title: Comparative in vitro and in vivo assessment of toxin neutralization by anti-tetanus toxin monoclonal antibodies.
abstract: Tetanus is caused by the tetanus neurotoxin (TeNT), a 150 kDa single polypeptide molecule which is cleaved into an active two-chain molecule composed of a 50 kDa N-terminal light (L) and a 100 kDa C-terminal heavy (H) chains. Recently, extensive effort has focused on characterization of TeNT binding receptors and toxin neutralization by monoclonal antibodies (mAbs). Toxin binding inhibition and neutralization is routinely assessed either in vitro by the ganglioside GT1b binding inhibition assay or in vivo using an animal model. These two assay systems have never been compared. In the present study, we report characterization of eleven mAbs against different parts of TeNT. The toxin inhibitory and neutralization activity of the mAbs was assessed in vitro and in vivo respectively. Our data demonstrated that seven mAbs bind to fragment C of the heavy chain, two mAbs react with the light chain, one mAb recognizes both chains and one mAb reacts with neither light chain nor fragment C. Six fragment C specific mAbs were able to inhibit TeNT binding to GT1b ganglioside in vitro but three failed to neutralize the toxin in vivo. One in vitro inhibitory mAb (1F3E3) was found to synergize with the in vivo neutralizing mAbs to reduce toxin lethal activity in vivo. Sequencing of the immunoglobulin heavy and light chain variable region genes revealed that the three in vivo neutralizing mAbs were derived from a common origin. Altogether, our data suggests that fragment C specific mAbs contribute to toxin neutralization in both systems, though some of the GT1b binding inhibitory mAbs may not be able to neutralize TeNT in vivo.
authors.affiliation: Department of Immunology; School of Public Health; Tehran University of Medical Sciences; Tehran, Iran; Immunology Research Center; Tabriz University of Medical Sciences; Tabriz, Iran; Department of Immunology; School of Medicine; Tabriz University of Medical Sciences; Tabriz, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Yousefi
authors.affiliation: Monoclonal Antibody Research Center; Avicenna Research Institute; ACECR; Tehran, Iran.
authors.forename: Roya
authors.initials: R
authors.lastname: Khosravi-Eghbal
authors.affiliation: Monoclonal Antibody Research Center; Avicenna Research Institute; ACECR; Tehran, Iran.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Reza Mahmoudi
authors.affiliation: Monoclonal Antibody Research Center; Avicenna Research Institute; ACECR; Tehran, Iran.
authors.forename: Mahmood
authors.initials: M
authors.lastname: Jeddi-Tehrani
authors.affiliation: Monoclonal Antibody Research Center; Avicenna Research Institute; ACECR; Tehran, Iran.
authors.forename: Hodjatallah
authors.initials: H
authors.lastname: Rabbani
authors.affiliation: Department of Immunology; School of Public Health; Tehran University of Medical Sciences; Tehran, Iran; Monoclonal Antibody Research Center; Avicenna Research Institute; ACECR; Tehran, Iran.
authors.forename: Fazel
authors.initials: F
authors.lastname: Shokri
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000276:immunology*; D001906:Botulism / Q000517:prevention & control*; D005732:Gangliosides / Q000378:metabolism; D008297:Male; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011485:Protein Binding; D016019:Survival Analysis; D013744:Tetanus Toxin / Q000037:antagonists & inhibitors*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D005732:Gangliosides; D013744:Tetanus Toxin; C016481:trisialoganglioside GT1
keywords: GT1b binding assay; in vivo neutralization assay; neutralizing mAbs; tetanus neurotoxin