Immunological and Molecular Diagnostics Flashcards
1
Q
Immunodiagnostics: Serology and Immunoassay
A
- exploiting immune system for diagnostic purposes - tend to talk about either doing serology or immunoassay
- serology: taking samples from animals and assessing immune system and response - may want to determine pathogen exposure (immune reactivity to that pathogen), vax response, immune disease (pathological Ab’s)
- Immunoassay: could be exploiting the immune system by stealing Ab’s and using them for diagnostic purposes
- labeling Ab’s so that when it binds to its target, you can observe the interaction
- use them against pathogens to see if they are present or other proteins as a biomarker
- can also use these Ab’s for immunophenotyping
2
Q
Sampling the Immune System:
(blood sample/tissue biopsy)
A
- generally taking a blood sample- usually clotted so you can collect serum and send off for measurement
- to look more at CELLS then you would want to look at using anticoagulant (avoid the blood clotting)
- biopsy or asperate is another alternative
3
Q
Serology
A
- quite common in vet practice
- measuring markers of INNATE IMMUNITY
- done in equine practice as a marker for an inflammatory process occurring
- loads of different acute phase proteins that can be measured
- for ADAPTIVE: really measuring Ab’s most often (stable and easy to measure)
- some kits allow us to measure cytokines- used a bit in research but starting to come into clinical practice
4
Q
Measurement of serum antibody
A
- may just want to see if there is an elevation in total Ab present?
- monoclonal gammopathy: Monoclonal gammopathy of undetermined significance (MGUS) is a condition in which an abnormal protein — known as monoclonal protein or M protein — is in your blood. The protein is produced in a type of white blood cell (plasma cells) in your bone marrow.
- MOST OF THE TIME looking for Antigen specific Ig
-good for checking horses coming from different countries: EIA (equine infectious anemia) –> if Ab positive, they are not allowed in the country as this is a virus that goes latent and they are likely persisitently infected
- herd that is BVDV free is needed for a farmer to join a health accreidatation scheme –> can do this by sampling a representative # of animals to see if it has been on the farm (if one has positive result- the virus is around)
- can get proof for appropriate RESPONSE to vax
5
Q
Diagnosis of FPT in Foals
A
- If we have a very expensive foal, want to make sure it doesnt die due to lack of MDA
- there are kits available to check
- can perform stable- side
- There is then a serum to top up if necessary
6
Q
Serum protein Electrophoresis
A
- sometimes use this particular method
- put the sample in the machine and it separates the proteins out depending on size
- left peak is for albumin (a lot in blood)
- right is albumin peaks (of more interest, particularly the gammaglobulin- where all the anitbodies really reside (immunoglobulin))
- Myeloma–> B cell tumor lurking in BM. turning out loads of antibody, from single clonal antibody so there is a very defined peak = Monoclonal gammopathy
-
Polyclonal Gammopathy: chronic persistent infection, turning out Ab much higher than they should be but it isnt curing the issue
*
7
Q
Measurement of antigen specific Ab
A
- Depends on what we are measuring to deem most appropriate
8
Q
Serology to determine response to vaccination
A
- make sure that animal has responded to vaccination
- you used to HAVE to do for rabies, but EU said stop to have comparibility across regions
- it is still highly recommended though!
9
Q
Serology testing for infection
A
- need to know what stage the animal is in that whole process to take effective sample
- acute phase of illness: antigen has caused enough tissue damage to cause clinical sign
- In comparison: antibody present shows a different profile
- usually in first 7 days, you wont be able to measure Ab as the plasma cells won’t be making antibodies yet
- Every day there is more Ab in the body and then there is a stable amount of antibody in the body and then the plateu phase lasts depending on the type of infection
- Diagnostic testing in an acute infection (e.g. puppy with parvo) - not helpful taking bloods for Ab due to lag, but will have to go to site of infection and look for pathogen. presence of antigen or DNA/RNA depending on pathogen
- past this point we can start testing for serology
- If you get a low titre of the thing you expect, test again 2-3 weeks later to check as the profile may go from log phase to plateau (dont want a false positive)
10
Q
Diagnostic testing
A
- careful when you use serology: because it is negative does not mean it is not infective (may be in the lag phase)
- Ab negative does not mean pathogen negative
- If we get a high titre, then that means that the animal was exposed to the pathogen, but not a good indicator of WHEN (days ago, weeks, years?)
- SO, we use ELISA a lot
11
Q
Using ELISA to determine exposure to a pathogen
A
- can measure antigen in biological samples in the early stages of infection or antibody in the blood
- Remember a negative serology does not mean it is not infected!
