Immunology Dr,Huda Ibrahim Antibody Diversity Immune Response Monoclonal Antibodies
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Teaching objectives
1- To understand the mechanism of immunoglobulin diversity. 2- To understand the concept of primary & secondary immune response. 3- To recognize monoclonal antibodies(MCA) . 4- To know the diagnostic & therapeutic applications of MCA .*
Antibody diversity
An individual produces a large number of Abs to cope with the vast number of different Ags. This Ab diversity is due to the Ig genes. Genes coding for the variable & constant portions of the chains are separate One or only few genes code for C region whereas many genes code for the V region.Immunoglobulin Diversity
The mechanism of Ig formation consists of 2 parts: 1-Synthesis of light chains(L). 2-Synthesis of heavy chains (H). The formation of L & H chains result in the formation of complete Ig .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 ).Ck : Constant kappaJ : Joining chain of 5 types (J1,2,3,4,5).IVS : Intervening segment (introns). *
Light 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 transferred 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 the removal of the L segment and the completion of the Kappa chain formation (Vk +J +Ck).Probabilities=30 V by 5 J by 1C=150
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Heavy chain formation
This process is controlled by genes on chromosome no.14.There are L ,50 different VH of gamma, mu, alpha, delta,or epsilon, 25 Diversity(D), 6(J) ,&CH. The formation starts by binding of one D & one J to one VH, forming VDJ (DNA rearrangement). Then the transcription & RNA splicing by the removal IVS .Then into the translation process and the removal of L segment.*
Structure of Immunoglobulin
*Production of Antibodies
Immune response is brought about by three types of cells 1- APC macrophages, and dendritic cells, 2- T Cell and 3- B cells The first step is capture and processing of antigens by APC and their presentation with the association of appropriate MHC molecule to T cells However some polysaccharides and simple molecules with repeating epitopes do not require T Cell participation*
Stages of Antibody mediated immune response
Contain two stages 1- The entry of antigen, its distribution and fate in the tissues and its contact with appropriate immunocompetent cells 2- The secretion of antibody, its distribution in tissues and body fluids and manifestations of its effects.*
Pattern of Antibody production.
A Lag Phase A Log Phase raise of antibody levels, Plateau A phase of Decline.*
Primary and Secondary Immune Responses
A single injection of antigen helps in sensitizing or priming of immunocpompent cell producing particular antibody than in the actual elaboration of high levels of antibody. Effective levels of antibody are usually induced by only subsequent injection of antigens.
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Primary and Secondary Response
The primary response is slow, sluggish and short lived with long lag phase and does not persist for long time The secondary response is prompt powerful and prolonged with short or negligible lag phase and with higher level of antibodies*
Types of Antibody Response,
Initial Antigenic Stimulus (Primary) Response Ig M Response is slow and short lived, Secondary Response Ig G Response is Prompt ,Powerful and Prolonged Higher level of Antibodies and Lasts longer,*
How long a Antibody be active
The duration of the lag phase and persistence of the antibody dependent on the nature of the antigen In diphtheria toxoid the lag phase in the primary response may be long as 2 -3 weeks*
Priming and Booster doses
With live vaccines a single dose is sufficient as multiplication of the organisms in the body provides a continuous antigenic stimulus that acts as both the priming and booster dose*
Fate of Antigens
Depends on the physical and chemical nature of antigens, dose and route of entryWhether induced primarily or secondarilyThe antigens introduced by IV are rapidly localized in the spleen, liver, bone marrow kidney and lungs Broken down by RES cells excreted in urineAbout 70 – 80 % eliminated in one or two days *Fate of Antigens…continue When antigens are introduced by subcutaneously are mainly localized in the draining lymph nodes only small amounts being found in the spleenThe pariculate antigens are removed from circulation in two phase – the first is antigens are engulfed by phagocytic cells broken down and eliminated *
Fate of Antigen…continue 2nd With the appearance of specific antibody the phase of immune elimination begins The antigen and antibody complexes are rapidly phagocytized results in disappearance of antigen from circulation
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Fate of Antigen…continue Immune Complexes can cause damage. Proteins eliminated in 1-2 weeks, Polysaccharides months to years. Pneumococcal polysaccharides up to 20 years.
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Immunoglobulin Switching.
Ig M specific for Antigen is produced. Switch to others Ig G - Ig A -Ig E But retain the same specificity, But carry different Biological activities*
Monoclonal Antibodies
Kohler and Milstein ( Nobel Prize 1984 )A single antibody forming cell or clone produces Antibodies against single antigen,Antibodies are usually polyclonal,Clone of Lymphocytes – Monoclonal antibodies. Useful in Diagnostic / Research work. *How Hybridoma Created.
Immunize Mice with Antigen, Obtain spleen cells , Mouse Myeloma cells Grow myeloma in HPRT medium Hypoxanthine,Phosphoribosyl transfeerase .Fuse cells & transfer fused cells to HAT medium (Hypoxanthine, Aminoptren and Thymidine Medium } Lead to formation of Hybridoma.*
Hybridoma Technology Produce Monoclonal Antibodies
What is Hybridoma? Fusion of Spleen cells + Myeloma Cells, Attains the capacity to produce 1. Antibody producing capacity. 2. Multiply indefinitely,*
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Evolution of monoclonal antibodies
*Diagnostic application
Detection of tumor related antigens e.g. B cell lymphoma Detection of colon cancer by using of radiological antibody against CEA Viral detection and subtyping HLA antigen detection Autoimmune disease Immunohistochemical applications in tissue sections.*
Therapeutic application
1-Antitumor :e.g., Herceptin used in ca breast (magic bullet therapy). 2-Immunosuppressive therapy :e.g., *Etanercept (chimeric MCA) & Adalimumab (humanized MCA) both used in rheumatoid arthritis*