Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Monoclonal antibodies that inhibit the enzyme activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

Three hybridoma cell lines secreting antibodies against human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) were produced. Purified IgG2b from these cell lines recognized a distinct band of Mr 28,000 on SDS/PAGE from the purified enzyme as well as a band of Mr 56,000 from the crude enzyme preparation. These three monoclonal antibodies inhibited 15-OH-PGDH activity to different degrees. Inhibition of the enzyme activity could be prevented by prior incubation of the enzyme with NAD+ but not with prostaglandin E2 (PGE2) or NADP+. Inhibition by monoclonal antibodies appears to be non-competitive with respect to NAD+ and PGE2. An increased concentration of antibodies alters the apparent Km for NAD+ but not for PGE2, further supporting the notion that the antibodies bind to the coenzyme-binding site. The availability of these monoclonal antibodies should be valuable for probing the structure of the active site.     

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.     

Isolation and characterization of a membrane-bound population of group B coxsackieviruses.

HeLa cells infected with several group B coxsackieviruses contain a previously undetected, virus-specific ribonucleoprotein particle which we designated membrane-bound virion (MBV). MBVs of B5 virus have a pronounced polygonal appearance and are slightly smaller than virions. The particles sediment more slowly (at about 107S) and have a lower buoyant density (about 1.30). They contain 35S virion RNA; only three, and not four, capsid proteins; and at least seven additional proteins with apparent molecular weights of 21,000 to 92,000. Three of the latter proteins appear to be of host origin; the rest may be precursors of virion capsid proteins. The RNA is resistant to digestion by RNase, and EDTA treatment disrupts the particle. MBVs are infectious, although significantly less so than virions. Cells infected with MBVs produce both types of progeny, virions and MBVs. In coinfected cultures, the yield of progeny is lower than in cells infected with virions alone, suggesting interference by MBVs. Synthesis of both types can be detected within 3.5 h after infection, and synthesis continues for 24 h.     

Anti-idiotype antibodies that mimic gp86 of human cytomegalovirus inhibit viral fusion but not attachment.

Human cytomegalovirus (CMV) infects cells by sequential processes involving attachment, fusion with the cell membrane, and penetration of the capsid. We used two monoclonal anti-idiotype that mimic one of the CMV envelope glycoproteins, gp86, to study its role in the early phases of CMV infection. Neither of two such antibodies inhibited virus binding to human embryonic lung (HEL) fibroblasts; however, both antibodies inhibited the fusion of CMV with HEL cells, as measured by an assay in which viral envelope is labeled with a fluorescent amphiphile (octadecyl rhodamine B chloride, or R18), resulting in increased fluorescence during fusion of virus with the cell membrane. Because these anti-idiotype antibodies were shown previously to bind to specific receptors on HEL cell membranes, these findings suggest that both gp86 and its cell membrane receptor may function in the fusion of human CMV with HEL cells.     

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.     

Purification of a HeLa cell receptor protein for group B coxsackieviruses.

Coxsackievirus B3 (CB3) firmly attaches to HeLa cells, forming a specific complex between the virus and its receptor on the cell surface. We extracted this virus-receptor complex (VRC) with the detergents sodium deoxycholate and Triton X-100. The VRC was identified by its sedimentation coefficient (140S), which was less than that of virions (155S). Formation of the VRC from cell lysates and 3H-CB3 occurred with the same specificity as did attachment of virions to cells, in that its formation was blocked by unlabeled CB3 but not by poliovirus. The VRC was purified 30,000-fold by differential and sucrose gradient centrifugation. Iodination with Na125I revealed that the purified VRC consisted of the normal CB3 proteins and one additional protein (RP-a) with an approximate molecular weight of 49,500. RP-a was eluted from the VRC and was shown to rebind with CB3 and CB1 virions but not with poliovirus type 1. We propose that Rp-a is a protein in the plasma membrane receptor complex which is responsible for the specific recognition and binding of the group B coxsackieviruses.     

