Immunology Exam 3 Flashcards
Ability of a particular antibody to combine with one antigen instead of another
Specificity
What does antibody specificity depend on?
Homologous antigen
The antigen that induces a specific antibody to be produced is known as
Homologous antigen
Two or more antibodies interacting with structurally similar antigens
Cross-reactivity
“Initial force of attraction that exists between an antibody Fab site and one epitope”
Affinity
What are the 4 types of noncovalent bonds can occur between antigens and antibodies?
- Ionic
- Hydrogen
- Hydrophobic
- van der Waals forces
The sum of all the attractive forces between an antibody and an antigen
Avidity
The force that keeps the molecules together
Avidity
What law states that free reactants are in equilibrium with bound reactants?
Law of Mass Action
The Rate of association will have what end product(s)?
An Antigen-Antibody complex
The Rate of dissociation will have what end product(s)?
A separated, free Antigen and free Antibody
K1 / K2 = ?
Keq
What does Keq measure?
The “perfect” fit - more complexes are formed while less are dissociating to free antigens and antibodies
DEFINITON:
Tendency of a particular antibody to combine with one antigen instead of another
Specificity
DEFINITION:
Antibodies which interact with structurally similar antigens
Cross-reactivity
DEFINITION:
Initial attraction between an Fab site and one epitope
Affinity
DEFINITION:
Sum of all antibody-eiptope pairs
Avidity
What are the three phases in which antibody-antigen interactions occur?
1) Primary
2) Secondary
3) Tertiary
What happens during the primary phase of antibody-antigen interaction?
The initial antibody-antigen binding
What occurs during the secondary phase of antibody-antigen interaction?
Aggregation of complexes to produce precipitation or agglutination (lattice formation)
What occurs during the tertiary phase of antibody-antigen interaction?
Reaction of body to immune complexes, such as inflammation, phagocytosis, etc
What occurs when antibodies and particulate antigens cross-link to form large, visible lattices?
Agglutination
What ways can we perform agglutination testing?
- Tube method
- Microtiter plate
- Slide
Tube method agglutination:
What grade would give one solid clump?
4+
Tube method agglutination:
What grade would result as several large clumps?
3+
Tube method agglutination:
What grade would result in numerous smaller clumps?
2+
Tube method agglutination:
What grade would barely discernable clumps be?
1+
Tube method agglutination:
What would a negative result be described as?
A smooth suspension, no clumping.
TRUE or FALSE
Antigens and antibodies do not have to have multiple binding sites
FALSE:
They must have multiple binding sites in order to interconnect/cross-link
What is zone of equivalence?
The number of antigens and antibodies are roughly equal to each other
What is prozone?
An excess of antibodies
What is postzone?
Too many antigens
What could happen if testing is performed in a prozone or postzone?
False-negative reactions
What is the first step for agglutination testing in the lab?
Sensitization - initial immunoglobulin binding.
Why is IgM used more often in agglutination?
It has 10 binding sites - more than the other immunoglobulins
What is the second step for agglutination testing in the lab?
Lattice formation - rearragement of antigen and antibody to form a stable lattice
This immunoglobulin reacts best between 30-37C
IgG
IgG is considered a _______ antibody
Warm antibody
This immunoglobulin reacts best between 4-27C
IgM
IgM is considered a ______ antibody
Cold antibody
Lattice formation is ideal in what pH?
6.7 - 7.2
What is the zeta potential?
Electrical charges on particles causing like particles to repel each other
TRUE or FALSE:
The following are ways to enhance lattice formation:
- Agitation of particles
- Centrifugation: forcing particles together
- Ionic strength saline
- Albumin
TRUE
What is direct agglutination used to detect?
Either antigens or antibodies
Term used for agglutination when RBCs are involved
Hemagglutination
Major difference between direct agglutination and passive agglutination
In direct agglutination, the target antigen or antibody is found naturally on tested particles.
In passive agglutination, antigens are put on carrier particles to improve the visibility of reactions.
Other names for passive agglutination
Indirect or particle agglutination
What is something we typically use passive latex agglutination to test for?
Rheumatoid factor - an autoantibody (anti-IgM) to the Fc portion of IgG
What is a positive passive latex agglutination test to Rheumatoid factor is associated with?
Rheumatoid arthritis and other autoimmune diseases
What does this image refer to?
Passive agglutination
Carrier particles used in passive hemagglutination
RBCs
Agglutination process in which antibodies are put on carrier particles in order to detect an antigen
Reverse passive agglutination
One of the main reasons we use reverse passive agglutination in the lab
To detect bacterial and fungal antigens in body fluids
-such as C. neoformans in CSF
What can cause a false positive reaction with reverse passive agglutination? Why?
Rheumatoid factor because the auto-antibody IgM will bind with the IgG antibodies on the carrier particles.
What does this picture depict?
Reverse Passive Agglutination
Difference between reverse passive agglutination and reverse passive hemagglutination
Reverse passive HEMagglutination uses RBCs as the carrier particle that the known antibody is attached to
Instance in which reverse passive hemagglutination is utilized in the lab
HBsAg detection
In this process, the carrier particle is a bacterial cell - typically Staph aureus - and the antibody is known.
Coagglutination
Why is Staphylococcus aureus the most frequently used bacteria for coagglutination?
Staphylococcus aureus has a high concentration of protein A in the cell wall allowing natural absorption to the Fc portion of the IgG antibody while keeping the Fab portion available for interaction with antigens
What is this image depicting?
Coagglutination
A lack of agglutination is an indicator of a positive reaction for this test
Agglutination inhibition
This picture represents what type of testing?
Agglutination inhibition
Does this image depict a positive or negative result for agglutination inhibition?
Negative result - agglutination inhibition is positive when no agglutination happens because patient antigen binds to the antibody being tested for causing the known antigen-coated particles in the test to remain unbound.
Does this image depict a positive or negative result?
Positive result
Type of agglutination testing:
- Known antibody
- Carrier particle with test analyte
- Unknown antigen
Agglutination inhibition
Type of agglutination testing:
- Antibody on laten
- Antigen unknown
Reverse passive agglutination
Type of agglutination testing:
- Antibody on bacteria
- Antigen unknown
Coagglutination
Type of agglutination testing:
-Identification of antibody of antigen
Direct agglutination
Type of agglutination testing:
- Antigen on latex particle
- Antibody unknown
Passive agglutination
The presence of cross-reactivity may cause what type of result?
False-positive
The presence of Rheumatoid factor may cause what type of result?
Fals-positive
Why would a delay in reading a slide test cause a false-positive?
Dried out antigen may look like agglutination
Prozone phenomenon may cause what type of result?
False-negative
Why would prozone phenomenon cause a false-negative result?
Too much patient antibody for the test
The combination of soluble antigen with soluble antibody to produce insoluble complexes which are visible refers to what type of testing?
Precipitation
Measurement of precipitation by light scattering can be done using 2 different methods. What are they?
Turbidimetry
and
Nephelometry
Name the method:
- Light is passed through a solution containing antibody and antigen
- A detection device is placed in direct line with the incident light
- The detection device measures the decrease in light intensity compared to a blank
Turbidimetry
Name the method:
- Measures light scattered at a particular angle from the incident beam
- The amount of light scattered is an index of the concentration of the solution
Nephelometry
What method is depicted in the picture?
Turbidimetry
What method is depicted in the picture?
Nephelometry
Which is more sensitive: nephelometry or turbidimetry?
Nephelometry
Precipitation reactions that occur in a support medium such as agar or gel
Passive immunodiffusion
What method is the picture depicting?
Ouchterlony Double Diffusion
With Ouchterlony double diffusion, what result means the 2 antigens are the same resulting in the pattern below.
Identity
AKA
Arc of identity
With Ouchterlony double diffusion, what result means the 2 antigens are completely different, creating crossed lines, like the pattern depicted in the picture?
Nonidentity
With Ouchterlony double diffusion, what is the result of spur formation like the one depicted in the picture?
Partial identity
Ouchterlony Double Diffusion:
The arc of identity signifies what type of result?
A positive result for the antigen being tested
Ouchterlony Double Diffusion:
Crossed lines means what type of result?
Negative- the unknown antigen is not the one the same as the known
Ouchterlony Double Diffusion:
What is the result of a spur formation?
The unknown antigen is not the same as the known but is similar (possibly some “like” epitopes) causing a partial identity.
Method of immunoelectrophoresis that is used to quantitate immunoglobulins present in body fluids
Rocket Immunoelectrophoresis
What is known and what is not known in rocket immunoelectrophoresis?
The antibody is known while the antigen is unknown
Rocket immunoelectrophoresis utilizes how many controls?
3 - low, medium, high antigen content
In rocket immunoelectrophoresis, the height of the rocket is ______ proportional to the amount of antigen in the sample
Directly proportional - the higher the rocket spike, the more antigen in that sample.
This method of electrophoresis uses agarose or cellulose acetate to detect antigen or antibody
Immunofixation electrophoresis
What disease uses immunofixation electrophoresis for diagnosis?
Multiple myeloma
When using Western Blot to test for HIV, what bands are we looking for in order to report as positive?
