Immunoassays

Question 1

What is the definition of affinity?

Answer 1

expression of the sum of all the interactions between an antibody binding site and its homologous antigenic determinant

Question 2

Does the term affinity apply to polyclonal antibodies or monoclonal?

Answer 2

Monoclonal only

Question 3

What is the definition of avidity?

Answer 3

Strength of multivalent antiserum to multivalent antigen

Question 4

Does the term avidity apply to polyclonal antibodies or monoclonal?

Answer 4

polyclonal only

Question 5

Which typically has a stronger “affinity”, polycloncal avidity or monoclonal affinity? Why?

Answer 5

Polyclonal avidity, because some antibodies are better than others so the combination leads to a stronger “affinity”

Question 6

What are the requirements for antibody cross reactivity?

Answer 6

Cross reacting groups need only be similar, not identical

Question 7

What are the two ways to eliminate cross reactivity?

Answer 7

Absorption (removing contaminant) and Affinity Chromatography (purifying the antibody)

Question 8

How are Monoclonal Antibodies made?

Answer 8

Made from a since cloned B-cell

Question 9

Preparation procedures for monoclonal antibodies?

Answer 9

1- Immunize the animal
2- Isolate the spleen cells from immunized animal
3- Fuse the spleen cells to plazmacytoma tumor cells
4- select for only thase cells that are hybrids of Tumor cells and B cells
5- Clone the hybridomas so that each single cell grows up independently
6- Select the individual clone with specificity that you are interested in

Question 10

Drawback from using monoclonal antibodies?

Answer 10

No avidity and lower affinity, also no coopertivity

Question 11

-omab

Answer 11

Mouse only

Question 12

-ximab

Answer 12

variable regions are from mouse and constant regions are human

Question 13

-zumab

Answer 13

only points of contact wth the antigen remain mouse

Question 14

-umab

Answer 14

all human, generally made through molecular biology techniques

Question 15

Procedure of an ELISA?

Answer 15

1- Antigen is stuck to the bottom of a well
2- Serum/ antibody is added
3- Incubation
4- Unbound antibody is washed away
5 Secondary antibody is added with enzyme covalently atttached
6- Visualize

Question 16

What immunoassay can be used to detect target within the cell?

Answer 16

Immunofluorescence

Question 17

What does flow cytometry tell you?

Answer 17

Immunofluorescence presence (cell type) and cell size. Can scan for one antigen or several antigens at once.

Question 18

What results are attained from Westerns?

Answer 18

Quantitative (amount of antigen) and Qualitative (MW of target)