Monoclonal antibodies distinguish synthetic peptides that differ in one chemical group

By using human calcitonin (hCT), human calcitonin-gene-related peptide (hCGRP), and a synthetic peptide with a sequence analogous to the 34 C-terminal amino acids of human preprocalcitonin (designated as PQN-34) as haptens in the generation of monoclonal antibodies, we assessed the role of amido and amino groups in paratope-epitope binding. By using peptide inhibition experiments and solid-phase immunoassays, monoclonal anti-hCT antibody CT07 and monoclonal anti-hCGRP antibody CGR01 were found to bind to an antigenic determinant located in the C-terminal segment of the hormones. These epitopes comprise the seven C-terminal amino acids of the hormones, and the presence of the hormone-ending carboxamide group was found to be essential for antibody binding. The corresponding heptapeptides, either bearing a carboxyl group or else linked to a glycine residue at their C-terminal part, failed to react with the antibodies. Moreover, these monoclonal antibodies did not bind to synthetic peptides analogous to the C-terminal region of the hormone precursor molecules that comprised the epitope site flanked by a peptide sequence. In an attempt to assess whether amido groups when present on the side-chain of amino acids may also modulate antibody binding, a monoclonal antibody referred to as QPO1 was produced and was found to recognize an antigenic determinant localized in the N-terminal region of the PQN-34 peptide bearing a glutamine residue as the N-terminal amino acid. The epitope was found to correspond to a topographic assembled site, and binding of QPO1 was found to be substantially dependent on the presence of the free amino and the side-chain amido groups borne by the N-terminal glutamine residue of this peptide PQN-34. In contrast to these findings, an antigenic determinant located in the internal sequence of calcitonin and recognized by monoclonal anti-hCT antibody CT08 was found to be expressed on the mature form of the hormone, as well as on synthetic peptides with sequence mimicking that of preprocalcitonin. These data should guide the choice of synthetic peptide haptens for the production of anti-protein antibodies.     

Characterization of a YAC-1 mouse cell receptor for group B coxsackieviruses.

A receptor on YAC-1 cells, a mouse T-lymphoma cell line, bound all six serotypes of the group B coxsackieviruses (CVB). In addition, the cells produced infectious virus. Each of the CVB competed for the same receptor on YAC-1 cells. CVB3 bound relatively slowly to YAC-1 cells (k = 4 x 10(-11) min-1 cell-1), and there were only 500 attachment sites per cell. A rabbit antiserum prepared against the HeLa cell receptor protein Rp-a specifically inhibited the binding of CVB1 and CVB3. A virus-receptor complex with CVB3 could be isolated from detergent (0.5% sodium deoxycholate, 1% Triton X-100)-solubilized YAC-1 plasma membranes. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the iodinated virus-receptor complex revealed a band with the same mobility as Rp-a. The results suggested that the YAC-1 receptor for CVB resembles that of the HeLa cell receptor.     

Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein

The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J. S. (1986) J. Biol. Chem. 260, 4378-4383) have been characterized. Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP. Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited. Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2. DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3. With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2. In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2. RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex. In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP. The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3. The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.     

Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells.

The six group B coxsackieviruses (CVBs) are highly prevalent human pathogens that cause viremia followed by involvement of different organs. Clinical and experimental evidence suggests that CVBs can induce kidney injury, but the susceptibility of human renal cells to these viruses is unknown. By using pure cultures of human glomerular and tubular cells, we demonstrated that all CVBs are capable of productively infecting renal cells of three different histotypes. Distinct pathogenic effects were observed. Proximal tubular epithelial cells and, to a lesser extent, glomerular podocytes were highly susceptible to CVBs; in both cases, infection led to cytolysis. In contrast, glomerular mesangial cells supported the replication of the six CVBs but failed to develop overt cytopathologic changes. Mesangial cells continued to produce infectious progeny for numerous serial subcultures (i.e., more than 50 days), especially with type 1, 3, 4, and 5 viruses. In the above cells, persistent infection induced the de novo synthesis of platelet-derived growth factor A/B and enhanced the release of transforming growth factor beta1/2. These two factors are important mediators of progression from glomerular inflammation to glomerulosclerosis. CVB replication appeared also to impair the phagocytic and contractile activity of mesangial cells. Loss of these properties--which are important in glomerular physiopathology--may contribute to the development of progressive nephropathy. The results show that CVBs induce distinct effects in different types of cultured renal cells and suggest that CVB infections may be associated with both acute and progressive renal injury.     

A monoclonal antibody specific for the cellular receptor for the group B coxsackieviruses.

