IMMUNOGLOBULINS (Ig)“Antibodies” Immunoglobulins are glycoproteins that are composed of 82-96% protein and 4-18% carbohydrate. They bind specifically (lock-and key) to antigens which stimulated their formation. The Igs are present at different body sites including serum, blood, extra-cellular fluid, CSF, saliva, tears, exocrine secretions (nasal , intestinal & breast milk), and on surface of lymphocytes. The serum protein electrophoresis shows that the Igs are located mainly at the globulin region. The Igs are produced from B cells that become plasma cells which synthesize & secrete thousands of antibody molecules per second.
Clonal Selection Theory: The antibodies are produced according to this theory which is not any more a theory, but almost a fact. This theory states that a single epitope stimulates a single clone of B cells that produce a single antibody specificity. Clone: A cluster of identical cells since they are all daughter cells of a single mother cell.
Basic Structure of antibody: Monomer: This is the basic structure of the antibody. It is composed of 2 pairs of identical polypeptide chains (i.e., the polypeptides in each pair are identical). In one pair, the polypeptide chains have twice the M.W. and twice the numbers of A.A. of the other pair. The first of pair polypeptides are called Heavy chains (H), while the second one are called Light chains (L). These chains are joined together by disulfide bonds (S-S). These bonds are between the H-L and H-H chains. The M.W. of H chain is 46 KD, and that of L chain is 23 KD.
Domains: The polypeptide chains are like threads folded on themselves. These folds are called domains and are held together by S-S bonds (intra-chain). Each domain is composed of 110 A.A. The sequences of these A.A. are of two types: 1.Variable (V): The domains located at the amino-terminal (H3N+) have variable (different) A.A sequences between the various antibody molecules. In each monomer, there is one V domain in the H chain (VH) and one V domain in the L chain (VL). The V domains of both the H & L chains determine the specificity of the antibody molecule.
2. Constant (C): All domains ,except the V , have constant A.A. sequences. The L chain have only one C domain (CL), but the H chain have 3 or 4 C domains (CH1, CH2, CH3 & CH4). These C domains are located toward the carboxy (COO-) terminal of the monomer. According to the C domains the H and L chains are subdivided into different types.
Types of H and L chains: H chains have 5 types which are gamma, muta , alpha, delta, and epsilon. According to these chains the antibodies are divided into 5 classes which are IgG, IgM, IgA, IgD, and IgE respectively. One type of H chain is present in each monomer. L chains have 2 types which are kappa and lambda. Each monomer have either kappa or lambda chains (never both). Generally in the body, the kappa: lambda ratio is 2:1.
Subtypes of H & L chains: According to minor differences in the constant regions, both H & L chains are subdivided into different subtypes. The subtypes of H chains include: Gamma : 1, 2, 3, & 4 (for IgG1, IgG2, IgG3 & IgG4 respectively). Mu: None Alpha: 1, & 2 (for IgA1, & IgA2 respectively). Delta : None. Epsilon : None The subtypes of L chains include: Lambda : 1, 2, 3, 4, 5 & 6 Kappa : None ISOTYPES = All types & subtypes of Igs.
Functions of Ig regions:Variable (VH & VL) : Are the Antigen binding sites via hypervariable areas which are more variable than the rest of the VH & VL regions. The areas are also called “ hot spots” or “ idiotypes”. In the VH there are 4 idiotypes, while in the VL 3 only. Only three idiotypes from each chain are shared in the actual binding with the AD residues; these idiotypes are called “paratopes”.These hot spots (paratopes) are the areas of intimate contact with the residues of the epitopes (Lock-Key according to sequences or configuration). Each idiotype is composed of about 10 A.A.Q. Idiotypes are AD of the Ig ( F? Or T?).Q. Anti-idiotypes can be prepared (F? Or T?).
Constant region: 1. Opsonization : Is the binging of IgG (IgG1 & IgG3 that are called Opsonin Igs) to an antigen which is then followed by phogocytosis via CH3. Also, binding of IgG to macrophages via CH3 results in arming of these cells. 2. NK cells: Bind to antigens (e.g. Cells or m.o.) via CH3 of IgG (CD16); part of ADCC (Antibody-Dependent Cell Mediated Cytoto.). 3. Complement fixation: Starts via C1 (C1q). IgM is good complement fixing antibody (+++), while IgG (+ - ++). All Ig subclaases can fix the C except IgG4. 4. Placenta cross: IgG is the only Ig that can cross the placental barrier; all subclassis can do this efficiently except IgG2 (not efficient).
Immunochemistry of Ig (enzymatic digestion by proteolytic enzymes):1. Papain: Digest the IgG monomer at a flexible area between CH1 & CH2 in front (toward H3N+ terminal) of the enter-H chains disulfide bonds. This area is called “Hinge Region”. The result is 3 fragments; two fragments are called Fab (Fragment of Antigen Binding) and the third one is named Fc ( Crystallizable Fragment). Fab = VH+VL+CH1+CL; Fc = CH2 + CH3 + Hinge R.
