Griffin:Antibody Basics

1) A protein target is chosen to develop an immunizing antigen towards a specific region of said protein.

• Common types of immunizing antigens
• a) 15-25 amino acid synthetic peptide
• b) 50-300 amino acid recombinant protein
• c) cell culture/in vivo derived immunizing fraction

2) Primary antibody; Monoclonal or Polyclonal?

      Monoclonal antibody production -single epitope=high specificity
              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
              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.

3) 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/myeloma fusions 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.

4) 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