Immunodiffusion/ELISA Flashcards
Immuno is associated with…
Diffusion is linked with…
Antibodies and antigens
Agar plates (antibodies/antigens dffuse from high concentrations to low)
Immunodiffusion determines presence of antigens and/or antibodies - solutions diffuse precipitin forms if reaction occurs.
Antibodies are
Glycoproteins - proteins that have sugars on it as well as blood proteins [immunoglobulins].
Produced by plasma cells - activated B cells due to a response to an antigen.
Y binds to antigen (& targets them for destruction by other immune system cells)
Antigens are
Any type of molecule that elicits an immune response
Ex: carbs, proteins, DNA, lipids can serve
•U•
Epitope
Where antibody binds to antigen
1:1 specificity - 1 antibody recognizes one antigen
Can be varied shapes because can be recognized by diff antibodies.
Antibody:
light chain \ / light chain
^
Variable portion
Heavy chain | | heavy chain
^
Constant portion
Composed of 2 heavy chains+ 2 light chains,
constant region - used by antibody to bind to cell of immune system; 5 classes: IgM,A,D,G,E,
variable region- place where antibody binds to antigen with epitope
IgG - monomer
Major antibody in serum
Crosses placenta
Long-lasting antibody/immunity
mom gives during pregnancy
IgA- monomer (blood) or dimer
Secretions - breast milk, genital, mucosal membranes, digestive tract, ears, eyes.
Protection as baby breastfeeds
Protect body surfaces exposes to outside foreign substances
IgM - monomer (receptor on B cells) or pentamer (units in blood)
First antibody we produce
Eliminates pathogens in early stages of B cell mediated immunity before IgG is sufficient
IgD - Monomer (receptor on B cells)
Antigen from environment and presents to immune system.
Can have IgD and IgM on B cell
IgE monomer
Do not have Allergies - low IgE
Seasonal, food, etc Allergies- high IgE
Binds to mast cells, which granulate, histamines released - watery eyes, hives, etc.
Serum
Clear to part of blood
- contains antibodies
No clotting factor
Precipitin
Rxn between antibody and antigen
Forms line
ELISA
Enzyme Linked Immunosorbent Assay
Positive result in ELISA
Antibody present within patient’s blood - associated w/ disease presence or encounter and recovery.
True or False - the body after recovery from disease can STILL make antibodies as an immune system response and prevention.
True
Amount of color in sample
Quantity of enzyme (amount of antigen present in OG sample)
- Centrifuge
Separates test sample - obtain fluid serum w/ antibodies
- Prepare Serial Dilution
Determine concentration of antibody in test sample’s serum.
3 diff. solutions to perform 3 serial dilutions (serum from patient and buffer)
3 & 4. ELISA Pretreated
With SLE antigens to bind to surface of well acting as protective coat - prevents antibody from sticking to wells.
When antibodies bind to antigen they do it without binding to wells themselves due to this step.
Positive Control
Primary antibody - first antibody to detect foreign molecule (antigen)
Negative control
Buffer
2 controls
R/o error and problems with reagents changing w/time & mistakes with ELISA procedure.
What happens if 2 positive responses are produced by both controls
Null results and assay must be completed again to produce accurate results
- Incubate
Allow sufficient binding between antibody - antigen to properly proceed w/ effective timing for.
- Wash plate
Unreacted antibodies need to be removed; prevent leftover antibody from binding w/ secondary antibody. (False positive)
- Secondary Antibody
Binds to primary antibody / source: diff. species than primary .
Secondary antibody distinguishes primary antibody as foreign.
