20. Sero Techniques- Secondary Reactions PDF

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IMMUNOLOGY SEROLOGY SEROLOGIC TECHNIQUES: SECONDARY REACTIONS PHASES OF IMMUNOLOGIC REACTIONS: 1. PRIMARY binding of Antigen and Antibody not easily detected because it is not visible to the eye Ex: Labelled immunoassays such as RIA, EIA, Chemiluminescence, Immunofluorescence 2. SECONDARY in Amino A...

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IMMUNOLOGY & SEROLOGY

SEROLOGIC TECHNIQUES: SECONDARY REACTIONS PHASES OF IMMUNOLOGIC REACTIONS: 1. PRIMARY - binding of Antigen and Antibody - not easily detected because it is not visible to the eye - Ex: Labelled immunoassays such as RIA, EIA, Chemiluminescence, Immunofluorescence 2. SECONDARY - conformation/change in Amino Acid chain - no need for labels because it is visible to the eye - Ex: Agglutination, Precipitation, Floculation, Neutralization, C-fixation test 3. TERTIARY - folding of Polypeptide chain - binding of Hydrogen & Hydrophobic bonds - takes place in vivo - Ex: Phagocytosis, Chemotaxis, Opsonization TERMS USED IN Ag-Ab BINDING:  AFFINITY - initial force of attraction between univalent Antigen & univalent Antibody  AVIDITY - sum forces of attraction between multivalent Antigen & multivalent Antibody - has stronger force & more stable bond  SPECIFICITY - follows the Lock & Key model INTRAMOLECULAR FORCES THAT BINDS TO Ag & Ab: 1. IONIC BONDS - exist between two opposite charge particles 2. HYDROGEN BONDS - exist between two polar molecules 3. HYDROPHOBIC BONDS - exist between two non-polar molecules 4. VAN DE WAELS FORCES - exist between electrons of Antigen & Antibody  LAW OF MASS ACTION - states that “Ag & Ab reaction are reversible”

↑ K1 = ↑ [Ag-Ab] = ↓[Ag] = ↓ [Ab] = ↓ K2 = ↑K K = K1 . = [ Ag-Ab ] K2 [Ag] [Ab]

 AGGLUTINATION / PRECIPITATION CURVE  LATTICE FORMATION

- by Marrack - network / crosslink of immune complexes - can be formed in Equivalent zone but can’t be found in Pro Zone & Post Zone TYPES OF SECONDARY REACTIONS: I. AGGLUTINATION - binding of particulates to Antigen to form large complexes seen as “Agglutinates” - Steps: 1. Sensitization = Antigen & Antibody binding 2. Lattice formation - Factors affecting Lattice Formation:  pH (6.7-7.2)  Temperature  Ionic strength of Medium - Ways to enhance Lattice Formation: 1. Add Low Ionic Strength Saline (LISS) or 22% Bovine Serum Albumin (BSA) 2. Add Enzymes such as Bromelin, Papain, Ficin 3. ↑ Viscosity (PEG, Polyvinyl, Pyrolidone) by Centrifugation or Agitation

Ag + Ab ↔ Ag-Ab complex

- there should be equilibrium so we can compute for K JOYCE ANN S. MAGSAKAY | BS MEDICAL LABORATORY SCIENCE

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OLFU VALENZUELA

IMMUNOLOGY & SEROLOGY

SEROLOGIC TECHNIQUES: SECONDARY REACTIONS Kinds of Agglutination: 1. DIRECT AGGLUTINATION - clumping of Antigen that is normally present in the particle - Ex: ABO, Widal, Weil-felix - (+) Result: with Agglutination 2. INDIRECT / PASSIVE AGGLUTINATION - clumping of Antigen that is attached to a Carrier - Ex: Latex, Charcoal, RBC - Antigen is not normally present in the particle - Unknown: Antibody - Ex: Slide-Agglutination, ASO, RF, SLE - (+) Result: with Agglutination 3. REVERSE INDIRECT / PASSIVE AGGLUTINATION - clumping of Antibody that is attached to a Carrier - Ab is not normally present in the particle - Ex: C-Reactive Protein (CR-P) Test - (+) Result: with Agglutination 4. AGGLUTINATION-INHIBITION - 1st step: Patient’s soluble Ag + Ab - 2nd step: Add Particulate Ag - (+) Result: without Agglutination 5. CO-AGGLUTINATION - the carrier used is a Bacteria - Ex: Protein-A of S.aureus - (+) Result: with Agglutination 6. ANTIGLOBULIN-MEDIATED AGGLUTINATION / COOMB’S TEST a. DIRECT ANTIGLOBULIN TEST (DAT) - in vivo sensitization of RBC b. INDIRECT ANTIGLOBULIN TEST (IAT) - in vitro sensitization of RBC II. PRECIPITATION - consist of soluble Antigen & soluble Antibody to form insoluble complex  Precipitation in Liquid: 1. TURBIDIMETRY - amount of Cloudiness in a solution - results to ↓ light intensity due to:  Light scattering  Light reflection  Light absorption

