Test 1: lecture 12 and 13 testing

Question 1

antibody test shows evidence of ___

Answer 1

PIE (protection (vaccine), infection or exposure)

Question 2

two main ways to make definitive diagnosis of an infection with an antibody test

Answer 2

4-fold increase in titer of non-class specific Ab

or

Detection of IgM antibodies (ideally paired with IgG)

Question 3

___ is the highest dilution of an assay to give a positive reaction

Answer 3

titer

Question 4

•Detecting a four-fold rise in titer requires___

Answer 4

acute and convalescent titers

2 weeks apart

(titer is the highest dilution that gives a positive reaction→ at lower dilutions they clump together and fall to bottom, at higher dilutions there are so little that they are all spread out, not clumping)

Question 5

are titers absolute or relative value

Answer 5

relative (has no unit)

Question 6

elevated liver and kidney values are common with ___

Answer 6

lepto

run agglutination antibody titer assay and again 2 weeks later

4x increase is positive for lepto

Question 7

___ indicated recent exposure or infection

Answer 7

IgM

Question 8

___ represents previous exposure

Answer 8

IgG

(secondary response)

Question 9

antigen test

Answer 9

• Detects a SPECIFIC antigen of a pathogen.
• In the presence of clinical disease detection of an antigen is considered diagnostic.
• Can cross react with similar targets (from closely related pathogens for example).

antigen titers can increase with treatment

Question 10

what is one down fall of the antigen test

Answer 10

pathogens can be very closely related

therefore antigen test can have cross react → react to wrong antigen

Question 11

weird shape of nose, run cryptococcus ___ test and it is + at 1:8. Treat. Rerun test and it is + 1:16. why the increase after treatment?

Answer 11

antigen test

by treating the fungus, it is broken down leading to more antigen and more response by body = swelling and higher antigen test results

antigen titers can rise with treatment

treat until you get 0 on titer

Question 12

what does a negative antigen test show

Answer 12

pathogen not there

antigen present, but not enough to detect

antigen present but bonded to antibody

technical error

Question 13

what does a + antigen test show

Answer 13

pathogen present

antigen present but viable microbe absent

cross reactive antigen present from another pathogen

technical error

poor assay specificity (false +)

Question 14

negative antibody test →

Answer 14

exposure to pathogen did not occur

too early in course of infection

severe immunosuppression

technical error

poor assay sensitivity (false negative)

Question 15

positive antibody test _>

Answer 15

previous natural exposure to pathogen

previous vaccination

cross- reactive

technical error

poor assay specificity → false +

Question 16

seronegative

Answer 16

used to screen blood donor

immunoassay studies are 0

antibody test → dog was never exposed to this pathogen and carries no antibodies for these diseases

Question 17

what kind of test do you use for blood donors and why?

Answer 17

antibody→ cast broad net

will show if every exposed, previous immunization

don’t use antigen→ cause antigens at very low levels can’t be detected and some diseases can lead to long term effects that would exclude the patient from being a blood donor

Question 18

why use titer for vaccination status

Answer 18

• Assess status prior to booster
• For Animals with Concurrent disease
• Assess immunity at > 24 weeks
• Unknown vax hx
• Assess prior to breeding
• To determine if animals are genetic “non-responders”
• Test in face of outbreak for quarantine purposes

Question 19

IFA (immunoflourescent Ab)

Answer 19

slide with antigen are covered with antibodies that have been linked to specific fluorescent, if the smear contains the antigen the corresponding antibody will bind, wash away anything unbound, use dark field microscopy to see what sticks

• Used to detect presence of a specific antigen directly in a sample (tissue or smear).
• Slide is incubated with fluorescently labeled Ab and the unbound Ab washed away.
• Darkfield microscopy with ultraviolet light is use to detect bound antibody.
• Can also be used for tumors, cell types etc.

Question 20

___ is a test where a slide with antigen are covered with antibodies that have been linked to specific fluorescent, if the smear contains the antigen the corresponding antibody will bind, wash away anything unbound, use dark field microscopy to see what sticks

Answer 20

IFA (immunofluorescent antibody)

Question 21

ELISA

Answer 21

enzyme linked immunosorbent assay

•Either Bidirectional Flow ELISA or Immunochromatography for in-house assay.

