Immuno - Final 1

drraythe's version from 2015-06-15 01:25

PowerPoint 12

Question Answer
Big signs of cancer in a needle aspirate?Big cells w/ big nuclei & mitotic figures in the nuc.
Why would they test a needle aspirate for CD3 & CD79a?They are T cell & B cell markers, respectively. Try to find the origin of the neoplasm
Does neoplasm = cancer?No, it just means new growth
First thing to ask yourself about an immuno-diagnostic test?Does it test ANTIBODY or ANTIGEN?
If tests antibody, it means...It means EXPOSURE (not infected or diseased)
If tests antigen, it means...It means you are finding AGENT (animal IS infected, doesn't mean it's diseased though unless it's showing signs)
What is serology?Measurement of antigen-antibody interactions for diagnostic purposes (USUALLY MEANS were trying to detect antibody in serum)
What interactions are serologic tests based on?Ag-Ab interactions
Basic mechanism of a serologic test?The basic mechanism of these tests is the detection of an unknown reactant (Ab) in body fluids by using a known reactant (Ag) … or vice versa (i.e. use a known Ab to detect an unknown Ag.)
Uses/benefits of im-diag tests? (3)1) CONFIRM OTHER TESTS
2) Antibody detection is test of choice for detecting EXPOSURE
3) SERUM is usually much safer than infected tissue
Is an immunodiag test qualifying or quantifying?Both - CAN BE QUANTITATIVE
How does an immunodiag test ID infectious agents?Uses an antibody to the agent
How does an immunodiag test monitor exposure?Monitor levels of antibody to the antigen over time
What can you type w/ an immunodiag test?Type tissues & blood
How can you classify types of neoplasms for therapy?Detect CD molecules
Can you ID tumors w/ immunodiags?Yes, by their antigens
How can you do surveillance w/ immunodiag test?Can test prevalence of exposure to agent
Can serosurveys be retrospective?Yes
How accurate is the immunodiag test?Very specific...lock & key fit. (Cross reaction CAN happen tho)
What can the ag-ab interaction be affected by?Forces of attraction, *concentration of the antigen & antibody
What is titration?Serum dilutions to measure the amount of Ab - HOW WE QUANTIFY AMT OF ANTIBODY
What is titer?Reciprocal of the highest dilution of a serum sample that did give a positive reaction
Ex) 1 mL serum + 10mL diluent =? What is its reciprocal?1:11...recip. is 11 (so the titer is 11 in this example)
2 examples of detection methods to know if a titer is a pos or neg?Precipitation or agglu test / ELISA detection
What does ELISA serum test look like?(Titer-well covered w/ known antigen) have positive control & neg control, look for color change of reagent sample & compare to pos & neg control
Can ELISA detect Ag or Ab?Can detect either!
If there is a 1:4 titer & a 1:512 titer, which has more Ab?The 1:512
What does a Primary Binding Test measure?Directly measures binding of antigen to antibody in vitro (ex: ELISA)
What does a secondary binding test measure?Measures RESULTS of antigen-antibody interactions (ex: agglu)
Of primary & secondary binding test, which is more sensitive?Primary
What is serum?Top fraction of blood AFTER clotting (contains serum proteins, no cells, no clotting factors)
Test samples are? (4)Serum, body cavity fluids, other fluids (CSF, synovial...), cells/tissues to probe w/ antibody when looking for antigen (whole blood, tissue section)
What are reagents?"A substance or mixture for use in chemical analysis or other reactions." examples: complement, polyclonal antibody, monoclonal antibody, anti-globulins (antibodies to antibodies - polyclonal or monoclonal), anti-serum (blood serum containing antibodies against specific antigens)
What is complement? Most efficient in hemolytic test?Normal constituent of all fresh serum. Fresh, unheated guinea pig serum is the most efficient in hemolytic tests
Poly vs mono clonal?If antigen has 4 diff epitopes - then the serum is polyclonal. If we want monoclonal, we use mouse spleen, to separate cells into singular antibodies, & make abs that recognize a single epitope only
What should you do every time for every test?Do a pos & neg control
What are the 2 kinds of immunofluorescence assays?Direct & indirect
What are the immunoenzyme assays?ELISA, Western Blot, Immunohistochemistry
Direct FA detects what? How?ANTIGEN. Detect by fixing Ag to slide, have flurochrome-labeled-Ab which is designed to detect an antigen, read by looking for fluoresce of green
Indirect FA detects what?ANTIBODY. So start w/ known antigen, add sample (serum)...if serum has antibody it attaches, then add fluorochrome-labeled-anti-Ig & read to see if positive
ANA test (anti-nuclear-antibody-test) is dir or indir? Looking for?Indirect. Looking for antibody against nuclear material
How is ANA test set up?Nuclear material is known (usually rate liver tissue) fix tissue to slide. Treat it so antibody can get into the cells, add pt serum - see if pt has Ab against nuc material. If there is Ab it will stick & not wash away, then have anti-globulin tagged w/flour tag + look under scope
Pos & neg controls of ANA test would be?Serum from known neg, serum from known neg
ELISA test. How are wells set up?All wells coated w/ antigen (if you want to detect Ab)...opposite if you wanna detect antigen.