- if you look for antigen/pathogen DNA after 5 days then may get false negative–> may be in log phase. serology would be more appropriate
12
Q
ELISA method for detection of Ab in serum
A
- need to put antigen in bottom (need to know what we are looking for)
- then add patients serum at a specific dilution and allow it to bind (water off anything that didnt bind)
- Add in our detection reagent which is antibody conjugated with an enzyme
- E in ELISA stands for enzyme
- add substrate and it will change color depending on if it is positive or negative
- but this only does tell us if it is + or - (amount of color isnt really quantitative) –> like a SNAP test
13
Q
Ab titre
A
- = DILUTION FACTOR
- titre helps us quantify a positive result
- how positive??
- keep testing each dilution until it is no longer positive
- titre that you report out is thae last postive dilution
- ex: serum A can be diluted out to 1/256 where as serum B can be diluted out to only 1/16
- but it is NOT a fraction! –> it is a dilution factor, so higher the number, the more Ab there is
14
Q
Immunofluorescence assay (IFA)
A
- way to identify things
- rather than enzyme, can use fluorescence detection on Ab
- typically better for more solid samples (tissue)
- ELISA is good for liquid samples (serum)
15
Q
Virus neutralisation test (VNT)
A
- absence of specific Ab
- bit more complicated to perform
- setting up a “mock” infection in the lab with the virus
- seeing if the animal can provide infection (having Ab from previous exposure) - rabies can be tested this way
- cells grown in culture that the virus can infect, deliberately infect the cells and will replicate to kill the cells
16
Q
Cytopathic Virus
A
- kills the cells after replicating in them
- as part of VNT
17
Q
Cytopathic Effect
A
- can see the cell death
- left: unaffected culture
- Right: affected culture–> some of the cells have died off and there are inclusion bodies present
18
Q
Positive Result of a VNT
A
- For the test, we bathe the cells in the patients serum before adding in the infective virus
- If that animal has made that specific virus antibody due to previous exposure then they will neutralize the virus and prevent it from infecting the cells
- they will remain alive! rather than in a negative result where the virus would infect and kill
19
Q
VN vs. ELISA
A
- VN tells us that not only is there an immune response present but it is actually PROTECTIVE
- If we see virus neutralization happening in the test tube, we can be pretty sure they will neutralize the virus in the animal
- can’t really be sure with ELISA (postiive in ELISA does not mean those Abs are protective or effective)
20
Q
Evaluation of T Cell responses
A
- Tend to measure Ab’s in serum bc they are quite robust
- can spin it down, send it to diagnostic lab and it will probably be ok by the time it gets there (3 or 4 days)
- it is only measuring one side of the adaptive immune system… so how do we measure T-cells? the cell mediated immunity? (would not last to be sent to lab..the cells will have died by then) - couldnt do a fucntional assay if sending by post
- but if you can get them there quickly enough: take the lymphocytes, stimulate them with antigen and look for presence of activation (express new things on cell surface, proliferate, but mainly produce cytokines!)
- if you add antigen and then see cytokines, they are reacting. and if they are reacting, they must have seen that pathogen in the past
21
Q
Bovine TB gamma- IFN test
A
- blood test for TB
- must send off very quickly!
- will culture it with mycobacterium antigen and then measure INF- gamma prodcution
- If the cytokine is produced, then antigen must have been present before as it is a reactor
22
Q
Immunodiagnostics for Allergy
A
- maybe we arent dealing with an infection based process
- we can measure IgE specific for allergies (this only picks up a type I!) or intra-dermal skin testing (can pick up immediate or delayed)
23
Q
Allercept Immunoassay
A
- lots of allergy tests out there
- this is the best one!
this is actually very specific for IgE - youll often get IgG produced to stuff due to environment (ex: things you inhale)
- IgE is the one that will cause you problems, not IgG, as this is what covers your mast cells
- This helps to avoid giving a false positive with IgG present as IgG is not pathogenic, so this is specific for IgE
- stick different allergens in different wells and then add serum from patient
- wash off after binding
- detection system is actually a cloned version of the Fc-epsilon receptor (which is actually the thing that is normally present on mast cells to capture IgE)
- recombinant protein that we can label and it does not bind to IgG
- add substrate which will then change color and tell us which well the IgE was binding to (dust, fleas, etc.) by intensity of color
24
Q
Caution: tests that measure IgG
A
- There are a lot of allergy tests out there
- -some of them aren’t good!
