RESUMO
Strains of Clostridium difficile produce toxins A and B that can cause diarrhoea and pseudomembranous colitis. Currently, there is no preventative therapy for this infection but antibodies to the toxins provide protection, therefore a toxoid-based vaccine is needed. To evaluate thermal stability, a lyophilized and liquid formulation of toxoids A and B were stored at a range of temperatures for 5 weeks. Changes in toxoid structures and immune responses in an animal model before and after the incubation period were assessed. The structural integrity and the immune responses to liquid formulations were affected when stored at 56°C but the lyophilized formulation was thermally stable and same treatment did not result in significant loss of immunological responses when immunized in an animal model.
Assuntos
Clostridioides difficile/imunologia , Toxoides/química , Animais , Chlorocebus aethiops , Cromatografia em Gel , Cricetinae , Estabilidade de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina A/sangue , Mesocricetus , Testes de Neutralização , Toxoides/efeitos adversos , Toxoides/imunologia , Células VeroRESUMO
Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision < or =20% CV and accuracy within +/-20% of the nominal values. Intra- and inter-assay coefficients of variation were within 9% and accuracies within +/-10% of the nominal values. The validated method was applied to the study of the cellular uptake of native and cationized anti-tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.
Assuntos
Fragmentos Fab das Imunoglobulinas/imunologia , Putrescina/química , Toxoide Tetânico/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Especificidade de Anticorpos/imunologia , Calibragem , Cátions/química , Endocitose/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Células HL-60 , Cavalos/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Focalização Isoelétrica , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Toxoides/imunologiaRESUMO
OBJECTIVE: To determine whether booster vaccination with a multivalent clostridial bacterin-toxoid would affect the sudden death syndrome (SDS) mortality rate among feedlot cattle. DESIGN: Field trial. ANIMALS: 83, 115 cattle at a Nebraska feedlot. PROCEDURE: Cattle arriving at the feedlot underwent routine processing according to established protocol. All cattle received a sequentially numbered ear tag and a 2-ml dose of a multivalent bacterin-toxoid designed to protect cattle against Clostridium chauvoei, C speticum, C novyi, C sordellii, and C perfringens types C and D. Approximately 90 days prior to slaughter, growth promotants were implanted in all cattle, and cattle were allocated to a treatment or control group on the basis of the last digits of their ear tag numbers. Cattle in the treatment group received a second 2-ml dose of clostridial bacterintoxoid; control cattle did not. RESULTS: Significant differences between groups in regard to crude, feeding pen, or SDS mortality rates were not detected. Sudden death syndrome mortality rate across both groups was 0.24%. If the SDS mortality rate in midwestern feedlot cattle was reduced > or = 40% by booster vaccination with a multivalent clostridial bacterin-toxoid, this experiment included enough animals to have a 90% probability of detecting that difference. CLINICAL IMPLICATIONS: Booster vaccination with a multivalent clostridial bacterin-toxoid does not affect SDS mortality rate among feedlot cattle.