Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Immunology > Quantitative Assays of Immunity > Flashcards

Quantitative Assays of Immunity Flashcards

1
Q

What type of forces hold Ag and Ab together?

A

non-covalent:

  1. Ionic
  2. Hydrogen
  3. Hydrophobic
  4. van der Wals
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is an ionic force?

A

attraction between opposite charged groups like O- in carboxylic acid (aspartic acid) with hydrogen in amino groups (lysine)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What three AA side chains form hydrophobic interactions?

A

Leucine, isoleucine, valine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

When leucine, isoleucine and valine interact with each other what happens?

A

They squeeze out water surrounding the reactants so water is order when it is around Ab or Ag and is disordered when excluded from the Ag-Ab interface

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

The strength of the interaction between Ab and Ag can be estimated by applying ___________.

A

Law of Mass action

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What measures the strength of an Ag-Ab interaction?

A

association/dissociation (Kon/Koff)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Typical Ab-Ag interactions have dissociation constants in the _______________range.

A

low nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the equation for association constant?

A

Ka= Kon/Koff = (Ag-Ab/) Ab*Ag

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the equation for dissociation constant?

A

Kd= Koff/Kon= Ab*Ag/(Ag-Ab)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Would the Kd be high or low for an Ab-Ag interaction with high affinity?

A

Low because there would be more complex than free Ag or Ab

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is the difference between affinity and avidity?

A

Affinity is the strength of the interaction between ONE combining site of the Ab and ONE Ag epitope.
Avidity is the summation of all interactions between combining sites of the Ab (2 for IgG, 5 for IgM) and all the identical epitopes of Ag

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Why is avidity NOT the arithmatic mean of the affinity of a single binding site?

A

Multivalent binding of Ag to each arm of the Ab confers an enhancing effect because when one combining site dissociates the other binding site is still bound and stabilizes the complex

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Avidity is only relevant when _______________________ are being considered.

A

repetitive antigens on a surface

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is cross-reactivity?

A

Sometimes an Ab directed toward a specific antigen can bind structurally related but not identical isotopes.
They will bind these epitopes with lower affinity then the EXACT epitope they are specific for.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is a monoclonal Ab?

A

An Ab derived from a clonal population of cells so it will have specificity for the same epitope on an antigen as its clones

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is a polyclonal Ab?

A

mixtures of different Ab that bind to different epitopes of the antigen. Some may bind to overlapping or the same epitope of the antigen.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What is the necessary condition for the precipitation of any soluble Ag?

A

More than one of its epitopes must interact with a bivalent Ab or polyclonal Ab

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

When a precipitate is forming a lattice of ________ is formed with increased ____ and decreased _______.

A

Ag-Ab complexes
size
solubility

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What are the two quantitative ways to measure the cloudiness of a mixture of Ag-Ab?

A
  1. Turbidimetry

2. Nephelometry

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Can a monovalent antigen for a precipitate?

A

No because there is no cross-linking effects

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Antigens with two or more identical epitopes can be precipitated by_____________./

A

Divalent Abs directed against the epitope (2 antibodies that bind the same antigenic epitope)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Ag expressing two or more different epitopes can be precipitated by ______________________________.

A

a mixture of divalent Ab (polyclonal)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What does the amount of immune precipitate depend on?

A

The Ag/Ab ratio

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What is it called when there is excess Ab, so weak or no precipitate forms?

A

Prozone

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What is it called when there is an antigen excess so no precipitate forms?

A

Post zone

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Only when Ag and Ab concentrations are ___________________________ does optimal precipitation occur. This is called the ___________________.

A

equivalent or close to equivalence (# of combining sites is equal to the # of epitopes)
Equivalence zone

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

Why would an excess of antigen (post zone) not allow for precipitation?

A

The antigen will bind monovalently so immune complexes will not form

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What is the precipitation reaction useful for studying?

A

serum sickness

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

What is tubidimetry?

A

the measure of the reduction of light intensity as a consequence of absorption, reflection and scattering.

30
Q

What is nephelometry?

A

the light that is scattered at a particular angle from the incident light beam

31
Q

What can the amount of light absorbed or scattered tell us about the substance?

A

the amount of Ag-Ab complexes formed

32
Q

What are nephelometry and turbidity used in the clinical lab to quantify?

