Immunoprecipitation

Definition of Immunology The study of the physiological mechanisms which enable the body to recognize materials as foreign and to neutralize, eliminate, or metabolize them without injury to the host tissue.

Overview of Immunodiagnostic Studies Antigen-antibody reactions for diagnosis of: infectious disease, autoimmune disorders, immune allergies, and neoplastic disease. These modalities also test for: blood groups and types, tissue and graft transplant matching.

Types of Tests Many methods of varying sophistication are used for immunodiagnostic studies

Types of Tests Tests for Visible Change Observable Reaction Name of Test Treponemal, heterophile, and cold agglutinin antibodies Clumping Particulate antigen reacts with corresponding antibody; antigen may be in form of RBCs (hemagglutination, latex, or charcoal coated with antigen). Agglutination Fungal antibodies, food poisoning, Quantitative immunoglobulins IgA, IgG Precipitates Soluble antigen reacts with corresponding antibody by ID or count. Precipitation Viral antibodies Complement activation, hemolysis Competition between two antigen-antibody systems (test and indicator systems) Complement fixation (CF) Antinuclear antibodies (ANAs); antimitochondrial antibodies (AMAs) Visible microscopic fluorescence Fluorescent-tagged antibody reacts with antigen-antibody complex in the presence of ultraviolet light. Immunofluorescence Hepatitis and human immunodeficiency virus (HIV) (screening) Color change indicates enzyme substrate reaction. Indirect EIA for quantification of an antigen or antibody enzyme and substrate Enzyme-linked immunosorbent assay (ELISA) Confirms HIV-1 Detection of antibodies of specific mobility Electrophoresis separation of antigen subspecies Immunoblot (eg, Western blot [WB]) Quantitative immunoglobulins IgA, IgM, C-reactive protein, anti-streptolysin O recorded in mg/dL or IU/mL Light scatter proportionately increases as numbered size of immune complexes increases. Measures either antigen or antibody in solution through the scattering of a light beam; antibody reagent used to detect antigen IgA, IgG, IgM; concurrent controls are run to establish amount of background scatter in reagents and test samples. Rate nephelometry

Antigen-antibody interaction Antigens are substances that stimulate and subsequently react with the products of an immune response. They may be: enzymes, toxins, microorganisms (eg, bacterial, viral, parasitic, fungal), tumors, or autoimmune factors. Antibodies are proteins produced by the body's immune system in response to an antigen or antigens. The antigen-antibody response is the body's natural defense against invading organisms.

Structure of an antibody

Antibody Structure V Light Chain Light Chain Heavy Chains Constant SS Antigen binding site V SS SS Light Chain Light Chain Constant Heavy Chains

Classes of antibodies IgG - A monomer - Most abundant antibody in blood. IgG easily leaves the circulatory system to fight infection and crosses the placenta conferring passive immunity to a fetus. IgD - A monomer - Found on the surface of B cells probably allowing recognition of antigens thus triggering differentiation into plasma and memory B cells IgE - A monomer - The least common antibody. The tails attach to mast cells and basophils. When antigens bind, they signal release of histamine. IgA - A dimer - Produced by cells in the mucus membranes to prevent attachment of pathogens. IgA is also found in many body secretions including milk. IgM - A pentamer - First antibody to appear following exposure to an antigen. Because it declines rapidly in the blood, high IgM levels indicate a current infection.

Three Distinct Phases of Antigen/Antibody Reactions Primary Phenomenon – involves the combination of an individual binding site on an antibody molecule with a single epitope or determinant site on an antigen Secondary Phenomenon – Lattice formation include precipitation, agglutination and complement fixation Tertiary Phenomenon – Detected by affect on tissues or cells, Inflammation, phagocytosis, deposition of immune complexes, immune adherence and chemotaxis

Detection of antigen-antibody interactions: Primary Phenomenon e.g. Enzyme linked immuono sorben assay (ELISA) Immuno flurescent antibody technique (IFAT) Radio immuno assay (RIA) Secondary Phenomenon : e.g. Agglutination tests Complement fixation tests (CFT) Precipitation tests Tertiary Phenomenon : e.g. Reaction not visible, detected by affect of reaction on tissues or cells

Antigen Binding Antigen 2 Antigen 1 Antigen 3 Variable Light Heavy

Ag-Ab interactions Bonds: Hydrogen Ionic Hydrophobic interactions Van der Waals forces Each bond is weak; many are strong To “hold” they must be close and complementary

Non-covalent binding

Features of the Antigen-Antibody Interaction Reversibility Non-covalent Interactions Affinity Measure of the strength of the binding Ease of association or dissociation Avidity Increase in affinity due to multivalent binding The summation of multiple affinities

Binding of the epitope in the antigen binding site POOR FIT GOOD FIT antibody combining site antigen determinant high attraction low repulsion high repulsion low attraction High Affinity Low Affinity

Antibody Affinity Affinity – the strength of the total noncovalent interactions between a single Ag-binding site on an Ab and a single epitope Low affinity Abs bind Ag weakly and dissociate readily High affinity Abs bind Ag more tightly and remain bound longer Ag + Ab Ab-Ag k1 k-1 K1 = forward (association) constant K-1 = reverse (dissociation) constant

Antibody Affinity K can be determined using equilibrium dialysis

K determined by equilibrium dialysis

Antibody Avidity Avidity – the strength of the total noncovalent interactions between a multivalent Ab and Ag The avidity of an antibody is a better measure of its binding capacity within biological systems than is the affinity of its individual binding sites High avidity can compensate for low affinity; eg. pentameric IgM often has lower affinity than IgG but its higher avidity (due to presence of 10 binding sites) may enable it to bind Ag more effectively

Antibody Avidity Y Keq = 104 Affinity Y 106 Avidity Y 1010 Avidity

Effect of multivalent interactions antibody Fab IgG IgG IgM effective antibody valence 1 1 2 up to 10 antigen valence 1 1 n n equilibrium constant (L/M) 10 4 10 4 10 7 10 11 advantage of multi-valence - - 10 3 -fold 10 7 -fold definition of bindng affinity affinity avidity avidity intrinsic affinity functional affinity

Factors Affecting Measurement of Ag/Ab Reactions Ab excess Ag excess Affinity Avidity Equivalence – Lattice formation Ag:Ab ratio Physical form of Ag

Tests Based on Ag/Ab Reactions All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

Immunoprecipitation Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody that specifically binds to that particular antigen. This process can be used to isolate and concentrate a particular antigen from a sample containing many thousands of different antigens.

