One problem with the study of antibodies was that they are heterogenous. For example, an electrophoresis pattern of an animal immunized against albumin (a homogenous protein) would show a spike of albumin and then several much smaller spikes of antibodies (meaning the albumin antibodies are polyclonal, or consisting of different subsets binding different sites on the same antigen). In multiple myeloma, tumorous plasma cells all secrete the tame type of immunoglobulin. This leads to a huge monoclonal spike of antibodies, since there will be huge amounts of a single antibody. The experiments below were all performed to determine the structure of antibodies. After all these experiments, the antibody structure shown above was determined.
|Electrophoretic Migration||Electrophoretic migration analysis was performed on serum from rabbits immunized with ovalbumin (resulting in ovalbumin, α, β and γ peaks), and for serum from rabbits immunized with ovalbumin but with ovalbumin antibodies removed (resulting in ovalbumin, α and β peaks). These results indicated that antibodies were some sort of gamma globulin.|
|Molecular Weight||To determine the molecular weight of this gamma globulin, it was migrated in a centrifugal field. Its migration was 7S, corresponding to a 150,000 dalton molecular weight.|
|Valence||Molecular analysis of immune precipitates between bacterial polysaccharide antigens and their specific antibodies showed a valence of 2.|
|Papain Cleavage||Cleavage of an antibody with papain yielded two different fragments that were separated using ion exchange chromatography: F(ab) and F(c). A whole antibody bound two antigen molecules, F(ab) bound a single antigen molecule (but could not precipitate) and F(c) formed crystals. F(ab) and F(c) each had a weight of 50,000 daltons. Thus, an antibody must contain 2 F(ab) fragments and 1 F(c) fragment.|
|Pepsin Cleavage||Cleave of an antibody with pepsin yields one fragment of 100,000 daltons capable of binding two antigen molecules and could precipitate.|
|Disulfide Cleavage||Cleavage of disulfide bonds yielded two products which could be separated based on their size: a heavy chain (50,000 daltons) and a light chain (25,000 daltons). An anti-L antibody reacted with Fab only. An anti-H antibody reacted with F(ab) and F(c). An anti-F(ab) antibody reacted with both H and L.|