Immunoglobulins - Serum proteins are against a number of important bacterial infections.

1. Immunoglobulins- Serum proteins are against a number of important bacterial infections. Introduction • All vertebrates possess immunoglobulin-like molecules. • Immunoglobulins are synthesized and secreted by end cells of the B cell lineage, i.e. plasma cell. • Immunoglobulins are largely confined to the broad and heterogeneous band of -globulins and show considerable diversity of structure and function.

2. Untreated antiserum Treated antiserum • The first evidence to show antibody is one of serum protein fractions • 1939, A. Tiselius and E. A. Kabat • To immunize rabbits with ovalbumin • Belong to -globulin fractionimmunoglobulin

3. Structure of Immunoglobulins • The typical immunoglobulin molecule is • Asymmetrically composed of four polypeptide chains • Linked by disulphide bridges • The larger chains are designated heavy(MW about 50~77 kDa) and the smaller light (MW about 25 kDa). • The two most important of their features in the immune response are specificity and biologic activity. • Several immunoglobulin fragments can be prepared using proteolytic enzymes and these have been of value in unravelling the functional activities of different parts of these molecules

4. Schematic diagram of structure of immunoglobulins

5. Papain digestion： • To cleave on the amino-terminal side of the inter-heavy chain disulphide bonds. • To yield two Fab（Fragment antigen-binding）fragments and one Fc（Fragment crystallizable）fragment. • Pepsin digestion ： • To cleave on the carboxy-terminal side of the inter-heavy chain disulphide bonds. • To yield a Fab dimer（F(ab' )2）and a rather smaller Fc fragment（pFc'）

6. A MW of 150,000 Da A MW of 100,000 Da A MW of 25,000 Da A MW of 45,000 Da A MW of 50,000 Da A MW of 50,000 Da

7. The immunoglobulin molecules are in Y configuration and have the flexible hinge region which permits considerable movement of the Fab arms. • Both light and heavy polypeptide chains consist of a series of similar subunits, designated domains. • 每一個domain約由110 amino acids及a single intrachain disulphide bridge堆疊而成之polypeptide chain。 • Light and heavy polypeptide chains之amino-terminal domain的氨基酸組成有較高之變化性，稱為variable region(可變區)；和可變區相較之下氨基酸組成變化低的domain，稱為constant region(不可變區)。 • light chain具有單一可變區及不可變區domains(VL及CL)；heavy chain具有單一可變區及三到四個不可變區domains (VH及CH1、CH2、CH3、CH4，其不可變區domain數目依immunoglobulin種類而定)。

8. Ribbon representation of an intact monoclonal antibody depicting the heavy and light chain

9. Qutternary structure immunoglobulin and interactions between domains • Extended peptide sequence between CH1 and CH2 of , , and  heavy chain • Poline rich sequence • Giving IgG, IgD and IgA segmental flexibility

10. Variable region為immunoglobulin與抗原接觸的部位(antigen-binding site)，其中具有三區hot spots (hypervariable regions，HVR1、HVR2、HVR3 or complementarity-determining regions, CDR1、CDR2、CDR3)。 • Affinity: the strength of binging or association of a single epitope for a single combining site • Avidity: indicate the average strength of binding

11. Diagram of an immunoglobulin light chain depicting the fold structure Held teogether by hydrophobic interaction and disulfide bond

12. Antigenic Deteminants on Immunoglobulin • Antibodies also are potent immunogens to induce an antibody response • Anti-Ig antibodies are powerful tools • Antigenic determinants (epitopes) on Ig: • Isotypic determinant: constant-region determinants, to distinguish each Ig class and subclass within a species • Allotypic determinant: be able to distinguish the subtle amino acid difference between the same set of isotype genes • Idiotypic determinant: be generated by conformation of amino acid sequences of heavy- and light-chain variable regions specific for each antigen

13. Individual determinant is called an idiotope. • The sum of the individual idiotope is the idiotype

14. The Classes and Subclasses of Immunoglobulins • 利用heavy chain constant region的結構和功能的不同可將Immunoglobulins分為IgG、IgA、IgM、IgD、IgE等五類；其中IgG及IgA具有subclasses。

