Immunological and Molecular Diagnostics Flashcards
(42 cards)
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
Intradermal skin testing
* 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
Intradermal skin test for DTH
* 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
Tuberculin Test for TB
- 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
Immunodiagnostics for Autoimmunity
* 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
Antibodies as detection reagents
* 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
Detecting Infection
* Using grown antibody to detect an infection or antigen in sample for diagnosis
31
SANDWICH ELISA method for detection of ANTIGEN
* 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
Immunohistochemistry: ELISA on a slide
* 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
Immunohistochemistry: BVDV
* 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
Direct IFA
* Rather than an ezyme, we can put a fluorescent marker on it
* ex: **leptospira organisms in urine**
35
Using labelled Ab's for measurement of a biomarker
* 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
Immunophenotyping
* 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
Molecular Diagnostics (pathogen)
* 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
Genotyping Pathogen and Epidemiological Studies
* 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
Molecular Diagnostics (Host)
* 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
Diagnostics- Host: PCR based techniques
* 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
Use of DNA genotyping
* 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
Mast Cells and KIT
* 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
