Modern Immunofluorescence Techniques
Day 5 Antibodies A Brief Review of Antibody Structure
The basic antibody is a dimer of dimer (2 heavy chain-light chain pairs) composed of repeats of a single structural unit known as the “immunoglobulin domain” A Brief Review of Antibody Structure
Primary structure Tertiary structure
Secondary structure
Quaternary structure Antibody Production The blood contains two types of white blood cell or leukocyte
Phagocytes ingest bacteria by endocytosis
Lymphocytes produce antibodies Production of Antibodies by Lymphocytes
A lymphocyte can produce only one type of antibody so a huge number of different types are needed Each lymphocyte has some of its antibody on its surface… The antigens of a pathogen bind to the antibodies in the surface membrane of a lymphocyte…
…This activates the lymphocyte. The active lymphocyte divides by mitosis to produce a clone of many identical cells
MITOSIS
The clone of cells starts to produce large quantities of the same antibody… … the same antibody needed to defend against the pathogen! Most microbes have more than one antigen on their surface, so…
…they stimulate more than one type of lymphocyte…
…resulting in the production of many different antibodies.
These are called polyclonal antibodies. Antibody Production: The Primary Response Step 1: Antigen Presentation Antigen Macrophages take in antigen by endocytosis The macrophage processes the antigen and attaches it to a membrane protein called a Macrophage MHC protein The MHC protein is moved to the MHC protein cell surface membrane by exocytosis so that the antigen is displayed on its surface. Step 2: Activation of Helper T-cell receptor Helper T-cells have receptors on their cell surface membranes which can bind to antigens presented by Helper T-cell macrophages. Helper T-cell binds to macrophage presenting the antigen
Macrophage sends a signal to activate the helper T-cell Step 3: Activation of B-lymphocytes
B-cells have antibodies in their cell surface membranes Antigens bind to the antibodies in the surface membranes of B-cells Antigen
Inactive B-cell
Antibody An activated helper T-cell with receptors for the same antigen binds to the B-cell
SIGNAL
The helper T-cell sends a signal to the B-cell, activating the B-cell. Step 4: Proliferation
The activated B-cell starts to divide by mitosis to form a clone of plasma cells.
Plasma cells are activated B-cells with a very extensive network of rough endoplasmic reticulum.
Plasma cells synthesis large amounts of antibody, which they excrete by exocytosis. The Secondary Response: Memory Cells
• If an antigen invades your body a second time, a much faster response occurs which produces much larger quantity of the required antibody.
•When activated B-cells are dividing during the primary response, some cells stop dividing and secreting antibody and become memory cells.
• Large numbers of memory cells remain in the body for a long time… • …they are capable of producing large amounts of antibody very quickly when stimulated. Antigen Antigen
Activate Activate Helper B-cell T-cell
Antibody Production: Macrophage Clone Summary
Memory Cell Plasma Cell Antibodies Polyclonal antibodies vs Monoclonal antibodies
Polyclonal antibodies: antibody preparations from immunized animals. Consist of complex mixtures of different antibodies produced by many different B cell clones
Monoclonal Antibody: homogeneous antibody preparations produced in the laboratory. Consist of a single type of antigen binding site, produced by a single B cell clone (later we’ll talk about how these are made). Polyclonal antibodies can form lattices with homogeneous, monomeric protein antigens because each antibody can interact with a different epitope on the antigen.
Monoclonal antibodies do not form lattices with homogeneous, monomeric proteins, because only they can bind to only one epitope on the antigen. When do antibody-antigen lattices form?
Polyclonal antibody and antigen with multiple Distinct epitopes
monoclonal antibody and antigen with repeating pattern of identical epitopes When do antibody-antigen lattices form?