ELISA means?Enzyme linked immunosorbant assay
If wells are coated w/ a known Ag in an ELISA, what do you do?Add pt serum w/ Ab to well. Wash off unbound Ab. add 2* antibody (antiglob.) which is conjugated w/ enzyme. Wash. add substrate to enzyme & look for color change
Antigen capture ELISA detects what? esp?Detect antigen. ESP heartworm antigen
Antigen capture ELISA setup?Well coated w/ known antibody, sample added, was off unbound antigen. Add antibody specific for antigen which is conjugated w/enzyme. Wash off unbound antibody. Add substrate, look for color change
What is a western blot used to ID?ID specific protein antigens in a complex Prot. mixture. OR id specific Ab to known protein antigen - DETECTS PROTEINS!!!!
How to do western blot?Take protein, apply to electrophoresis, separate complex protein into separate proteins, blot onto nitrocellulose paper (they stay put) then we can probe w/ specific Ab to detect antigen, or probe w/ pt serum (has antibodies) to detect antigen. Either way, probe, then wash, then use detection system (like antiglobs labeled w/ an enzyme)
Common things Western blot is used to detect?HIV, FIV, Glanders (burkholderia mallei)
Positive result of western?Looking for a band on the paper
How can you do a very specific allergy test?Western for pt serum to have IgE against allergen protein
ImmunoHISTOchemistry (IHC) what is being detected?(So looking for an ANTIGEN) Ab against particular antigen can be conjugated to an enzyme & used to detect specific antigen in TISSUE SECTIONS
Most common enzyme used in Immunohistochemistry (IHC)?Horseradish peroxidase
How do you do an Immunohistochemistry (IHC)?Tissue section treated w/enzyme-labeled antibody, washed, incubated w/enzyme substrate to cause a color change wherever ab is bound to ag (brown color), can visualize using a light microscope for color change
What is the neg control in IHC?IHC w/ irrelevant control antibody. (Or a diff, unrelated section of tissue)
(CQ) Polyclonal goat anti-bovine IgA means?Goat antibody from several B-cell clones that all recognize epitopes of bovine IgA
(CQ) Polyclonal origin means what about where it came from?More than 1 B-cell
(CQ) Direct FA on a cow for BVD virus was neg, this means...The cow did not have detectable BVD virus in the blood
(CQ) What does titer specifically test for?Titer = antibody
(CQ) Direct FA on blood from cow for BVD virus was done, the cultured cells fluoresces, what is the correct interpretation of the test?Cow as detectable BVD virus in blood this is a positive test result
(CQ) Direct FA test for rabies on a skunk’s brain came back as positive. What does this mean?Skunk has rabies virus in its brain
(CQ) An ELISA plate is coated w/parvo virus Ag. Dog’s serum is added to well & wash. Next step is...add enzyme-labeled mouse anti-dog antibody
(CQ) The next step in an ELISA after washing is to add substrate to the enzyme. Color changes yellow in well where dog serum is added. Means...Dog’s serum contains antibody to parvo virus
(CQ) A section of liver from a lamb is prepared for IHC & is probed w/mouse monoclonal antibody for an antigen, coxiella. Fetal liver shows color change in come cells after enzyme added. This means..Fetal liver contained antigen to coxiella
Precip test summaryAbs combine w/ SOLUBLE AGs in solution of gel, resulting complexes may precipitate
Agglu test summaryIf Ags ARE PARTICULATE (bact, or RBC) then Abs may make them clump/agglu. (ex: coombs)
Neutralization test summaryEstimate ability of Ab to neutralize the biological activity of an antigen, when mixed w/ it in vitro
Complement fixation...Lysis summaryIf Ab can activate classical complement pathway & the Ag is on the cell surface, cell lysis may result
Precip tests require what type of antigen? What is the precipitate?Soluble antigen. Ab-Ag complexes
What is the zone of equivalence?When there is a good conc of Ab & Ag (approx. equal) promotes maximal binding, so you can see the precip in a precip test
What is the pro & post zone?In precip test. Pro =Ab excess Post =Ag excess (can’t visualize precip either way)
What is double immunodiffusion?In agarose gel wells - center has Ag of interest. Ag diffuses out & gets less & less concentrated...put serum samples in wells surrounding center. Serum will also diffuse (& get less concentrated) when Ag & serum meet in zone of equivalence, you see a line of precip. **Guarantees zone of equivalence
What can double immunodiffusion be used to test?"Agar gel immunodiffusion test" coggins test - test for ANTIBODIES TO equine infectious anemia virus (EIAV)
What is AGID?Agar gel immunodiffusion test - type of double immunodiffusion test
AGID test is more _________ but less _________ than ELISAMore specific, less sensitive. It is a SPin test. Rules IN a disease, may have false negatives.
