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cellular basis of immunity > 15 - immunoassays > Flashcards

15 - immunoassays Flashcards

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1
Q

2 methods of using antibodies in diagnostics

A

tracking pathogens in their natural environment

looking at what circulating biomarkers are present in the blood

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2
Q

qualitative measurements

A

allow you to look at where things are distributed

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3
Q

quantitative measurements

A

allow you to measure how much antigen is present

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4
Q

qualitative immunoassay formats (4)

A 
qualitative:
Ouchterlony Immunodiffusion
 Immunofluorescence
 Immunoelectron gold microscopy
 Western Blotting
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5
Q

ouchterlony immunodiffusion (OID)

A

detects antibody-antigen interactions

allows indentification of similarities between antigenic properties of two unknown solutions

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6
Q

indication of +ve OID test

A

precipitin lines seen in agar gel

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7
Q

method of OID

A

place both into a plate of agar and allow them to diffuse towards each other

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8
Q

uses of OID test

A

forms basis of allergen tests

Has the patient got circulating antibodies against an allergen?
Is there more than one source of allergen causing the problem?

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9
Q

why is formation of immune complexes (antigen-antibody) important

A

larger bodies are easier for macrophages to destroy

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10
Q

problem with too little antigen in OID testing

A

antibody concentration too high
immune complexes cannot form
no agglutination
no precipitation

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11
Q

problem with too much antigen in OID testing

A

antibody concentration too low
antigen excess
steric hinderance
immune complexes cannot form

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12
Q

in OID, if precipitin lines fuse completely into 1 smooth arc…

A

then the test has shown the presence of

identical antigenic determinants in the sample

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13
Q

in OID, if the precipitin lines cross but without any interaction…

A

then the two solutions dont share any common determinants detectable by antiserum
they react but are not related

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14
Q

OID:

if the precipitin lines fuse, but an additional spur forms towards the well containing solution…

A

the the two solutions share some common determinants but solution A has additional unique determinant detected by the antiserum

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15
Q

western blotting

A

means of looking at antigenic similarity
specific antibodies used to identify particular antigens in a mixture of proteins that have been resolved and transferred to a membrane

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16
Q

protein separation technique in western blotting

A

SDS-PAGE

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17
Q

method of western blotting

A

separation of proteins from mixture using SDS-PAGE
transfer by electroblotting
incubate with specific antibodies for the target antigen
unbound antibodies washed away
location of bound antibodies identified using colour marker

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18
Q

what does PAGE stand for

A

POLY-ACRYLAMIDE GEL ELECTROPHORESIS

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19
Q

speed of migration of in an electrical field depends on…

A

the dimension, form and charge of the molecules

high molecular weight proteins found at the top of the gel

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20
Q

use of SDS

A

provides molecules with negative charge

so charge does not affect separation

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21
Q

why do we denature proteins in western blotting

A

to break disulphide bridges

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22
Q

SDS-PAGE in 1D

A

one dimension of separation

all given same -ve charge, proteins separate according to molecular weight

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23
Q

SDS-PAGE in 2D

A

2 dimensions of separation:

1- separate proteins on basis of pH - according to charge
2 - separation according to molecular weight - SDS electrophoresis

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24
Q

use of secondary antibody in western blotting

A

used as a recording molecule

e. g.
- conjugate anti-mouse antibody to fc domain of primary antibody that is being inserted and tested
- enzyme on secondary antibody will convert substrate to a different colour