Not with monoclonal antibody and antigen with multiple distinct epitopes MONOCLONAL ANTIBODY PRODUCTION Formation and Selection of Hybrid Cells
• Hybridoma: the B cell X myeloma cell – To be produce by using polyethylene glycol (PEG) to fuse cells – The myeloma cells: immortal growth properties – The B cells: to contribute the genetic information for synthesis of specific antibody – Selected by using HAT medium (hypoxanthine, aminoprotein, and thymidine) • Myeloma cells are unable to grow • B cells are able to survive, but can not live for extended periods Hybridomas are hybrids between a non-transformed antibody producing B cell and a transformed cell (myeloma) that can grow continuously in culture. Two different pathways to synthesis nucleotide in mammalian cells
(Folic acid analog) Myeloma cells used in hybridoma technology are double mutants, they lack the HGPRTase and lose the ability to produce Ig HAT selection is used to select for growth of hybrids and against the growth of the parental myeloma. (Most common screening techniques are ELISA and RIA)
Low concentration High concentration (1-20 ug/ml) (1-10 mg/ml) Cloning hybridomas from fusion Plate at limiting dilution (<1 cell/well) in 96 well plates. Allow clones to expand. Expand positive well and test for production of antibody of desired specificity in culture supernatant Cloning hybridomas from fusion Plate at limiting dilution (<1 cell/well) in 96 well plates. Allow clones to expand. Expand positive well and test for production of antibody of desired specificity in culture supernatant
Production of antibodies 1. Polyclonal antibodies – a mix of many different antibodies that recognize different determinants on an antigen. This mix makes standardization of assays difficult.
Step 1. Animal is inoculated with Step 2. Blood containing the antibody several doses of antigen along with blood cells and other proteins is collected from the over several weeks to animal. induce production of antibodies.
Step 4. Blood sample is centrifuged to sediment the blood Plasma containing cells antibodies and other proteins
Pelleted blood cells
Step 5. Plasma is heated to 65o C to destroy contaminating Polyclonal antibodies proteins leaving the antibodies intact Polyclonal antisera
Can be generated by repeated immunization of animal (rabbit) with antigen (with adjuvant). polyclonal antibodies are a complex mixture of antibodies directed against different epitopes and that differ in their affinity for the antigen.
Polyclonal antisera
Polyclonal antisera are mixtures of immunoglobulins and other plasma proteins. For cleaner results antibodies are best purified. This is done by affinity purification Polyclonal antisera
Peptide affinity purification removes non-specific IgGs Polyclonal antisera
A “dirty” way is to purify IgGs by virtue of their affinity of protein A or protein G which are surface proteins of streptococci and Staphylococcus aureus respectively. Polyclonal antisera Protein A can bind with strong affinity the Fc portion of immunoglobulin of certain species: Immunoglobulin Species Protein A Binding
Human IgG, IgG2, IgG4 Strong Human IgG3, IgD None Human IgM, IgA Weak
Mouse IgG1 Weak Mouse IgG2A, IgG2BIgG3 Strong Mouse IgM None
Rat IgG1 Weak Rat IgG2A, IgG2B None Rat IgG2C Strong Goat IgG1 Weak Goat IgG2 Strong Bovine IgG1 None Bovine IgG2 Strong Guinea Pig IgG Strong Rabbit IgG Strong Chicken IgG (IgY) None Validation of antibodies A variety of different assays have been developed to detect antibody-antigen interactions.
Some of these are based on the tendency of antibody-antigen complexes to come out of solution called “precipitation reaction”.
Some are based on the ability of antibodies to stick cells together, called an “agglutination reaction”. Double-diffusion assay (Ouchterlony method)
antigen antibody
Antigen & antibody embedded in agar gel atop glass slide Line of precipitation
Precipitin Reaction: precipitates form most efficiently when antibody and antigen are at similar concentrations: the Equivalence Zone Antibody-excess Equivalence Antigen-excess zone zone zone Antibody binding to cells can bind cells together into a large clump: an “agglutination reaction”
Wells contain sheep red blood cells (SRBC) and increasing concentration of anti-SRBC antibodies Affinity between two macromolecules can measured using a biosensor Technique: Surface Plasmon Resonance Instrument: Biocore
-Resonance units are proportional to the degree of binding of soluble ligand to the immobilized receptor. (or soluble antibody to immobilized antigen, as shown here) - Determining the amount of binding at equilibrium with different known concentrations of receptor (antibody) and ligand (protein antigen) allows you to calculate equilibrium constants (Ka, Kd).
-Rate of dissociation and association (koff, kon) can also be calculated. Affinity refers to strength of binding of single epitope to single antigen binding site.
But antibodies have 2 or more identical binding sites.
Most antigens are multivalent.
What is impact of valence on strength of binding? Avidity (strength of binding) is influenced by both Affinity (Ka of single binding site) x Valence of interaction (number of interacting binding sites)
Antibody-antigen interactions are multivalent when both the antibody and the antigen have multiple binding sites.