1. Antibodies (Immunglobulins (Igs) A- Definition: Immunoglobulins are a group of proteins (gamma globulins) produced by the body from B lymphocytes and plasma cells in response to presence of foreign bodies (antigens). There are 5 basic types: G, M, A, E and D. All of immunoglobulins have a similar basic structure. B- Basic structure of immunoglobulins: The basic unit of all Igs molecules consists of 4 polypeptide chains linked by disulfide bonds: a) 2 polypeptide chains of low molecular weight called light chains (L) b) 2 polypeptide chains of high molecular weight called heavy chains (H)

2. Disulfide bonds in Ig: 1. Inter-chain disulfide bonds - heavy(H) and light (L) chains are linked together by disulfide bond. In addition, the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions. 2. Intra-chain disulfide bonds - Within each of the polypeptide chains (within H and L chains) there are also intra-chain disulfide bonds. Enzyme called “papain” cleaves Ig into two fragments: Fab fragment and Fc fragment. Fab fragment is the part of antibody molecule which combine with antigens.

3. Basic structure of Ig

4. Immunoglobulin classes The Igs can be divided into five different classes, based on differences in the amino acid sequences in the heavy chains. Ig G a) IgG is the major Ig in serum:70-75% of serum Ig is IgG b) IgG is composed of one basic unit (monomer), i.e of low Mol. weight c) Placental transfer: IgG is the only class of Ig that crosses the placenta. In addition, Ig G is also transferred from mother to baby in breast milk. Transplacental and breast milk-derived Ig G offer the baby a passive immunologic protection until the endogeenous Ig G is produced. The half life of Ig G is about 30 days and with prolonged breast feeding the infant can get additional protection.

5. IgA 1. Structure - IgA is a monomer in serum but found in secretions is a dimer as presented in the Figure. In diameric IgA, the molecules are joined by a small polypeptide chain called J chain linked to Fc regions. - When IgA is found in secretions:, it also has another protein associated with it called the secretory piece or T piece. The secretory piece helps IgA to be transported across mucosa and also protects it from degradation in the secretions. 2. Properties a) IgA is the 2nd most common serum Ig (10-15%). b) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. c) can’t pass placenta

6. IgM 1.Structure IgM normally exists as a pentamer. So it has the highest molecular weight. The five units are linked by small polypeptide chain called J chain 2. Properties a) IgM is the third most common serum Ig (5-10% of total Ig). b) IgM is the first Ig be made by a virgin B cells when it is stimulated by antigen. c) It can’t cross placenta

7. IgE 1. Structure: IgE exists as a monomer. 2. Properties: a) is the least common Ig in serum b) It is largely responsible for immunity against parasites. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. c) Ig E, in the presence of antigen, binds to mast cells and basophils causing release of histamine and other substances from mast cells. These substances result in allergic manifestations. Ig D: IgD exists only as a monomer. IgD is found in low levels in serum; its role in serum uncertain.

8. Ig EIg D I g M Ig AIg G 184 970160 Mass (KD) trace Less than 1 5-1010-1570-75% of total Ig NO yesCross placenta NO yes In breast milk NO yes NOIn secretions yesNO Binding with mast cells

9. Electrophoresis: It is the movement of charged molecules in an electrical field towards the oppositely charged electrode. It is used for separation of proteins for dignosis of some diseases such as immunity disease, genetic diseases ( such as Hb S and Hb C diseases. The rate of migration of charged molecules depends on: a - amount of charge on the molecule b- molecular weight (density) of the molecule. Serum protein electophoresis: In the standard method, serum samples are applied to support medium ( agarose gel) on plate close to the cathode end. Both ends of support medium is immersed in alkaline buffer (pH 8.6), so proteins in this pH will be negatively charged.

10. 2) The support strip is connected to two electrodes and a current is passed throughthe strip to separate proteins. 3) As serum proteins are negatively charged, they will move toward anode (positive electrode). Serum proteins move according to their density and amount of charge then arranged into 5 bands: albumin moves faster to the anode followed by α1 globulin, α2 globulin, β globulin and γ globulins.

11.  In case of decreased serum albumin (hypoalbuminuria), the albumin band becomes less dense. This occurs,for example, in advanced liver disease as liver is the site of albumin synthesis. The separated proteins are made visible by staining. 4) Density of each band is directly proprtional to its serum concentration, so albumin will show the most dense band ( serum albumin is 3.5-5.5 gm%, while globulins 1.8-3.3 gm%).