Griffin:Antibody Basics: Difference between revisions
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*b) 50-300 amino acid recombinant protein | *b) 50-300 amino acid recombinant protein | ||
*c) cell culture/in vivo derived immunizing fraction | *c) cell culture/in vivo derived immunizing fraction | ||
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II) Primary antibody; Monoclonal or Polyclonal Production | II) Primary antibody; Monoclonal or Polyclonal Production | ||
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avidity means a polyvalent reagent; may be specific, although higher | avidity means a polyvalent reagent; may be specific, although higher | ||
chance of sensitivity. | chance of sensitivity. | ||
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III) Animal host types for polyclonal or monoclonal production. | III) Animal host types for polyclonal or monoclonal production. | ||
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*c) rodent (mouse, rat, hamster); small, user friendly animal for spleenectomy and B cell fusion to produce monoclonal Ig. | *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. | *d) chicken; unique non-mammal, polyclonal IgY from yolk sack. | ||
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IV) Antibody purification | IV) Antibody purification | ||
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*a) peptide antigen affinity column | *a) peptide antigen affinity column | ||
*b) Protein A or G column | |||
Revision as of 21:18, 10 September 2008
I) 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
II) Primary antibody; Monoclonal or Polyclonal Production
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.
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/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.
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
