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Microbiology & Immunity > 32- Immunology in the Clinic and Research Lab > Flashcards

32- Immunology in the Clinic and Research Lab Flashcards

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1
Q

what is an immunoassay?

A

analytical technique that uses the specific interaction between antibodies and antigens – one of which is typically labelled/ tagged to allow its detection

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2
Q

list the different types of immunoassays

A

radioimmunoassay

enzyme linked immuno-sorbent assays/ ELISA - direct, indirect, capture(sandwich)

luminescent immunoassay

ELISpot

SDS-PAGE and Western Blotting

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3
Q

what enzymes are used for ELISA?

A

horseradish peroxidase
alkaline phosphatase

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4
Q

what does direct ELISA do?

A

measures antigen concentration

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5
Q

describe how direct ELISA works?

A

antigen immobilised at bottom of microplate well

enzyme-conjugated antibody added - complementary antibody-antigen binding

substrate for antigen added - reacts with enzyme, produces a measurable colour signal proportional to the amount of antigen present

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6
Q

what does indirect ELISA do?

A

measures antibody concentration

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7
Q

what does capture/ sandwich ELISA do?

A

measures antigen concentration - especially low concentrations

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8
Q

how does indirect ELISA work?

A

antigen immobilised at bottom of microplate well

primary antibody added (from sample) - complementary binding interactions with capture antigen

secondary antibody conjugated with enzyme added - binds to exposed Fc region of primary antibody

enzyme substrate added, reacts with enzyme, produces a measurable colour signal proportional to the amount of primary antibody present

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9
Q

advantage of indirect ELISA over direct?

A

amplified signal from multiple secondary antibodies binding to different epitopes of each primary antibody, higher sensitivity

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10
Q

how does capture/ sandwich ELISA work?

A

antibody immobilised at bottom of microplate well

target antigen added - binds to bound antibody, and any unbound antigen is washed off

second enzyme conjugated (e.g. Biotin antibody) antibody added - binds to different epitope of target antigen

substrate for enzyme - e.g. Streptavidin - added, reacts with enzyme Biotin, produces a measurable colour signal

absorbance of colour signal can be measured, proportional to concentration of antigen

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11
Q

advantage of capture/ sandwich ELISA?

A

concentrates low conc antigens with high affinity antibodies, requires two antibodies reacting with different antigen epitopes

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12
Q

applications of ELISA/ immunoassays?

A

quantifying specific antigens/ antibodies in samples

monitoring immune responses

diagnosing infections and diseases

hybridoma supernatant screening

detection of exposure to infectious agents

measuring vaccine efficacy and immune responses

screening and detection & research and diagnostics

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13
Q

what does ELISpot measure?

A

the frequency of cytokine-secreting cells at the single-cell level

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14
Q

describe how ELISpot works?

A

immobilised cytokine specific antibodies at the bottom of a well

cytokine-producing immune cells - e.g. T cells - with a mix of different effector functions added

T cells are activated secrete cytokines which bind to their complementary antibodies

second enzyme conjugated antibody added which binds to bound cytokine

substrate for enzyme is added - reacts with enzyme, produces an insoluble coloured spot

each spot represents one cell - microscopic imaging allows visualisation of cytokine production from each individual cell under different conditions

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15
Q

use for ELISpot?

A

monitor vaccine efficacy and cellular responses vaccines generate

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16
Q

what does SDS PAGE do?

A

separates proteins based on their molecular weight on a polyacrylamide gel

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17
Q

what does a Western blot do?

A

detects presence of specific proteins in a sample using antibodies

18
Q

how does SDS PAGE work?

A

proteins are denatured using SDS and reducing agents - then loaded on a polyacrylamide gel and exposed to an electric field

proteins migrate across the gel matrix - smaller proteins/ lower molecular weight migrate further

19
Q

how does Western blotting work?

A

cell is lysed, and produces all its proteins under treatment - cellular proteins denatured with SDS and loaded on polyacrylamide gel

smaller proteins will accumulate further up the gel, larger proteins lower down = form protein rich accumulations

proteins removed from gel and transferred into a type of membrane - e.g. nitrocellulose

labelled antibodies - e.g. with alkaline phosphatase - are used against proteins of interest in the protein mix

proteins bind to antibody, trigger alkaline phosphatase activity = produce a visual band of the protein corresponding to a region in the (nitrocellulose) membrane
- e.g. smaller protein = further at the bottom of the membrane
- e.g. bigger protein = at a higher membrane level

20
Q

advantage of Western blotting over other methods - e.g. ELISA?

A

detects presence/ absence of a protein, its expression (high/low intensity of expression), if a protein is modified/ degraded, determine the amount that has been degraded/ modified

with degraded/ fragmented proteins - methods like ELISA may still bind and produce a colour signal as it’s still present in the sample, but won’t tell you that the protein is modified

21
Q

uses of SDS-PAGE and Western blotting?

A

antibody-antigen detection = identifies specific proteins/ antibodies in a sample

determines molecular weight of proteins

quantifies protein concentration by comparing band intensity to known standards

detects protein degradation by identifying fragments

22
Q

what is MACS/ Magnetic-Activated Cell Sorting?

A

purification of immune cell subsets antibody-coated magnetic beads, isolates specific immune cell subsets based on surface markers

23
Q

method behind MACs?

A

magnetic beads are coated with antibodies specific to surface markers on target cells

cell sample is incubated with magnetic beads for a time - then sample is placed in a
magnetic field, and unbound cells are washed away and the remaining

24
Q

what are lateral flow tests for?

