Antibody-based techniques

Question 1

what is antibody-based technique

Answer 1

is a technique to characterize antibody, characterize sample using antibody and purification of antibody

Question 2

what are technique for antibody-based technique

Answer 2

• precipitation technique
• immunochromatography
  -immunoblotting technique
  -agglutination analysis

Question 3

explain more about precipitation technique

Answer 3

• there is formation of high molecular weight antigen-antibody complex that resulting in insoluble precipitate

Question 4

what does amount of precipitate depends on in precipitation technique

Answer 4

• amount of antigen and antibody
• molar ration of antigen:antibody
  -valence : maximum number of antibody that can bind antigen

Question 5

What happens at: Ab excess ? Equivalens?
Ag excess?

Answer 5

-Ab excess: precipitate increase and no formation of stable complex

-Equivalens: precipitate is at optimal

-Ag excess: precipitate decrease and each ab has two ag epitope to bind to

Question 6

what is the different btn small and large antigen

Answer 6

small ag: less epitope are available for ab to bind to.

large ag: more epitope for ab to bind to

Question 7

compare valence and epitope

Answer 7

number of valence has to be less than number of epitopes

Question 8

which value of valenceis required for precipitate formation

Answer 8

valence has to be greater than 2

Question 9

state if precipitate is going to be formed

Answer 9

1) Small hapten + mAb ? no precipitate
2) Small hapten + pAb? no precipitate
3) Large antigen + mAb? no precipitate, it can be but mAb has no other ab to bind epitope of antigen
4) Large antigen + pAb? precipitate, highest probability of multiple Ab binding

Question 10

what are precipitation technique

Answer 10

• single radial immunodiffusion: ab is added to the gel and sample is added too, then sample/ antigen will diffuse out of the gel and form precipitate

-rocket electrophoresis

Question 11

what happen if the concentration of ag is increased in single radial immunodiffusion

Answer 11

ag will diffuse before t reach the equivalent

Question 12

when and what is single radial immunodiffusion is used

Answer 12

-antibody-antigen interaction form precipitate
- to measure diameter of precipitate

Question 13

how does rocket electrophoresis work

Answer 13

gel containing ab is added to gel and then ag will be added then they are run in electrophoresis the precipitate is formed. the surface area of precipitate is direct proportional to the number of antigen.

by measuring the height of formed precipitate the concentration of unknown sample is determined through standard curve

Question 14

what is advantage of rocket electrophoresis

Answer 14

it increase resolution

Question 15

where is immunochromatograph usesd for

Answer 15

• purification of ab

Question 16

what is dos choosing method depends on

Answer 16

property of the ab its specificity and its stability

Question 17

how do you test purification of antibody specificity

Answer 17

we use antigen chromatograph
first ag is immobilized on the beads, ab is added but specific ab will bind then unspecific ab will be washed out and bind ab will elute

Question 18

how do you test purification of antibody without basing on specificity

Answer 18

first protein A or G column are added, then ab id added in the solution. unbound ab are washed out and bound ab are eluted

Question 19

what is the condition for testing purification of antibody without basing on specificity

Answer 19

low pH

Question 20

what is solid-phase absorption

Answer 20

is used to get rid of unwanted antibodies by using unwanted reactivity

how: by immobilizing protein that binds to unwanted ab, then unwanted anb will bind, wanted ab will be collected

Question 21

how is Purification of other proteins using Abs works

Answer 21

ab will be immobilized on chromatogram to purify ag

Question 22

what is the problem of affinity chromatography

Answer 22

strong ab-ag interaction that can lead to denature of samples

Question 23

how does Purification of antibody fragments works?

Answer 23

use His tag to purify under mild condition (pH 6)

Question 24

Immunoblotting techniques

Answer 24

used for HIV test

• you take protein/HIV and transfer it in the membrane
• patient serum/ sample is added
  -ab detection is added
• if there is ab bound to the HIV protein in ways of protection means the patient is positive

Question 25

what is the use of Immunoblotting techniques

Answer 25

• identify ag a certain ab react with
• identify molecule that interact with antigen

Question 26

what is immunoprecipitation/ SDS-PAGE

Answer 26

identify which protein ab binds to

Question 27

what is Agglutination techniques

Answer 27

is clumping of the particle

note is different from precipitate

Question 28

where is Agglutination techniques used

Answer 28

to determine blood group