Indirect Methods

Question 1

What are the most widely used serologic methods?

Answer 1

• serum neutralization
• complement fixation
• enzyme linked immunosorbent assay
• hemagglutination inhibition

Question 2

Antibody titers

Answer 2

Expressed as the reciprocal of the highest serum dilution causing a positive observable antigen-antibody reaction

Question 3

What specimen is collected for serologic tests?

Answer 3

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

Question 4

Non-hemolyzed serum sample

Answer 4

Frozen at -10 - 20 C and shipped immediately, or sent later with a second sample drawn from the same animal

Question 5

Positive serologic result

Answer 5

Considered to be a 4 fold rise in titer over a 2-3 week period

Question 6

A good serum specimen will contain no ______

Answer 6

RBCs

Question 7

Serum neutralization

Answer 7

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

Question 8

Serum neutralization procedure

Answer 8

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

Question 9

Neutralization titer

Answer 9

Expressed as the reciprocal of the highest dilution at which the virus infection is blocked

Question 10

What 3 tests give a titer as a result?

Answer 10

• serum neutralization
• ELISA
• indirect immunoflurescence

Question 11

ELISA

Answer 11

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

Question 12

Indirect immunofluorescence

Answer 12

The specimen is the serum antibody

• virus is known and standardized
• fluorescent labeled antiglobulin is used to detect serum antibody bound to antigen

Question 13

Agar gel immunodiffusion

Answer 13

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

Question 14

Double diffusion test

Answer 14

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

Question 15

Double diffusion test is used to determine relationship between _____

Answer 15

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

Question 16

Example of a double diffusion test

Answer 16

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

Question 17

Antibodies to EIA in equines are indicative of infection, no need to do a ______

Answer 17

Titer

- same for all lenti viruses

Question 18

Certain viruses are capable of agglutinating suspensions of ________

Answer 18

Mammalian and avian erythrocytes

- antibodies detected against these viruses may inhibit agglutination

Question 19

Hemagglutination

Answer 19

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)

Question 20

Hemagglutination inhibition

Answer 20

• 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

Question 21

Hemagglutination inhibition titer

Answer 21

Obtained by multiplying the highest dilution of serum that just inhibits hemagglutination by the number of hemagglutinating units of virus involved

Question 22

Problems with hemagglutination inhibition

Answer 22

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

Question 23

It is necessary to absorb the test serum with _______ in order to remove natural hemagglutinins

Answer 23

Erythrocytes

Question 24

False positive may be obtained if there is excessive ______

Answer 24

Delay in reading some hemagglutination inhibition tests

Question 25

HAI titer

Answer 25

The highest dilution of serum (Ab) that prevents hemaggluination

Question 26

What indicates agglutination?

Answer 26

Smooth or jagged shield of cells, or an irregular button