Immunology And Serology: Unlabeled Immunoassays PDF

Central Philippine University | College of Medical Laboratory Science MEDICAL LABORATORY SCIENCE INTERNSHIP KEY CONCEPTS MODULE IMMUNOLOGY AND SEROLOGY I. INTRODUCTION TO S...

Central Philippine University | College of Medical Laboratory Science MEDICAL LABORATORY SCIENCE INTERNSHIP KEY CONCEPTS MODULE IMMUNOLOGY AND SEROLOGY I. INTRODUCTION TO SEROLOGY A. Serology 1 UNLABELED IMMUNOASSAYS 1. Latin sero, serum 2. The study of constituents of serum, the straw-colored fluid component of whole blood. 3. The branch of science dealing with the measurement and MODULE OUTCOMES characterization of antibodies, antigens, and other immunological At the end of this unit, the learner should have been able to: substances in body fluids or serum. 1. Explain accurately the principle of the various unlabeled immunoassays. 4. Serology employ in vitro antigen-antibody reactions to identify such 2. Interpret and correlate correctly serology results. antigens or antibodies 3. Identify properly the clinical laboratory application of the different B. Antigens and Antibodies unlabeled immunoassays. 1. Antigens are foreign substances (immunogens) that can stimulate the production of antibodies 4. Discuss comprehensively factors affecting antigen-antibody reactions. 2. Antibodies are glycoproteins (immunoglobulins) produced in 5. Distinguish correctly precipitation and agglutination. response to an antigenic challenge. Antibodies can be found in blood plasma and body fluids C. Antigen-Antibody Reactions INTRODUCTION 1. Primary union of antigen and antibody depends on affinity and Immunoassays are widely employed for the detection of a wide range of avidity substances – bacteria, viruses, toxins, allergens, and others. They are a. Sensitization is the basic reaction of an antigen and antibody generally classified into two groups, unlabeled and labeled immunoassays. binding. While some kinds of labels like enzymes and radioactive substances are b. Lock and key theory: Ag and Ab must be complementary to one used in labeled immunoassays, unlabeled immunoassays do not rely on another c. Non-covalent interactions occur between an antigen and an labels. Most unlabeled immunoassays are based on the principles of antibody (each bond is weak; many are strong) precipitation and agglutination. Though unlabeled immunoassays are quick d. Forces that participate in Ag-Ab reactions and easy to perform, less time-consuming, and usually do not require i. Ionic bonding or Electrostatic force – occur between special equipment they are less specific, less sensitive, and have more oppositely charged particles interferences. This is why they are often used as screening tests. ii. Hydrogen bonding - binding of H to an electronegative atom such as oxygen or nitrogen iii. Hydrophobic interactions – attraction between polar groups iv. Van der Waals force – a weak attractive force between an electron orbital of one atom and the nucleus of another atom MODULE 1 | Immunology and Serology 1  e. Affinity D. Sensitivity VS Specificity i. Strength of the interaction between a single antibody site 1. Analytical sensitivity is the ability of a test to detect very small and a single epitope amounts of a substance ii. Initial force of attraction that exists between a single Fab site 2. Clinical sensitivity is the ability of a test to give positive result if on an antibody molecule and a single epitope or determinant patient has the disease (“no false negative results”) site on the corresponding antigen 3. Analytical specificity is the ability of a test to detect substance iii. Strength of attraction depends on the specificity of antibody without interference from cross-reacting substances to a particular antigen 4. Clinical specificity is the ability of test to give negative result if iv. Cross-reactivity can also occur – antibodies become patient does not have the disease (“no false positive results”) capable of reacting with antigens that are structurally similar E. Immunoassays to the original antigen. The original stimulating antigen is a 1. General term that refers to many techniques that involve antigen- better fit than cross-reacting antigen. antibody reactions performed in the clinical laboratory f. Avidity 2. Can be qualitative or quantitative i. A measure of the overall stability of an antigen–antibody 3. Can be labeled or non-labeled complex a. Non-labeled: precipitation, agglutination ii. Measure of overall bonding between multivalent antibodies b. Labeled: assay is named according to attached label and multivalent antigens i. RIA – radioactive substance iii. Avidity is influenced by both the valence of the antibody and ii. ELISA – enzymes the valence of the antigen iii. FIA – fluorescent compounds iv. Avidity is the sum of the forces binding multivalent antigens F. Phases of Antigen/Antibody Reactions to multivalent antibodies 1. Primary Reaction 2. Law of Mass