Immunoprecipitation tests The antigen and antibody are in soluble form Combine to form a visible precipitate Complete precipitation occurs at certain Ab/Ag concentrations Antigen Antibody

Precipitation Curve Prozone – antibody excess, many antibodies coat all antigen sites- results in false negative Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

Precipitation Curve

Precipitation Reactions Ab + soluble Ag Lattice Precipitate Precipitation techniques Tube precipitation test Gel diffusion Single radial Double Immunoelectrophoresis Countercurrent electrophoresis (CEP),

Tube precipitation test

Gel diffusion Immunodiffusion tests Antigen and antibody placed in separate wells cut in gel Solutions diffuse and meet between the wells Results in line of precipitation at zone of optimal proportion

Double Diffusion Note: A line of precipitation has formed between the center well and wells 3 & 5. This indicates there is antigen/antibody specificity between the center well and these two wells. Usually a known antigen or known antibody is placed in the center and test serum is placed in the peripherial wells. Antigens and antibodies will diffuse and at some point optimal concentrations will occur and if the antigen is specific for the antibody a precipitate line will form. Example: Has this patient ever had rubella, or diptheria? If they have their serum will contain antibodies against the disease. Put patient serum in the center. Put the disease agents (antigens) in wells 1 – 5, and allow to diffuse. A precipitation line between wells indicates that the patient has had that disease

Double Diffusion Ab and Ag diffuse towards each other Used for determining antigenic relationship between substances Identity Non-identity Partial identity

Double Diffusion 1 2 Ab

Double Diffusion

Identity If wells 1 and 2 contain two identical antigens and well 3 has antibodies to that antigen, a reaction of identity results as all antibodies react with what is in both antigen wells indicating that the contents of both wells are closely related. X X Anti X Y Anti X

Identity: What You Really See

Double Diffusion

Non-identity If wells 1 and 2 contain two separate antigens and well 3 has antibodies to both antigens, a reaction of non-identity results indicating that either X and Y are different or that well 3 has a mixture of two antibodies in it. X Y Anti X Y Anti X Anti Y

Non-identity: What You Really See

Double Diffusion

Partial Identity If wells 1 and 2 contain two separate antigens and well 3 has antibodies to both antigens, a reaction of partial identity may result if all antibodies react with X, but only some react with Y. indicating that X and Y are related in some way. X Y Anti X Y Anti X Anti Y

Non-identity: What You Really See

Radial immunodiffusion test Radial immunodiffusion test is quantitative Antibody is added to liquid agar that is allowed to harden Created a uniform antibody concentration Antigen added to wells cut in gel Diffusion outward forms concentration gradient Ring forms at antigen-antibody precipitation Standards can be used to construct standard curve to establish concentration

Single radial immunodiffusion Ag

Single radial immunodiffusion

Single radial immunodiffusion

Single radial immunodiffusion Interpretation Diameter of ring is proportional to the concentration Quantitative Ig levels Ag Ab in gel Ag Concentration Diameter2

Single radial immunodiffusion

Single radial immunodiffusion Exercise 1

Materials Standard Single radial immunodifusion plates Moisture chamber Micropipettes

Specimen Collect whole blood without anticoagulant and allow to clot at room temperature. Separate serum by centrifugation within 2-3 hours after collection.

Method Remove plates from refrigerator to room temperature approximately 30 minutes before filling wells. Do not open bag until ready for use. If excess moisture is present, remove plate from its bag and remove cover until evaporation has dried the surface and wells. Replace cover until used. For best results, three wells should be filled with reference sera for each plate. Location of each should be noted.

Preparing the standards (serial dilution) Label four microtest tubes: 1:2, 1:4, 1:8, and 1:16. Using a micropipette, add 50 microliters of Buffer to each tube. With a fresh pipet tip, add 50 microliters of “Standard” to the tube labeled 1:2, Mix. Transfer 50 microliters of the 1:2 dilution to the tube labeled 1:4, Mix, and so on.

Performance of Test Deliver specimen or standard (5 µl) to well by placing the pipette tip at the bottom of the well. Allow the well to fill to the top of the agar surface. Avoid bubbles to ensure proper volume and diffusion of sample. Visualization may be aided by placing the plate on dark background. Mark time of completion on plate cover and replace cover. Replace plate in bag and reseal carefully. Incubate plates upright on a flat surface at room temperature (20 to 24oC) over 48 hours for End Point readings.

Calibration Curve Using the reference sera determine their ring diameters to the nearest 0.1mm. Using regular graph paper plot the concentration on the X axis and the zone diameters squared on the Y axis for each protein for End point readings. Draw a straight line of "best fit" between the four points.

Results Measure diameters of precipitin zones to within 0.1mm. Variations in incubation times of more than 30 minutes will produce changes in diameters, especially those at higher levels of antigen except when plates have reached endpoint. Determine the concentration of each unknown or specimen protein from the reference curve find the corresponding concentration.

Results Ag Concentration Diameter2