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25
Scedosporium prolificans
An ‘emerging’ fungal pathogen of immuno-compromised/AIDs patients source is often pot plants
26
problems with Scedosporium prolificans
resistant to many anti-fungal drugs | causes lesions in CNS of those infected
27
virulence factor of Scedosporium prolificans
heavy melanisation | gene disruption removes enzyme to make black pigment --> reduced virulence
28
immunofluorescence
qualitative and widely used technique can be direct or indirect antibodies get tagged with fluorescent reagents apply UV: reagents fluoresce at different absorbances
29
examples of fluorescent reagents
fluorochromes FITC: Fluorescein IsoThioCyanate (green) TR: Texas Red
30
immunofluorescence uses
detect localisation of molecules of interest --> e..g distribution of an antigen identify pathogenic organisms in tissues
31
importance of clusters of differentiation (CD) in immunofluorescence
antibodies tell you where the relevant CD is localised on outside of immune cell
32
indirect immunofluorescnce
use of secondary antibody which is conjugated to fluorochrome secondary antibody binds to Fc domain of primary antibody which bins to antigen
33
direct immunofluorescnce
fluorochrome attached directly onto antibody of interest
34
immuno-electron gold microscopy
qualitative technique | antibodies tagged with gold particles (instead of fluorochrome) are used to detect molecules of interest
35
uses/importance of immuno-electron gold microscopy
- allows localisation of antigen-antibody interactions at the nm scale (better resolution) - powerful method of visualising intracellular antigens and organelles
36
importance of fungal antigen
only produced when pathogen is in active germination or polarisaed (hyphal) growth good biomarker
37
ELISA: | Enzyme-linked Immunosorbent Assays
quantitative technique detects antigens/antibodies using enzyme-substrate reactions alows high throughput screening of antibodies
38
how are the enzymes coupled to the antibodies in ELISA
either directly (to the antibody) or indirectly (to the anti-immunoglobulins)
39
2 main ELISA formats
1. Plate-Trapped-Antigen (PTA)-ELISA | 2. Double-Antibody-Sandwich (DAS)-ELISA
40
example of anti-immunoglobulin use in ELISA
. Goat anti-mouse peroxidase conjugate
41
how is quantification in ELISA achieved
using standard calibration curves | known amount of antigen on plate and colour reaction
42
what separates IgG from IgM
different heavy chains | IgM is a pentamer and is therefore really good at pulling down the antigen
43
fusarium
common plant pathogen can infect humans e.g. fusarium nail infection
44
PTA-ELISA
plate trapped antibody -ELISA
45
results of PTA-ELISA of fusarium
probe with specific antibody | absorbance increases over time where fungus is present and antibdy binds
46
Rhizoctonia solani
Ubiquitous soil-borne plant pathogen worldwide distribution and wide host range prevents any growth in soil if infected Difficult to control using chemicals
47
why is Rhizoctonia solani difficult to control using chemicals
1. Soil dwelling 2. Widespread resistance to fungicides 3. Soil fumigants are banned (Class II ozone depletors)
48
what is the most effective way to deal with Rhizoctonia solani
to ‘evade’ the pathogen altogether do not use in contaminated soils test soils for infestation prior to sowing with susceptible crops
49
R. solani-specific mAbs
monoclonal antibdies raised specifically against the fungal pathogen - Rhizoctonia solani antigen is secreted by live cells only
50
examples of R. solani-specific mAbs
mAb EH2 - protein epitope mAb EE1 - carbohydrate epitope
51
procedure to diagnose Rhizoctonia solani
Stimulate active growth using baits 2. Detect R. solani using specific mAbs 3. Recover positive isolates for further testing
52
method to construct a baiting molecule
Take soil sample and put in petri dish with lid Bait used to bait pathogen out of the soil Colonise bait to detect pathogen using monoclonal antibodies Coat microtitre plate with 1st antibody Bathing solution Pathogen present will secrete antigen Process double antibody ELISA
53
mass extinctions of what seen
amphibians Examples = frogs, toads, salamanders
54
hypervirulent lineage of fungus
causing extinction of amphibian species | 500 species of frog lost over last 10 years
55
chytrid fungus (Bd)
aquatic, motile fungus that causes severe disease in amphibians. causes chronic disease of the skin --> Chytridiomycosis
56
life cycle of chytrid fungus (Bd)
lasts 4-5 days starts as tiny zoospore that swims around until burrowing and penetrating skin of host develops into a thallus which matures into a sporangium grows and bursts to release more zoospores, chronic re-infection or infection of other organisms
57
Chytridiomycosis
Colonises keratinised skin layer. Hyperplasia + Hyperkeratosis = Disrupted electrolyte balance Ultimately leads to cardiac arrest and death. amphibians require skin to breathe and drink
58
hyperplasia
enlargement of organ tissue due to cellular proliferation.
59
hyperkeratosis
thickening of the skin
60
diagnosis of chytridiomycosis
difficult due to ambiguous symptoms qPCR is gold standard histology important
61
lateral flow assay
similar to a pregnancy test dipstick assay 2 lines appear if you infected with disease of interest semi-quantitative point-of-care test
62
quantitative immunoassay formats (2)
quantitative: PTA-ELISA and DAS-ELISA Lateral-Flow Assay

cellular basis of immunity (21 decks)

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