Figure 3.1.2. Antibody titres of rabbit antisera. The antisera were collected from immunized rabbits and antibody titres measured by indirect ELISA as described under Methods (section 2.2.3.1).
3.1.2.1.3 Purification of α-LA, HRP and human erythrocyte specific polyclonal IgG
The IgG fractions were purified from rabbit sera by ammonium sulphate precipitation followed by negative ion exchange chromatography employing DEAE-cellulose. The antisera were decomplemented, IgG fraction precipitated by 40% (w/v) ammonium sulphate saturation in PB, pH 7.2 and the precipitate dialysed against the same buffer. The preparation thus obtained was dissolved in 1 mL of PB, pH 7.2 and loaded on to a DEAE-cellulose column equilibrated with PB. An inverse sigmoidal elution profile was obtained as shown in Figure 3.1.3 B. Figure 3.1.3. Purification of IgG. Panel A. Reducing SDS-PAGE of human erythrocyte ghost. Panel B. Elution profile of IgG from a DEAE-cellulose column. Panel C. Non-reducing SDS-PAGE profile at various steps of purification. Lane 1: molecular weight markers, lane 2: decomplemented serum, lane 3: 40% (w/v) ammonium sulphate fraction, lane 4: supernatant after the …show more content…
Rabbit IgG half molecules could be generated by using sodium sulphite as the reductant and DTNB as the blocking agent for the released sulphydryls from the inter H chain disulfide (Chan & Wasserman, 1993). For this purpose, 2 mg/mL rabbit antibody solutions were reduced with varying concentration of [i] sodium sulphite (200-500 mM) in presence of 2.5 mM DTNB in Tris EDTA buffer, pH 8.8 for 2 h at 37°C followed by dialysis against the same buffer and [ii] DTNB (2.5-20 mM) and 500 mM sodium sulphite in Tris EDTA buffer for 2 h at 37°C. The formation of half molecules was assessed by non-reducing
3.1.2.1.3 Purification of α-LA, HRP and human erythrocyte specific polyclonal IgG
The IgG fractions were purified from rabbit sera by ammonium sulphate precipitation followed by negative ion exchange chromatography employing DEAE-cellulose. The antisera were decomplemented, IgG fraction precipitated by 40% (w/v) ammonium sulphate saturation in PB, pH 7.2 and the precipitate dialysed against the same buffer. The preparation thus obtained was dissolved in 1 mL of PB, pH 7.2 and loaded on to a DEAE-cellulose column equilibrated with PB. An inverse sigmoidal elution profile was obtained as shown in Figure 3.1.3 B. Figure 3.1.3. Purification of IgG. Panel A. Reducing SDS-PAGE of human erythrocyte ghost. Panel B. Elution profile of IgG from a DEAE-cellulose column. Panel C. Non-reducing SDS-PAGE profile at various steps of purification. Lane 1: molecular weight markers, lane 2: decomplemented serum, lane 3: 40% (w/v) ammonium sulphate fraction, lane 4: supernatant after the …show more content…
Rabbit IgG half molecules could be generated by using sodium sulphite as the reductant and DTNB as the blocking agent for the released sulphydryls from the inter H chain disulfide (Chan & Wasserman, 1993). For this purpose, 2 mg/mL rabbit antibody solutions were reduced with varying concentration of [i] sodium sulphite (200-500 mM) in presence of 2.5 mM DTNB in Tris EDTA buffer, pH 8.8 for 2 h at 37°C followed by dialysis against the same buffer and [ii] DTNB (2.5-20 mM) and 500 mM sodium sulphite in Tris EDTA buffer for 2 h at 37°C. The formation of half molecules was assessed by non-reducing