1
Q
What were the first immunoassays developed?
A
Precipitation and agglutination were the first immunoassays developed
2
Q
What do immunoassays rely on to work?
A
Rely on the multiple binding sites of both antibody and antigen
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
4
Q
Define affinity
A
Affinity
- Strength of binding of one Fab with one epitope on an antigen
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
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)
7
Q
Define avidity
A
- The number of binding sites times the affinity
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
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
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
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
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
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
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
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
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
17
Q
Precipitin Reactions
- what are some examples of immunodiffusion gel precip methods?
A
- Double-diffusion gel precipitation
- Single-diffusion precipitation (radial immunodiffusion)
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
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
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
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
- What is a line of partial identity?
| - What feature does the line have that the others lack?
- 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
Ouchterlony and Relative Concentrations
| - higher concentration of antigen will form a line closer or further from the antibody well?
- The antigen with the higher concentration would form a precipitin line closer to the antibody well
27
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?
- Quantitative
- Single diffusion of the antigen occurs in an agarose gel containing antibody
- Antibody concentration is thus held at a constant
28
Radial Diffusion
- what does the size of the precip circle depend on?
- how is a standard curve prepared?
- 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
Radial Diffusion: Fahey Method
- how long does this take?
- how do you calculate the diameter?
- how is the calculation plotted?
- 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
Radial Diffusion: Mancini or end-point method
- describe what happens
- how long does this take?
- how do you calculate the diameter?
- Reactants are allowed to come to equilibrium
- - 24–72 hrs
- The square of the diameter is directly proportional to the concentration
31
Radial Diffusion
- what antibodies does it measure?
- what antibodies can't it measure
- - why?
- 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
Light Scatter—Enhancement of Precipitation: Turbidometry
- how does it collect its measurements?
- what does it measure?
- 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
Light Scatter—Enhancement of Precipitation: Nephalometry
- how does it relate to turbidometry
how does the measurement process work?
- 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
Light Scatter—Enhancement of Precipitation: Nephalometry
| - what all can this measure?
- Immunoglobulin concentrations, IgG, IgM, IgA, and IgE, as well as kappa and lambda light chains are measured this way
35
Turbidometry and Nephalometry
| - what has been implemented to increase their sensitivity?
- Both techniques have been made more sensitive with the use of laser light as the light source
36
Agglutination
- what was the first disease diagnosed with agglutination?
- why is it not a sensitive test?
- why is it more sensitive than precip?
- 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
Passive Agglutination
| - why does this occur?
- Increased sensitivity of agglutination over precipitation is the reason for the development of passive agglutination
38
Passive Agglutination
- describe the process
- give an example
- 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
Agglutination with Charged Particles
| - why is IgM better for agglutination of these particles than IgG?
- reach between the antigen binding sites of IgM
| - Numerous binding sites
40
Agglutination with Charged Particles
- what role does pH play?
- how does charge affect the titer?
- pH of the reaction is important because pH affects charge
| - If charge increased, the apparent titer would decrease
41
Agglutination with Charged Particles
| - what are 5 methods used to improve binding of charged particles?
- 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
Agglutination
- when would you add AHG to a reaction?
- what does it do to the particles?
- 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
What are the 5 types of agglutination?
- Direct agglutination
- Passive agglutination
- Reverse passive agglutination
- Hemagglutination
- Agglutination inhibition assays
44
Types of Agglutination
Define:
- Direct agglutination
- Passive agglutination
Direct agglutination
- Antigen is naturally part of the particle
Passive agglutination
- Antigen not normally part of the particle
- Also called indirect
45
```
Types of Agglutination
Define:
- Reverse passive agglutination
- Hemagglutination
-- what are the different types?
```
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
Hemagglutination—DAT
- what does this detect the presence of?
- in what conditions might this occur?
```
- 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
Hemagglutination—DAT
- why is it considered "direct"?
- what components is DAT made up of?
- - what reaction occurs?
- 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
Indirect Antiglobulin Test
| - what does this test for?
- Looks for the presence of the antibody in a patient, or for blood group Antigens on a patients cells
49
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?
- 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
Agglutination Inhibition
| - what are the options for antigen source?
- Competition between kit-supplied particle antigen and patient antigen for supplied antibody
Immunohematology and Serology
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