh and protected AChE from heat inactivation.

Antibody to lactate dehydrogenase IV. Enzyme-inhibiting and inhibition-interfering components

1. 1. The immunochemical properties of chicken antibodies to rabbit and mouse lactate dehydrogenase ( L-lactate: NAD + oxidoreductase, EC 1.1.1.27), isoenzyme 5, were studied. 2. 2. These antibodies were separated into several distinct fractions by rate zonal ultracentrifugation in a sucrose density gradient and by differential elution from DEAE-cellulose. 3. 3. Immunoglobulins were separated which showed high titers when measured by passive hemagglutination of sheep erythrocytes tanned with rabbit or mouse lactate dehydrogenase but which caused little or no enzyme inhibition. Other immunoglobulins were capable of inhibiting the enzyme but caused little or no passive hemagglutination. 4. 4. Noninhibitory antibodies were shown to interfere strongly with the activity of inhibitory antibody. The removal of noninhibitory antibody yielded anti-lactate dehydrogenase with greatly increased specific activity (45-fold) as measured by enzyme inhibition.