Immunology 6 Lecture - Immunological Assays

Front 1

Region of Antibody responsible for recognizing defined epitopes

Back 1

Variable Region

Front 2

Structure of Immunoglobulin

Back 2

Heterodimers consisting of heavy chains and light chains;

Some Isotypes of Ig can exist as monomers while others can exist as polymers. IgM commonly exists as a pentameric polymer. IgA can exist as a dimeric polymer

Front 3

Immunofluorescence Microscopy: Steps

Back 3

1. Label Ab with fluorochrome (FITC) which will reflect a certain color under UV light.

2. Incubate tissue with labeled Ab.

3. Ab should bind to Antigen so only the cells with Antigen should have labeled Ab attached

4. View with UV light so only labeled Ab are shown

Front 4

Immunofluorescence Microscopy: Uses

Back 4

1. Identify a particular cell type based on Ab binding to a known molecule on that cell type

2. Study the intracellular localization of a particular molecule the Ab has affinity for

Front 5

Immuno Electron Microscopy: Uses

Back 5

This technique can in finer detail than Immunofluorescence Microscopy, locate a molecule that a labelled Ab has affinity for.

Front 6

Immuno Electron Microscopy: Steps: How it differs from Immunofluorescence Microscopy

Back 6

Instead of labelling the Ab with FITC(fluorochrome) and visualizing the incubated sample with UV light as in regular Immunofluorescence Microscopy, the Ab is labeled with a gold particle and visualized under an Electron Microscope. The gold particles appear as black dots in the image.

Front 7

Immunohistochemistry: Uses

Back 7

In order to use traditional Immunofluoresence Microscopy the tissue samples being tested must be prepared in a certain way. If however, you have a fixed sample (like old tissue sample fixed with formalyn) you will be unable to use Immunofluorescence Microscopy. Immunohistochemistry allows you to test the fixed tissue samples

Front 8

Immunohistochemistry: Steps

Back 8

Instead of labeling Ab with FITC (fluorochrome), attach an enzyme to the Ab that has the ability to change color of the tissue being "stained with Ab" from colorless to colored.

Front 9

Indirect Immunofluorescence: Uses

Back 9

In the other forms of Immunofluorescence microscopy used an actual tissue from the patient was used but in this case we are talking about INDIRECT IF where the major difference is that we are trying to find circulating Ab in a patient.

Front 10

Indirect Immunofluorescence: Steps

Back 10

1. Take patients serum
2. Incubate serum with Normal human skin
3. In order to find where patient derived Ig binds to need to use a secondary labeled (FITC) antibody that recognizes human IgG and add that t the above mixture.
4. Then can visualize where the labeled secondary Ab are by using UV light

Front 11

Antibody Titer: Steps

Back 11

1. Take Serum from Pt.
2. Make Serial Dilutions of Serum.
3. Add Sample of each serial dilution of serum to normal tissue.
4. Add labeled secondary Ab against human Ig into tissue/serum sample
5. Visualize at which point signal from secondary Ab is gone.
6. The serial dilution ratio used just before the signal is gone is the Antibody titer.

For example: If a 1:40 dilution of a serum still shows a signal but a 1:160 serial dilution (the next dilution in the series) does not show a signal under UV light, then the antibody titer is 1:40.

Front 12

Antibody Titer: Uses

Back 12

Acts as an indicator of Disease activities. If you have higher amounts of circulating Ab then you should have higher titers so antibody titers can indicate disease activity. In this example 1:160 titer would be "higher" in comparison to 1:40.

Front 13

Immunoblotting: Western Blot: Uses

Back 13

To see which Antigens are recognized by particular Antibodies

Front 14

Immunoblotting: (Western Blot) Steps

Back 14

1. Take the tissue (with antigens that Ab likely recognizes) and homogenize it so that you have a mixture of the tissue associated proteins
2. Separate the tissue mixture by weight (using SDD-Page)
3. Transfer a copy of the gel onto a membrane to be used as a template
4. Add the Antibody of which you are looking for the particular Antigen
5. The Ab should bind to the Antigen
6. Add secondary Ab labeled with enzyme to see the localization of the first Antibody which should be bound to the Antigen
7. The Serum Ab should be bound to an area of specific weight which represents the area of the Antigen involved because it is the colored spots on the membrane that contain the antigen-ab complex.