Specific Immunoassays

Question 1

3 immunoassays?

Answer 1

Binding assay (ELISA)
Precipitation assay (rocket immunoelectrophorisis)
Agglutination assay

Question 2

Binding assay information?

Answer 2

Immobilised substance
binding between antibody and antigen detected
Detection system
Competitive/ Non competitive

Question 3

Precipitation assay?

Answer 3

Soluble substance and antibody interact, resulting event is assessed

Question 4

Agglutination assay?

Answer 4

Particulate substance and antibody interact, resulting event is assessed

Question 5

Typical binding assays

Answer 5

Western blotting
Immunohistochemistry
Enzyme immunoassay

Question 6

Western blotting?

Answer 6

Protein mixture

Detection of ag-ab interaction

Question 7

Sample prep for western blotting?

Answer 7

Cells/tissues/viruses/bacteria
Extract of solublise protein: specific buffer
Filtration techniques to form protein mixture

Question 8

What is indirect immunophloresence?

Answer 8

Primary antibody binds to antigen on cells or tissues sections
Secondary antibody- flourophore conjugate specific to the primary antibody gets used in conjugation with flourescent microscope for detection

Question 9

what is Immunohistochemistry?

Answer 9

Detection of protein expression
Detection of protein localisation 
In tissues (histo) or cells (cyto)
Primary antibody binds to antigen 
Secondary antibody linked to enzyme
Addition of substrate leads to colour change 
Detection by microscope

Question 10

Competitive immunoassays?

Answer 10

Used to detect large or small molecules: antigens or haptens
one site assays:mAb is used speicfic for 1 epitope on ag or hapten
One of the two reagants is limiting: either antigen or antibody
Monitor antigen or antibody

Question 11

Competitive ELISA?

Answer 11

Incubated limited amount of mAb with sample (contains antigen )
Perform ELISA
The higher the concentration of antigen in the original sample the lower the signal at the end of the assay

Question 12

Non competitive assays?

Answer 12

Reagants are in excess
Rapid reactions
Either 1 site (one mab)
or 2 sites (two mab)

Question 13

1 site non competitive immunassay?

Answer 13

Enzyme conjugate antibody is used

Question 14

two site non competitive immunassay?

Answer 14

Large antigens
2 different epitopes:
Detection antibody—>labelled for detection
Capture antibody—>captures antigen

Question 15

Precipitation assays?

Answer 15

Interaction between antigens and antibodies
Due to multivalent nature of antigens and antibodies
—> Precipitate formation
—>Molecule cross linked—>lattice

Question 16

What happens in the precipitation reaction?

Answer 16

Best when polyclonal antibodies and soluble antigens are at equal concentration
Precipitates form due to increase in size of immunocomplexes
When aggregates exceed a critical volume, spontaneous precipitation occurs.
This is the zone of equivilance
Carried out in solution or in agar

Question 17

What happens in soluble antigen antibody interactions?

Answer 17

precipitation ring test-carried out in a small tube

Question 18

What happens in immunoprecipitation?

Answer 18

1. Isolate protein of intrest
2. Isolate protein from tissue
3. Add specific antibody
4. Add protein
5. Centrifuge
6. Wash
7. Western blotting

Question 19

Immuoprecipitation in gels?

Answer 19

Lab class and seminar to follow 
Ab-Ag precipitate forms line, that are visible in agar
1.Radial immundiffusion
2.Double immundiffusion
3.Rocket immunelectrophoresis

Question 20

Agglutination assays?

Answer 20

Easy to perform
Inexpensive
Small amount of antibody needed

Question 21

Difference between agglutination and Precipitartion

Answer 21

In agglutination, the antibody is insoluble before added to together
In precipitation, Antigen is initially soluble;antibody binding to it createsa lattice and makes it insoluble