Antibody-based techniques Flashcards
what is antibody-based technique
is a technique to characterize antibody, characterize sample using antibody and purification of antibody
what are technique for antibody-based technique
- precipitation technique
- immunochromatography
-immunoblotting technique
-agglutination analysis
explain more about precipitation technique
- there is formation of high molecular weight antigen-antibody complex that resulting in insoluble precipitate
what does amount of precipitate depends on in precipitation technique
- amount of antigen and antibody
- molar ration of antigen:antibody
-valence : maximum number of antibody that can bind antigen
What happens at: Ab excess ? Equivalens?
Ag excess?
-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
what is the different btn small and large antigen
small ag: less epitope are available for ab to bind to.
large ag: more epitope for ab to bind to
compare valence and epitope
number of valence has to be less than number of epitopes
which value of valenceis required for precipitate formation
valence has to be greater than 2
state if precipitate is going to be formed
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
what are precipitation technique
- 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
what happen if the concentration of ag is increased in single radial immunodiffusion
ag will diffuse before t reach the equivalent
when and what is single radial immunodiffusion is used
-antibody-antigen interaction form precipitate
- to measure diameter of precipitate
how does rocket electrophoresis work
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
what is advantage of rocket electrophoresis
it increase resolution
where is immunochromatograph usesd for
- purification of ab
what is dos choosing method depends on
property of the ab its specificity and its stability
how do you test purification of antibody specificity
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
how do you test purification of antibody without basing on specificity
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
what is the condition for testing purification of antibody without basing on specificity
low pH
what is solid-phase absorption
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
how is Purification of other proteins using Abs works
ab will be immobilized on chromatogram to purify ag
what is the problem of affinity chromatography
strong ab-ag interaction that can lead to denature of samples
how does Purification of antibody fragments works?
use His tag to purify under mild condition (pH 6)
Immunoblotting techniques
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
what is the use of Immunoblotting techniques
- identify ag a certain ab react with
- identify molecule that interact with antigen
what is immunoprecipitation/ SDS-PAGE
identify which protein ab binds to
what is Agglutination techniques
is clumping of the particle
note is different from precipitate
where is Agglutination techniques used
to determine blood group
