Immunology Lab 3

Question 1

serum Ab titer

Answer 1

measure of relative strength and/or amount of specific Ab in a sample (typically serum)

Question 2

serial dilutions

Answer 2

series of sequential dilutions of a substance in a solution

Question 3

How are dilutions expressed?

Answer 3

volume of the sample in relation to the total volume

Question 4

total volume

Answer 4

volume of sample plus the volume of the buffer/diluent

Question 5

How do you make a 1:5 dilution of a 2mL sample?

Answer 5

2+x = 10
x = 10-2
x = 8 mL

add 8mL diluent to 2mL sample

Question 6

In a two fold (2X) dilution series where tube #1 contains a 1:5 dilution, what dilution will tube #6 contain?

Answer 6

1:160

Question 7

Which test is more sensitive - agglutination or ELISA?

Answer 7

ELISA - by a lot!

Question 8

What might an increase in titer over a two week period mean clinically?

Answer 8

this might indicate that the patient is fighting an ongoing infection

Question 9

What dilution factor would you use for low-titered sera?

Answer 9

two-fold (2)

Question 10

What dilution factor would you use for high-titered sera?

Answer 10

ten-fold (10)

Question 11

ELISA

Answer 11

enzyme-linked immunosorbent assay (uses enzyme-substrate), typically chromogenic (color-producing)

Question 12

RIA

Answer 12

radio-immunoassay (uses radioactive isotopes)

Question 13

RAST

Answer 13

radioallergosorbent test (uses radio-labeled antiglobulin)

Question 14

immunofluoresence

Answer 14

self-explanatory (uses fluorochrome)

Question 15

Advantages of ELISA

Answer 15

1. Pretty damn sensitive (not as much as RIA)
2. Inexpensive
3. No radioactivity necessary
4. Portable (for field use) (think SNAPs, pregnancy, etc)

Question 16

Clinical Applications of ELISAs

Answer 16

1. Monitor vaccination status, quantify immunoglobulins
2. Detect wide range of viruses and antiviral Abs
3. Detects bacterial and parasitic infections (salmonella, heartworm, trichinella, brucella)

Question 17

Most common anti-globulins (3)

Answer 17

alkaline-phosphatase
peroxidase
beta-galactosidase

Question 18

TiterCHEK

Answer 18

the ELISA we used in lab to look at antibodies specific to canine parvovirus and distemper virus

Question 19

Two Main Ways We Can Use ELISAs

Answer 19

1. Quantification of Specific Antibodies (antiGEN is adsorbed to well and you add serum to determine if the patient has Ab specific for that antigen)
2. 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)

Question 20

Wash Steps in ELISA

Answer 20

important for the removal of unbound antibody/enzyme-linked so that not everything pops up positive! (color development in the negative control)

Question 21

Why would you determine titer prior to revaccination?

Answer 21

avoid potential adverse immune reactions

negative titers indicate the need for revaccination (less than 16 for distemper or less than 80 for parvovirus)

Question 22

Titers in Clinic

Answer 22

monitor maternal Ab, tailor vaccine programs, avoid reimmunizing high risk breeds or individuals (ie cancer/chemo/allergies/etc), check unknown vax hx

Question 23

fluorochrome

Answer 23

molecule or dye that emits light in the visible range following excitation of light in the UV range

Question 24

fluorescein

Answer 24

green fluorochrome

Question 25

phycoerythrin

Answer 25

red fluorochrome

Question 26

2 Systems for Detection of Immunofluoresence

Answer 26

1. Fluorescence Microscopy
2. Flow Cytometry

Question 27

direct fluorescence

Answer 27

antibody labeled with fluorochrome reacts directly with antigen on a specimen

example: rabies

Question 28

indirect fluorescence

Answer 28

anti-immunoglobulin labeled with fluorochrome binds to an unlabeled antibody that is specific to the antigen on the specimen

example: systemic lupus erythematosus

Question 29

Flow Cytometry

Answer 29

label cells/microbes with fluorescent tagged Ab and then use an instrument to determine the percent of cells expressing the fluorescent molecule

Question 30

Advantages of Flow Cytometry

Answer 30

1. Can evaluate many cells in a short time
2. Can use different fluorochrome tags to evaluate different molecules of interest at once
3. Can isolate pure populations of cells
4. Can evaluate relative density of the cell surface molecules based on staining intensity