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IMSE311 WEEK 7 Antibody Structure AND Functions
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Immunology and Serology (IMSE311)
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Our Lady of Fatima University
Academic year: 2022/2023
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IMSE311 (MIDTERMS)
WEEK 7: ANTIBODY STRUCTURE AND FUNCTIONS
B CELL ACTIVIATION
- Part of humoral branch of immune response (production of the antibodies)
- Subsequent stages: ✓ Pro b cell ✓ Pre b cell ✓ Mature b cell ✓ Immature b cell ✓ Activated b cell ✓ Plasma cell (to release or secrete antibodies)
- When b- cells are stimulated by an antigen, they will go differentiation and the product is antibody production, or immunoglobulins, memory b- cells.
- CD19, CD24, CD45R -clusters of differentiation or surface markers that remain on the cell surface throughout the subsequent developmental stages.
- CD25 - surface protein, found on both activated b cells and t cells and acts as a receptor for IL
I MMUNOGLOBULINS
- Glycoprotein found in the serum/plasma portion of the blood
- Composition ✓ 82-96%→ polypeptides (protein) *Majority ✓ 2-14%→ carbohydrates
- Electrophoresis (pH 8) → Immunoglobulins appear primarily in the gamma region (located in the negative electrodes or cathode region). ✓ Gagamit ng Agarose gel w/ pH 8.
- Part of humoral branch of immune response. (secretion or production of antibodies)
- They play an essential role during <Antigen recognition = and in biological activities related to immune response such as <opsonization= (preparation of phagocytosis) and <complement activation= ->> Not all IG can perform complement activation, only IgM and IgG aka complement fixation.
Classification of immunoglobulins:
- <Ig= → <Immunoglobulin
- GAMDE (IgG, IgA, IgM, IgD, IgE)
SERUM ELECTROPHORESIS
Albumin – fastest (anode region) - Most abundant protein (laging naka pwesto sa left side ng serum electrophoresis activity) - Most predominant region - (Thick bond) 60% in blood components Has different globulins: alpha globulin, a2 globulin, beta globulin and on the right side ay gamma globulin (lagging nasa dulo and nakikita yung mga immunoglobulins)
✓ a1 globulin: alpha1 antitrypsin, alpha fetoprotein ✓ a2 globulin: ceruloplasmin, haptoglobin, a2 macroglobulin ✓ beta globulin: transferrin, hemopexin, complement system, fibrinogen, lipoprotein (consist of LDL, HDL, VLDL)
- possible magkaroon ng abnormal pattern. Beta gamma bridging, nagkakaroon ng curve and kapag nagkaroon ng beta gamma bridging, these are the condition of liver cirrhosis
- Spike pattern- kapag biglang tumaas sa may gamma region, means of multiple myeloma ( benz jones protein ) ( elevated ang plasma protein ng patient ) ✓ Benz jones protein- will precipitate for about 60pC and will dissolved at 80pC
TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS
All immunoglobulin molecules are made up of a basic 4 chain polypeptide unit that consists of 2 large chains called heavy or H chains and 2 smaller chains called light or L chains.
These chains are held together by non-covalent forces and Disulfide linkage/bond
Structure of Immunoglobulins was first described by 2 scientists. (Gerald Edelman and Rodney Porter) ✓ They decide to work with IgG molecule because it’s easy to collect compare to others. ✓ IgG – Most predominant Ig in the blood
- Edelman’s work centered on using the analytical ultracentrifuge to separate out immunoglobulins on the basis of molecular weight.
Sedimentation rate is directly related to molecular weight.
The higher the sedimentation rate, the higher the molecular weight, the larger the molecule (vice versa)
- He found that intact IgG molecules had a sedimentation coefficient of 7S (Svedberg unit).
- On obtaining a purified preparation of IgG. Edelman used 7Molar urea to unfold the molecule.
- Once unfolded, the exposed sulfhydryl bonds could be cleaved by a reducing agent such as 2-Mercapthoethanol.