2. NEPHELOMETRY JOYCE ANN S. MAGSAKAY

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- amount of the Light scattered when it passes to a solution - usually @ 10-70° - Ex: IgG, IgM, IgD, IgE, C4, C2, C1 inhibitor, RF Kinds of Nephelometry: a. KINETIC - measures light scattering before the equivalent zone b. ENDPOINT - wait for the equivalent zone first before measuring the light scattering  Precipitation in Gel: - we used Agar or Agarose because it’s matrix is Neutral (with 0.3-1.58%) Factors affecting Precipitation in Gel:  Size of particles  Temperature  Gel viscosity  Amount of Hydration  Interaction between matrix & reactants  STANDARD CURVE - used to measure the exact amount of Antigen a. ENDPOINT / MANCINI METHOD - after 48hrs, read the diameter of the ring using Vernier caliper, then square it - plot the curve in a Graphing paper b. KINETIC / FAHEY & MCKELVEY METHOD - after 18hrs, measure the diameter of the ring using Vernier caliper, but you don’t have to square it - plot the curve in a Semilog paper Kinds of Precipitation in Gel: 1. PASSIVE IMMUNODIFFUSION - you just let Antigen & Antibody across the gel on their unknown - Sources of Errors:  Underfilling or Overfilling of the holes/wells  Incorrect incubation of time & temperature  Spilage of sample over the gel  Specific contamination a. SINGLE DIFFUSION - by: Oudin - only one diffuses the gel (Antigen) - establishes relationship with Antigen & Antibody

b. RADIAL IMMUNIDIFFUSION (RID) - modified single diffusion test BS MEDICAL LABORATORY SCIENCE | OLFU VALENZUELA

IMMUNOLOGY & SEROLOGY

SEROLOGIC TECHNIQUES: SECONDARY REACTIONS - Reagent: Antibody (incorporated to the gel) - Unknown: Antigen - Process: 1. Add the Px’s sample in the holes called “Wells” 2. Incubate 3. Once the Antigen & Antibody meet @ the Equivalent zone, they will form a Precipitin ring c. DOUBLE DIFFUSION IN 1 DIMENSION - by: Oakley & Fulthorpe - determines the relationship of single Antigen & single Antibody - both Antigen & Antibody diffuses d. DOUBLE DIFFUSION IN 2 DIMENSIONS / OUCHTERLONY TECHNIQUE - by: Ouchterlony - determines the relationship of two or more Antigen - Process: 1. Add the Px’s sample w/ Antigen on the small wells (the Ab is placed on the central well) 2. Incubate for 12-48hrs 3. When Antigen & Antibody meet at the Equivalent zone, it will form Precipitin line  Line of Identity: Smooth arch  Line of Partial Identity: Spur  Line of Non-Identity: Cross line 2. ROCKET IMMUNOELECTROPHORESIS / LAUREL IMMUNOELECTROPHORESIS - by: Laurel - known as “One Dimension Electroimmunodiffusion” - Radial immunodiffusion w/ applied electric current - Application: used for measurement of Antibody & Complement in other fluids - Reagent: Antibody - Unknown: Antigen - Process: 1. Add the Px’s sample 2. Run a Standard curve in different concentration 3. Buffer ph: 8.6 4. When Antigen & Antibody meet at the Equivalent Zone, they form a Rocket-shaped line

- Application: Used to detect Immunodeficiency & Immunoproliferative diseases - Reagent: Antibody - Unknown: Antigen - Process: 1. Add the Px’s sample to the holes called “Through” 2. Incubate for 18-24hrs 3. When the Antigen & Antibody meet at the Equivalent zone, it will form Precipitin Arch 4. Compare the Standard & Px’s sample in terms of Location, Intensity, & Shape 4. COUNTERCURRENT IMMUNOELECTROPHORESIS - Double diffusion but w/ applied voltage - pH: 8.6 - Application: Used to detect Autoantibodies to Infectious agents 5. IMMUNOFIXATION ELECTROPHORESIS - by: Alfer & Johnson - allows the reagent Antigen to be separated in the gel through Electrophoresis - modification of this process is “Westernblot” - Process: 1. Add Px’s sample directly on the surface of the gel 2. When the Antigen & Antibody meet at the Equivalent zone, Immunoprecipitates / Precipitin bond will be formed - Sources of Errors:  Application of current in the wrong direction  Incorrect pH of the buffer  Excess Antigen or Antibody  Amount of current is weak or too strong

3. IMMUNOELECTROPHORESIS - by: Grabar & Williams - Double diffusion w/ applied electric current JOYCE ANN S. MAGSAKAY | BS MEDICAL LABORATORY SCIENCE

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