lecture 9

Question 1

ELISA

Answer 1

detection of antigen (SNAP test)

sample w antigens (potentially) + labeled detector antibodies (conjugate) mixed!
capture antibodies to membrane so stuck
sample mixture added across membrane and antibody - antigen complexes bind to the stuck antibodies
wash buffer and enzymes goes back over from device activation and remove unbound antibodies - antigen complexes
colored area appears on membrane when positive

Question 2

immunochromatography

Answer 2

lateral flow assay
detection antibody is conjugated (stuck) to colloidal gold (pink) or colloidal selenium (blue) rather than an enzyme

1. smaple containing antigen applied to sample pad
2. antigen binds to antibody that is conjugated to nanoparticles on conjugate pad
3. immune comlpexes (antigen bound to antibody) are stuck by antibodies - colored line! on antigen capture pad
4. control compare line of antibodies bound to antigens w no nanoparticles
5. unbound excess reagents pass through the absorption pad

Question 3

immunohistochemistry assay

Answer 3

used to detect pathogens in tissues

place enzymatic tagger on antibody into test and if binds to antigen on cell then will see enzymatic color

Question 4

direct immunofluorescence test

Answer 4

FA - using antibody to test for antigen - produce titer

fluorescent tagged detection antibody specific to antigen is poured over sample
wash and examine - any unbound antibodies are washed
read using a fluorescent microscope - fluorescence = positive result of antigen in tissue!

TGE, rota, herpes, rabies

Question 5

western blotting

Answer 5

detect if ANTIBODY present using specific proteins of pathogen/cell of interest

1. separate proteins using electrophoresis - separates by size and charge
2. transfer proteins to membrane
3. probe membrane with addition of patients serum (which has potential antibodies)
4. antibody specific to a protein of the pathogen will show dark band

Question 6

titer

Answer 6

expression of concentration or level of antibodies or viruses
titer testing uses serial dilutions to obtain semi-quantitative information from a series of positive/negative results
titer corresponds to highest dilution factor that still yields a positive reading - higher numbers the better protection - amount of antibodies that will show up in dilution

Question 7

precipitation assay

Answer 7

shows how much antibody to antigen ratio produces optimal zone of proportions (highest precipitate)

Antigen - antibody equivalence produces lattice formation and precipitation
prozone = antibody excess leads to a negative result
postzone = antigen excess leads to negative result

Question 8

principle of radial immunodiffusion

Answer 8

RID - type of precipitation assay

1. serum placed in gel containing an anti-Ig antibody
2. If and anti-Ig precipitate together and form visible ring
3. size of ring is proportional to the [Ig] in serum!

clinical uses:
characterize class of immunoglobulins in hyperglobulinemia (dogs, cats, horses)
IDENTIFY FAILURE OF TRANSFER FOR PASSIVE IMMUNITY (mom to baby)

Question 9

principle of agar gel immunodiffusion

Answer 9

AGID - type of precipitation assay

1. antigen solution placed in central well and diffuses into surrounding agar
2. serum placed into surounding wells (has potential antibody!)
3. where the antigen and antibodies find eachother an opaque white line forms - precipitate! shows zone of equivalence for immune complexes

antigen placed in center well as control to travel to the antibodies in surrounding wells and look for pct line bw circles

used for COGGINS test (equine infectious anemia) -> arched pct line = positive - uh oh call state vet!

Question 10

latex agglutination

Answer 10

latex beads are coated with antibody dog IgG and rolled over serum (containing antibody IgG?) - testing for animals antibodies

positive: clumping or agglutination - patient has anti-dog igG
negative: smooth

can be used to detect rheumatoid factor (RF) in dogs w polyarthritis

Question 11

Coombs’ test (direct antiglobulin test)

Answer 11

agglutination test for anti-erythrocyte antibodies

Immune mediated hemolytic anemia (IMHA)

• production of autoantibodies to RBC membrane antigens will cause hemolytic anemia (adaptive imm problem)
• add antiglobulin (coombs’ reagent) to detect RBC autoantibodies
• if autoantibodies (indicator of disease) present then the RBC will agglutinate/pct after addition of antiglobulin
• use multiple antiglobulin dilutions to account for pro and post zone effects (want to reach equilibrium)
• no agglutination = negative

autoantibodies on dzd patient RBC + antiglobulin against species-specific antibody = agglutination

Question 12

titration

Answer 12

serum dilution are used to measure amount of titer of antibody in the serum

amount of serum compared to the total volume of the dilution - serum:dilution -> the higher the number that still produces result the more protected the animal is against antigen bc has antibodies

measurement of level of antibodies in a serum testing in increasing dilutions of the serum for antibody reactivity (binding to antigen)

Question 13

titer

Answer 13

inverse of the highest dilution of serum that still gives a reaction in an immunological test (antibody to antigen)

compare titer result of test on dog to the parameter and if higher then it is protective, if lower than the dogs system (current vaccination status) is not efficient to defend a pathogen infection - need booster

Question 14

endpoint titer

Answer 14

inverse of highest dilution of serum giving a positive test result

Question 15

making a serial dilution of serum

Answer 15

start w 0.5mL saline per tube and 0.5mL serum into first tube then pipet down line from previous tube - getting ratio

Question 16

most common tests in which titers are used

Answer 16

SN/VN - serum or virus neutralization
HI - heamagglutination inhibition assay
IFA - indirect fluorescent assay
CF - complement fixation
other

Question 17

four fold rise

Answer 17

two serial dilutions
in titer (or greater) from acute to convalescent serum is diagnostic for ACTIVE INFECTION

samples must be tested in parallel and by same lab

Question 18

acute serum is collected

Answer 18

early after onset of illness

initial collection

Question 19

convalescent serum collected

Answer 19

14-21 days later

remember! antibodies show up in system 14 days after antigen in system

Question 20

flow cytometry

Answer 20

measurement of single cells in a fluid stream based off: size, complexity, fluorescence intensity

can be used on multiple sample types:
bacteria/microorganisms
latex beads/nanoparticles
cells from peripheral blood, tissues, CSF

cells suspended through flow chamber and pass through single file, get shot by light source, and sorted into differenet tubes - allow us to see what cells are in serum?

Question 21

cluster of differentiation (CD) molecules

Answer 21

define cell function/subtype (especially in B/T cells)

monoclonal antibodies are available to many common CD molecules (canine CD4, bovine CD11c)

antibodies can be conjugated to diff fluorescent markers and flow cytometry can be used to determine the frequency of labeled populations (antibodies + CD)