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cellular basis of immunity > 15 - immunoassays > Flashcards

15 - immunoassays Flashcards

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
Q

Scedosporium prolificans

A

An ‘emerging’ fungal pathogen of immuno-compromised/AIDs patients

source is often pot plants

26
Q

problems with Scedosporium prolificans

A

resistant to many anti-fungal drugs

causes lesions in CNS of those infected

27
Q

virulence factor of Scedosporium prolificans

A

heavy melanisation

gene disruption removes enzyme to make black pigment –> reduced virulence

28
Q

immunofluorescence

A

qualitative and widely used technique

can be direct or indirect

antibodies get tagged with fluorescent reagents
apply UV: reagents fluoresce at different absorbances

29
Q

examples of fluorescent reagents

A

fluorochromes

FITC: Fluorescein IsoThioCyanate (green)
TR: Texas Red

30
Q

immunofluorescence uses

A

detect localisation of molecules of interest –> e..g distribution of an antigen
identify pathogenic organisms in tissues

31
Q

importance of clusters of differentiation (CD) in immunofluorescence

A

antibodies tell you where the relevant CD is localised on outside of immune cell

32
Q

indirect immunofluorescnce

A

use of secondary antibody which is conjugated to fluorochrome
secondary antibody binds to Fc domain of primary antibody which bins to antigen

33
Q

direct immunofluorescnce

A

fluorochrome attached directly onto antibody of interest

34
Q

immuno-electron gold microscopy

A

qualitative technique

antibodies tagged with gold particles (instead of fluorochrome) are used to detect molecules of interest

35
Q

uses/importance of immuno-electron gold microscopy

A
  • allows localisation of antigen-antibody interactions at the nm scale (better resolution)
  • powerful method of visualising intracellular antigens
    and organelles
36
Q

importance of fungal antigen

A

only produced when pathogen is in active germination or polarisaed (hyphal) growth
good biomarker

37
Q

ELISA:

Enzyme-linked Immunosorbent Assays

A

quantitative technique
detects antigens/antibodies using enzyme-substrate reactions
alows high throughput screening of antibodies

38
Q

how are the enzymes coupled to the antibodies in ELISA

A

either directly (to the antibody) or indirectly (to the anti-immunoglobulins)

39
Q

2 main ELISA formats

A
  1. Plate-Trapped-Antigen (PTA)-ELISA

2. Double-Antibody-Sandwich (DAS)-ELISA

40
Q

example of anti-immunoglobulin use in ELISA

A

. Goat anti-mouse peroxidase conjugate

41
Q

how is quantification in ELISA achieved

A

using standard calibration curves

known amount of antigen on plate and colour reaction

42
Q

what separates IgG from IgM

A

different heavy chains

IgM is a pentamer and is therefore really good at pulling down the antigen

43
Q

fusarium

A

common plant pathogen
can infect humans
e.g. fusarium nail infection

44
Q

PTA-ELISA

A

plate trapped antibody -ELISA

45
Q

results of PTA-ELISA of fusarium

A

probe with specific antibody

absorbance increases over time where fungus is present and antibdy binds

46
Q

Rhizoctonia solani

A

Ubiquitous soil-borne plant pathogen
worldwide distribution and wide host range
prevents any growth in soil if infected
Difficult to control using chemicals

47
Q

why is Rhizoctonia solani difficult to control using chemicals

A
  1. Soil dwelling
  2. Widespread resistance to fungicides
  3. Soil fumigants are banned (Class II ozone depletors)
48
Q

what is the most effective way to deal with Rhizoctonia solani

A

to ‘evade’ the pathogen altogether
do not use in contaminated soils
test soils for infestation prior to sowing with susceptible crops

49
Q

R. solani-specific mAbs

A

monoclonal antibdies raised specifically against the fungal pathogen - Rhizoctonia solani
antigen is secreted by live cells only

50
Q

examples of R. solani-specific mAbs

A

mAb EH2 - protein epitope

mAb EE1 - carbohydrate epitope

51
Q

procedure to diagnose Rhizoctonia solani

A

Stimulate active growth
using baits

  1. Detect R. solani using specific
    mAbs
  2. Recover positive isolates for
    further testing
52
Q

method to construct a baiting molecule

A

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
Q

mass extinctions of what seen

A

amphibians

Examples = frogs, toads, salamanders

54
Q

hypervirulent lineage of fungus

A

causing extinction of amphibian species

500 species of frog lost over last 10 years

55
Q

chytrid fungus (Bd)

A

aquatic, motile fungus that causes severe disease in amphibians.

causes chronic disease of the skin –> Chytridiomycosis

56
Q

life cycle of chytrid fungus (Bd)

A

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
Q

Chytridiomycosis

A

Colonises keratinised skin layer.
Hyperplasia + Hyperkeratosis = Disrupted electrolyte balance
Ultimately leads to cardiac arrest and death.

amphibians require skin to breathe and drink

58
Q

hyperplasia

A

enlargement of organ tissue due to cellular proliferation.

59
Q

hyperkeratosis

A

thickening of the skin

60
Q

diagnosis of chytridiomycosis

A

difficult due to ambiguous symptoms
qPCR is gold standard
histology important

61
Q

lateral flow assay

A

similar to a pregnancy test dipstick assay
2 lines appear if you infected with disease of interest

semi-quantitative point-of-care test

62
Q

quantitative immunoassay formats (2)

A

quantitative:
PTA-ELISA and DAS-ELISA
Lateral-Flow Assay

cellular basis of immunity (21 decks)

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