Ab-Ag Interactions Flashcards
Ab AVIDITY
Strength of multivalent antiserum to multivalent Ag
Most Ags have numerous EPITOPES and are also mulltivalent –> Multiple Ab species that can bind to several of these epitopes
NOT TESTED ON THIS WORD
Absorption
The use of cross reacting material to remove the activity that causes the cross reactivity
Affinity Chromatography
Used to purify many immunological reagents
Monoclonal Ab
A single clone of one B-cell is produced by fusing that cell to a tumor cell and isolating the clone with the Ab specificity of interest
NO HETEROGENEITY
Have low affinity, but no avidity
Steps to making a monoclonal Ab
Immunize an animal
Isolate spleen cells from immunized animal
Fuse spleen cells to cancer cells
Select only those that are hybrids
Clone the hybrids
Select clone with specificity that you are interested in
Problem with using non human Abs in humans
Have an immune response to non-human Abs
Chimeric
Variable regions from mouse
Constant from humans
Humanized
Only the points of contact with Ag remain mouse
Human monoclonal
Made through molecular biology techniques
Immunoassays - ELISA
- Ag stuck to bottome of well
- Ab is added, allowed to incubate, excess Ab washed
- 2nd Ab that has enzyme linked molecule (that binds to 1st Ab) added
- Incubation, and excess 2ndary washed
- 2ndary Ab detected by adding a chemical reagent that turns color in presence on En bound to 2ndary Ab
Immunofluorescence
Tissue or cells are reacted with antisera specific for a cell marker or pathogen
Unbound antiserum is removed by washing
A 2nd Ab specific for the 1st Ab is added and allowed to bind
Attached to 2nd Ab is a fluorescent molecule
Cells viewed through microscope
Fluorescence Activated Cell Sorter - Flow cytometry
Mixture of cells are labeled with fluorescent Abs
Analyze cells with lasers and sort
Western Immunoblot
- Mixture of proteins such as cell lysates or serum are separated electrophoretically
- Separated proteins bound to nitrocellulose paper
- Ab to protein of interest then added, wash unbound, add 2nd Ab
- Ab detected by En-linked 2ndary Ab
Possible constant light chain regions
Kappa and lambda
Epitope
Part bound by Ab
Types of Epitopes
Linear
Discontinuous
What is meant by Abs are HETEROGENOUS
Difference in how and where Abs bind to same Ag
Polyclonal Abs
Abs made from diff B-cells for same Ag
Uses of Abs in Lab
-Abs are used to measure many biological parameters
How to make Abs more specific
- Affinity purify
- Absorb non-specific antisera
- Monoclonal Ab
Affinity Purify Ab
Make Ab more specific by purifying the Ab you want
- Add Ag with beads
- Add diff Abs
- Wash off Abs not attached to Ag and bead
- Remove bead from Ab
- Left with specific Ab
Absorb Cross Reacting Ab
Remove the contaminated Ab
- Pour mixture of Ab into Ag attached to beads (that arent interested in)
- Pull out Abs not attached
- Pulled out purified Ab
Serum Sickness
Immune reaction to Ab (usually not our own)
- We are sometimes given Abs (that are foreign) to help, but build up Abs against foreign Ab
- Next time injected will have serum sickness