A

serum proteins such as:

  1. classes of Ig
  2. Complement components (C3,C4,C1 inhibitors)
33
Q

What is the sensitivity of turbidity and nephelometry?

How can sensitivity of precipitation reactions be improved?

A

low- about 1-5micrograms/ml

Sensitivity can be improved by performing the precipitation in semisolid support like agar

34
Q

How is immunodiffusion performed in agar?

What is the name of this test?

A

The Ag and Ab are placed in separate wells and allowed to migrate by diffusion or driven by electric current (immunoelectrophoresis)

When the Ab and Ag interact they form a lattice and a line of precipitate is formed.

This is called the Ouchterlony test.

35
Q

What is Ouchterlony double diffusion used to identify?

What are the drawbacks of this method?

A

Identify Ag in the biological fluid.

There is low sensitivity and it is time consuming (72 hours)

36
Q

What is the radial diffusion assay used for?

How does it work?

A

It is used to quantify the amount of Ab or Ag.
An agar gel that contains Ab and fill wells with Ag, some with known quantities of Ag others (prob from serum) with unknown quantity.
Use ring diameters formed around the well to plot a standard curve of “diameter vs. amount in well”.
The curve can determine the “unknown’ amounts of antigen.
(this can also be done in reverse where the agar has antigen and the substance in the well is Ab)

37
Q

How will the lines on the Ouchterlony test form if the antigens are:

  1. the same
  2. when they share NO common epitopes
  3. when they share some common epitopes
A
  1. it is “identity” and the precipitate will form a V
  2. it is “non-identity” and the two test antigens will form a cross pattern
  3. It is “partial identity” where the lines will partially cross because some antibodies will be cross-reactive and others won’t be
38
Q

How would agglutiation appear on the bottom of a test tube?

A

It would be spread out along the walls of the test tube (vs. a button formed at the bottom when not agglutinated)

39
Q

When are the two most common scenarios for using an agglutination test?

A
  1. in Ob/Gyn for Rh+ tests

2. Before giving a blood transfusion

40
Q

What is the necessary condition for agglutination to occur?

A

The surface of the particle must express multiple epitopes and the Ab combining sites must be aboue to encompass the spatial distance between the two particles

41
Q

What is the distance between 2 RBCs determined by?

A

The repulsion force of their negative charges (zeta-potential)

42
Q

What is the only Ab that can agglutinate RBCs?

A

IgM because it is a large enough molecule to span the distance between two RBCs.

43
Q

People with blood type A have what Abs?

A

Anti-B

44
Q

RBCs belonging to a person with blood type A can be agglutinated only with the serum harvested from ____________________.

A

a person belonging to blood group B that makes anti-A

45
Q

At low dilutions of serum, will agglutination occur?

A

No because the Ab conc is too low

46
Q

If a Rh negative woman has children with a Rh+ man, what could occur in the children?

A

The first child would be fine, but subsequent RH+ children may have hemolytic disease because the mother’s serum will make anti-Rh antibodies that are able to cross the placenta

47
Q

What is used to stop the Rh- mothers serum from attacking Rh+ RBCs in subsequent pregnancies?

A

At the first pregnancy, the woman is given RhoGAM which suppresses her immune system to not attack the Rh+ blood cells (so she doesnt form antibodies against them)

48
Q

What is done in the direct Coombs Test to detect the mothers anti-Rh IgG in the baby’s blood?

A

IgG can’t agglutinate RBCs on their own.
Anti-human IgG is added to the baby’s blood.
If agglutiation occurs then the baby will have hemolytic disease because this means they already have Ab on their RBCs (Anti-Rh Ab)

49
Q

What is the indirect Coombs test for detecting whether the mother;s anti-Rh+ Ab are attacking the baby’s RBCs?

A

Use the mother’s serum and add known Rh+ blood to it. Then add anti-human IgG antibodies. If there is agglutination, then the mother is making Anti-Rh+ Abs

50
Q

What is immunoblotting (Western Blotting)?

What disease is it used to test for?

A

It is using electrophoresis to separate Ag mixtures according to molecular size and charge. Then you can incubate them labeled Abs against the Ag of interest.
It is frequently used to check for anti-HIV antibodies.

51
Q

After the separation of proteins by electrophoresis, they are trasfered from the polyacrylamide gel on the nitrocellulose paper followed by incubation of the paper with _______________________.

A

labeled Ab directed toward the Ag of interest.