After such treatment, Gerald Edelman subjected again this cleaved/unfold antibody perform analytical ultracentrifugation, and 2 fraction has been given, 1 is 3 and 1 is 2
Edelman concluded that 3 fraction has approximately with molecular weight of 50,000 Daltons , he designated is as the H-chain.
The 2 fraction with a molecular weight of 22,000 Daltons is the L- chain.
Won a Nobel prize in 1972
ANTIBODY DIGESTION
PAPAIN DIGESTION
- Rodney Porter’s work was based on the use of the proteolytic enzyme papain to cleave antibody (equal size) structure (monomeric structure). ✓ Cleave antibody into 3 pieces in equal size. The 3 pieces has sedimentation coefficient of 3 represent a molecular weight of 45,000 to 50,000 daltons
- He then subjected the IgG to Carboxymethyl cellulose ion exchange chromatography. ✓ 1 fragment spontaneously crystallized at 4∞C. ▪ Called the FC region of antibody , or the fragment crystallizable region. ▪ Has no antigen binding ability ▪ Now known to represent the carboxy terminal halves of 2 heavy chains that held together by ss bonding ✓ The remaining 2 identical fragments were found to have antigen-binding capacity. ▪ Called the FAB (Fragment Antigen Binding) region
PEPSIN DIGESTION
- Alfred Nisonoff used pepsin to obtain additional evidence for the structure of IG.
- This proteolytic enzyme was found to cleave IgG at the carboxy- terminal side of the interchain disulfide bonds.
- 1 single fragment with a molecular weight of 100,000 d and all the antigen-binding ability, known as F(ab).
- An additional fragment called FC’ (composed of 2 heavy chain ) was similar to FC except that it disintegrated into several smaller pieces.
-> Papain digestion successfully cleaved antibody into 3 portions or fragments. -> Pepsin digestion successfully cleaved antibody into 2 portions or fragments
FAB region- variable region (conjunction with the location of amino terminal) (End terminal) FC region- constant region (located at carboxyl terminal)
- There is no such thing that 2 light chains are bind together (ABNORMAL); multiple myeloma (benz jones protein)
- The basic monomeric structure of an antibody has 2 vibrations
LC- light chain HC- heavy chain
- IgG, IgA, IgD = 2 domains in their LC
- IgG, IgA, IgD = 4 domains for HC
- IgE (epsilon) & IgM (mu) = 2 on LC, 5 on HC
- All Ig has the same number of domains for light chain (VL & CL). They only differ on the heavy chains
Papain Antibody Digestion 2Fab and 1Fc - Each Fab Fragment →1 L chain and ½of H chain - Fc→ 2 halves of H chain
Pepsin Antibody Digestion 1F(ab)2 and 1Fc - 1F(ab)2 → 2 full L chain and two halves of H chain - Fc→ 2 halves of H chain
NATURE OF LIGHT CHAINS
- The structure of light chains is not fully discovered until the discovery of
an abnormal protein produce ( bence jones protein ) by patients with
multiple myeloma.
✓ There is an over proliferation of plasma cells that produces
an abnormal protein composed of 2identical light chains
bonded together.
- Filtered out by the kidney released through urine
- Analysis of several Bence-Jones proteins revealed that there were 2 main types of L chains -> Bence-Jones Protein – composed of 2identical light chains, excess in the blood, filtered by the kidney, glomerulus part of the kidney, release through urine. -> Precipitates at 60pC and dissolves at 80pC– A way to extract this protein
- Designated as kapa (on chromosome 2) and lambda(λ)on chromosome
- Each contained between 200 (kappa) and 220 (lambda) amino acids, and from position number 111 on (the amino terminus is position number 1), it was discovered that each type had essentially the same sequence.
- This region was called the constant region
- And the amino-terminal end was called the variable region
HEAVY CHAIN
- The first approximately 100 amino acids at the amino terminal end constitute the variable domain.
