Precipitation And Agglutination Reactions PDF 6/27/2024

6/27/2024 Precipitation and Agglutination Reactions CHAPTER 10 Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each stud...

6/27/2024 Precipitation and Agglutination Reactions CHAPTER 10 Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES Unit Objectives are your study guide (not this PowerPoint) Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  6/27/2024 Chapter Overview Serology Testing requirements Testing phases Antigen–antibody binding Precipitation curve Immunoturbidimetry and nephelometry Passive immunodiffusion techniques Electrophoretic techniques Agglutination reactions Instrumentation Quality control and result interpretation Specimen Requirements for Serology Serology Study of fluid components in the blood, especially antibodies Liquid portion of blood minus coagulation factors Serum Most frequently encountered specimen in immunologic testing Separated from other components of a blood specimen via centrifuge Collection Tubes Red top (no additives) or Tiger top (red/gray) – Gel barrier Storage (if testing Between 2°C and 8°C for up to 72 hours is delayed) Frozen at –20°C or below 2  6/27/2024 Causes for rejection of specimen Hemolyzed Clotted specimen Wrong tube or container Tube not labeled Inadequate quantity Tube mislabeled Phases of laboratory testing Three phases of laboratory testing Pre-analytical—specimen collection, transport and processing Analytical—testing Post-analytical—testing results transmission, interpretation, follow-up, retesting. Pre- and post-analytical errors are estimated to constitute 90% of errors. Analytic errors around 10%. Impact of errors can potentially lead to a serious patient problem. Inadequate or improper patient care Inconvenience to patient Misdiagnosis Harm to patient Death 3  6/27/2024 Antigen/Antibody Complexes This Photo by Unknown Author is licensed under CC BY 3 Phases of Antigen/Antibody complexes Combination of binding site on antibody with a single epitope on antigen. Primary phenomenon Reversible - occur in milli- seconds. Not easily detectible - hard to measure. Secondary precipitation, agglutination, complement fixation. easily detectable phenomenon most serological tests based inflammation, phagocytosis, deposition of immune complexes, immune adherence, chemotaxis Tertiary phenomenon all in vivo skin tests measure 4  6/27/2024 3 Types of antigen/antibody reactions. 1. Precipitation - soluble antigen + soluble antibody = visible complexes. 2. Agglutination - particulate antigens aggregate to form larger complexes when specific antibody is present. 3. Complement fixation -triggers the classical complement pathway to detect antibody or antigen present in the patient’s serum. Antigen–Antibody Binding: Affinity Initial attraction force between a single Fab site on an antibody molecule and a single epitope on an antigen Strength of attraction depends on specificity of antibody for antigen Cross-reacting antigens have lower affinity. 5  6/27/2024 Antigen–Antibody Binding: Avidity Is the sum of attractive forces between an antigen and an antibody Strength with which a multivalent antibody binds a multivalent antigen Measure of the overall stability of an antigen- antibody complex Antigen–Antibody Binding: Law of Mass Action K = [AgAb]/[Ab][Ag] Free reactants are in equilibrium with bound reactants. Value of K depends on the strength of binding between antibody and antigen. The higher the value of K: The larger the amount of antigen–antibody complex The more visible or easily detectable the reaction 6  6/27/2024 Precipitation Soluble antigen is combined with soluble antibody to produce insoluble complexes that are visible. Precipitation Reactions Require antigen and antibody to have: Multiple binding sites for one another Equivalent concentrations 7  6/27/2024 Precipitation Curve: Zone of Equivalence Number of multi-valent sites of antigen and antibody are approximately equal. For optimal precipitation, reactions must be run in the zone of equivalence. Prozone phenomenon (antibody excess) Precipitation Antigen combines with only one or two antibody molecules. No cross-linkages are formed. Curve: False-negative reactions may occur as a result of high Prozone antibody concentration. If a false-negative reaction is suspected, diluting out the antibody and performing the test again may produce a positive result. 