Question 22

indirect elisa

Answer 22

antigen bound to bottom of well

add serum from patient (may contain antibody)

wash away extra stuff

enzyme linked secondary antibody (or antiglobulin→ antibody that binds to antibody)

wash away

add chromogenic substrate that binds to secondary antibody

color change→ antibody was in serum = +

Question 23

sandwich ELISA

Answer 23

•Capture Ab is coated on plate and Ag in sample is bound.••Unbound antigen is washed a detection Ab is added.••Finally a labeled antiglobulin is added for detection.

sandwich looks for antigen

capture antibody is coated on bottom of plate

add serum that might have the antigen in it

wash away every thing else

add enzyme linked antibody to that antigen we are looking for

wash everything else away

add labeled anti-globulin ( antiglobulin→ antibody that binds to antibody) that reacts to 2nd antibody(changes color)

Question 24

what type of ELISA is for antigen identification

Answer 24

sandwich

•Capture Ab is coated on plate and Ag in sample is bound.••Unbound antigen is washed a detection Ab is added.•Finally a labeled antiglobulin is added for detection.

Question 25

•Capture Ab is coated on plate and Ag in sample is bound.••Unbound antigen is washed a detection Ab is added.••Finally a labeled antiglobulin is added for detection.

Answer 25

sandwich elisa looking for a specific antigen

Question 26

* Ab is detected in sample when it is bound to antigen that is coated on the plate. * Plates are washed of unbound Ab. * Secondary Ab (or antiglobulin) with reporter is bound to sample Ab.

Answer 26

indirect ELISA | (looking for antibodies)

Question 27

Bidirectional flow ELISA

Answer 27

snap test matrix is pre-coated with specific antibodies add sample, that has enzyme linked antibodies added to it if antigen is present in sample will bind to the antibodies on the matrix and the enzyme linked antibodies will keep it from getting swept away the substrate then moved across the cleaned matrix and blue dot = +

Question 28

competitive ELISA

Answer 28

color change is **inversely proportional** to AB or Ag level no color change = + well is bound with antigen, add sample, if sample has antibodies will bind to the bound antigen, then add lab derived enzyme linked antibodies, if there are no antibodies in the original sample lab Ab will bind and if there was antibodies in the original sample lab Ab will not bind if lab sample Ab bind there will be a **color change = negative**

Question 29

\_\_\_ has color change is inversely proportional to Ab level in serum

Answer 29

competitive elisa negative result = color change positive result = no color change

Question 30

why use competitive elisa

Answer 30

not species specific other antibody ELISA need antiglobulins that are against the species Ig where competitive just needs lab derived antibody

Question 31

AGID

Answer 31

* Based on the passive diffusion of soluble Ab and Ag through agar gel. * When “positive” precipitation occurs. * Inexpensive but subjective. 4= negative **put lab derived antigen in the center, in wells around put +/- control and serum** **precipitation line = +**

Question 32

serum neutralization

Answer 32

* Typically used for viral diagnosis. * Observation of virus challenged cell cultures in the presence of test serum. * If cells **do not become infected** then there **is antibody** in the serum. * Results can be titered. have specific cell line, add in virus, if the cells become infected → no antibodies in cell line. If cells do not become infected → + antibodies

Question 33

have specific cell line, add in virus, if the cells become infected → no antibodies in cell line. If cells do not become infected → + antibodies

Answer 33

serum neutralization used for viral diagnosis

Question 34

complement fixation

Answer 34

if serum has antibodies, RBC will pellet and no lysis have serum, heat it up to kill complement add lab derived antigen, if serum has antibodies already present will bind then will add lab derived complement if Ab:Ag present, complement will bind then add in sheep RBC and antibodies to sheep RBC if complement is bound because antibodies are present in original sample→ RBC will settle into a pellet, no lysis (complement was used up → nothing happens to added RBC)

Question 35

have serum, heat it up to kill complement add lab derived antigen, if serum has antibodies already present will bind then will add lab derived complement if Ab:Ag present, complement will bind then add in sheep RBC and antibodies to sheep RBC if complement is bound because antibodies are present in original sample→ RBC will settle into a pellet, no lysis (complement was used up → nothing happens to added RBC)

Answer 35

complement fixation test