- The remaining amino acids can typically be divided up into 3 or more constant regions with very similar sequences
- Constant regions of the H chain are unique to each class and give each immunoglobulin type its name. ✓ IgG has a/an = Gamma chain <ɣ= ✓ IgM has a/an= Mu chain <μ= ✓ IgE has a/an= Epsilon chain <ɛ= ✓ IgD has a/an= Delta chain <ẟ= ✓ IgA has a/an= Alpha chain. <α=
ANTIBODY VARIATIONS
- Isotype – a unique amino acid sequence that is common to all molecules in a given species. Eg. Ig has the same amino acid sequence.
- Allotype – minor variation of this sequences that are present in some individuals but not others. (IgG)
- Idiotype – variations in variable regions that give individual antibody specificity
- Constant region of allotype – reason why we have subclass on particular Ig.
- IgG subclass (4 subclass) -> IgG 1, IgG 2, IgG 3, IgG 4.
- IgA subclass -> IgA 1, IgA (2 subclass) 2
HINGE REGION
- Responsible for antibody’s flexibility.
- The segment of H chain located between the CH1 and CH2 regions is known as the hinge region.
- Mainly compose of an amino acid proline (allows for flexibility, it makes the antigen binding site work independently) ✓ IgD, IgA, IgG = has hinge regions ✓ IgE & IgM = no hinge region
- Basic immunoglobulin structure:
- Through valence we can identify the binding sites ✓ Monomer: Has 2 antigen binding sites ▪ IgG, IgD, IgE Serum IgA and IgM (surface of b cells), ✓ Dimer: Has 4 antigen binding sites ▪ Secretory IgA ✓ Polymer: more than 4 antigen binding sites ▪ IgM (Pentamer) total of 10 ✓ IgM – monomer if they are attached to a b-cell, and pentamer if seen in secretions like serum and plasma ✓ IgA – monomer when they are in serum portion of blood, dimer in structure if they are in secretions
CLASSIFICATIONS OF IMMUNOGLOBULINS
IgG:
- IgG is the most predominant immunoglobulin in humans.
- Half like in serum about 23 days
- There are 4 major subclasses , with the following distribution: IgG1, 67%; IgG2, 22%; IgG3, 7%; and IgG4, 4% ✓ IgG1 – predominant ✓ IgG – least dominant
- These subclasses differ mainly in the number and position of the disulfide bridges between the γ chains.
- Can cross the placenta: except IgG 2. ✓ IgG 1 is the most efficient in crossing the placenta.
- Complement fixation : would be the IgM, except IgG 4 (can’t). ✓ IgG 3 is the most efficient in complement fixation, followed by IgG 1, and IgG 2
- Opsonization (Can help in phagocytosis, IgG can act as opsonin)
- Neutralization of toxins and viruses
- Participation during agglutination and precipitation. Serological reactions or clumping reactions would be IgM ✓ IgG is best in precipitation than agglutination
- Number of the Disulfide bonds ✓ 2- IgG 1 ✓ 4- Ig 2 ✓ 5- IgG 3 ✓ 2- IgG 4
IgM (largest)
- IgM is known as a macroglobulin , because it has a sedimentation rate of 19 S , which represents a molecular weight of approximately 970,.
- The half-life of IgM is about 10 days. ✓ Half life connected with serum is about 6 days
- The pentamer form is found in secretions (serum or plasma), while the monomer form occurs on the surface of B cells.
- The 5 monomeric units are held together by a <J chain= or <joining chain= (Bukod kay IgM kasama din yung IgA particularly the secretory IgA)
- Found also on the cell surface of B-cells. (Monomeric form)
- Considered as the most primitive immunoglobulins ✓ If the patient has contracted infection, most likely IgM increases followed by IgG after 1, 2 days or week of an infection. ✓ If a patient is positive in IgM, the person is suffering on the early stage of disease (acute stage) (first to appear is IgM)
- Functions include complement fixation, Agglutination, opsonization, and toxin neutralization. ✓ Classical complement pathway- antibody dependent ✓ Can also participate in precipitation reaction, but IgG is better ✓ IgM is very effective in complementation and agglutination compered to IgG
antigen remains behind to direct further synthesis. - Comparable to induced fit model in enzyme model.
