[M] Lec 02.3: Intro to Sero (Immunologic Reactions) Flashcards
Refers to
● Their basis is the primary Ag-Ab reaction.
● It measures the antigen binding capacity of antibodies.
● It measures low molecular weight antigen such as bovine serum albumin and hapten.
Primary Ag-Ab Reaction
Classification
- Farr Technique/Ammonium Sulfate Precipitation
- Equilibrium Dialysis
- Labeled Immunoassay: Radio Immunoassay (RIA), Enzyme Immunoassay (EIA), and Immunofluorescence
Assay (A.K.A. Fluorescence Immunoassay).
Primary
- Specific
- General
A. Enzyme Immunoassay (EIA)
B. Enzyme-Linked
Immunosorbent Assay (ELISA)
- B
- A
T or F:
○ In the laboratory, we now use Labeled Immunoassays.
○ We rarely use Agglutination and Precipitation tests because Labeled Immunoassays are more sensitive.
True
Arrange labeled immuno assays according to sensitivity (most to least)
- RIA
- EIA
- IFA
- Use a precipitating reagent, particularly Ammonium Sulfate to visualize the reaction.
- Use a dialysis device composed of two chambers—chamber A and chamber B— with a semipermeable membrane between them
A. Equilibrium dialysis
B. Farr Technique .
- B
- A
- Radio labels; beta or gamma counters
- Enzyme labels; ELISA reader
- Fluorescent labels; fluorescent microscopes
A. EIA
B. RIA
C. IFA
- B
- A
- C
Refers:
- Farr Technique and Equilibrium Dialysis are commonly
performed to measure below molecular weight antigen— like HAPTEN AND BOVINE SERUM ALBUMIN. - Bovine Serum Albumin + I131 → (I131BSA) + Anti-BSA → no visible reaction
○ Bovine Serum Albumin will be coupled to a Radioisotope (I131), forming a complex.
○ The complex, when added with the antibody will produce no visible reaction.
Farr Technique
Important for precipation in Farr Technique
Ammonium sulfate
Determines the antigen binding capacity of antibody in Farr technique
Amount of I131
Refers:
- Reference method for FT4 (Free Thyroxine)
- A dialysis device is used, which is divided into two chambers:
Chamber A and Chamber B.
○ Cellulose is often used as a semi-permeable membrane. - During the initial state of testing, antigens are placed in Chamber B, while antibodies are placed in Chamber A
Equilibrium dialysis
Refers to reactions that are visible
Secondary
Classify the ff reactions:
- Precipitation
- Agglutination
- Complement Fixation
- Neutralization (In Vitro)
Secondary
Arrane from sensitive to least sensitive serologic tests
- Neutralization (In Vitro) - most sensitive
- Complement Fixation
- Agglutination
- Precipitation - least sensitive
Define the ff secondary serologic texts
- The combination of SOLUBLE AG WITH AB to produce an INSOLUBLE COMPLEX.
- The antigen involved is insoluble; has a stronger reaction compared to precipitation
- Fixation of complement by antigen antibody (immune) complex.
- When test serum is mixed with standard amount f toxin/ virus, there is decrease of toxicity of toxin or infectivity of virus.
- Precipitation
- Agglutination
- Complement fixation
- Neutralization
Two types of precipitation medium
Liquid and semi-liquid gel
- Slide Precipitation, Tube Precipitation, Capillary Precipitation makes use of capillary tube, Interfacial ‘ring’ Test uses test tubes
- Immunodiffusion, electroimmunodiffusion
- Liquid
- Semi-solid gel
Refers to
- Non-treponemal/non-specific test for syphilis
○ It is non-specific because RPR only detects reagin. - Principle: FLOCCULATION
- Detection of antibody called Reagin
- Uses Choline-chloride instead of heat inactivation. Choline-chloride is used to inactivate complement.
Slide precipitation (RPR)
Study the procedures nalang
Pls
Match (Slide precipitation)
- Medium and large clumps
- No clumping or very slight roughness
- Small clumps
A. Weakly reactive
B. Non-reactive
C. Reactive
- C
- B
- A
Tests we can do in RPR
- NOT exact amount of value since titer only relies on highest dilution with positive reaction
- We only detect the presence or absence of the antigen or the antibody
- Determines the exact amount by using machine/equipment
- Semi-quantitative
- Qualitative
- Quantitative
Qualitative RPR
- SCREENING TEST and DETECTING ANTIBODY
- CONFIRMATORY TEST and DETECTING ANTIGEN
- Reactive/Non
- Positive/Negative
In semi-quantitative RPR, dilute specimens with what buffer?
