Immunoglobulins: Structure, Classification and Determination

In this article we will discuss about Immunoglobulins:- 1. Structure of Immunoglobulins 2. Classification of Immunoglobulins 3. Electrophoretic Determination 4. Quantitative Determination.

Structure of Immunoglobulins:

a. The basic unit of all immunoglobulin molecules consists of 4 polypeptide chains linked by disulfide bonds shown in Fig. 30.1

b. There are two identical heavy (H) chains (MW 53,000-75,000) and two identical light (L) chains (MW 23,000).

c. Immuno-globulins composed of more than one basic monomeric unit are termed polymers.

Important examples are IgA dimers (2 units), IgA trimers (3 units) and lgM pentamers (5 units).

d. The polypeptide chains are not straight sequences of amino acids but are folded 3-dimensionally with disulphide bonds to form areas called domains.

e. The part of the antibody molecule which combines with antigens is formed by a few amino acids in the F region of H and L chains.

f. Papain cleaves IgG in the presence of cysteine into 3 fragments: 2 Fab fragments (MW 52,000) and an FC fragment (MW 48,000). The Fab fragments bear the entire antibody combining site for the antigen.

g. There are two major types of L chains in man, the Kappa (к) and lambda (λ) chains. About 70 per cent of the human immu­noglobulin molecules carry к light chains and 30 per cent carry λ light chains. In IgG, the H chain is termed a gamma (γ) chain; in IgA, an alpha (α) chain; in IgM, a mu (µ) chain; in IgD, a delta (δ) chain; and in IgE, an epsilon (ɛ) chain.

h. Carbohydrate residues are attached to the polypeptide chains. 2 carbohydrate units per molecule of IgG and 3 per molecule of IgM. The carbohydrate residues include D-mannose, D-galactose, L-fructose, D-acetylneuraminic acid and glucosamine.

i. Each chain is divided into specific domains or regions that have structural and func­tional significance. The half of the light chain (L) towards the carboxy terminus is termed as the constant region (CL), while the amino terminal half is the variable re­gion (VL) of the light chain.

About one quarter of the heavy (H) chain at the amino terminus is termed as variable region (VH), and the other three quarters of the heavy chain are referred to as the constant regions (CH¹, CH², CH³) of that H chain.

j. The portion of the immunoglobulin mol­ecule which binds the specific antigen is formed by the amino-terminal portions (variable regions) of both the H and L chains, i.e., the VH and VL domains. The domains of the Protein Chains do not sim­ply exist as linear sequences of amino ac­ids but form globular regions with second­ary and tertiary structure.

Classification of Immunoglobulins:

On the basis of electrophoretic, immunologic and ultra-centrifugal studies, the immuno-globulins have been divided into 5 groups:

i. IgG:

(a) This is the major antibody-containing frac­tion which comprises 80 per cent of the gamma globulins.

(b) It is a single basic immunoglobulin unit with y heavy chains.

(c) Its molecular weight is 150,000-160,000 and it contains 2-4 per cent carbohydrates.

(d) It has the slowest electrophoretic mobility and is distributed in the extracellular fluid and is capable of crossing the placenta.

(e) Its normal level in blood is 570-1,920 mg/dl

ii. IgA:

(a) It has a molecular weight of about 180,000-400,000 and its S rate is 6.6-13.

(b) It has a higher content of carbohydrate (5- 10 per cent).

(c) It is present in high concentrations in the blood, in saliva and tears and in the secre­tions of gastrointestinal tract.

(d) It is single basic immunoglobulin unit with heavy a chains. Secretory IgA is made up of 2 basic units connected by a J chain.

(e) A 60,000 MW molecule called transport piece (t piece) is attached to the Fc por­tion. This is necessary for the transport of IgA molecules into the lumens of exocrine glands. Secretory IgA plays an important role in host defense mechanisms against viral and bacterial infections. IgA does not cross the placenta.

(f) Its normal level in blood is 70-400 mg/dl.

iii. IgM:

(a) It contains 576 amino acids and has a mass of 950,000 Daltons.

(b) It is the first antibody to be formed in a new born animal or human.

(c) IgM (with IgD) is the major immunoglobu­lin expressed on the surface of B cells.

(d) It’s carbohydrate content is 10-12 per cent. It is dissociated into subunits designated IgMs. Each monomer is composed of two L chains and two H chains (µ) with 2 com­bining sites, so that the intact molecule has 10 combining sites.

(e) IgM does not cross the placenta.

(f) The basic units of it are connected by disulfide bond bridges and small polypep­tide J chain.

Its normal level in blood in males is 30- 250 mg/dl.

Its normal level in blood in females is 30- 300 mg/dl.

iv. IgD:

(a) No antibody activity is associated with IgD.

(b) Its normal level in blood is 0-8 mg/dl.

v. IgE:

(a) It is present in the serum in very low con­centrations as a single basic unit with heavy E chains.

(b) Its molecular weight is about 190,000 (8S). Half of patients with allergic diseases have increased serum IgE levels.

(c) The specific interaction between antigen and IgE bound to the surface of mast cells results in the release of inflammatory mast cell products such as serotonin and hista­mine.

(d) Its normal level in blood is < 0.025 mg/dl.

Electrophoretic Determination of Immunoglobulins:

In 1937, Tiselius performed the separation of pro­teins in electrical fields. Owing to the relative com­plexity of his method, zone electrophoresis has re­placed electrophoresis in a free solution.

Zone Electrophoresis:

Serum or other biologic fluid samples are placed on the cellulose acetate and sepa­rated by electrophoresis for 90 minutes using alkaline buffer solutions. The strips are then stained and scanned. This procedure separates normal serum pro­teins into 5 major electrophoretic bands (albumin, α₁-globulin, α₂-globulin, β- globulin, and γ-globulin).

This electrophoresis is very useful for the diagnosis of human para-protein disorders such as multiple myeloma and hypo­gammaglobulinemia. In hypogamma­globulinemia, the decrease in serum γ- globulin is easily detected.

Immunoelectrophoresis (IEP):

a. It consists of both electrophoretic separa­tion and immune precipitation of proteins.

b. A glass slide covered with buffered mol­ten agar (pH 8.2) is required to perform the test.

c. An antigen (serum) well and an antibody trough are cut in the agar after the agar is cooled.

d. The serum sample is placed in the well and the various proteins are then separated in an electrical field.

e. Antiserum is next placed in the trough and allowed to diffuse for 18-24 hours in the direction of the separated proteins.

f. When the various protein fractions meet the migrating antibody, precipitin lines are formed. These lines are stained or pho­tographed to make a permanent record.

g. The absence of immunoglobulin classes or the presence of abnormal immu­noglobulin molecules are detected by this process even in very low concentrations that are not apparent by zone electrophore­sis.

Quantitative Determination of Immunoglobulins:

a. The radial diffusion technique is utilized for the quantitative determination of immuno-globulins.

b. Wells are cut in an agar plate impregnated with a specific antiserum directed against a single human immunoglobulin class.

c. A circular precipitin ring will form after the human serum proteins placed in the well diffuse through the agar.

d. The diameter of the precipitin ring is pro­portionate to the concentration of serum immunoglobulin.

e. The level is determined by comparing the diameter of the unknown serum to that of a standard containing known levels of immuno-globulins.

f. This test does not differentiate between normal and abnormal immunoglobulin molecules as does the immuno-electrophoresis procedure.

g. The normal concentrations of the 3 major immunoglobulin classes are as follows:

IgG : 570-1920 mg/dl.

IgA : 70-400 mg/dl.

IgM : Males : 30-250 mg/dl

Females : 30-300 mg/dl