Radial immunodiffusion is...Specific AB incorporated evenly though agar (same conc) (the KNOWN) then test sample solution placed in well & allowed to diffuse out (the UNKNOWN). Precip will form around antigen well where OPTIMAL proportions an AG & AB meet. (DIAMATER OF RING =AMOUNT OF antigen) SO CAN QUANTIFY!!!
Is radial immunodiffusion is qualitative or quantitative?Quantitative
Ex of radial immunodiffusion?Test failure of passive transfer in foal - quantitative measurement of how much IgG
Agglu test is...Look for agglu/clumping since Abs can cross-link & form clumps. ALSO dependent on conc of Ab & Ag
Ex of agglu test?Coombs test (ab on RBCs of animal?)
Most efficient Ab for agglutinating?IgM
What particles can you use in agglu?RBCs are commonly used, come bacterial cell surfaces, & latex beads coated w/ things (Ag, Ab, etc)
If use RBC in agglu test, its calledHemagglutination test
Agglu can detectUnknown antigens, or detect or quantity circulating antibodies
Can you use agglu to determine titers?Yes. Determine highest dilution that still causes clumping
Ex of what agglu test can be used to test for brucella canis?Whole killed bact on card -add animal’s serum. Have a positive control. If serum has Ab, it will clump
Latex bead agglu works how?Coat bead w/ Ab to particular antigen. Beads clump around antigen & visualize agglu
Eex of what latex agglu can be used to detect?Test to detect rheumatoid factor in dogs
Hemagglu test was figured out when ppl saw...Some viruses naturally can attach to RBCs. can use this to develop hemagglu test.
Hemagglu done by...Red cells sensitized w/ Ag. Fix Ag on RBC surface. React w/ pt serum. If pos, complexes on surface of RBCs cause them to clump
Hemagglu determines specific what?Specific antibody
Coombs is a kinda test?Anti-glob test (ex of hemagglu test)
What is a hemagglu inhibition test?ID antibodies against a virus that can cause RBC aggly -so ass ab & see if we can inhibit agglu. (IF NO AGGLU, THAT'S A POSITIVE)
Neutralization tests do what?Estimate ability of Ab to neutralize bioactivity of Ag when mixed w/ it in vitro
Serum neutralization test is used to detect what?Abs in pt serum that neutralizes viruses/bact toxins
How is serum neutralization test done?Living cell systems inoculated w/ serum w/ Ab & w/ Ag - if neutralizing ability intact, no adverse effect to this test
How specific & sensitive are serum neutralization tests?Highly for both
Ex of serum neutralization test?BVDV SN test for antibody
Positive result of serum neutralization test?NO cell lysis
(CQ) ELISA wells coated w/ antigen, & animal serum dilutions of 1:2, 1:4, 1:8, 1:16, & 1:128. If a positive control well turns yellow, & yellow change in 1:2-1:16, what is the interpretation of the test?The cat has an antigen antibody titer of 16
(CQ) Animal might be infected. Pigs serum incubated w/ antigen infected cells, cells then probed w/ florescent anti pig IgG, test designed to detect what in pt?Antibodies
What is "the RFFIT"RFFIT = Rapid fluorescent foci inhibition test
What is RFFIT used for?Rabies serology test for vaccine titers in people
What type of test in the Rabies RFFIT test?Serum neutralization (inhibition) test.
What does Rabies RFFIT test?Measures ability of rabies-specific antibodies to neutralize rabies virus & prevent the virus from infection cells
What are the antibodies tested for in the Rabies RFFIT test?RVNA "rabies virus neutralizing Abs"
How diluted is the serum for the Rabies RFFIT test?5 fold (one part serum, 4 part dilutent), then further
What is the serum dilutions mixed w/ in the Rabies RFFIT test?Mixed w standard amt of live virus (slide 94)
Equation of sensitivity of a test?Sensitivity of a test = (# of true positives / # w/ the dz (TP+FN) ) / x100 = %
Equation of specificity of a test?Specificity of a test = (# of true negs / # w/o the dz (TN+FP) ) / x100 = %
When would you want a test w/ high sensitivity?You can trust the negatives (test positives may not be reliable) DON'T WANT TO MISS ANY POSITIVES ex: blood transfusion test for aids
When would you want a test w/ high specificity?You can trust the positives (test negatives may not be reliable) (can do specific test after a sensitive to see for sure)
If you are testing cattle for brucella abortus, do you do sensitivity or specificity first/second?Initial = high sensitivity, second = specific
If ELISA FIV test shows positive, what do you do next?That was a sensitivity, now do a specificity (Western blot)