A 50-kilodalton receptor protein (Rp-a) for the group B coxsackieviruses (CB) was isolated in a virus-receptor complex from detergent-solubilized HeLa cells (J. E. Mapoles, D. L. Krah, and R. L. Crowell, J. Virol. 55:560-566, 1985). It was used as an immunogen for preparation of a mouse monoclonal antibody (RmcB) which protected HeLa cells and Buffalo green monkey kidney cells from infection by all six serotypes of CB. RmcB did not protect HeLa cells from infection by poliovirus, echovirus 6, or coxsackievirus A18. This monoclonal antibody differed in receptor epitope specificity from a previously isolated antibody (RmcA) (R. L. Crowell, A. K. Field, W. A. Schleif, W. L. Long, R. J. Colonno, J. E. Mapoles, and E. A. Emini, J. Virol. 57:438-445, 1986) which blocked receptors only for type 1 CB (CB1), CB3, CB5, and echovirus 6. RmcA and RmcB recognized two distinct saturable receptors on HeLa cells, designated HR2 and HR1, respectively. Human rhabdomyosarcoma (RD) cells have the HR2 receptor for CB3-RD (a variant of CB3), but lack the HR1 receptor for CB3. Therefore, RD cells were resistant to infection by CB3. Although binding of CB3-RD to the HR2 receptor on RD cells can lead to infection, binding of CB3-RD to the HR2 receptor on HeLa cells did not lead to infection. Apparently, both CB3 and CB3-RD use only the HR1 receptor for infection of HeLa cells. Thus, a given virus may use two distinct receptors to bind to cells when only one virus-receptor interaction leads to infection.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.     

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.     

Properties of the deoxycholate-solubilized HeLa cell plasma membrane receptor for binding group B coxsackieviruses.

Physical and chemical properties of deoxycholate-solubilized HeLa cell plasma membrane receptors for binding group B coxsackieviruses were determined. Receptors eluted from Sepharose 4B with an apparent molecular weight of 275,000 and sedimented with an S value of between 14.7 and 4.9 and a buoyant density of 1.06 to 1.10 g/cm3. Virus-binding activity was destroyed after treatment with proteases, glycosidases, and periodate but was unaffected by lipases or reducing or alkylating agents. Additionally, lectins, including concanavalin A, adsorbed receptors and inhibited virus attachment. The composite data suggested that glycoprotein is an integral part of the receptors for binding virus.     

Monoclonal antibodies to bovine blood group antigens

Hybridomas were made by fusing mouse myeloma cells with spleen cells from mice immunized with bovine red cells. Sixteen cloned lines which secreted haemolytic monoclonal antibodies reacting with antigens in the A, B, F, Z and S blood group systems were established; one of the antibodies identified a new factor in the B system. Extensive tests on red cells from 1000 animals indicated that several of the antibodies are suitable for use in routine blood typing; others are of potential use for genetic studies of the bovine blood group systems.     

Monoclonal antibodies that disrupt poliovirus only at fever temperatures.

Three of thirty-six monoclonal antibodies were found to cause irreversible inactivation of type 1 poliovirus at 39 degrees C but not at 37 degrees C. Neutralization at 37 degrees C depended on aggregation and was reversible by acid-induced deaggregation; at 39 degrees C, the virions (N antigenic, 160S) were disrupted to empty capsids (H antigenic, 100S), and neutralization was irreversible. The rate of antibody-dependent conversion of N to H antigen increased steeply between 37 and 39 degrees C.     

Monoclonal antibodies that recognize different membrane proteins that are deficient in Rhnull human erythrocytes. One group of antibodies reacts with a variety of cells and tissues whereas the other group is erythroid-specific.

1. Rhnull human erythrocytes lack all of the antigens of the Rh and LW blood group systems and have abnormal shape and an increased osmotic fragility. In this paper two murine monoclonal antibodies raised against intact human erythrocytes were used to investigate further the abnormalities in these cells. BRIC 125 reacts weakly with Rhnull erythrocytes and BRIC 69 does not react at all. The results showed that BRIC 125 reacts with a component of Mr 47,000-52,000 which has a substantial content of N-glycans. In contrast, BRIC 69 reacted with a band of Mr 31,000 together with a very diffuse band of Mr 35,000-52,000. Treatment of BRIC 69 immunoprecipitates with endoglycosidase F/peptidyl-N-glycosidase F resulted in the loss of both BRIC 69 reactive components and the appearance of a new band of Mr similar to that of the Rh(D) polypeptide. 2. BRIC 125 had a broad reactivity with cells in peripheral blood, whereas the reactivity of BRIC 69 was confined to erythrocytes. BRIC 125, but not BRIC 69, reacted with human kidney tissue and bound to endothelium in peritubular capillaries, arteries and veins as well as the epithelial tissue of distal tubules. BRIC 125 stained haemopoietic cells, foetal hepatocytes and megakaryocytes in foetal liver and sinusoidal cells, hepatocytes and portal tracts in adult liver. In contrast, BRIC 69 reactivity was confined to haemopoietic cells in foetal liver. The BRIC 125 epitope has a wide tissue distribution, suggesting the occurrence of a related group of polypeptides which have a general functional role on cell surfaces. 3. Rhnull erythrocytes are deficient in at least four different membrane polypeptides.