Immunology Lab 3 Flashcards
serum Ab titer
measure of relative strength and/or amount of specific Ab in a sample (typically serum)
serial dilutions
series of sequential dilutions of a substance in a solution
How are dilutions expressed?
volume of the sample in relation to the total volume
total volume
volume of sample plus the volume of the buffer/diluent
How do you make a 1:5 dilution of a 2mL sample?
2+x = 10
x = 10-2
x = 8 mL
add 8mL diluent to 2mL sample
In a two fold (2X) dilution series where tube #1 contains a 1:5 dilution, what dilution will tube #6 contain?
1:160
Which test is more sensitive - agglutination or ELISA?
ELISA - by a lot!
What might an increase in titer over a two week period mean clinically?
this might indicate that the patient is fighting an ongoing infection
What dilution factor would you use for low-titered sera?
two-fold (2)
What dilution factor would you use for high-titered sera?
ten-fold (10)
ELISA
enzyme-linked immunosorbent assay (uses enzyme-substrate), typically chromogenic (color-producing)
RIA
radio-immunoassay (uses radioactive isotopes)
RAST
radioallergosorbent test (uses radio-labeled antiglobulin)
immunofluoresence
self-explanatory (uses fluorochrome)
Advantages of ELISA
- Pretty damn sensitive (not as much as RIA)
- Inexpensive
- No radioactivity necessary
- Portable (for field use) (think SNAPs, pregnancy, etc)
Clinical Applications of ELISAs
- Monitor vaccination status, quantify immunoglobulins
- Detect wide range of viruses and antiviral Abs
- Detects bacterial and parasitic infections (salmonella, heartworm, trichinella, brucella)
Most common anti-globulins (3)
alkaline-phosphatase
peroxidase
beta-galactosidase
TiterCHEK
the ELISA we used in lab to look at antibodies specific to canine parvovirus and distemper virus
Two Main Ways We Can Use ELISAs
- Quantification of Specific Antibodies (antiGEN is adsorbed to well and you add serum to determine if the patient has Ab specific for that antigen)
- Quantification of Specific Antigen (Capture Assay) (antiBODY is adsorbed to well and you add serum to determine if patient has the antigen (disease) for that specific antibody)
Wash Steps in ELISA
important for the removal of unbound antibody/enzyme-linked so that not everything pops up positive! (color development in the negative control)
Why would you determine titer prior to revaccination?
avoid potential adverse immune reactions
negative titers indicate the need for revaccination (less than 16 for distemper or less than 80 for parvovirus)
Titers in Clinic
monitor maternal Ab, tailor vaccine programs, avoid reimmunizing high risk breeds or individuals (ie cancer/chemo/allergies/etc), check unknown vax hx
fluorochrome
molecule or dye that emits light in the visible range following excitation of light in the UV range
fluorescein
green fluorochrome
