Computational protocol: LT-IIb(T13I), a Non-Toxic Type II Heat-Labile Enterotoxin, Augments the Capacity of a Ricin Toxin Subunit Vaccine to Evoke Neutralizing Antibodies and Protective Immunity

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Protocol publication

[…] Levels of isotype and subclass anti-RiVax Ab in serum, saliva, lung lavage, and feces were measured by ELISA. Nunc Immulon 2HB polystyrene 96-well microtiter plates (ThermoFisher Scientific) coated with 100 µL RiVax (5 µg/mL) per well were incubated overnight at RT. To determine total IgA concentrations, plates were coated with 100 µL unlabeled goat anti-mouse IgA specific Ab (1 mg/mL) (Southern Biotechnology Assoc., Birmingham, AL). After blocking with PBS containing 0.15% Tween-20 and 1% bovine serum albumin (Amresco, Solon, OH), serial two-fold dilutions of serum or secretion samples were added in duplicate and plates were incubated overnight at RT. Plates were washed with PBS-Tween and incubated at RT for 4 h with the appropriate alkaline phosphatase-conjugated goat anti-mouse Ig isotype or subclass-specific Ab (Southern Biotechnology). Plates were washed and developed with nitrophenyl phosphate substrate (Amresco) and the reaction was terminated by the addition of 100 µl/well of 2N NaOH. ELISA plates were read on a VersaMax microplate reader at 405 nm wavelength and analyzed with SoftMax Pro 5.4 (Molecular Devices, Sunnydale, CA). Concentrations of Ag-specific and total IgA Ab were calculated by interpolation of calibration curves generated using a mouse Ig reference serum (ICN Biomedicals, Aurora, IL). Salivary IgA responses are reported as the percentage of RiVax-specific IgA in total IgA to compensate for variation in salivary flow rate.Anti-RTA ELISAs were performed using previously established methods . Briefly, wells of Nunc Maxisorb F96 microtiter plates (ThermoFisher Scientific) were coated overnight with 100 µL RTA (1 µg/mL) in PBS (pH 7.4) prior to being treated with sera from the immunized mice. Horseradish peroxidase-labeled goat anti-mouse IgG-specific polyclonal Ab (Southern Biotechnology) was employed as a secondary detection reagent. ELISA plates were developed using the colorimetric detection substrate 3,3′,5,5′-tetramethylbenzidine (Kirkegaard & Perry Labs, Gaithersburg, MD) and were analyzed using a SpectroMax 250 spectrophotometer and Softmax Pro 5.2 software (Molecular Devices). [...] Mice immunized by the i.d. route with RiVax and adjuvants were observed every 24 h for reactivity at the immunization site. For gross morphologic analysis, indurations resulting from i.d. immunization were measured daily for 14 days using digital calipers. Skin sections were collected for histological analysis from separate groups of mice at a time point 7 days after i.d. immunization. Skin sections were fixed overnight in 10% buffered formalin, embedded in paraffin, and sliced into sections of 5 µm thickness. Unstained sections were employed for subsequent immunofluorescence analysis. Other sections were stained with hematoxylin and eosin (H&E) for light microscopic analysis. For light micrographs of H&E stained sections, images were acquired using a Nikon Eclipse E600 Epifluorescence microscope at 40x and 200x magnification. Unstained sections were processed for immunostaining . Briefly, Ag retrieval of sections was performed by treating the sections for 20 min at 95°C in 10 mM citrate buffer (pH 6). After cooling to RT, sections were washed in PBS and blocked with 2% (W/V) powdered nonfat milk for 1 h at RT, followed by additional washes in PBS. Sections were incubated overnight at 4°C with dilutions (1∶100) of primary rat anti-mouse CD45 and rabbit anti-mouse collagen 1 Ab in 1% (W/V) powdered nonfat milk. After washing in PBS, sections were incubated for 1 h at RT with 1∶500 dilutions of chicken anti-rat Alexa647 and chicken anti-rabbit Alexa488 secondary Ab (Invitrogen). After washing in PBS, sections were mounted onto glass slides using SlowFade Gold mounting medium containing DAPI nuclear stain (Invitrogen) and analyzed using a Zeiss Axioimager fluorescence microscope at 200x magnification. Fifteen random images were obtained throughout each section and the number of CD45+ immune cells and the total number of nuclei were counted using Axiovision and ImageJ software. […]

Pipeline specifications

Software tools SoftMax Pro, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus, Escherichia coli