Action governs the reversibility of the antigen-antibody a. Non-covalent interactions occurring between an antibody and a reaction. univalent antigen a. Law of Mass Action states that free reactants are in equilibrium b. Tests to detect this reaction are technically difficult, complex, with bound reactants. expensive, may require special equipment and are time b. All Ag-Ab binding is reversible consuming (immunofluorescence, radioimmunoassay, c. KA = Association rate = K1 = Ab-Ag complex immunoenzymatic assays, chemiluminescent immunoassay) Dissociation rate K2 (free Ab) (free Ag) c. Primary tests are more sensitive than secondary/tertiary tests d. The higher the equilibrium constant (KA), the more will be the 2. Secondary Reaction amount of Ab combining with Ag a. Interactions that occur between antigen and antibodies when e. The higher the value of KA, the larger the amount of antigen– the antigens are multivalent antibody complex and the more visible or easily detectable the b. Antigen and antibody form cross links or lattice formation to reaction is. create larger molecules that are easily detectable f. As the strength of binding, or avidity, increases, the tendency of c. Conformation of the amino acid chain resulting from interchain the antigen–antibody complexes to dissociate decreases, and hydrogen bonding the value of K2 decreases. This increases the value of K1. MODULE 1 | Immunology and Serology 2  d. Methods used to detect these reactions are quick and easy to 5. Serial Dilutions perform, less time consuming and usually do not require special a. A serial dilution is any dilution where the concentration equipment (precipitation, agglutination, complement fixation) decreases by the same quantity in each successive step. e. Methods are less specific, less sensitive and have more b. When performing serial dilutions in the clinical laboratory one interferences must know the dilution of each tube. 3. Tertiary Reaction c. Serial dilutions are mutiplicative. If you know the dilution factor a. Antigen/antibody reactions are not visible but is detected by the you multiply each successive tube by it and can easily effect the reaction has on tissue or cells determine the concentration of each tube. b. Involves the folding of polypeptide chains through hydrophobic d. Example: If a 1/8 dilution of the stock solution is made followed and hydrogen bonds by a 1/6 dilution what is the final dilution? c. Occur as biologic reactions (phagocytosis, opsonization, Answer: The final dilution is 1/8 × 1/6 = 1/48 chemotaxis, immune adherence, cellular degradation, 6. Doubling Dilutions inflammation) a. Series of dilutions, each a doubling dilution of the previous one G. Dilution b. This is a series of ½ dilutions. Each successive tube will ½ the 1. Titer is the number of antibody molecules per unit volume of serum, amount of the original concentrated solution. giving an indication of antibody concentration. Titer is read at c. Each tube contains 0.2 mL of diluent. Patient serum (0.2 mL) is highest dilution of serum that gives a reaction with antigen. added to tube one. If this is done 6 times, what you would end 2. A dilution involves two entities: up with? a. Solute, the material being diluted 1st dilution = 1/2 b. Diluent, the medium making up the rest of the solution 2nd dilution = 1/2 × 1/2 = 1/4 3. Expressed as a fraction 3rd dilution = 1/4 × 1/2 = 1/8 a. 1/Dilution 4th dilution = 1/8 × 1/2 = 1/16 b. Amount of Solute/Total Volume 5th dilution = 1/16 × 1/2 = 1/32 4. Simple Dilutions 6th dilution = 1/32 × 1/2 = 1/64 a. An algebraic equation can be set up to find the total volume, the amount of solute, or the amount of diluent needed to make a dilution. II. PRECIPITATION b. Example: 2 mL of a 1:20 dilution is needed to run a specific A. General Information serological test. How much serum and how much diluent are 1. Simplest method for detecting antigen-antibody reactions needed to make this dilution? 2. Soluble antibody (precipitin) combines with soluble antigen + Volume Solute soluble antibody (in proper proportions) resulting to insoluble 1/20 = X/2 mL complexes (precipitate) 20 (X) = 1 (2mL) 3. Precipitation occurs at the interface of antigen and antibody X = 0.1 mL reagents Volume diluent = Total volume – Volume solute 4. First noted in 1897 by Kraus, who found that culture filtrates of = 2 mL – 0.1 mL enteric bacteria would precipitate when they were mixed with = 1.9 mL specific antibody. MODULE 1 | Immunology and Serology 3  5. Lattice Hypothesis 2. Named after Orjan Ouchterlouny, a Swedish scientist a. Formulated by Marrack 3. Used to determine relationships between antigens and antibodies b. Ab has more than 1 valence and therefore may be found with 4. Can be used to determine if a specific antibody/antigen is present multivalent Ag to form a coarse lattice. As they combine, this in serum results in a multimolecular lattice that increases in size. 5. “Double” because both Ag and Ab diffuse c. Reaction is influenced by the quantities of antigen and antibody 6. Principle: present a. Ag and Ab are placed in wells in the gel i. Ag and Ab should be in correct proportions for maximum b. Ag and Ab diffuse towards each other precipitation to occur c. Precipitation bands form at zone of equivalence ii. Zone of equivalence (Ag and Ab are about equal): point at d. Location of bands depend on concentration and rate of diffusion which the most antibody is precipitated by the 7. Basic Patterns in Double Diffusion least amount of a. Identity: smooth curve, Ags are identical antigen b. Non-identity: intersection, Ags not identical (no common iii. Prozone (Ab excess) antigenic determinants) and Postzone (Ag c. Partial Identity: spur formation, Ags not identical but possess excess): insufficient common determinants reactive sites on antigen for lattice formation, lead to false-negative results B. Types of Precipitation Reactions 1. Precipitation reactions in agar gel or semi-solid medium a. Passive Immunodiffusion techniques D. Single/Linear Diffusion i. Double immunodiffusion 1. James Oudin was the first to use gels for precipitation reactions ii. Single Diffusion 2. Principle: iii. Radial Immunodiffusion (RID) a. Antibody was incorporated into agarose in a test tube. b. Electrophoretic Techniques b. Antigen was layered on top i. Countercurrent immunoelectrophoresis (CIE) c. As the antigen moved down into the gel, precipitation occurred ii. Immunoelectrophoresis d. Precipitation moves down the tube in proportion to the amount iii. Immunofixation electrophoresis of antigen present iv. Rocket electrophoresis E. Radial Immunodiffusion (RID) 2. Fluid-phase precipitation 1. A modification of the single-diffusion technique a. Turbidemetry 2. Principle b. Nephelometry a. Antibody is uniformly distributed in the support gel C. Double Diffusion b. Antigen is applied to a well cut into the gel. 1. aka Ouchterlouny technique MODULE 1 | Immunology and Serology 4  c. As the antigen diffuses out from the well, Ag-Ab combination G. Immunoelectrophoresis (IEP) occurs until a ring of precipitate is formed (zone of equivalence) 1. Combination of electrophoresis and double diffusion d. Diameter of the ring is measured 2. Used for the detection of free light chain proteins and as a screening e. Diameter of the ring can be compared to a standard curve procedure to detect Ig classes obtained by using antigens of known concentration. 3. Useful for ID of monoclonal proteins; used to confirm BJP 3. Principal RID Methods 4. Principle a. Fahey-McKelvey RID a. Antigens undergo electrophoresis i. aka Kinetic RID b. Current is stopped ii. Incubate for 18 hours c. Trough is cut in the agar and filled with antibody iii. Diameter of precipitation ring proportionate to log of Ag d. Antibody and antigen diffuse through agar concentration e. A precipitin arc is then formed at the equivalence point of b. Mancini RID antigen and antibody i. aka Endpoint RID f. A normal control serum is performed simultaneously ii. Incubate for 24 hours (IgG) and 50-72 hours (IgM) g. Any changes in size, shape or intensity of arcs from the normal iii. Area (d2) of precipitation ring proportionate to Ag serum indicate abnormality concentration H. Immunofixation Electrophoresis (IFE) F. Countercurrent Immunoelectrophoresis (CIEP) 1. Protein electrophoresis + immunoprecipitation 1. Aka counterimmunoelctrophoresis (CIE) or eletroprecipitation 2. IFE replaced IEP due to IEP’s complexity 2. Can be used to detect abs to infectious agents and microbial 3. IFE More sensitive than electrophoresis antigens 4. Detects the presence of immunoglobulin in serum or urine 3. Been replaced by easier to perform assays 5. Principle 4. Principle: a. CHON (Ag) separated through electrophoresis a. Gel poured into plate b. Cellulose acetate strips with anti-serum is placed over b. 2 wells are cut c. Abs diffuses into the electrophoresis gel and combine with Ags c. Ag is added to one well and Ab is added to another well forming a precipitate d. Plate is placed in an electric field at pH 8.6 (shortens the time d. Gel is washed and stained required to produce precipitation) I. Rocket Electrophoresis e. Antibody and antigen migrate toward each other 1. aka Laurell Technique i. Ag (-) migrate towards Anode(+) 2. Similar to RID but electrophoresis is used to speed formation of ii. Ab (+) migrate towards Cathode (-) precipitate f. Precipitate forms if antigen is present for the corresponding 3. Principle antibody a. Ab incorporated in agar plate 5. Interpretation: b. Add Ag in well cut in gel a. High Ag: precipitation band closer to Ab well c. Plate is electrophoresed b. High Ab: precipitation closer to Ag well d. Ag migrate and combine with gelled Ab in the shape of a rocket c. No Ab against Ag: no precipitation band e. Measure height of rocket f. Distance of migration (height of rocket) = Ag concentration MODULE 1 | Immunology and Serology 