1	CLSC320 Principles of Immunology Diagnostic Laboratory Immunology Program for Clinical Laboratory Science Unit - 10 Principles of Antigen- Antibody Reactions
2	Unit 10 - Guidelines Reading assignment: Pages 212 - 227 of textbook Learning objectives: Those listed on page 213 of textbook Key terms: Those listed on pages 213 - 214 of textbook
3	Conditions for Maximum Reaction Factors that will contribute to maximum binding of antigen by antibody: 1.specificity of antibody for antigen 2.close proximity of antigen and antibody 3.high affinity (forces of attraction) and high avidity (strength of binding) of antibody for antigen 4.presence of approximately equal numbers of epitopes and idiotypes 5.appropriate concentration of antigens and antibodies 6.appropriate environmental conditions
4	Primary Phase of Ab-Ag Interactions antigen + antibody Uepitope binds to idiotype UAg-Ab complex formed is undetectable unless one of reactants is labeled Ag-Ab complex Umaximum Ag-Ab complex formed when equal ratio of Ag & Ab present (zone of equivalence)
5	Secondary Phase of Ab-Ag Interaction Umultivalent Ag binds to bivalent Ab Ag-Ab complex + Ag-Ab complex Ag-Ab complex Ucross-linking of complexes occurs to produce: agglutination - if particulate antigen precipitation - if soluble antigen
6	Factors Affecting Ab-Ag Interactions Structural Characteristics Š Physiochemical Factors Š Environmental Factors
7	Structural Characteristics Specificity Š Antigen Accessibility Š Antibody Structure Š Valence of Antigen Š Ag to Ab concentration
8	Specificity of Antigen-Binding Sites 1. it is the sequence of amino acids in the idiotype that determines whether or not the antigen (epitope) will bind with antibody (idiotype) 2. if there is specificity between Ag and Ab they are said to be complementary to each other 3. cross-reactivity is when specific antibodies react with: Utwo dissimilar antigens that share common epitopes Uepitopes with structural similarities to epitope that initially stimulated the immune response Uantigens from other species (heterophil Ag=s & Ab=s) i.e.Ab=s to Rickettsia rickettsia agglutinate Proteus vulgaris
9	Antigen accessibility & Antibody Structure Antigen accessibility: eit is the arrangement of the constant regions and the hinge region that allows the antibody to join with the epitope Antibody structure: ethe number of antigen-binding sites determines the size of Ag-Ab complexes: Utwo FAB sites found on: IgG, IgE, and IgD Ufour FAB sites found on: IgA Uten FAB sites found on: IgM
10	Valence of Ag & Conc. Of Ab-Ag Valence of Antigen: eit is the of identical epitopes contained on an antigen Umultivalent (>1 identical epitope) antigens bind much stronger with antibodies than do univalent antigens Uthe size of Ag-Ab complexes is largely dependent on the valence of antigens Concentration of Antigens & Antibodies: ethe number of antigen-binding sites (thus ab=s) and the number of epitopes (thus antigens) influences the formation of Ag-Ab complexes. Uzone of equivalence: the concentration of antigen and antibody are such that maximum Ag-Ab complexes are formed
11	Zone of Equivalence ethe number of antigen-binding sites (thus ab=s) and the number of epitopes (thus antigens) influences the formation of Ag-Ab complexes.
12	Prozone Reaction the concentration of antibody is greater than that of antigen so lattice formation is inhibited giving false negative results
13	Postzone Reaction the concentration of antigen is greater than that of antibody so lattice formation is inhibited giving false negative results
14	Physiochemical Factors Affinity and Avidity Š Binding Forces
15	Affinity & Avidity eforces or binding energy between an antigen (epitope) and antibody (idiotype) that gives stability to Ag-Ab complex Uaffinity: intrinsic force of attraction between one idiotype and one epitope Uavidity: the sum of affinities when antibodies bind with multivalent antigens to form Ag-Ab complexes.
