title: An alternative method for purifying and detoxifying diphtheria toxin.
abstract: Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 μg/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated (mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoid obtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however.
authors.affiliation: Lab. of Research and Development of Immunobiologicals for Veterinary Use, Institute Butantan, São Paulo, Brazil.
authors.forename: Fernando
authors.initials: F
authors.lastname: Fratelli
authors.forename: José
authors.initials: J
authors.lastname: Abrahão-Neto
authors.forename: Aline Tojeira Prestia
authors.initials: AT
authors.lastname: Caricati
authors.forename: Monamaris Marques
authors.initials: MM
authors.lastname: Borges
authors.forename: Rosalvo
authors.initials: R
authors.lastname: Guidolin
authors.forename: Celso Pereira
authors.initials: CP
authors.lastname: Caricati
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D003603:Cytotoxins / Q000737:chemistry / Q000302:isolation & purification*; D004165:Diphtheria / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D004167:Diphtheria Toxin / Q000737:chemistry / Q000302:isolation & purification*; D004168:Diphtheria Toxoid / Q000276:immunology / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D006801:Humans; D009500:Neutralization Tests; D011092:Polyethylene Glycols / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D016060:Sorption Detoxification / Q000379:methods*; D013997:Time Factors; D014709:Vero Cells
chemical_list: D003603:Cytotoxins; D004167:Diphtheria Toxin; D004168:Diphtheria Toxoid; D011092:Polyethylene Glycols; C029483:Blue-Sepharose; C028210:monomethoxypolyethylene glycol; D012685:Sepharose