8  6/27/2024 Precipitation Curve: Postzone Postzone phenomenon (antigen excess) Small aggregates are surrounded by excess antigen. No lattice network is formed. The presence of a small amount of antibody may be obscured, causing false-negative results. Test may be repeated about a week later with another specimen to give more time for antibody production. If the test is negative again, it is unlikely that the patient has the antibody. SENSITIVITY TECHNIQUE APPLICATION PRINCIPLE (μg AB/ML) Immunoturbidimetry Immunoglobulins, complement, C- 1–10 Light that is scattered at an angle is measured, and Nephelometry reactive protein, other serum proteins indicating the amount of antigen or antibody present. Radial immunodiffusion Immunoglobulins, 10–50 Antigen diffuses out into gel that is infused with antibody. complement Measurement of the radius indicates concentration of antigen. Ouchterlony double Complex antigens, such as fungal 20–200 Both antigen and antibody diffuse out from wells in a gel. The diffusion antigens lines of precipitate formed indicate the relationship of antigens. Immunoelectrophoresis Differentiation of serum proteins 20–200 Electrophoresis of serum is followed by diffusion of antibody from the wells. Immunofixation Over- or underproduction Variable Electrophoresis of serum is followed by direct application of electrophoresis of antibody antibody to the gel. Comparisons of Precipitation Reaction Techniques 9  6/27/2024 Immunoturbidimetry and Nephelometry Automated methods Immunoturbidimetry Measures reduction in light intensity Nephelometry Measures light scatter as immune complexes form Radial Immunodiffusion (RID) A manual, single-diffusion technique Antibody is in the support gel, and antigen is placed in a well cut into the gel. Antigen diffuses out and reacts with antibody to form a ring of precipitation around the well. End-point (Mancini) method – reaction goes to completion Kinetic (Fahey) method–faster; measurements taken before reaction is complete RID Endpoint Method Results The square of the diameter is proportional to the antigen concentration. Patient results are determined from a standard curve. 10  6/27/2024 Ouchterlony Diffusion A double-diffusion technique Wells are cut into a gel, and both antigen and antibody diffuse out radially. A line of precipitate forms where antigen and antibody meet in equivalent amounts. Three possible patterns: Identity (arc) partial identity (one line extends) Nonidentity (line cross X) Immunofixation Electrophoresis (IFE) A double-diffusion technique Proteins in patient serum are electrophoresed, then antibody is applied directly to the gel. Precipitates form where antigen–antibody combination has taken place. It is performed to visualize increased or decreased production of antibody classes and to differentiate monoclonal and polyclonal immunoglobulins. 11  6/27/2024 Agglutination Agglutination is the visible aggregation of particles caused by combination with specific antibody. A two-step process Sensitization (initial binding) Antigen and antibody unite through binding of antigenic determinant sites; fast and reversible Lattice formation (creation of large aggregates) Visible aggregates form as multiple antigen and antibody molecules bind to create a stable lattice Produced by antibodies called agglutinins Phases of Agglutination 12  6/27/2024 Types of Agglutination Reactions 1. Direct agglutination 2. Passive agglutination 3. Reverse passive agglutination 4. Agglutination inhibition Direct Agglutination Uses particles with naturally occurring antigens to test for antibodies in patient serum Bacteria Widal test A rapid screening test used to determine the possibility of typhoid fever Uses Salmonella O (somatic) and H (flagellar) antigens Red blood cells (RBCs) Hemagglutination ABO typing 13  6/27/2024 Grading of Agglutination Reactions Passive Agglutination Uses particles that are coated with antigens not normally found on their surfaces: Erythrocytes, latex, gelatin, silicates Process: Antigen is attached to the carrier particle. Agglutination occurs if patient antibody is present. 14  6/27/2024 Passive Agglutination (continued) Examples are tests for: Rheumatoid factor Antibodies to Group A Streptococcus antigens Antibodies to viruses such as rubella Heterophile antibody in infectious mononucleosis Reverse Passive Agglutination Process: Antibody is attached to the carrier particle. Agglutination occurs if antigen is present in the patient sample. Common applications Rapid identification of antigens from infectious agents (e.g., Staphylococcus aureus, Streptococcus Groups A and B, rotavirus, and Cryptococcus neoformans) Detecting soluble antigens in urine, spinal fluid, and serum 15  6/27/2024 Comparison of Passive and Reverse Passive Agglutination  Passive Agglutination  Reverse