Summary auto-generated
This study demonstrates that concanavalin A (Con A), a lectin that binds specific carbohydrates, can be used to purify hepatitis B antigen from human serum. Con A was applied as a soluble reagent or covalently linked to Sepharose beads to selectively precipitate and capture hepatitis B antigen. When serum containing hepatitis B antigen was mixed with Con A, the antigen precipitated and could be recovered after redissolution in mannopyranoside solution. Using Con A-Sepharose affinity chromatography, approximately 82% of contaminating serum proteins were removed while hepatitis B antigen was retained and subsequently eluted. The purified antigen was further processed by molecular exclusion chromatography and examined by electron microscopy, revealing three morphological forms: 40 nm spherical particles, 22 nm spherical particles, and filamentous rods. All three forms bound Con A, indicating exposed carbohydrate-binding sites on their surfaces. The purification method preserves biological activity better than existing procedures that use proteolytic enzymes or extreme pH conditions, which can damage larger particle forms.
Key findings
- Concanavalin A selectively binds hepatitis B antigen through terminal carbohydrate residues, enabling effective precipitation and purification from serum
- Con A-Sepharose affinity chromatography removed approximately 82% of contaminating serum proteins while retaining hepatitis B antigen
- All three morphological forms of hepatitis B antigen (40 nm spheres, 22 nm spheres, and filamentous rods) possess accessible Con A-binding sites
- The affinity chromatography method preserves antigen biological activity better than proteolytic enzyme or extreme pH-based purification procedures
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Abstract
Concanavalin A (Con A), a lectin isolated from jack beans, binds specifically to saccharides with terminal α-D-mannopyranosyl, α-D-glucopyranosyl or -D-fructofuranosyl residues (Goldstein, Hollerman & Merrick, 1965a; Goldstein, Hollerman & Smith, 1965b; Goldstein & So, 1965). Glycoproteins, present in human serum (Leon, 1967; Morse, 1968) or representing structural components of membranes of mammalian cells (Inbar & Sachs, 1969) or of enveloped viruses (Oram et al. 1971; Becht, Rott & Klenk, 1972; Calafat & Hageman, 1972; Klenk, Rott & Becht, 1972) react with Con A. Recently, Cawley (1972) observed that Con A partially precipitated hepatitis B antigen. Results presented here extend this finding and show that the interaction between hepatitis B antigen and Con A may be utilized as a step in purifying the distinct morphological forms of this antigen.
Four vol. of serum containing hepatitis B antigen were mixed with increasing amounts (0.2 to 1.0 vol.) of a solution of Con A (1 g in 26.4 ml of saturated NaCl; Miles-Yeda Limited, Rehovoth, Israel).