52
Q

What are the two ways Ab against Ag of interest can be labeled when using Western Immunoblot?

A
  1. Radioactively

2. Enzymatically - that catalyze colorless substrate to a colored product (used more nowadays)

53
Q

If anti-HIV antibodies are present on the Western Blot, what does this mean?

A

The person is HIV positive

54
Q

In a typical western blot, what is the enzymatically labeled molecule?

A

The anti-human IgG antibody because it will bind to the Ab of interest, then be converted to colored product to visualize

55
Q

What do Radioimmunoassays and ELISAs measure?

A

quantitative measure of the amount of an Ag or Ab in a solution by specific binding with corresponding Ab or Ag and comparing it to a standard dilution

56
Q

What are some of the Ags and Abs used in ELISA/RIA testing?

A 
HIV Ag or Ab
HepABC Ag or Ab
Rubella
HTLV I and II Ab
T. gondii
57
Q

What is non-competitive ELISA?

A
  1. Solid Ag is in the well
  2. Serum (with Ab to the Ag) is added
  3. Ab binds to Ag if there is Ab present
  4. Enzyme-labeled anti-human IgG is added
  5. Color development can determine Ab presence
58
Q

What is a competitive RIA assay?

A
  1. Ab is in the well
  2. Add 4 labeled Ag, no unlabeled–> 100% radioactivity
  3. Add 4 labeled Ag, 4 unlabeled__>50%
  4. Add 4 labeled Ag, 12 unlabeled –> 25%

More unlabeled added, the less labeled will bind and the less radioactivity there will be. You can then generate a standard curve to determine Ag levels in serum

59
Q

What is a “real life” example of an ELISA?

How does it work?

A

A pregnancy test.

  1. Pee on the stick.
  2. If it contains hCG it will complex with colloidal gold-linked mouse anti-hCG
  3. bound and unbound anti-hCG will diffuse up the strip to a test region where it complexes with OTHER mouse anti-hCG
  4. Unbound Ab continues to diffuse up the strip to the control region where there is goat anti-mouse Ab
  5. Colloidal gold molecules form a line in one or both windows (neg and pos)
60
Q

What is flourescence microscopy used to detect?

A

the presence of Ag in fixed tissue OR live cell suspension

61
Q

How do you perform the direct method of floursecence microscopy?

A
  1. Tag Ab with fluorescence

2. It will bind to Ag fixed to a microscope slide

62
Q

How do you perform indirect method of fluorescence microscopy?

A

Detect Ab in patient serum know to be specific for an Ag present in a cell smear by fluorescently labeling anti-human IgG

63
Q

What does the indirect method of fluorescence allow you to visualize?

A

The places in the cell tissue where the Ag is localized (while also revealing the presence of Ab)

64
Q

What is flow cytometry?

A

A system that detects the presence of fluorescent agent on a target and the intensity of the fluorescence.
Can sort cells into different types based on their fluorescence.

65
Q

How does FACS (fluorescent activated cell sorting) work?

A

Cells are incubated with different fluorescent labeled Ab then pass through a laser to excite the fluorescence. As they pass the charged plates, they are sorted into different reservoirs based on the type of antibody

66
Q

How are monoclonal Ab produced in a lab?

A
  1. Mice are immunized with AgX
  2. The cells are fused with “immortalized” aka tumor cells from a mouse myeloma
  3. Screen for anti-Agx antibodies
  4. Allow differentiation of clones
67
Q

What is a problem with how monoclonal Ab are created now?

A

They use mice cells so they make mice Ab/ This may cause serum sickness in humans

68
Q

A new approach is allowing the engineering of antibodies from human Ab genes. What is the first drug developed in this manner?
What is this drug an antibody against?

A

Humira which is an anti-TNFa antibody

69
Q

What is phage display to isolate Ab?

A
  1. The Ab variable domain or Fab is attached to the surface of a phage.
  2. The phage binds Ag displayed on plastic
  3. The phages that bind Ag clonally expand
  4. The clone genes can be selected and used to rebuild complete Ab with that variable region
70
Q

How do scientists try to ensure monoclonal Ab are not dangerous for humans?

A
  1. They make chimeric Ab- rodent variable region is transfered to human constant region
  2. Humanized Ab- rodent HVregion transfered onto human V-region framework segments and connected to Human constant region

Immunology (25 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions