Indirect Methods Flashcards
What are the most widely used serologic methods?
- serum neutralization
- complement fixation
- enzyme linked immunosorbent assay
- hemagglutination inhibition
Antibody titers
Expressed as the reciprocal of the highest serum dilution causing a positive observable antigen-antibody reaction
What specimen is collected for serologic tests?
Blood samples in a sterile, sealed unit (B-D Vacutainer)
- specimen should clot at room temperature before overnight refrigeration
- serum is then decanted from clot into a sterile centrifuge tube and centrifuged at 2,500 xg for 20-30 min
Non-hemolyzed serum sample
Frozen at -10 - 20 C and shipped immediately, or sent later with a second sample drawn from the same animal
Positive serologic result
Considered to be a 4 fold rise in titer over a 2-3 week period
A good serum specimen will contain no ______
RBCs
Serum neutralization
Based on inhibition of viral replication by specific antibody
- the titer is expressed as the neutralization index, or the highest dilution that protects 50% of the test host against a precalculated viral dose
Serum neutralization procedure
100 TCID50 of virus is used against varying 2-fold dilutions of serum
- cell layers are rinsed with methanol, and stained with Giemsa
- stained wells indicate lack of viral cytopathic effect, clear wells lack cells as a result of viral cytopathic activity
Neutralization titer
Expressed as the reciprocal of the highest dilution at which the virus infection is blocked
What 3 tests give a titer as a result?
- serum neutralization
- ELISA
- indirect immunoflurescence
ELISA
Known antigen bound to a solid substrate
- serum antibodies (specimen) are then reacted washed and detected by a secondary antispecies antibody conjugated to an enzyme
Indirect immunofluorescence
The specimen is the serum antibody
- virus is known and standardized
- fluorescent labeled antiglobulin is used to detect serum antibody bound to antigen
Agar gel immunodiffusion
Cut 2 round wells 5 mm in diameter and 1 cm apart in a layer of agar in a petri dish
- one well filled with antigen, the other with antiserum –> reactants diffuse out radially
- circular concentration gradient is established for each reactant that will eventually overlap
- known as double diffusion test
Double diffusion test
If several antigen-antibody mixtures are used, each component is unlikely to reach optimal proportions in the same position –> separate line of precipitation is produced for each interacting set of antigens and antibodies
Double diffusion test is used to determine relationship between _____
Antigens
- if 2 antigen wells are set up and 1 antibody well then lines will form between each antigen well and the antibody well
- if 2 lines are completely confluent, then the 2 antigens are considered identical
- if the lines cross over, the antigens are different
- if the lines merge with spur formation, then partial identity exists
Example of a double diffusion test
Coggins test
- detects presence of antibodies against equine infectious anemia virus
- extract of infected horse spleen or cell culture antigen is reacted with the serum of horses
- occurrence of the line of precipitate constitutes a positive reaction
Antibodies to EIA in equines are indicative of infection, no need to do a ______
Titer
- same for all lenti viruses
Certain viruses are capable of agglutinating suspensions of ________
Mammalian and avian erythrocytes
- antibodies detected against these viruses may inhibit agglutination
Hemagglutination
Detection of virus induced hemagglutination may be used as a preliminary when attempting to identify a virus
- is a direct test, but lacks specificity
- include some mycoplasmas (M. gallisepticum)
Hemagglutination inhibition
- alpha procedure: amount of virus added to each tube is kept constant, while the serum tested is serially diluted
- beta procedure: add a standard amount of antiserum to each tube while serial dilutions are made of a virus suspension of known hemagglutinating activity
Hemagglutination inhibition titer
Obtained by multiplying the highest dilution of serum that just inhibits hemagglutination by the number of hemagglutinating units of virus involved
Problems with hemagglutination inhibition
Presence in test serum of nonantibody hemagglutination inhibitors
- some are carbs that may be destroyed by treatemnt of the test serum with bacterial neuraminidase
- lipoproteins that may be removed by absorption of serum with washed kaolin, or destroyed by trypsin treatment
It is necessary to absorb the test serum with _______ in order to remove natural hemagglutinins
Erythrocytes
False positive may be obtained if there is excessive ______
Delay in reading some hemagglutination inhibition tests
