Griffin:Antibody Basics

I) A protein is an important element of the cellular world. A fragment of a protein of interest is chosen to develop an immunizing antigen.

Common types of immunizing antigens include;

• a) 15-25 amino acid synthetic peptide
• b) 50-300 amino acid recombinant protein
• c) cell culture/in vivo derived enriched fraction

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II) Primary antibody; Monoclonal or Polyclonal Production

Monoclonal antibody production -single epitope=high specificity, low avidity

• a) Smaller animal conserves materials since the animal eventually

gets sacrificed. Mouse or hamster is immunized over time, then the spleen is harvested from the sacrificed animal. Ig producing B Cells from the spleen are immortalized by fusing with a myeloma cell in culture; the B Cell/myeloma fusion is known as a hybridoma and secretes a single clone type of Ig.

• b) Monoclonals detect a single epitope; hence mono-clonal. High

specificity, low avidity.

Polyclonal antibody production -multiple epitopes=high avidity, high sensitivity

• a) Larger animals produce more blood. Goat or rabbit is immunized

over time and blood is drawn on a routine basis. Serum fraction of the blood undergoes purification by affinity column or protein A/G column. The total circulating IgG derived from humoral immunity is isolated from the animal blood and recognizes many parts of the antigen.

• b) Polyclonals detect several epitopes; hence poly-clonal. High

avidity means a polyvalent reagent; may be specific, although higher chance of sensitivity.

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III) Animal host types for polyclonal or monoclonal production.

Common animal types

• a) rabbit; large, unique animal with enough blood to draw and isolate total polyclonal IgG. Also suitable for spleen fusions (immortalization) to produce monoclonal Ig.
• b) goat; large, hearty animal with enough blood to draw and isolate total polyclonal IgG.
• c) rodent (mouse, rat, hamster); small, user friendly animal for spleenectomy and B cell fusion to produce monoclonal Ig.
• d) chicken; unique non-mammal, polyclonal IgY from yolk sack.

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IV) Antibody purification

Monoclonal purification

• a) protein-G affinity column
• b) hybridoma cell cutlure supernatant
• c) ascites fluid from peritoneal cavitiy of a mouse

Polyclonal purification

• a) peptide antigen affinity column
• b) Protein A or G column

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