MONOCLONAL ANTIBODY
- AKA as hybridoma technique
- George Kohler and Cesar Milstein discovered a technique to produce antibody arising from a single B cell. ✓ They figured out how to produce a hybrid cell that produces a very specific antibody that will react with the specific antigen. That’s why we have diff. antibody reagent.
- In result, this discovery plays an important role in revolutionized serological testing
- Kohler and Milstein’s technique fuses an activated B cell with a myeloma cell that can be grown indefinitely in the laboratory.
- Myeloma cells (cancer cell) ➔ lacks an enzyme HGPRT (Hypoxanthine- guanine phosphoribosyl transferase)
- An important enzyme to synthesize nucleotides from hypoxanthine and thymidine.
- Where unable to manufacture nucleotides from hypoxanthine and thymidine, since they do not have HGPRT enzyme.
HYBRIDOMA PRODUCTION
- 1 pathway, which builds DNA from degradation of old nucleic acids, is blocked. ✓ They are using mice
- The other pathway, which makes DNA from new nucleotides, is blocked by the presence of aminopterin.
- The remaining hybridoma cells are diluted out and placed in microtiter wells, where they are allowed to grow.
- Each well, containing 1 clone, is then screened for the presence of the desired antibody by removing the supernatant.
- Once identified, a hybridoma is capable of being maintained in cell culture indefinitely, and it produces a permanent and uniform supply of monoclonal antibody that reacts with a single epitope.
✓ Plasma cells do not undergo replication ✓ Cancer cells can replicate ✓ Hybridoma has characteristics of both spleen and myeloma cells, it can produce an antibody ✓ Myeloma cells die in the process because they do not have HGPRT enzyme
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IMSE311 WEEK 7 Antibody Structure AND Functions
Course: Immunology and Serology (IMSE311)
43 Documents
Students shared 43 documents in this course
University: Our Lady of Fatima University
Was this document helpful?
IMSE311 (MIDTERMS)
WEEK 7: ANTIBODY STRUCTURE AND FUNCTIONS
B CELL ACTIVIATION
• Part of humoral branch of immune response (production of the
antibodies)
• Subsequent stages:
✓ Pro b cell
✓ Pre b cell
✓ Mature b cell
✓ Immature b cell
✓ Activated b cell
✓ Plasma cell (to release or secrete antibodies)
• When b- cells are stimulated by an antigen, they will go differentiation
and the product is antibody production, or immunoglobulins, memory
b- cells.
• CD19, CD24, CD45R -clusters of differentiation or surface markers that
remain on the cell surface throughout the subsequent developmental
stages.
• CD25- surface protein, found on both activated b cells and t cells and
acts as a receptor for IL2
IMMUNOGLOBULINS
• Glycoprotein found in the serum/plasma portion of the blood
• Composition
✓ 82-96%→ polypeptides (protein) *Majority
✓ 2-14%→ carbohydrates
• Electrophoresis (pH 8.6) → Immunoglobulins appear primarily in the
gamma region (located in the negative electrodes or cathode region).
✓ Gagamit ng Agarose gel w/ pH 8.6
• Part of humoral branch of immune response. (secretion or production
of antibodies)
• They play an essential role during <Antigen recognition= and in
biological activities related to immune response such as <opsonization=
(preparation of phagocytosis) and <complement activation=
->> Not all IG can perform complement activation, only IgM and
IgG aka complement fixation.
