MadSci Network: Immunology

Re: Experiments which led to the elucidation of the structure of the antibody

Date: Tue Nov 21 17:52:13 2000
Posted By: Ingrid Dodge, Grad student, Immunology
Area of science: Immunology
ID: 974234761.Im

Dear John:

Thank you for your interesting question. It was actually something that I was asked about on my PhD program qualifying exam. The first descriptions of antibodies (or gamma-globulins) were from Tiselius and Kabat, in 1939 (J. Exp. Med 69:119). They immunized rabbits with ovalbumin (chicken egg white protein) and ran the immunized serum on a gel. They saw four protein bands (globulins): albumin, alpha-globulin, beta-globulin and gamma-globulin. They then did an insightful experiment, where they adsorbed the serum against ovalbumin first, then ran the resultant "depleted" serum on a gel. They found that the gamma-globulin peak was dramatically reduced. This experiment pointed out that there is some globulin in serum that binds directly to antigen.

The next step forward in our understanding of the immunoglobulin molecule came from ultracentrifugation and digestion experiments in the 1950s and 1960s. Rodney Porter and Gerald Edelman used ultracentrifugation to separate the immunoglobulin protein fraction into a 19S (high molecular weight) and a 7S (low molecular weight) fraction. Porter then used papain digestion of the 7S IgG molecule to yield the now well-known 2Fab and 1Fc per whole IgG molecule (these were resolved by gel electrophoresis). The Fc, incidentally, was called Fc because it was a fragment that readily crystallized if chilled. Alfred Nisonoff also digested the antibody molecule, but with pepsin, which yields F(ab')2 (which was about 100kD, a little over twice the size of the 45kD Fab fragment) and no Fc, because the Fc is digested away. These researchers also noted that the Fab and F(ab')2 portions of the molecules could precipitate antigen, but the Fc could not, so the Fab and F(ab')2 contained the antigen-combining region of the molecule. After separating the immunoglobulin molecule into the antigen- combining portion and the crystallizable portions, Edelman and Porter then went on to separate the molecule into heavy and light chains by mercaptoethanol reduction and alkylation, which cleaves intrachain disulfide bonds. These experiments demonstrated that IgG was composed of two 50kD heavy chains and two 25kD light chains. For their contributions to our understanding of the immunoglobulin molecule, Porter and Edelman shared the Nobel Prize in 1972.

So Porter, Edelman and Nisonoff elucidated the components of the immunoglobulin molecule in the experiments above, but the function of each portion remained unclear. Porter addressed this question, too, in a series of seminal experiments. He immunized goats with rabbit Fab fragments and rabbit Fc fragments, then asked whether the goat antisera would react with the rabbit heavy or light chains. He found that the Fab antisera reacted with both the heavy and light chains, while the Fc antisera reacted with only the heavy chains. Based on this obeservation, Porter and Edelman proposed that the IgG molecule consists of two identical disulfide-bonded heavy chains which are further disulfide-bonded to two identical light chains. This insight has proven to be accurate up to today.

The next step in determining protein structure, beyond chain resolution, is to sequence the protein. Initial attempts at sequencing were hindered by a lack of sufficient quantities of protein. A human cancer, multiple myeloma, ultimately solved this problem. Multiple myeloma is a cancer of plasma cells, which are antibody-secreting B cells, and the myeloma proteins can account for up to 95% of the serum immunoglobulin. Many myeloma patients produce an excess of light chains, which can be filtered out of the blood by the kidney and end up in the urine. These light chains in the urine, referred to as Bence-Jones proteins, provided enough protein for the first light chains to be sequenced. When these light chains were compared, it was noted that the amino terminal half was variable, while the carboxy terminal half was constant and came in one of two flavors lambda or kappa. Heavy chains were also sequenced from myeloma patient serum and compared. Again, there was a variable amino terminal portion, and one of five constant regions: alpha, gamma, mu, delta or epsilon.

Finally, the immunoglobulin molecule was solved by X-ray crystallography. I believe this was first done by LJ Harris in 1992 (Nature 360:369), but I may be wrong. The polymeric structure of IgM was contributed by electron micrography by Davis, Roux, and Shulman (1988, European J. Immunology 18: 1001). (These are cool pictures - you should check them out sometime).

These are the fundamental experiments that I know of that elucidated structure of the antibody molecule. I hope it is helpful!


Ingrid Dodge, Mad Scientist

My favorite textbooks on immunology are:

Paul, WE Ed. Fundamental Immunology, 4th Ed. New York: Lippincott-Raven Pub., 1999.

Kuby, J. Immunology, 3rd Ed. New York: WH Freeman and Co., 1997 (from which I drew much of this discussion, as well as Paul above, and my history of immunology class notes).

Janeway and Abbas also have good introductory textbooks.

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