16	Binding Forces eforces that bind an antigen (epitope) and antibody (idiotype) are relatively weak and results in complexes that can be broken apart relatively easily: UIonic attraction: oposite charged portions of the molecules are attracted to each other UHydrogen bonds: bonds that form between negatively charged atoms and positively charged atoms of polar molecules (COO^- and H⁺) UHydrophobic bonds:(most important one) bonds that form between nonpolar molecules causing elimination of water molecules
17	Environmental Factors - Summary pH Š Temperature Š Ionic strength Š Zeta Potential
18	Environmental Factors Hydrogen ion concentration (pH): 6.5 to 7.5 Temperature: Ionic strength: Zeta potential: on next slide changes in pH may cause ionization of amino acids and may lead to changes configuaration in Ag or Ab or both certain classes of antibodies react most strongly at specific temperatures: IgG: IgM: 37^oC 4^oC - 27^oC E temperatures above 40^oC may denature proteins, alter idiotypes, and decrease Ag-Ab complex stability low salt concentrations promote specific Ag-Ab binding thus low ionic strength (LISS) solutions used to promote Ag-Ab interactions in blood bank testing
19	Zeta Potential - the electrical potential that forms between particles which prevents them from coming in close proximity to each other. - when antibodies and antigens are suspended in a saline solution the zeta potential keeps them apart for a distance of 25 nm. Antibody classes can span a distance of: IgG: IgM: 14 nm 35 nm - thus IgG antibodies must either have the zeta potential lowered or the antigens must be forced closer together (centrifugation) before agglutination will occur - another way to visualize that IgG has in fact attached to the antigen is to add anti-Human Globulin (antibodies to IgG) which then link the attached IgG to form Ag-AB compexes
20	Ways to Lower Zeta Potential Utreat with proteolytic enzymes: papain ficin: Uuse colloidal diluents 22% albumin Uuse low ionic strength diluents LISS
21	Manifestations of Ab-Ag Reactions ePrimary manifestation: eSecondary manifestation: Ubonds form between the idiotype and epitope Ureaction only detected if a label has been attached to one of the reactants UAg-Ab complexes formed are reversible Ucross linking of Ag-Ab complexes occurs forming lattices Uprecipitation or agglutination is visible UAg-Ab complexes formed are non-reversible and insoluble
22	Detection of Ab-Ag Interactions - 1 Testing Techniques used to Detect Primary manifestations: eA labeled reactant (Ag or Ab) is used to detect reaction Uenzymes alkaline phosphatase Ufluorescent dyes: fluorescein or rhodamine Uradioactive isotope: ⁴²K ¹³¹I ¹²⁵I ⁵⁷Co ¹⁴C 124 hours 2 life 8.1 days 2 life 60 days 2 life 270 days 2 life 5,730 years 2 life eTechniques using labels are: Uextremely reliable Usensitive Uversatile
23	Examples of Testing Techniques - 1 Testing Techniques used to Detect Primary manifestations: UFluorescent Immunoassays (FIA) URadioimmunoassays (RIA) UEnzyme Immunoasssays (EIA) UFluorescence-Activated Cell Sorting (FACS) UComplement fixation techniques fluorescent dye used as label radioactive isotope used as label enzyme used as label and reacts with a substrate to produce a colored product fluorescent dye tagged antibodies used in flow cytometry to separate cell populations next slide LLLLLL
24	Examples of Testing Techniques - 2 Testing Techniques used to Detect Primary manifestations: UComplement fixation techniques Mwhen certain antibodies form Ag-Ab complexes complement is bound to the complex Man indicator cell (sheep or human RBC=s) can then be added and the amount of hemolysis is inversely proportinate to amount of Ab or Ag present in the initial reaction.
25	Detection of Secondary Ab-Ag Reactions & Examples of Tests eSince the end result of this stage is visible precipitation or agglutination, no labels are necessary for detection of end products. eExamples of Techniques are: UImmunoprecipitin techniques when soluble mulivalent antigens form insoluble Ag-Ab complexes that are not visually detected Mturbidimetry MNephelometry when Ag-Ab complexes form the cloudiness of the solution changes when Ag-Ab complexes form the light passing through the solution is scattered and the amount of light at right-angles is measured
26	Examples of Tests for Secondary Ab-Ag Reaction Detection - 1 eExamples of Techniques are: [continued] UImmunoprecipitin techniques - cont=d MImmunodiffusion techniques MImmunoelectrophoretic techniques when Ag-Ab complexes form in a support media such as agarose gel they form visible lines of precipitation when complex antigens are seperated using electrophoresis followed by Ag-Ab complexes forming in a gel media and show up as precipitin lines
27	Examples of Tests for Secondary Ab-Ag Reaction Detection - 2 UAgglutination techniques when particulate mulivalent antigens form Ag-Ab complexes that are visually detected MDirect agglutination MPassive agglutination MReverse Passive agglutination MAgglutination Inhibition NOTE: we will be looking at each of the preceeding techniques in more detail in upcoming units.
28	End of Principles of Ag-Ab Reactions Press the ESC key to end program