A

antibodies specific to an antigen detect its presence in a sample - mostly associated with quick diagnosis of an infectious disease

presence of absence of bands will indicate an infection

25
Q

what is flow cytometry for?

A

to analyse the physical and chemical characteristics of cells/ particles in fluid as they pass through a laser

measure cell size and granularity through forward and side scatter

26
Q

what is FACs/ flow-activated cell sorting for?

A

allows for the physical separation of cells based on their fluorescence characteristics

27
Q

how does FACS work?

A

cells pass through a laser and the nozzle is vibrated - individual cells in droplets which become charged as they pass through a laser/ electric field

charge is detected by machine, identifies cells that meet a certain characteristics and directs their collection into separate collection tubes based on distinct characteristics

allows for physical separation of cell populations with high purity and precision

28
Q

list types of samples analysed

A

blood serum
blood cells
urine
synovial fluid
saliva
mucus
cerebrospinal fluid

29
Q

what types of disease do we often look for in analysing samples with immunology tests?

A

transplant compatibility
immunodeficiency
allergy
autoimmunity
malignancy

30
Q

what’s the importance in lab immunology in transplant rejection?

A

PCR based-typing of HLA-alleles ensures the best MHC match between donor and recipient to reduce risk of immune transplant rejection

recipient immune system will reject anything that is non-self, and the highly polymorphic nature of HLA genes makes donor & recipient MHC matching very hard

PCR based typing ensures the best match possible

31
Q

what’s the importance of lab immunology in autoimmune diseases?

A

flow cytometry can quantify and analyse cell populations based on the presence of specific cell surface markers using fluorescent-labelled antibodies

  • in XLA = identify and quantify number of mature B cells in suspected XLA patient using CD19 marker fluorescently-labelled antibodies
  • HIV monitoring to quantify lymphocyte subsets
  • tracking antigen-specific T cell responses using fluorescent MHC complexes
32
Q

how is lab immunology (flow cytometry) used for XLA?

A

with XLA/ X-linked agammaglobulinemia there’s an inability to generate mature B cells

CD19 marker on B cells can be identified and quantified by flow cytometry using fluorescently labelled antibodies

33
Q

how is flow cytometry useful for clinical HIV monitoring?

A

monitor lymphocyte subsets in patient using monoclonal antibodies that target specific cell surface markers CD3, 4, 8

percentage and absolute count of each marker subset determine by FACS - helps determine immune status of HIV patient

34
Q

how is lab immunology used in the treatment of neutrophil immunodeficiencies like CGD? - use? method?

A

for disorders like chronic granulomatous disease/CGD or neutropenia - can measure neutrophil function, serves as a diagnostic tool, monitors treatment

method:
- DHR123 is a non-fluorescent compound phagocytosed by activated neutrophils
- neutrophils are then stimulated with PMA to produce ROS and undergo oxidative burst
- ROS produce oxidise DHR 123 to rhodamine 123 which is a green fluorescent compound, can be detected and quantified using flow cytometry

impaired neutrophil function = lack of ROS production = reduced fluorescence – indicative of neutrophil-related immunodeficiencies

35
Q

how is lab immunology (nephelometry) used in analysing antibody levels in immunodeficiency? - method for nephelometry?

A

nephelometry - high throughput automated method which quantifies serum Ig levels

beam of light passes through a solution containing antigen-antibody complexes = complexes scatter light in various directions

nephelometry instruments detect scattered light and quantify light intensity

intensity of scattered light is directly proportional to the conc of antigen-antibody complexes in the sample

36
Q

what is nephelometry?

A

high throughout automated method that quantifies serum Ig levels

37
Q

what is nephelometry used for?

A

analysing serum samples to determine levels of specific Igs

precise quantification of different antibody classes – info on immune status

abnormalities in antibody levels can indicate various immunodeficiencies/ infections

monitors effectiveness of treatments aimed at modulating immune responses

38
Q

how is the RAST test used to diagnose allergies?

A

suspected allergen is bound to an insoluble material - patient’s serum is added

antibodies specific to the allergen will bind to the insoluble-antigen complex

then labelled anti-human IgE antibody is added - will bind to the antibodies bound to the allergen

amount of radioactivity is proportional to the existing patient serum IgE for the allergen

39
Q

how is immunofluorescence used for diagnosing/ detecting SLE? - method?

A

immunofluorescence detects specific antibodies, and 98% of SLE patients exhibit anti-nuclear antibodies

method:
- human cell lines are cultured and fixed
- patient serum with suspected anti-nuclear antibodies are added to the fixed cells
- if the antibodies are present - patient serum will bind to specific antigens within cell nucleus
- slides are probed with fluorochrome-labelled anti-Ig antibodies = exhibit fluorescence under microscope

40
Q

two types of antibody therapies?

A

polyclonal antibodies
monoclonal antibodies

41
Q

how are polyclonal antibodies used as antibody therapy?

A

intravenous Igs derived from pooled human serum can be used to treat antibody deficiencies and modulate immune responses

42
Q

how are monoclonal antibodies used as antibody therapy?

A

developed to treat cancer, chronic inflammatory conditions, infectious diseases

can bind and block disease process or mediate immune responses - e.g. initiating ADCC

conjugating toxins or radionuclides to monoclonal antibody for direct treatment delivery and killing of cancer cells

Microbiology & Immunity (37 decks)

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