15. IgG、IgD、IgE為monomer；IgA為dimer；IgM為pentamer。 • J chain： • A small acidic protein，15 kDa，其結構與light and heavy chain無關。 • 由plasma cell分泌而來。 • J chain is disulfide-bonded to the penultimate cysteine residue in the tail piece of theαorμchain。主要用來聚合immunoglobulin monomer。 • Secretory component： • A single polypeptide，about 70 kDa，僅可與IgA聚合在一起；由黏膜皮層細胞產生，這些細胞會將血液中的IgA抓住，使其與secretory component結合後轉移進入分泌系統。 • 其結構中具有高量的carbohydrate；氨基酸組成和 J chain及immunoglobulins沒有相似處。 • 利用非共價鍵的接出方式與游離或被鍵結的IgA相接。

19. IgG • 具有γ1、γ2、γ3、γ4 四種heavy chains，κ或λlight chain；因此有IgG1(60-70%)、IgG2(14-20%)、IgG3(4-8%)、IgG4(2-6%)四種次種。 • IgG accounts for about 75-80% of the total serum immunoglobulin in normal adult and is the abundant antibody produced during secondary humoral immune responses in the blood。 • IgG1、IgG2、IgG3可活化classical complement pathway，IgG2可額外活化alternative complement pathway。

20. IgM • 具有μheavy chain，κ或λlight chain。 • 為pentamer，具有10 antigen-binding sites，五個單元由一個J chain連結在一起。 • 重鏈具有四個constant regions。 • IgM accounts for only about 10% of serum immunoglobulin，is the key immunoglobulin of the primary response and an efficient activator of the classical complement pathway，and is the most common immunoglobulin expressed on the surfaces of B cells。 • 因為分子很大，無法由循環系統進入組織液中，但因其對poly-Ig recoptor有高親和性，因此在IgA deficiency時會出現在分泌系統中與secretory piece連結扮演IgA的角色。

21. IgA • 具有α1、α2兩種heavy chains，κ或λlight chain；因此有IgA1、IgA2(兩者存在的比例為5：1)兩種次種。 • 釋出時為dimer (secretory IgA, 最多可為pentamer)加上secretory component游離於組織及血液中，其間具有J chain連接；如果為membrane-bound form則為monomer。

22. IgA accounts for only about 10-15% of serum immunoglobulin • The major immunoglobulin of external secretions • The most abundant antibody class was found in saliva, tear, intestinal mucus, bronchial secretions, milk, prostatic fluid, and other secretions。 • 不會活化classical complement pathway，但可以協助alternative complement pathway中C3 convertase的stabilization。 • Specific Fc receptors for IgA have been observed but are not well charaterized。

23. Formation of secretory IgA: • Dimer IgA binds to a poly-Ig receptor • Internalized by receptor-mediated endocytosis • Ploy-Ig receptor is enzymatically cleaved • Secretory component bound to the dimer IgA

25. IgE • 具有εheavy chain，κ或λlight chain，about 180 kDa，為所有Ig量最少的一種(約佔0.004%)，為偵測allergic disorder(過敏性失調)之臨床標準指標值。 • 重鏈具有四個constant regions。 • 會與mast cells及basophils表面higt-affinity Fc receptor (FcεRI)接合，當有特殊抗原出現時，會刺激該細胞釋出inflammatory mediators。 • 某些細胞(such as eosinophils)表面受器FcεRII可與IgE接合(親和性低)，對於parasitic worms感染之免疫性有重要關係。

26. Allergen cross-linkage of receptor-bound IgE on mast cell induces degranulation, causingrelease of substances that mediate allergic manifestations

27. IgD • 具有δheavy chain，κ或λlight chain，在血液及體液中出現濃度相當低。 • 目前研究發現在B細胞表面具有IgD，與IgM相連在一起；扮演B細胞表面的antigen receptor。 • 其與其他免疫球蛋白間之功能尚未明瞭。