Peroxidase conjugated protein a

Among all the isolates E. tarda ED-BDU1 was found to be highly resistant, so we constructed a genomic DNA expression library for our local isolate. E. tarda strain ED-BDU1 was constructed using λZAPII library (Strategene, San Diego, CA). Briefly 5 x 10⁴ pfu of library construct was used for amplification. Validation of the entire library was carried out by PCR. A λZAPII library consisting of 3- to 5-kb random fragments of E. tarda ED–BDU1 was screened to identify phage that expressed gene products reactive with rabbit polyclonal antibodies specific for E. tarda. The secondary antibody was peroxidase-conjugated Protein A (Sigma, St. Louis, MO) diluted 1:2000. After primary and secondary antibody treatment, the NC membrane was developed with the chromogenic substrate 4-chloro-1-napthol (Sigma, St. Louis, MO).

Two young healthy rabbits weighing 3kg were selected and their sera were pretested for E. tarda antibodies prior to inoculation. Live antigens were prepared from 24 hours old E. tarda ED-BDU1 culture (1-2 X 10⁸/ml). The cultures were centrifuged at 10,000 x g for 10 mins and dissolved in Phosphate buffered saline (PBS) and the cells count was determined by using a Neubauer counting chamber. Briefly, 1-2 x 10⁸ cells were injected subcutaneously into the rabbits on day 1, followed by booster inoculums on days 14 and 28. The level of antigen-specific circulating antibodies was determined by titrating the serum samples in ELISA. For performing serum dilution in ELISA, wells were coated with 100μl carbonate coating buffer (pH 7.2) containing 1μg of sonicated antigen and stored at 4⁰C overnight. Then the plate was proceeding to wash with phosphate-buffered saline with Tween 20 (PBST) (8mM Na₂HPO₄, 150mM NaCl, 2mM KH₂PO₄, 3mM KCl, 0.05% Tween^® 20, pH 7.4) and blocked with 200μl/well of 4% non-fat milk in PBST for 1 h at 37°C. Sera were diluted from starting dilution of 1:25 to 1:51200 in triplicate and incubated for 1 h at 37°C. Bound IgG was detected using peroxidase-conjugated Protein A (Sigma, St. Louis, MO), followed by development using 100μl of ortho phenylenediamine (OPD). Optical densities (OD’s) were read at 490nm using an ELISA reader (Bio-Rad, Hercules, CA, USA).