Humoral Immunity

1. Humoral Immunity Lecture 7

2. Antibody Mediated Immunity ( Humoral Immunity) ImmunoglobulinsStructure and Function

3. Antibodies • The immunoglobulins (Ig) or antibodies are a group of glycoproteins present in the serum and tissue fluids of all mammals, make up 20% of plasma proteins. • Secreted by plasma cells, which are B cells actively fighting exogenous antigen. • Soluble, protein molecules that react specifically with the antigen that stimulated their production. • Considered part of the humoral immune response.

4. Antibody mediated immunity is important mainly: ◦ Against capsulated bacteria ◦ Prevention of some viral infections ◦ Toxin-induced disorders

5. Mechanism of Antibody production 1- When an antigen is inoculated into tissues, it is carried through lymphatic vessels to neighboring lymph nodes ( become hyperplastic and increase in weight). 2- The antigen is taken up by antigen presenting cells ( macrophages) where it is processed through partial enzymatic degradation. 3- B-lymphocyte is the cell type that responds to the processed activated antigen.

6. Mechanism of Antibody production cont…. 4-On stimulation of these lymphocytes by the activated antigen, they will divid rapidly and differentiate into large cells called immunoblasts. 5- The immunoblasts will further differentiate into plasma cell (antibody producing cells) or memory cells.

7. Basic Structure Of Immunoglobulins Although different immunoglobulins can differ structurally they all are built from the same basic unit. A. Heavy and Light Chains - All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (25kD) and two identical heavy chains (50-70 kD). B. Variable (V) and Constant (C) Regions - both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. 1. Light Chain - VL (110 aa) and CL (110 aa) 2. Heavy Chain - VH (110 aa) and CH (330-440 aa)

8. Basic Structure Of ImmunoglobulinsCont….. • C) Disulfide bonds: there are 2 types • disulfide bonds between the 2 heavy chains (H-H), and other two bonds join with the adjacent heavy chain (H-L). • disulphide bonds present between the acids of the same chain forming loops, which are called domains D). Domains - 3D images of the immunoglobulin molecule shows that it is not straight. It is folded into globular regions each of which contains an intra-chain disulfide bond. These regions are called domains. 1. Light Chain Domains - VL and CL 2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)

9. Basic Structure Of Immunoglobulins Cont…….. E) Hinge Region - The region at which the arms of the antibody molecule forms a Y is called the hinge region because there is some flexibility in the molecule at this point. F) Oligosaccharides - Carbohydrates are attached to the CH2 domain in most immunoglobulins.

11. Ag Binding Complement Binding Site Binding to Fc Receptors Placental Transfer Immunoglobulin Fragments: Structure/Function Relationships

12. - Antigen binding - These fragments were called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. - An antibody is able to bind a particular antigenic determinant because it has a particular combination of VH and VL.

13. Treatment of basic Ig molecule with papain enzyme: produce three fragments Papain Fc Fab A) Fab- Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain. B) Fc - Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain.

14. Pepsin F(ab’)2 Treatment of basic Ig molecule with pepsin enzyme Treatment of antibody molecule with pepsin results in the production of two fragments, one Fc and one divalent large fragment containing 2 Fab regions linked by disulfide bonds that is termed Fd or Fab2

15. Fab Fc S VL CL S S S CH3 S S CH2 CH1 VH S S F(ab)2 Pepsin cleavage sites - 1 x (Fab)2 & 1 x Fc Papain cleavage sites - 2 x Fab 1 x Fc Domain Structure of Immunoglobulins Domains are folded, compact, protease resistant structures Light chain C domains k or l Heavy chain C domains a, d, e, g, or m

16. Why do antibodies need antibodiy? The (Fab)2 fragment can - • Detect antigen • Precipitate antigen • Block the active sites of toxins or pathogen-associated molecules • Block interactions between host and pathogen-associated molecules But Fc fragment carries various reactions as: • Complement fixation • Opsonization • Placental transfer • Binding to mast cells

17. Valency • The valency of antibody refers to the number of antigenic determinants that an individual antibody molecule can bind ( combining capacity of an antibody with antigen). • The valency of all antibodies is at least two and in some instances more (divalent or multivalent).

18. The response to an antigen (Ag) in terms of specific antibody production over time Initially the levels of each unique antibody are extremely low, as soon as the stimulation events occur and the plasma cell begins producing antibodies the Titer (concentration or quantity/volume) of a unique antibody begins to rise.

19. Antibody production over time Continue…….. • It takes about 2 weeks for the Ab level to peak. • Once the foreign antigen is removed, antibody production slowly returns to a low level, while memory plasma cells remain in the system.

20. Antibody production over time Cont…….. • When the original antigen again appears in the host these memory cells respond rapidly and produce higher levels of antibodies. • This “remembering response" is why we remain immune to many diseases for a long time. • The secondary exposure to the antigen may be natural or it may be artificial in the case of Booster vaccinations.

21. Antibody Functions: Function in several ways ◦ Activation of complement ◦ Stimulation of inflammation ◦ Agglutination ◦ Neutralization ◦ Opsonization

22. Classes of Antibodies • A single type of antibody is not sufficient for the multiple types of invaders to the body. • The class involved in the immune response depends on: • - the type of foreign antigen, • - the portal of entry, • - and the antibody function needed. • 5 different classes of antibodies: • IgG, IgM, IgA, IgD, IgE

23. Human ImmunoglobulinTypes • Immunoglobulins can be classified by the type of heavy chain that they have to 5 types. • gamma () IgG • alpha (α) IgA, • mu ( μ ) IgM • delta ( δ) IgD • epsilon ( ε) IgE

24. Human Immunoglobulin Types cont…… • Immunoglobulins can also be classified by the type of light chain that they have. Light chain types are based on differences in the amino acid sequence in the constant region of the light chain, there are 2 types of light chains • 1. Kappa light chains (κ) • 2. Lambda light chains (λ) The Ig molecule contains either (κ) or (λ), but never both.

25. IgG1, IgG2 and IgG4 IgG3 IgG • Properties: • Major serum Ig 75% (systemic immunity) • Monomeric unit • Four subclasses are known. • Major Ig in extravascular spaces (neutralize bacterial toxins) • Placental transfer • Fixes complement • Enhance phagocytes - opsonization • Binds to Fc receptors • NK cells - ADCC

26. J Chain Cµ4 IgM • Properties • Does not cross the placenta • Consisting of five monomers joined by a single J chain • First Ig made by fetus and B cells, short lived, its presence indicates recent infection. • possesses 5-10 antigen binding sites hence they are efficient agglutination, complement fixation.

28. Comparison between primary & secondary response