Passive Agglutination Agglutination Inhibition Based on competition between particulate and soluble antigens for limited antibody-combining sites Lack of agglutination = positive reaction Used to detect hapten antigens (e.g., illicit drugs such as cocaine or heroin) Hemagglutination inhibition — uses RBCs Used to detect antibodies to certain viruses (e.g., rubella, influenza, and respiratory syncytial virus) that can bind to RBCs and agglutinate them 16  6/27/2024 Principle of Agglutination Inhibition Principle of Hemagglutination Inhibition 17  6/27/2024 Comparison of Agglutination Reactions TYPE OF REACTION PRINCIPLE RESULTS Direct agglutination Antigen is naturally found on a particle. Agglutination indicates the presence of patient antibody. Passive (indirect) Particles are coated with antigens not Agglutination indicates the agglutination normally found on their surfaces. presence of patient antibody. Reverse passive Particles are coated with reagent Agglutination indicates the antibody. presence of patient antigen. Agglutination inhibition Haptens are attached to carrier Lack of agglutination is a particles. Particles compete with positive test, indicating the patient antigens for a limited number of presence of patient antigen. antibody sites. Hemagglutination Red blood cells spontaneously Lack of agglutination is a inhibition agglutinate in presence of certain positive test, indicating the viruses. presence of patient antibody. Agglutination and Precipitation Animation 18  6/27/2024 Instrumentation Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) Patient sample is incubated with latex beads coated with analyte and reagent antibody. If amount of analyte in patient sample is high, analyte will prevent antibody from binding to beads and less turbidity results. Measures small analytes such as therapeutic drugs (e.g., digoxin). Quality Control and Result Interpretation Avoid cross-reactivity by using a monoclonal antibody directed against an antigenic determinant unique to a particular antigen. Store reagents properly, check expiration dates, and follow manufacturer’s instructions. Account for sensitivity and specificity of specific test kits used. Be aware that a negative result does not rule out presence of the disease or antigen. 19  6/27/2024 Summary Precipitation involves the combination of soluble antigen with soluble antibody to produce insoluble complexes that are visible. Union of antigen and antibody depends on the affinity between one antibody-binding site and a single epitope on an antigen. Avidity is the sum of all attractive forces occurring between multiple binding sites on antigen and antibody. Summary (continued_1) Concentrations of antigen and antibody that yield maximum binding represent the zone of equivalence, where the multivalent antigen and antibody sites are approximately equal. In the prozone, antibody is in excess; precipitation or agglutination cannot be detected. In the postzone, antibody has been diluted out, antigen is in excess, and the reaction is undetectable. 20  6/27/2024 Summary (continued_2) Precipitation techniques include nephelometry, passive immunodiffusion techniques (radial immunodiffusion and Ouchterlony double diffusion), and electrophoretic techniques (immunofixation electrophoresis). Agglutination is the process by which particulate antigens react with antibody to form large clumps. Summary (continued_3) Agglutination occurs in two steps: (1) sensitization and (2) lattice formation. In direct agglutination, antigens are found naturally on the indicator particle. In passive agglutination, antigens are artificially attached to such a particle. Reverse passive agglutination is so called because antibody is attached to the indicator particle. 21  6/27/2024 Summary (continued_4) Agglutination inhibition is based on competition between antigen-coated particles and soluble patient antigen for a limited number of antibody sites. Agglutination represents a negative test. In hemagglutination inhibition, presence of antibody to certain viruses inhibits agglutination of RBCs by these viruses. Summary (continued_5) Immunoturbidimetry is an automated method that measures reduction in light intensity as immune complexes form. Nephelometry is an automated technique that measures light scatter by free antigen, free antibody, and antigen-antibody complexes in solution to determine the amount of antigen in a patient sample. 22  6/27/2024 Postamble READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. USE THE UNIT OBJECTIVES AS A STUDY GUIDE All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 23