5  J. Fluid-Phase Precipitation B. Direct Agglutination 1. Photometric measurement of the quantity of cloudiness or turbidity 1. Uses antigens found naturally on surface of cells/particles in a solution caused by suspended particles 2. Examples: Blood typing (Hemagglutination), Antiglobulin test, 2. Used to quantitate immunoglobulin, complement and immune Widal’s test complexes 3. Antiglobulin Technique 3. Principle: a. aka Coomb’s test a. Serum substance reacts with specific antisera and forms b. Tool for detection of IgG Abs insoluble complexes. c. IgG Abs are non agglutinating Abs b. Light is passed through suspension. d. IgG molecules are small and cannot link one Ag binding sites c. Scattered or reflected light is proportional to number of insoluble on one RBC to another RBC complexes e. Uses anti-human globulin (AHG) 4. Nephelometry f. Direct Antiglobulin Test (DAT): detects in vivo sensitization of a. More sensitive than turbidimetry RBC with IgG and/or C’ b. Direct measurement of light scattered by particles suspended in g. Indirect Antiglobulin Test (IAT): detects “in vitro” sensitization a solution C. Passive Agglutination c. Light scattering proportional to size and amount of immune 1. aka Indirect agglutination complexes formed 2. Carrier particle is coated with antigen 5. Turbidimetry 3. Antigen is attached to the carrier particle, and agglutination occurs a. Measurement of light transmitted through a suspension of if patient antibody is present. particles 4. Carrier particles: latex, bentonite, charcoal, RBC b. The more immune complexes are formed, the greater the 5. Detects presence of Antibody decrease in light that can pass through 6. Ex: ASOT, RPR, test for RF D. Reverse Passive Agglutination 1. Carrier particle is coated with Antibody III. AGGLUTINATION 2. Detects presence of Antigen A. General Information 3. Antibody is attached to the carrier particle, and agglutination occurs 1. Reaction between “particulate Ag and Ab” or “particulate Ab and if patient antigen is present. Ag” producing clumping large enough for direct observations 4. Ex: CRP 2. Process divided into 2 steps: E. Agglutination Inhibition a. Sensitization 1. Principle b. Lattice formation! a. Ab reagent + Unknown Ag (sample) 3. Classification of Agglutination Reactions: b. Add particulate Ag a. Direct agglutination c. Interpretation: b. Passive agglutination i. (+) = No agglutination c. Reverse passive agglutination ii. (-) = Agglutination d. Agglutination inhibition 2. Ex: Urine HCG, ABH in body fluids MODULE 1 | Immunology and Serology 6  F. Flocculation c. Interpretation: If the lethal dose fails to kill the test animal, 1. A specific type of precipitation that occurs over a narrow range of neutralizing Ab to the virus (or toxin) is known to be present in antigen concentrations. the test serum 2. The process by which individual particles aggregate or precipitate 4. In vivo toxin neutralization tests 3. The antigen consists of very fine particles. a. Dick’s test 4. Uses fine particles of antigen to detect antibody in patient’s serum i. Susceptibility test for Scarlet fever (SF) ii. Test area: arm + toxin iii. Control area: arm + toxoid IV. OTHER UNLABELED IMMUNOASSAYS iv. Interpretation: A. Complement Fixation Test Redness with dick’s toxin = susceptible to SF 1. Used to detect antibody in patient sera No redness with dick’s toxin = immunity 2. Applicable to IgM only b. Schultz-Charlton reaction 3. Once the reference method for detecting many antibodies i. Diagnostic test for Scarlet fever 4. Largely replaced by methods that are easier to perform ii. (+) for SF if rashes fade (blanching phenomenon) upon 5. Used to detect antibodies to viruses, rickettsia, and fungi injection of SF antitoxin 6. Principle: c. Schick’s test a. Patients serum + reagent Ag iii. Susceptibility test for C. diphtheriae b. Add complement c. Add Ab sensitized RBC d. Interpretation: FURTHER READING i. Negative result: Lysis Stevens, C. D., & Miller, L. E. (2016). Clinical Immunology and Serology: If Ab is absent, complement will be unfixed. If complement A Laboratory Perspetive. FA Davis. is not fixed by specific Ag-Ab reaction, it will combine with PER handbook: A Review Manual for Clinical Laboratory Examinations indicator system = lysis McPherson, R. A., Msc, M. D., & Pincus, M. R. (2021). Henry's clinical ii. Positive result: Lack of hemolysis diagnosis and management by laboratory methods E-book. Elsevier If Ab is present it will bind to the reagent Ag. Complement is Health Sciences. fixed on this Ag-Ab complex and unable to combine with indicator system = no lysis B. Neutralization Tests 1. Virus and toxin neutralization tests used if precipitation and agglutination tests are unsuccessful 2. In this assay, neutralizing antibodies inhibit biological activity of a target 3. Principle a. Lethal dose of virus (or toxin) + serum. b. Mixture is injected into test animal MODULE 1 | Immunology and Serology 7