Glycine NaCl
Give me the CRP of the ff
- 1+1 (1:2)
- 1+3 (1:4)
- 1+7 (1:8)
- 1+15 (1:16)
- 1+31 (1:32)
- 12
- 24
- 48
- 96
- 192
- Best example is RPR
- We can use centrifuge to speed up the reaction as compare to slide precipitation where we ise mechanical rotator
- Can be performed using capillary tube or test tube
A. Slide precipitation
B. Ring precipitation
C. Tube precipitation
- A
- C
- B
Define terms of precipitation
- Refers to the reactant– the antigen or antibody
- Refers to the direction of the migration
- Direction of the migration is either upward or downward
- Refers to radial movement (from center
inner to outer) - Either the antigen is moving or the
antibody is moving to meet to form
precipitation - Both the antigen and antibody are moving to meet to form precipitation
- Diffusion
- Dimension
- Single dimension
- Double dimension
- Single diffusion
- Double diffusion
Refers to:
- example of precipitation reaction, therefore this is classified under secondary Ag-Ab reaction
- Precipitation reactions that is why these tests are not commonly performed in laboratory and are obsolete and usually needed for board exam purposes only
Immunodiffusion tests
Factors that can affect immunodiffusion tests
○ Size of particles
○ Size of antigen
○ Temperature of the reaction
○ Viscosity of the gel
○ Amount of hydration
Match
- Only targeted antigen is moving, direction of movement is radially
- Only antigen is moving
- Both the antigen and antibody is moving
- Antigens B and C are reactive with the patient’s serum in the center well
A. Single Diffusion, Single Dimension (Oudin test)
B. Single Diffusion, Double Dimension (Radial
Immunodiffusion)
C. Double Diffusion, Single Dimension (Modified Oudin with Oakley & Fulthorpe)
D. Double Diffusion, Double Dimension (Ouchterlony
Technique)
- B
- A
- C
- D
Two methods in single diffusion, double dimension
- Fahray
- Mancini
Refers to
- Precipitation reaction
- Classified under secondary antigen antibody reaction
- Also known as Immunoelectrophoresis
- Electrophoresis’ purpose is to enhance the mobility of the reactants. To increase the movement and mobility of the antigen and antibody towards one another
Electroimmunodiffusion Tests
Electroimmunodiffusion Tests
- Also known as Laurel Immunoelectrophoresis or Rocket electroimmunodiffusion; Based on single electroimmunodiffusion so there is only one reactant moving
- Also known as countercurrent immunoelectrophoresis or cross immunoelectrophoresis; Both antigen and antibodies are moving.
- Counterpart of the radial immunodiffusion; Only one reactant is moving and is double dimension
- Developed by Grabar and Williams; Both antigen and antibodies are moving; Double dimension means the direction or movement is
radially.
- Single Electroimmunodiffusion, Single dimension
- Double Electroimmunodiffusion, Single Dimension
- Single Electroimmunodiffusion, Double dimension
- Double Electroimmunodiffusion, Double dimension
- The antibody/ anti-serum is placed in the trough
- The antibody/ anti-serum is placed directly on thegel’s surface (agar)
A. Immunofixation
B. Immunoelectrophoresis
- B
- A
Types of agglutination
- Direct Agglutination
- Indirect/Passive Agglutination
- Reverse Passive Agglutination
- Agglutination Inhibition
- Hemagglutination
Clinical application of agglutination
- Coombs’ Test
- Bentonite Test
- Latex Fixation Test
- Rose Waaler Test
Types of agglutination
- Agglutination of natural antigen
- Antibody is artificially attached to a particulate carrier
- Uses antigen that is artificially attached to a particulate carrier
- Agglutination of RBC due to antibody (e.g. in vivo sensitization), viruses, bacteria, or other biologic substance.
- Based on Blocking Principle; The homologous antigen inhibits the agglutination of antigen
coated particle
- Direct
- Reverse
- Indirect
- Hemagglutination
- Agglutination Inhibition
Uses of direct agglutination
- Detection of antigen
- Detection of antibody
HCG is an example of which type of agglutination
Agglutination inhibition
Types of hemagglutination
- Positive result (+): Carpet/Mat cells or Hemagglutination
- Pased on blocking principle
- Useful in the diagnosis of Influenza and mumps
- Useful in the diagnosis of influenza, mumps, and German measles
- Involved are natural antigens
- Indirect
- Hemagglutination inhibition
- Virus inhibition
- Virus hemagglutination inibition
- Direct hemagglutination
Two systems involved in complement fixation
○ Test system/Bacteriolytic system
○ Indicator system/Hemolytic system
Main components of complement fixation
- Known Ag reagent (ex. beef heart extract, bacterial Ag)
- Complement from Guinea pig serum (Best Source)
- Hemolysin or Amboreceptor
- Indicator cells (Sensitized Sheep RBCs)
Best source of complenent
Guinea Pig Serum
Complement fixation
- Positive result
- Negative result
A. No hemolysis
B. Hemolysis
1, A
2. B
Types of toxin neutralization
- Tertiary antigen-antibody reaction
- Secondary antigen-antibody reaction
- In vivo
- In vitro
Types of virus neutralization
- Pock Reduction Test (In Ovo)
- Plaque Reduction Test
- Metabolic Inhibition Test
- Tissue Culture Technique
