L:10 Immunoassays/Flow Cytometry

Question 1

Define affinity in terms of antibodies. How is avidity different?

Answer 1

Strength of binding on 1 site of a molecule to its ligand.

Avidity is the strength of binding of multiple sites of a molecule to its ligand

Question 2

What does immunodiffusion do? What are its downfalls?

Answer 2

It can detect antibody specific for a particular antigen, useful for simple antigen mixtures.
It has poor sensitivity and is non-quantitative.

Question 3

What is immunoelectrophoresis? What are its downfalls?

Answer 3

It inolves electrophoretic separation of antigens. Can be used for more complex antigen-mixtures.
It has poor sensitivity and is non-quantitative

Question 4

What is nephelometry?

Answer 4

It is used to determine levels of IgM, IGG, and IgA from a serum sample. It is performed by measuring scattering of light as it is passed through a diluted serum sample at an angle. The amount of light scatter is measured and compared to the amount of scatter from a known normal sample.

Question 5

Define titer. What is an antibody titer?

Answer 5

The quantity of a substance required to react with or to correspond to a given amount of another substance. Antibody titers is a measurement of how much antibody an organism has produced that recognizes a particular antigen

Question 6

What is the direct Coomb’s Test?

Answer 6

Direct is a test to detect Ab or complement proteins on the surface of RBCs collected directly form a patient. These Abs are indicative of autoimmune hemolytic anemia.

Question 7

What is an agglutination titer?

Answer 7

The highest dilution of a serum which causes clumping of microorganisms or other particulates

Question 8

What is hemaglutination? What are the benefits?

Answer 8

Use of RBCs in an antibody titer. If antibodies bound to RBCs are specific for antigen, they will form a mat and sink. If not, they will fall independently as a pellet

Question 9

What is the indirect Coomb’s test?

Answer 9

Used to detect very low concentrations of RBC-specific antibodies present in a patient’s plasma. It is used for crossmatching before blood transfusions are given. It is also used to detect atypical Abs in the serum of pregnant women.

Question 10

What is the monospot test?

Answer 10

a hemagglutination test that is diagnostic for Epstein-Barr virus. Very similar to an indirect Coomb’s test. Uses sheep RBCs.

Question 11

What is a complement fixation assay?

Answer 11

Anti-serum is mixed into sample, and binding is allowed to occur. Complement proteins are thrown in, and if there are the right IgA, IgM, etc antibodies, then the complement will be used up. If they are not present, the protein will still be present.
Can also be used with RBCs. If RBC is lysed, then + test because proteins were used up and cell remained intact. If Abs are not present, complement will lyse the RBC

Question 12

What is convalescent serum?

Answer 12

Serum from a person who has recuperated from a particular infection, which may be of use in treating a person with the same infection. Typically has low IgM and high IgG pathogen-specific antibody levels.

Question 13

What is acute-phase serum?

Answer 13

Serum collected from the blood of a patient that is actively infected with the pathogen. Typically has high IgM and low IGG pathogen-specific antibody levels. Also often contains high levels of mannase-binding protein and C-reactive protein.

Question 14

What is a primary antibody test?

Answer 14

Test sample that may or may not contain antibody specific for a particular antigen

Question 15

What is a secondary antibody?

Answer 15

an Ab that will detect the primary Ab, typically conjugated to an indicator molecule.

Question 16

What is a clone?

Answer 16

a cell population that has descended form a single cell. All are essentially identical to the mother cell

Question 17

What does mono mean?

Answer 17

Single, in this case witha single antigen-specificity

Question 18

WHat is a hybridoma?

Answer 18

A Cloned hybrid cell line (B cell fused to a tumor cell) that produces antibody against a specific antigenic determinant

Question 19

What is a monoclonal antibody?

Answer 19

a preparation of Abs that was produced by a cloned hybridoma. EAch Ab molecule is identical.

Question 20

What is affinity chromatography for Abs?

Answer 20

Antigens are placed in column with gel bead matrix. Antibodies with antigen specificity will bind to antigens and matrix and remain in tube. All others will wash out. You then release selected Abs from matrix with ellusion buffer.

Question 21

What is western blot analysis?

Answer 21

complex mixture of antigen is separated on a gel with current.
Antibodies with some sort of marker molecule or enzyme bound to them are added to the gel and bind to the antigen, marking it.

Question 22

What is ELISA?

Answer 22

Enzyme-linked immunosorbent Assay. Quantitative measure of Ab specificity. IT is very sensitive, quantitative and has high throughput. You bind antigens to wells, then wash antibody serum through. Antibodies will bind to the antigens they are specific for. The solution is washed away and unbound Abs will be lost. Chemical coloring makes remaining, bound antibody visible and this can be quantitatively read.

Question 23

What is a flow cytometer?

Answer 23

used to determine surface phenotype of cells. Use marked antibodies that have specificity for the substance you are looking for and mix them with cells. Run fluid of cells through tube with laser passing through it.
Forward scatter is size of cell
Side scatter detects granularity (lymphocytes smooth, neutrophils granular)
Can check for color emission from marked antibodies also.

Question 24

Would lymphocytes have a high or low side-scatter?

Answer 24

low. They are relatively smooth.

Question 25

What is FACS?

Answer 25

Fluorescence-Activated Cell Sorting. Uses a flow cytometer that can identify and collect a specific cell population for experimental or clinical use.