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Immunohematology and Serology > Chapter 6 -Agglutination and Precipitation Reactions > Flashcards

Chapter 6 -Agglutination and Precipitation Reactions Flashcards

1
Q

What were the first immunoassays developed?

A

Precipitation and agglutination were the first immunoassays developed

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2
Q

What do immunoassays rely on to work?

A

Rely on the multiple binding sites of both antibody and antigen

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3
Q

Differentiate between a precipitation reaction and a agglutination reaction

A
  • If the antigen is soluble, the reaction is called precipitation. It is a crosslinking of a soluable Ag to create an insoluable precipitate that is visable
  • If the antigen is a particulate, the reaction is called agglutination. Crosslinking of particulate antigens to form larger complexes that are also visable
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4
Q

Define affinity

A

Affinity

- Strength of binding of one Fab with one epitope on an antigen

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5
Q
  • What is Ka?

- What is the calculation for it?

A
  • Ka is affinity constant
  • Ka = [AbAg]/[Ab][Ag]
    • [AbAg] is the concentration of the Ab-Ag complex
    • [Ab] is the free Ab
    • [Ag] is the free Ag
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6
Q

Affinity

  • what happens to the complexes when affinity is higher?
  • what happens to the sensitivity of the reaction when the affinity is higher?
    • why does this happen?
A
  • The higher the affinity, the more of the antibody and antigen that is complexed
  • The higher the affinity, the more sensitive the reaction
    • because less of the antigen and antibody will be in the uncomplexed form (free)
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7
Q

Define avidity

A
  • The number of binding sites times the affinity
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8
Q

Avidity

  • of IgG
  • of IgM
    • in the case of IgM, why is this important?
A
  • IgG with its two binding sites has an avidity of two times the affinity
  • IgM with its ten binding sites has a theoretical avidity of ten times its affinity constant
    • Important because IgM average weaker affinity than IgG, since IgG is usually produced after somatic mutational events
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9
Q

Define Equivalence

A

Equivalence

  • Antigen and antibody meet at a concentration where the number of paratopes (antibody binding sites) approximately equals the number of epitopes
  • precip/agglutination will form
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10
Q

Define prozone

- what test result will you get in this state?

A
  • when there is too much antibody in relation to the number of antigens
  • there is no need for bridges to form, so little to no precip/agglutination will form
  • false negative
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11
Q

Define Post-zone

- what test result will you get in this state?

A
  • when there is more antigen than antibody. all of the binding sites are full, so cross-linking can’t occur.
  • no precip/agglutination will form
  • false negative will occur
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12
Q
  • How do we combat the false results for prozone?

- How do we combat the false results for post-zone?

A
  • In prozone, the patient’s serum can be diluted and tested again to reach equivalence
  • In postzone reaction, the patient’s blood can be drawn again later to allow an increase in titer to bring the reaction to the zone of equivalence
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13
Q

Immunodiffusion Techniques

- how does radial immunodiffusion work?

A
  • antibody incorporated in agar
  • a well of the antigen is made in the agar
  • the antigen will diffuse into the agar
  • a ring of precipitate will occur where the Ab and Ag reach equivalence
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14
Q

Immunodiffusion Techniques

- how does double immunodiffusion work?

A
  • a well of Ab is added to an agar matrix
  • a well of Ag is added to an agar matrix
  • a straight line of precipitate will occur where the Ab and Ag reach equivalence
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15
Q

Precipitation

  • how sensitive are these tests?
  • why?
A
  • Least sensitive serological technique
    • Requires visualization of substances from solution
    • Not amplified by the antigen being on a particle
    • Not amplified by linking to a more visible reaction. Measuring limit of usually ~20 µgs/ml
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16
Q

Precipitin Reactions

  • what type of antigens are these reactions typically used for?
  • what type of information is gleaned from these reactions?
A
  • Important assays for fungal antigens and for some research purposes
  • Give information on
    • Relatedness of antigens
    • Minimum number of antigen and antibody pairs that is difficult to garner in any other way
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17
Q

Precipitin Reactions

- what are some examples of immunodiffusion gel precip methods?

A
  • Double-diffusion gel precipitation

- Single-diffusion precipitation (radial immunodiffusion)

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18
Q

Ouchterlony

  • define
  • describe the process
A
  • the process by which both antibody and antigen diffuse through agar or agarose
  • A precipitin line forms where diffusion brings the relative concentrations of antibody and antigen to equivalence
  • therefore double diffusion
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19
Q

Ouchterlony

  • what does it allow for the determination of?
    • what are the three different types?
A
  • Allows for determination of antigenic relatedness of an unknown test material with known antigen
    • Identity
    • Partial identity
    • Nonidentity
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20
Q

Ouchterlony

  • qual or quant procedure?
  • what type of infection does this type of testing help to diagnose?
A
  • Qualitative procedure

- Utilized clinically in the diagnosis of some fungal infections including coccidiomycosis

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21
Q

Ouchterlony

  • what type of disorder did this used to diagnose?
  • what type of testing has replaced this?
A
  • Was also used in the laboratory for analysis of serum from patients with autoimmune disorders
  • Has largely been replaced by enzyme immunoassays
22
Q

Ouchterlony

  • how many wells are used in this method?
  • what happens when they meet?
A
  • Two different antigen preparations are placed in different wells and antiserum is placed in a third well
  • Antigens and antibodies diffuse in a circle out of the well
  • When antibody and antigen meet at equivalence they form a precipitin line
23
Q

Line of Identity

  • define
  • why does the arc occur?
A
  • When the two different antigen preparations contain the same antigen, a line of identity will form
  • The arc shape of this line is the result of the circular shapes of the diffusion from each well and the reagents reaching each other at equilibrium
24
Q

What is a line of nonidentity?

A
  • Two wells are filled with different antigens and when they diffuse, they form lines that cross which is called a line of nonidentity
25
Q
  • What is a line of partial identity?

- What feature does the line have that the others lack?

A
  • Two molecules share one epitope and antibody to this epitope will give a line of identity
  • However, one of the molecules has an additional epitope so a spur is formed on the precipitin line
26
Q

Ouchterlony and Relative Concentrations

- higher concentration of antigen will form a line closer or further from the antibody well?

A
  • The antigen with the higher concentration would form a precipitin line closer to the antibody well
27
Q

Radial Diffusion

  • qual or quant technique?
  • what kind of diffusion is it (single/double)?
  • what advantages does this technique have when it comes to the antibody?
A
  • Quantitative
  • Single diffusion of the antigen occurs in an agarose gel containing antibody
  • Antibody concentration is thus held at a constant
28
Q

Radial Diffusion

  • what does the size of the precip circle depend on?
  • how is a standard curve prepared?
A
  • Diameter of the circle is related to the concentration of the antigen
  • A standard curve is prepared that uses three concentrations of the antigen
29
Q

Radial Diffusion: Fahey Method

  • how long does this take?
  • how do you calculate the diameter?
  • how is the calculation plotted?
A
  • Diffusion proceeds for 18 hours
    • Diameter is proportional to the log of the concentration
    • Plotted using semilog paper with diameter on the arithmetic axis and concentration on the y-axis
30
Q

Radial Diffusion: Mancini or end-point method

  • describe what happens
  • how long does this take?
  • how do you calculate the diameter?
A
  • Reactants are allowed to come to equilibrium
    • 24–72 hrs
  • The square of the diameter is directly proportional to the concentration
31
Q

Radial Diffusion

  • what antibodies does it measure?
  • what antibodies can’t it measure
    • why?
A
  • To measure IgG, IgM, and IgA levels and complement

- Serum concentrations of IgE and IgD are not high enough to be read by this method

32
Q

Light Scatter—Enhancement of Precipitation: Turbidometry

  • how does it collect its measurements?
  • what does it measure?
A
  • Initial cloudiness is measured by passing a light through the solution
  • Amount of scatter is proportional to the concentration of the molecules in the lattice structures
  • Measures the light that gets directly across the solution
  • Measures the amount of light that is lost
33
Q

Light Scatter—Enhancement of Precipitation: Nephalometry
- how does it relate to turbidometry
how does the measurement process work?

A
  • Similar to but more sensitive than turbidometry
  • The light is not measured directly across from the light source; it is measured at a 10–90˚ angle from the light (70˚ typically used) source
34
Q

Light Scatter—Enhancement of Precipitation: Nephalometry

- what all can this measure?

A
  • Immunoglobulin concentrations, IgG, IgM, IgA, and IgE, as well as kappa and lambda light chains are measured this way
35
Q

Turbidometry and Nephalometry

- what has been implemented to increase their sensitivity?

A
  • Both techniques have been made more sensitive with the use of laser light as the light source
36
Q

Agglutination

  • what was the first disease diagnosed with agglutination?
  • why is it not a sensitive test?
  • why is it more sensitive than precip?
A
  • typhoid fever
  • Agglutination is not very sensitive because nothing amplifies the actual antibody–antigen reaction
  • It is more sensitive than precipitation because the larger antigen particles enhance visualization
37
Q

Passive Agglutination

- why does this occur?

A
  • Increased sensitivity of agglutination over precipitation is the reason for the development of passive agglutination
38
Q

Passive Agglutination

  • describe the process
  • give an example
A
  • The coating of red blood cells or inert latex beads with soluble antigens
    Example:
  • Latex particles coated with antibody molecules with their Fcs facing out to detect rheumatoid factor
  • Rheumatoid factor is an autoimmune disease antibody that binds to the Fc of IgG
39
Q

Agglutination with Charged Particles

- why is IgM better for agglutination of these particles than IgG?

A
  • reach between the antigen binding sites of IgM

- Numerous binding sites

40
Q

Agglutination with Charged Particles

  • what role does pH play?
  • how does charge affect the titer?
A
  • pH of the reaction is important because pH affects charge

- If charge increased, the apparent titer would decrease

41
Q

Agglutination with Charged Particles

- what are 5 methods used to improve binding of charged particles?

A
  • Use of low ionic strength media (LISS)
  • Increased viscosity media (reduces water of hydration)
  • Altering the temperature - IgM best between 4 and 27˚C; IgG best at 37˚C
  • Treatment of the red cells with enzymes to decrease surface charge
  • Agitation or centrifugation to increase interaction
42
Q

Agglutination

  • when would you add AHG to a reaction?
  • what does it do to the particles?
A
  • Sometimes IgG antibodies do not cause agglutination due to repulsion, so antihuman immunoglobulin is added
  • Links the Fc regions of the IgG molecules that are bound to the particles together
43
Q

What are the 5 types of agglutination?

A
  • Direct agglutination
  • Passive agglutination
  • Reverse passive agglutination
  • Hemagglutination
  • Agglutination inhibition assays
44
Q

Types of Agglutination
Define:
- Direct agglutination
- Passive agglutination

A

Direct agglutination
- Antigen is naturally part of the particle
Passive agglutination
- Antigen not normally part of the particle
- Also called indirect

45
Q 
Types of Agglutination
Define:
- Reverse passive agglutination
- Hemagglutination
-- what are the different types?
A

Reverse passive agglutination

  • Antibody is attached to the particle, not antigen
    • These particles detect antigen rather than antibody
  • Agglutination assays using red blood cells
    • DAT: direct antiglobulin test
    • IAT: indirect antiglobulin test
46
Q

Hemagglutination—DAT

  • what does this detect the presence of?
  • in what conditions might this occur?
A 
- Demonstrates the presence of AB or C on an individual's RBC
Conditions:
- Autoimmune hemolytic anemia
- Hemolytic disease of the newborn
- Drug RBC sensitization
- Transfusion reaction
47
Q

Hemagglutination—DAT

  • why is it considered “direct”?
  • what components is DAT made up of?
    • what reaction occurs?
A
  • Called direct because cells tested as they come out of body (in vivo)
  • Red blood cells from patient + antibody to human IgG
    • The antiglobulin bridges the gap between the RBCs and agglutination occurs
48
Q

Indirect Antiglobulin Test

- what does this test for?

A
  • Looks for the presence of the antibody in a patient, or for blood group Antigens on a patients cells
49
Q

Indirect Antiglobulin Test

  • what are the steps used to detect whether or not a pregnant woman has antibody to Rh?
  • in what other reaction can this test be used for?
A
  • Rh-positive RBC + mom’s serum (37˚C is best temperature for IgG binding)
  • Then add antihuman immunoglobulin
  • Used to see if mom has antibody to Rh
  • Also can be used to test for compatibility for transfusion
50
Q

Agglutination Inhibition

- what are the options for antigen source?

A
  • Competition between kit-supplied particle antigen and patient antigen for supplied antibody

Immunohematology and Serology (8 decks)

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