Rabbit Antisera Analysis

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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
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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

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