- p 24
- gp 41
- gp 120 / gp 160
If at least 2 of these are seen, then the result is positive
What is the ionic cloud that prevents RBCs from getting very close to each other?
Zeta potential
What type of bond uses attraction of oppositely charged particles (postive to negative)?
Ionic bond
What antigen-antibody phase is the aggregation of complexes to produce precipitation or agglutination?
Secondary
Antibody-antigen binding is dynamic, reversible, and governed by what?
Law of mass action
What type of bond is the interaction between electron clouds?
Van der Waals forces
What type of bond is the interaction between nonpolar molecules except for water molecules?
Hydrophobic bond
In which antigen-antibody phase does the initial antigen-antibody binding occur?
Primary phase
Which antigen-antibody phase includes the aggregation of complexes to produce precipitation or agglutination?
Secondary phase
Which antigen-antibody phase is the body’s reaction to immune compexes?
Tertiary phase
What type of bond uses hydrogen to bridge between atoms and molecules?
Hydrogen bond
What is the ionic cloud that surrounds individual RBCs and keeps them from getting really close to each other?
Zeta potential
Term for when antigen and antibody combine by means of diffusion?
Passive immunodiffusion
What is the term for the ability of an antibody to combine with one antigen instead of another?
Specificity
TERM:
When antibodies interact with structurally similar antigens
Cross reactivity
TERM:
The initial force of attraction between an antibody Fab site and one epitope
Affinity
Term:
The sum of all attractive forces between antibody-antigen
Avidity
TERM:
An IgM or large molecular weight molecule
Macroglobulin
TERM:
Excess antibody
Prozone
TERM:
Excess antigen
Postzone
TERM:
Where tests must be performed in order to avoid false negatives
Zone of equivalence
What occurs during the first phase of agglutination?
Sensitization: initial binding of antibody with antigen; binding is reversible
What occurs during the second phase of agglutination?
Lattice formation: rearrangement of antigen and antibody to form a stable lattice
TERM:
When antibodies and particulate antigens cross-link to form visible lattices
Agglutination
TRUE or FALSE:
Agglutination tests include direct, passive, reverse passive, coagglutination, and agglutination inhibition
TRUE
Name the agglutination tests that include RBC carriers
(3)
- Direct
- Passive
- Reverse passive hemagglutination
What testing method detects antibody or antigen; AGN + ABY = clumping
Direct agglutination
What is a hemagglutination test?
Antigens or antibodies attached to red cells
What testing procedure includes the following:
- Antibody is unknown
- Antigen on latex beads
- ABY + AGN = clumping
Passive latex agglutination
Soluble antigen is bound to carrier particles such as latex or charcoal. The carrier particles agglutinate in the presence of corresponding antibody.
Passive latex agglutination
Autoantibody (anti-IgM) to the Fc portion of IgG
Rheumatoid factor
What occurs when antibodies and particulate antigens cross-link to for visible lattices?
Agglutination
What testing procedure includes the following:
- Antibody is unknown
- Antigen on latex beads
- antigen + antibody = clumping
Passive latex agglutination
TRUE or FALSE:
A positive rheumatoid factor test always indicated the patient has rheumatoid arthritis
FALSE: RF can be present in patients with diseases other than rheumatoid arthritis
Which testing method includes the following:
- Antigen in unknown
- Antibodies are on latex beads
- Antibody + antigen = clumping
Reverse passive latex agglutination
What is a problem caused by rheumatoid factor when performing reverse passive agglutination?
False positives because RF binds to the known IgG on latex beads
Which testing method includes the following:
- Antibody is unknown
- Antigens are on RBCs
- AGN + ABY = clumping
Passive hemagglutination
Which testing method includes the following:
- Antigen is unknown
- Antibodies are on RBCs
- AGN + ABY = clumping
Reverse passive hemagglutination
Which testing method includes the following:
- Antigen unknown
- Antibodies Fc portion are attached to protein A on Staph aureus
- AGN + ABY = clumping
Coagglutination
Which testing method includes the following:
- Uknown patient antigen and known antibody are incubated together
- Known antigen on latex particles are added to sample
- Positive test = NO clumping
- Negative test = clumping
Agglutination inhibition
For this test method, lack of agglutination is an indicator of a positive reaction.
Agglutination inhibition
What do the following represent:
-Prozone, Rheumatoid Factor, & delay in reading a slide test
Sources of error associated with agglutination procedures
What is the agglutination endpoint (titer) when performing 2-fold dilutions of serum?
The highest dilution that shows agglutination
What does a 4-fold rise in a patient’s titer mean?
Clinically significant. Patient’s condition is getting worse.
What is the equation for the law of mass action?
Keq
=
K1
________
K2
