The body can produce antibodies to what may be a limitless array of antigens, each antibody reacting specifically with the antigen that initiated the immune response, leading to its appearance in the serum. Determining the structure of the molecule that can impart so much diversity and the structural basis of for the specificity -- was for years one of the central problems of biology.
Now puzzle is almost complete.
Terms antibody and immunoglobulin are interchangeable.
The antibody molecule is a monomer composed of four polypeptide chains: two identical heavy chains and two identical light chains held together by interchain disulfide bonds. There are several classes (isotypes) of immunoglobulin molecules, and they differ in size and number of monomers.
Antibody activity is present in the serum [ the fluid portion of the blood that is left after the blood has been allowed to clot]. Plasma, in contrast, is the fluid portion of the blood, containig unreacted clotting factors.
Serum contains many other proteins besides immunoglobulin, but antibodies are found in the gamma globulin fraction.
The type of experiment that was used to discover that Igs are in the gamma globulin fraction of serum is described below:
Rabbit was immunized with egg albumin (OVA)
The serum was analyzed by electrophoresis. [see pattern in textbook]
Next, the serum was combined with the OVA antigen and precipitated antibody-antigen complexes were removed by centrifugation.
Following electrophoresis, the gamma globulin fraction of the serum was greatly diminished in size, demonstrating that the OVA-specific Igs were located in this fraction.
Biochemical Analysis of Immunoglobulins
Some of the first biochemical analyses were performed using proteases. In particular, Igs were treated with either papain or pepsin.
When Ig is treated with papain and the resulting fragments are separated on a carboxymethyl cellulose column, 3 fragments are obtained.
2 fragments were of equal mw ~50,000D (daltons)
- these fragments could bind antigen and, therefore, were termed Fab fragments. (fragments of antigen binding)
It had been known for some time that the antibody molecule had two antigen binding sites, and this was physical conformation of that finding.
A third fragment, with a molecular mass of ~80,000 D did not bind antigen, but crystalized at cold temperatures and, therefore, was called Fc for the (crystallizable fraction).
In contrast, when Ig is treated with pepsin, a single large fragment is yielded that possesses antigen-binding capacity with two antigen binding sites. This large fragment contained both Fab arms of the Ig molecule and, therefore, was termed the F(ab')2 fragment. In addition, the Fc portion of Ig is cleaved into numerous small pieces with pepsin treatment.
See figures in text of Papain and Pepsin treatment.
Chain Dissociation Studies
When Ig is treated with mercaptoethanol, disulfide bond are reduced. If you then alkylate, the disulfide bonds cannot reform. Then the resulting polypeptide chains can be analyzed:
Results:
2 kinds of chains
One chain with a molecular mass of ~20,000D - light chains
Other chain with a molecular mass of ~50,000D - heavy chains
From the relative concentrations of each, it was determined that the monomeric Ig molecule contains four chains, two H and two L, held together by disulfide bonds.
See figure in text.
Data from the chain dissociation / proteolytic cleavage studies combined suggested that the L chains and part of the H chains were located in the Fab fragments. The remainder of the H chain was located in the Fc fragment.
Importance of Myeloma Proteins in Ig Analysis
Antibodies in serum (antiserum) are extremely heterogeneous. They have a variety of molecular masses and reflect antibody specificities to many different antigens. This heterogeneity was one of the major stumbing blocks in the study of the chemistry of antibodies.
Myeloma proteins
Multiple myeloma - is a disease in which there is a malignant transformation in a single plasma cell that normally produces antibody. The transformed cell, now a cancer cell, multiplies in an uncontrolled manner while continuing to secrete its Ig product into the serum. The result is a very high concentration of only one kind of Ig (the myeloma protein) in the serum of the patient. In some patients, up to 95% of serum Ig is represented by the myeloma protein. The myeloma protein molecules are physically homogeneous, being the products of the progeny of a single clone of B cell. Therefore, high concentrations of identical Ig molecules can be obtained from the serum of such patients. Fortunately, the Ig product was normal in these patients, even though the cell producing it was abnormal.
In addition, multiple myeloma patients excrete high concentrations of light chain Ig in urine. This excreted Ig is termed Bence Jones protein. Therefore, the urine of multiple myeloma patients is a source of homogeneous light chain.
The Bence Jones proteins and the myeloma proteins were subjected to amino acid sequence analysis in many laboratories across the world.
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