be wary of the ones saying they can detect food hypersensitivities… hasnt really been proven to be true
25
Q
Intradermal skin testing
A
- can be done often instead of serology
- inject different allergen at each site and then a bit later measure each site
- good way of detecting immediate type hypersensitivity
26
Q
Intradermal skin test for DTH
A
- Delayed type hypersensitivity
- more like a Type IV (cell mediated hypersensitivity)
- can’t just measure it after a coffee (have to do a few days later)
27
Q
Tuberculin Test for TB
A
- delayed type hypersensitivity if present
- what we do in people
- looking for influx of inflammatory cells and T-cell reaction
- in cattle, we compare both avian and bovine to make sure we arent killing cattle that have just been exposed to pigeon mycobacterium TB - want to make sure they are actually reactors to M. bovis
28
Q
Immunodiagnostics for Autoimmunity
A
- dogs are the main one that get autoimmunity
- IMHA (immune mediated haemolytic anemia)
- ANA= anti-nuclear antibody test
- SLE: systemic lupus erythematosus
- acetylcholine antibodies in myasthenia gravis
- Thyroglobulin autoantibodies for hypothyroidism
29
Q
Antibodies as detection reagents
A
- can buy these commercially
- raised against certain antigens (sometimes a pathogen or biomarker)
- or a cell surface marker for immunohistochemistry (BVDV in PI calf ear notch sample)
- quite often can use antibodies to diagnose and detect infection
30
Q
Detecting Infection
A
- Using grown antibody to detect an infection or antigen in sample for diagnosis
31
Q
SANDWICH ELISA method for detection of ANTIGEN
A
- use a capture antibody that is specific to the thing that we are looking for
- canine parvo virus Ab can be used as base
- then add serum from animal having hemorrhagic gastroenteritis
- let it bind, wash off what isnt bound, then need a second Ab with enzyme label (wash off what isnt bound) and then add substrate to look for a color change)
32
Q
Immunohistochemistry: ELISA on a slide
A
- ELISA needs a liquid sample
- If it is a tissue sample, we can use the same principal, but rather put the tissue biopsy sample on a slide
- put on antibody that recognizes the pathogen, is enzyme labeled
- Add substrate and then observe for color change on the slide
33
Q
Immunohistochemistry: BVDV
A
- ELISA on a slide is how we check for peristenetly infected calves of BVDV
- then need to label the calves if they are positive
- Use BVDV antibody to label and then substrate will turn it a pink/brown (right)
34
Q
Direct IFA
A
- Rather than an ezyme, we can put a fluorescent marker on it
- ex: leptospira organisms in urine
35
Q
Using labelled Ab’s for measurement of a biomarker
A
- we can use an antibody to look for proteins, not just pathogens!
- ex: FeLV/FIV Snap Test
- or CLP: canine pancreatic lipase (can tell you if they have pancreatitis)
- Some of these tests can be more complicated though where you need a whole kit and machine (chemiluminescence and radioimmunoassay) - uses Ab’s to measure things in different blood samples. just a bit more complicated than SNAP test
36
Q
Immunophenotyping
A
- H & E stain and then the use of specific Abs to identify different cell types
- Better than going with just a visual under the microscope as this will tell you exactly what the cell is
- ex: can tell whether it is a T cell or B cell lymphoma
- T-cell lymphoma has a much worse prognosis!
- Sometimes can use for malignant tumors and really recognize what cell source they are from as it will be difficult to tell alone
- ex: melanoma (Ab will cause cells in sample to stain brown)
37
Q
Molecular Diagnostics (pathogen)
A
- Molecular Diagnostics can be used alongside immunoassay to diagnose samples and pathogens
- using much more molecular than immunological assays now
- We can identify pathogens specific code of nucleic acid, typically DNA, but also need to adapt this technique so we can pick up foreign RNA in the sample (for RNA viruses as well)
- using quantitative PCR method
- using primers and probes that are very specific to the code that is unique to that pathogen
- or sometimes: we are doing whole genome sequencing and then attempting to match those with a pathogen
38
Q
Genotyping Pathogen and Epidemiological Studies
A
- we use this not to just identify species, but also to find out a little bit more about those organisms
- allows us to sequence pathogens VERY SPECIFICALLY
ex: different strains of Influenza virus or the toxin producing strains of E.Coli (VTEC) - Helps us to track infection as well- sequence to help us locate the spread (ex: foot and mouth disease outbreak in the population or if a farm recently acquired TB in the herd, where has it come from?)
39
Q
Molecular Diagnostics (Host)
A
- Sometimes we are looking at DNA in the animal itself, not just foreign nucleic acids
- can look for mutations
- genetics is becoming more common with companion animals- need to see if they are prone to disease or have possible mutations
- Monogenetic disorders are caused by a mutation in a single gene. The mutation may be present on one or both chromosomes (one chromosome inherited from each parent)
- penetrance: if you have that particular susceptibility gene, do you always get the disease or only 10% of the population that has it shows disease?
40
Q
Diagnostics- Host: PCR based techniques
A
- once we have amplified it, we need to analyze it
- is it normal? abnormal? –> different systems to do this
- sometimes just big or small? –> gel electrophoresis
41
Q
Use of DNA genotyping
A
- becoming much more common, requested by clients
- need to know about this as owners will always come to you having looked it up
- If there is a breeding program, you may want to try and rid of a disease by doing this before breeding
42
Q
Mast Cells and KIT
A
- Mast cell tumors have a mutant version of KIT (stem cell factor receptor)
- the ones with the KIT mutation tend to be more resistant to chemotherapy
- has a large insertion mutation
- amplify it and then run on gel
- mutant one is too big, won’t run as far on electrophoresis and therefore most likely has an insertion mutation and most likely a worse prognosis
