Varughese, M

Varughese, M., A. 22). Here, we describe MAbs that we raised against PA83 and LF and screened for his or her ability to neutralize the activities of the toxins. Characterization of the epitopes of PA83 identified by the MAbs enhances our knowledge of the protein. Passive administration of polyclonal antisera against PA83 prevents spore illness in guinea pigs (15, 19): we display the neutralizing activities of MAbs against PA83 and LF protect mice against a lethal spore challenge with the toxinogenic Sterne strain. MATERIALS AND METHODS Hybridoma production. BP2 mice were immunized subcutaneously (s.c.) with 20 g of PA83 or LF in 100 l of NaCl (150 mM) emulsified with 100 l of total Freund’s adjuvant. Four s.c. booster injections in incomplete Freund’s adjuvant were given 1, 3, 5, and 7 weeks later on. Fifteen days later on, mice received the last intraperitoneal booster of antigen (10 g). Splenocytes were collected 3 days later on and immortalized by fusion with mouse myeloma cells (x63Ag8-653) (16). Cells were cultivated on 24-well plates, and the tradition medium was screened by enzyme-linked immunosorbent assay (ELISA) for PA antibodies. Hybridomas from positive wells were cloned by limiting dilution and plated on microtiter plates. Positive clones recognized by ELISA were expanded and used to inject mice for ascites production. Antibodies were purified by precipitation with ammonium sulfate. ELISA. We used a modified version of a standard ELISA to test for the presence of antibodies in tradition supernatants (7). Microtiter plates (Nunc, Roskilde, Denmark) were incubated over night at 4C with 1 g of antigen/ml of phosphate-buffered saline (PBS). Plates were washed four instances with buffer A (0.1% Tween 20 in PBS), and supernatants were diluted in buffer B (0.5% gelatin in buffer A). The plates were incubated for 2 h at 37C and washed again, and horseradish peroxidase-labeled rabbit Lys05 anti-mouse antibodies (Bio-Rad Pasteur, Marnes-la-Coquette, France) were added for 1 h at 37C. Then, the plates were washed with buffer A, and freshly prepared 0.2% orthophenylenediamine (Dakopatts A/S, Glostrup, Denmark) and 0.03% H2O2 in 0.1 M citrate buffer (pH 5.2) were added to each well. The peroxidase reaction was stopped by adding 3 M HCl, and the optical denseness was measured at 490 nm. The dissociation constants were identified as previously explained by Friguet et al. (11). Phage display techniques. The PhD-7 phage display kit (New England Biolabs, Beverly, Mass.) was utilized for the biopanning experiments essentially as recommended by the manufacturer. Briefly, MAb 48.3 at a concentration of 100 Lys05 g/ml in carbonate buffer (0.1 M NaHCO3 [pH 8.6]) was immobilized in 96-well plates with gentle agitation for 15 h at 4C. The covering solution was eliminated, and each well was filled with obstructing buffer (0.1 M NaHCO3 [pH 8.6], 5 mg of bovine serum albumin/ml, 0.02% NaN3) for 1 h at 4C. Then, the blocking remedy Rabbit Polyclonal to GFP tag was discarded, and the wells were washed six instances with Tris-buffered saline (TBS) (50 mM Tris-HCl [pH 7.5], 150 mM NaCl) Lys05 containing 0.1% vol/vol Tween 20. We added 4 1010 phages from the original library in TBS-0.1% Tween 20 and remaining them to absorb for 30 min at space temperature. Unbound phage was eliminated by washing with TBS comprising 0.1% Tween 20. The bound phages were eluted by the addition of 0.2 M glycine-HCl (pH 2.2) and 1 mg of bovine serum albumin/ml for 10 min with subsequent neutralization having a 1:10 volume of 1 M Tris-HCl (pH 9.1). An aliquot of the eluted phage was kept for.