[20] Distinction of epitopes by monoclonal antibodies

Stähli, C.; Miggiano, V.; Stocker, John; Staehelin, Theophil; Häring, P.; Takács, B.

doi:10.1016/0076-6879(83)92023-2

Cited by 107 publications

(40 citation statements)

References 2 publications

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“…Because 3E2 was considered an essential formulation component, binding inhibition assays were performed to determine whether candidate MAbs inhibited its binding to antigen. Inhibition of 3E2 binding by candidate MAbs was not observed based on comparison of test OD values to that of control 3E2 competed against itself (Table 3) (43). At the completion of preliminary testing in mice, ␣GP25-200 MAb 3H2, ␣P23 MAb 1E10, and ␣CSL MAb 3E2 had consistently demonstrated the greatest efficacy, individually and combined, and were selected for further study.…”

Section: Resultsmentioning

confidence: 99%

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Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

2000

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The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocystchallenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes.

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Section: Resultsmentioning

confidence: 99%

“…The ability of MAbs to inhibit binding of each other to C. parvum was determined by ELISA using biotin-labeled and unlabeled MAbs (43). For biotinylation, bioreactor-derived MAb (2 mg/ml in PBS) was incubated with Sulfo-NHSbiotin (Pierce) according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

2000

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“…: 1.5 -3.2 pmol mg-' min-'). To get a higher yield of antigenspecific hybridomas the mice were injected with 30 pg pure antigen, 4, 3 and 2 days before fusion [21]. Spleen cells were fused with a non-secreting NSO-1 myeloma cell line (M. R. Clark, B. Wright and C. Milstein, unpublished data quoted in 1231) which was kindly donated by C. Milstein's laboratory, according to standard methods [23].…”

Section: Immunization Protocol and Hybridoma Productionmentioning

confidence: 99%

Two different adenylyl cyclases in brain distinguished by monoclonal antibodies

Mollner

Pfeuffer

1988

European Journal of Biochemistry

124

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Four monoclonal antibodies, raised against the 11 5-kDa adenylyl cyclase from bovine brain EMBO J. 4, 3675-36791 have been selected and designated BBC-1 to BBC-4. BBC-1 and BBC-3 are highly specific for the 115-kDa enzyme from bovine brain. The two other antibodies, BBC-2 and BBC-4, recognize an additional 150-kDa adenylyl cyclase in bovine brain, but also in brain tissue from other species. In membranes from lung and myocardium (bovine and rabbit) only the 150-kDa species is detected by the crossreacting antibodies BBC-2 and BBC-4. The two adenylyl cyclases from brain can be separated by calmodulin-Sepharose: only the enzyme of 115 kDa but not that of 150 kDa was retained by the affinity resin and could be stimulated by Ca2+/ calmodulin. The data obtained with these antibodies of defined specificity provide for the first time direct evidence for the presence of two distinct adenylyl cyclase species in brain tissue.Over the past ten years considerable progress has been made with respect to isolation and characterisation of the components of the hormonally regulated adenylyl cyclase. For several of these proteins (8-adrenoceptors, G, and Gi proteins) even the primary structure is now available [l -41. The catalytic unit has also been purified to homogeneity from several sources thanks to the specifity of the hypotensive drug forskolin [5 -81. This made the reconstitution of the entire signal transduction chain from its individual components possible [9, lo]. Both the calmodulin-sensitive enzyme from brain and the calmodulin-insensitive enzyme from myocard appear to be glycoproteins, although the significance of this finding remains elusive. While both the Ca2+-sensitive and insensitive enzymes are hormonally stimulated and coupled via G, proteins to hormone receptors, the enzymes from bacteria, yeasts and other lower eucaryotic organisms like protozoa and slime molds are not stimulated by hormones but seem to be regulated by G proteins at least in some instances [l 11. In contrast the adenylyl cyclase from mammalian sperm is not stimulated by hormones, guanyl nucleotides or even forskolin [12], although the latter is supposed to act through the catalytic subunit itself [13, 141. An idea how these different adenylyl cyclases may be related to each other could be obtained by monoclonal or polyclonal antibodies directed against distinct epitopes of the protein. Reports on antibodies against the components of the adenylyl cyclase system are extremely scarce, most probably because of their poor antigenicity. In some cases these drawbacks had to be circumvented by the formation of antibodies against haptens consisting of synthetic peptides corresponding to defined regions of their primary structure [15] clonal antibodies have been raised against the soluble adenylyl cyclase of Neurospora crassa which, however, did not crossreact with the enzymes from testicular cytosol or from turkey erythrocyte membranes [16]. Here we describe the production and characterisation of monoclonal antibodies raised against the homogeneo...

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“…DT or CRM, under examination. The epitope specificity of the Mabs was determined by a competitive solid-phase antibody binding assay, according to Ivanyi [27] and Stahli et al [28].…”

Section: Radioimmunoassaysmentioning

confidence: 99%

Conformational changes in diphtheria toxoids

Bigio¹,

Rossi²,

Nucci³

et al. 1987

FEBS Letters

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Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRMlOOl, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mabl 1.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM 197, CRM 176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.

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[20] Distinction of epitopes by monoclonal antibodies

Cited by 107 publications

References 2 publications

Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

Two different adenylyl cyclases in brain distinguished by monoclonal antibodies

Conformational changes in diphtheria toxoids

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