2. Pepsin: Digest the IgG monomer behind the disulfide bond (toward COO- terminal) of the hinge region. The result is 2 fragments; one is F(ab)2 and the second is small negligible portion. The F(ab)2 = 2 Fabs + Hinge Region. F(ab)2 has clinical & laboratory applications. Example: Therapeutic use when I.R. to Fc portion is unwanted.
Polymers: Are composed of more than one monomer; 2 monomers= Dimer, 3= Trimer, 4= Teramer, 5= Pentamer, 6= Hexamer. Examples: IgM in the serum is usually pentamer and rarely hexamer, while on lymphocyte surface is monomer. Also, secretory IgA (IgA2 in exocrine secretions) is usually dimer and some times trimer, while in the serum is monomer.
IgM pentamer structure: It is composed of 5 IgM monomers linked covalently by disulfide bonds and only one J (joining) chain of 15 KD M.W. Also, the J-chain is linked by –S-S- to the main structure.
Secretory IgA (IgA2): It is composed of 2 IgA monomers that are linked covalently by J-chain (15 KD) with non-covalent binding of Sc (Secretory) component of 70 KD. Its light chains are present in opposite position to the usual one , and linked together by –S-S- bonds (L to L), but not to the H chains (as usual). The Sc component is produced by the epithelial lining of the mucosal surfaces and its function is to assist in the transfer (crossing) of secretory IgA across the epithelial barrier.
Items
IgGIgA
IgM
IgD
IgE
H chain class
gamma
alpha
mu
delta
epsilone
H chain subclasses
1, 2, 3, 4
1, 2
none
none
none
M.W.
150,000
160,000 or 400,000
900,000
180,000
190,000
Sedimentation coefficient (S)
6 - 7
7
19
7 - 8
8
Percentage (%) in serum
75%
15 %
10-15%
0.2%
0.004%
Serum Concentration (mg/dl)
1000
200
120
3
0.05 or 10-15 IU / ml
Serum half life (days)
23
6
5
3
2
Items
IgGIgA
IgM
IgD
IgE
Complement fixation
+
0
++++
0
0
Placental transfer
+
0
0
0
0
Mast cells/ basophils degranulation
IgG4 only
0
0
0
++++
Bacterial lysis
+
+
+++
?
?
Antiviral Activity
+
+++
+
?
?
Immune Response
Secondary (mainly)
Secondary (uncommon)
Primary (mainly)
Secondary (rare)
Secondary)uncommon)
Heavy cain M.W. Carbohydrate %
50,000 4
55,000 10
70,000 15
62,000 18
70,000 18
items
IgGIgA
IgM
IgD
IgE
Common Molecular form
Monomer
Dimer or Monomer
Pentamer or Monomer
Monomer
Monomer
Antigen receptor on B cells
-
-
+
+
-
J chain
-
+
+
-
-
Secretory piece (component)
-
+
-
-
-
IgG Functions:
1. Opsonin: It enhances phagocytosis (opsonization) by coating bacteria and attaching by its Fc portion to Fc receptors on phagocytic cells. 2. Precipitin: It precipitates soluble antigens. 3. Incomplete-Haemolysin: Does not cause haemolysis by complement fixation and activation as Anti-Rh. 4. Complement Fixing Antibody. 5. NK Fc receptor (CD16) which functions as ADCC. 6. Neutralizing antibody as anti-toxin,& anti-viruses. 7. Present in C.S.F. and extracellular fluids. 8. Can Cross the Placents . 9. IgG4 reaginic antibody in allergy (mainly ingestants). 10. Present in active diseases as Brucellosis. 11. Generally against protein antigens except IgG2 which is anti-CHO. 12. The major antibody in secondary immune response (anamnestic I.R.).IgA Functions:
1.Local immunity at mucous surfaces (coat m.o., prevent adherance & entery). It is synthesized locally by submucosal plasma cells (80% of plasma cells in MALT synthesize IgA). It protects 400 sq.m. of mucous membranes. 2. Present in mucous secretions (RT, GIT,GUT) and in tears,sweat,saliva, colostrum& milk. 3. IgA1 (monomeric) is mainly present in the serum, while IgA2 (di- or trimeric) is present as secretory antibody. 4. IgA1 can be inactivated by an IgA protease produced by Gonococci, Pneumococci, & H. Influenzae, while IgA2 can not be inactivated.IgM Functions:
1. Agglutinin antibody that agglutinates particle antigens.2. Haeagglutnin as Natural isohaemagglutinin (Anti-ABO).3. Complete haemolysin.4. Good complement fixing ِ ِ5. Antigen Receptor on mature lymphocytes (B cells) together with IgD.6. Main antibody of primary immune response.*IgD Functions: 1. It is co-expressed with IgM on the suface of mature B lymphocytes. Like IgM functions as an antigen receptor. 2. It may act as antibody against certain antigens as insulin receptor...etc. IgE Functions: 1. It plays a pathogenic role in type I (immediate) hypersensitivity. It has Fc receptor (Fc-epsilon) on mast / basophil cells. 2. High levels are also detected during parasitic infestations mainly helminthic (e.g. Ascariasis). 3. It is monmeric, does not fix complement, and does not cross the placenta.