Classification of immunoglobulins:
• <Ig= → <Immunoglobulin
• GAMDE (IgG, IgA, IgM, IgD, IgE)
SERUM ELECTROPHORESIS
Albumin – fastest (anode region)
• Most abundant protein (laging
naka pwesto sa left side ng
serum electrophoresis activity)
• Most predominant region
• (Thick bond) 60% in blood
components
Has different globulins: alpha1
globulin, a2 globulin, beta globulin
and on the right side ay gamma globulin (lagging nasa dulo and nakikita
yung mga immunoglobulins)
✓ a1 globulin: alpha1 antitrypsin, alpha fetoprotein
✓ a2 globulin: ceruloplasmin, haptoglobin, a2 macroglobulin
✓ beta globulin: transferrin, hemopexin, complement system,
fibrinogen, lipoprotein (consist of LDL, HDL, VLDL)
• possible magkaroon ng abnormal pattern. Beta gamma bridging,
nagkakaroon ng curve and kapag nagkaroon ng beta gamma bridging,
these are the condition of liver cirrhosis
• Spike pattern- kapag biglang tumaas sa may gamma region, means of
multiple myeloma (benz jones protein) (elevated ang plasma protein ng
patient)
✓ Benz jones protein- will precipitate for about 60pC and will
dissolved at 80pC
TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS
• All immunoglobulin molecules are made up of a basic 4 chain
polypeptide unit that consists of 2 large chains called heavy or H chains
and 2 smaller chains called light or L chains.
• These chains are held together by non-covalent forces and Disulfide
linkage/bond
• Structure of Immunoglobulins was first described by 2 scientists.
(Gerald Edelman and Rodney Porter)
✓ They decide to work with IgG molecule because it’s easy to
collect compare to others.
✓ IgG – Most predominant Ig in the blood
1. Edelman’s work centered on using the analytical ultracentrifuge to
separate out immunoglobulins on the basis of molecular weight.
- Sedimentation rate is directly related to molecular weight.
- The higher the sedimentation rate, the higher the molecular weight, the
larger the molecule (vice versa)
• He found that intact IgG molecules had a sedimentation coefficient of
7S (Svedberg unit).
• On obtaining a purified preparation of IgG. Edelman used 7Molar urea
to unfold the molecule.
• Once unfolded, the exposed sulfhydryl bonds could be cleaved by a
reducing agent such as 2-Mercapthoethanol.
• After such treatment, Gerald Edelman subjected again this
cleaved/unfold antibody perform analytical ultracentrifugation, and 2
fraction has been given, 1 is 3.5S and 1 is 2.2S
• Edelman concluded that 3.5s fraction has approximately with molecular
weight of 50,000 Daltons, he designated is as the H-chain.
• The 2.2S fraction with a molecular weight of 22,000 Daltons is the L-
chain.
• Won a Nobel prize in 1972
ANTIBODY DIGESTION
PAPAIN DIGESTION
•
Rodney Porter’s work was based on the use of the proteolytic enzyme
papain to cleave antibody (equal size) structure (monomeric structure).
✓ Cleave antibody into 3 pieces in equal size. The 3 pieces has
sedimentation coefficient of 3.5S represent a molecular
weight of 45,000 to 50,000 daltons
• He then subjected the IgG to Carboxymethyl cellulose ion exchange
chromatography.
✓ 1 fragment spontaneously crystallized at 4°C.
▪ Called the FC region of antibody, or the fragment
crystallizable region.
▪ Has no antigen binding ability
▪ Now known to represent the carboxy terminal
halves of 2 heavy chains that held together by ss
bonding
✓ The remaining 2 identical fragments were found to have
antigen-binding capacity.
▪ Called the FAB (Fragment Antigen Binding) region
PEPSIN DIGESTION
•
Alfred Nisonoff used pepsin to obtain additional evidence for the
structure of IG.
• This proteolytic enzyme was found to cleave IgG at the carboxy-
terminal side of the interchain disulfide bonds.
• 1 single fragment with a molecular weight of 100,000 d and all the
antigen-binding ability, known as F(ab)2.
• An additional fragment called FC’ (composed of 2 heavy chain) was
similar to FC except that it disintegrated into several smaller pieces.
-> Papain digestion successfully cleaved antibody into 3 portions or
fragments.
-> Pepsin digestion successfully cleaved antibody into 2 portions or
fragments
FAB region- variable region (conjunction with the location of amino
terminal) (End terminal)
FC region- constant region (located at carboxyl terminal)
• There is no such thing that 2 light chains are bind together
(ABNORMAL); multiple myeloma (benz jones protein)
• The basic monomeric structure of an antibody has 2 vibrations