Immunoglobulin DiversityThere are about 10 trillions different Ig specificities in the human body which is called “ repertoire” (i.e., equipped with all the possibilities of antigens present in nature). This is achieved by different mechanisms by the immune system and controlled genetically.The B cells differentiate and mature to plasma cells that synthesize and secrete Igs. The mechanism of Ig formation consists of 2 parts:1. Synthesis of light chains (L).2. Synthesis of heavy chains (H).The combination of L & H chains result in the formation of complete Ig. Each of the L & H chains are formed from two portions (Variable & constant regions) which are joined together by joining (J) region or portion. The regions are all formed under the control of genes.
L : Leader Vk : Genes controlling the peptides that form the Kappa chain. There are 30 different types of variable region of Kappa chain ( VK1,2,3,...30). C : Constant K : Kappa J : Joining chain of 5 types (J1,2,3,4,5). IVS : Intervening segment (intron).
Kappa Chain Formation: The first process in the formation of Kappa chain is the rearrangement which is the binding of one J chain with one VK (brought together). Then the C region will bind. This process occurs at the DNA. Then there is transcription process on the RNA and the formation of mRNA which will be transfered to the ribosome for protein synthesis (Ig synthesis). Also, there is a process of removal of all the IVS segments which is called RNA splicing (result in L + VK + J + CK). The last process is the translation of these information by removal of the L segment and the completion of the Kappa chain formation (VK + J + CK). Probabilities = 30 V X 5 J X 1 C = 150
Antibody Diversity: This process depends on: 1. Multiple gene segments. 2. Their arrangement into different sequences. 3. The combing of different L & H chains in the assembly of Ig molecules. 4. Mutation. 5. Junctional diversity (D) ; applies to the H chain only.
Isotype switching: During the immune response the plasma cells switch from producing IgM to IgG or any other class. This could occurs directly or in a programmed downward process (e.g. Mu to gamma 1 , then gamma 2 ..etc...).There is no change of the V region and the L chain, but the C region is replaced . So, there is no change in the specificity, but in the class of Ig. This occurs through the process of DNA rearrangement and RNA splicing.
Heavy chain formation: This process is controlled by genes on chromosome No. 14. Comparable to that of L. There are L, 50 different VH of gamma, mu, alpha, delta, or epsilon, 25 Diversity (D), 6 J, and CH. The formation starts by binding of one D and one J the to one VH forming VDJ. The transcription and RNA splicing by the removal of IVS. Then into the translation process and the removal of L segment. N.B. : Lambda light chain is controlled by genes on chromosome No. 22. Also is has 4 constant (C) segments.
Monoclonal AntibodiesDiscovery:George Kohler and Cesar Milstein published in Journal “Nature” in 1975 a new technique for antibody production. The technique is named “Hybridization” for the production of monoclonal antibodies in tissue culture.
Principles of the technique: 1. Immunization of a mouse with an antigen (X). 2. Spleenocytes are taken from the immunized mouse. 3. Hybridize using poly Ethylene Glycol (PEG) the spleen cells (which synthesize immunoglobulin, but can NOT live in tissue culture) with mice non-secretory myeloma cell lines (which do NOT synthesize Ig, but can live in tissue culture). 4. Hybrid cells (of tetraploid chromosomes) are cultured in fluid named RPMI in which Ig are secreted by these cells, the collected as supernatants.
Tissue Culture: 1. Microtitre plates of 96 wells are used. 2. In each well ONE hybrid cell will grow and form ONE clone of cells. ONE cell ONE clone 3. This clone secretes ONE type of antibody of a single specificity to ONE epitope that is why it is named Monoclonal Antibody. 4. The non-hybridized myeloma cells are killed by HAT, while the non-hybridized spleen cells die spontaneously in 5-7 days
Applications of MCA: I. Diagnostic: 1. Leucocyte identification. 2. Lymphocyte subset determination. 3. HLA detection and typing. 4. Detection of different microbes (bacteria, viruses, fungi & parasites). 5. Detection of tumour markers or antigens. 6. Determination of concentration of hormones. 7. Typing of lymphomas and leukaemias. 8. Detection of different cardiac or other tissues markers as cardiac myosin in myocardial infarction. 9. Tissues immunohistochemical applications.
II. Therapeutic:1. Anti-Tumour as anti-tumour antigens or anti-idiotypes for lymphomas. Antibodies labelled with toxin as Ricin or radioactive material can be used against tumours and named ‘Magic Bullet’ or ‘War Head’.2. Immuno-suppressive therapy in organ transplantation, autoimmune diseases, hypersensitivity, and treatment of graft versus host disease (GvHD).3. Control and treatment of infertility as using anti-hCG.4. Treatment of drug toxicity as digitalis intoxication.
CD Markers (Clusters of Designation or Differentiation): CD markers are a group of special molecules on the surface of body cells. There are more than 400 CD markers. They are detected by specific monoclonal antibodies which share with them the name All body cells have one or more of these markers. However, they are most important or useful for the identification and differentiation of W.B.C. and their subsets. Examples: CD4 = Helper T-cells; CD8 = Cytotoxic T-cells; CD56 = NK cells …etc. MCA as anti-CD4, Anti-CD8, Anti-CD56.. etc