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Immunology Jenny > Practical lessons > Flashcards

Practical lessons Flashcards

1
Q

Antigen

A
  • Auto (self)
  • Allo (other)
  • Xeno (alien)
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2
Q

Epitope

A

Antigenic determinant

Two types:
- Conformational (recognized by BCR)
- Linear (recognized by TCR)
(and neoantigenic determinant - created by proteolysis)

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3
Q

Immune complex

A

Soluble antigen-antibody complex
- Overproduction: Hypersensitivity
=> ABO, Rh, hemolytic anemia, SLE, serum sickness

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4
Q

Serum electrophoresis

A

Detection of Ab as a protein

  • Separate serum proteins by electrophoresis
  • Gamma-globulins (Ab’s) move toward negative electrode, rest move toward positive)
  • Densitogram: se bilde ipad
  • γ-globulins increase w/o M peak on densitogram in case of chronic liver failure, longstanding inflammation and polyclonal gammopathy
  • M-peak γ on densitogram: plasma cell myeloma, monoclonal gammopathies
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5
Q

Turbidimetry

A

Measures protein levels in serum using light intensity

- More sensitive than nephelometry

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6
Q

Nephelometry

A

Measure protein levels in serum using light dispersion intensity (“scatter”)
- Less sensitive than turdbidimetry

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7
Q

Precipitation methods

A

Precipitation: insoluble immune complexes due to physical/chemical processes in lab

  • Need equivalance of Ag’s and Ab’s to get precipitation
  • Types
    a) Ring precipitation
  • Ring = zone of equivalance (in test tube)
    b) Radial immunodiffusion
    c) Electrophoresis+immunodiffusion
  • Immune-electrophoresis
  • Immunfixation
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8
Q

Radial immunodiffusion

A

Radial immunodiffusion (cheap, not recently used)

  • Precipitation reaction of Ab and Ag in agarous gel
    1) Simple
  • Patient Ag’s in wells
  • Known Ab’s in gel
  • Ag’s diffuse into gel during incub => ring precipitation where Ag-Ab complexes (bigger ring=bigger conc Ag)
    2) Radial double
  • Ag in one well, Ab in another well
  • Diffuse toward each other
  • Precipitation line where Ag-Ab complexes formed (more Ag=line toward Ab well)
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9
Q

Immune-electrophoresis

A

Combination of:

  • Electrophoresis: + on left side, - on right side
  • Immunodiffusion: Patient serum above, normal human serum below
  • Middle: horse serum with polivalent antibodies
  • Compare patient and normal serum
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10
Q

Immunofixation

A

To detect one antibody isotype

  • Sample (any body fluid) is run by electrophoresis in six => separation proteins
  • Add monospecific antibodies
  • Precipitation
  • Fix and stain
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11
Q

Application of immunoserology methods

A
  • Blood group determination
    a) Traditional slide/tile method
    b) Quick bedside test
    c) Micro column method in lab
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12
Q

Homing

A

Wandering in target tissue

- Tissue specific cytokines

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13
Q

Methods based on agglutination + fields of use

A

Used when antigen is a particulate

  • Antibody titer: highest dilution (lowest concentration) where we can see agglutination
  • Types:
    a) Direct: Ab-Ag on cells (AB0)
    b) Indirect: 2ndAb-Ab-Ag (2nd binds to Fc) (Rh)
    c) Passive: haptens or antigens bind to RBCs or latex particles
  • IgM most effective agglutinin
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14
Q

Labeling of diagnostic antibodies

A

1) Covalent conjugation to marker molecules
- Enzymes
- Radioisotopes
- Colloidal gold
- Fluorochrome
2) Biotinylated antibody
- Marked streptavidin can bind to it

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15
Q

ELISA

A

Biological body fluid sample. Marked w/enzyme
Types:
1) Indirect (Bottom: Ag-Ab-2ndAb)
2) Sandwich (Bottom: Ab-Ag-2ndAb)
3) Competitive
- Measure Ag in sample
- Bottom: Ag coat-Ab not bound by sample Ag-2nd Ab
- Antibodies attached to sample Ag’s are washed away
- More antigen = less color (opposite of 1 and 2)

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16
Q

ELISPOT

A

“Enzyme-linked immuno spot”. Isolated cells.

  • Well coated with capture Ab’s
  • Add cells (some are secreting, some not)
  • Secretion product attach to Ab’s
  • Wash away cells
  • 2nd Ab conjugated w/enzyme added
  • Where there was secretory cells we see spots of insoluble reaction product
  • *ELISPOT and ELISA good for tuberculosis test (IFNγ)
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17
Q

Western blot

A

Isolated proteins

1) Proteins separated by SDS PAGE
2) Blotted (transferred) onto a membrane
3) Detected by immune detection (membrane: protein-Ab-2nd Ab w/enzyme)

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18
Q

RIA

A

“Radioimmunoassay”

  • Detection of radioactively labeled Ab’s
  • Test for Ag’s
  • Competitive method: (Radiolabeled Ag’s and patient serum Ag’s compete for the same Ab’s)
  • More patient Ag => weaker sign (radiolabeled Ag’s kicked out and washed away)
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19
Q

IRMA

A

“Immunoradiometric assay”

  • Detection of radioactively labeled Ab’s
  • Test for Ag’s
  • Sandwich-method (Tube surface: Ab coat-Ag-2ndAb w/radioactive labeling
  • More antigen => stronger sign
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20
Q

Immuno(histo)chemistry

A

Labeled (chemistry) antibodies (immuno) attach to specific parts of a tissue (histo)

  • Direct: Labeled Ab bind to protein of interst
  • Indirect: Labeled 2nd polyclonal Ab’s bind to primary monoclonal Ab’s which are bound to protein of interest
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21
Q

Lateral flow test

A

Biological body fluid sample (eg urine - pregnancy)

  • Fast, cheap and easy
  • Pregnancy test: hCG
    1) Plastic strip - porous membrane - control band (anti-Ig Ab), test band (antigen-specific Ab/anti-hCG Ab) and colored Ab-covered (anti-hCG) latex bead attached
    2) If pregnant: hCG attaches to latex bead -> migrates to test band (anti-hCG Ab band) => 2 lines on pregnancy test (control+test line)
  • Can also be used for HIV
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22
Q

Principles of flow cytometer and cytometry

A

Fast and quantitative laboratory methods for multiparametric analysis of single cells

  • Can examine many cells (10^5-10^6) in a short time (1-2 min) and is objective
  • Use fluorescence labeled antibodies (direct/indirect)
  • Measure forward scatter (size) and side scatter (granulation)
  • Can also tell rel. fluorescence intensity and time-dependency of these parameters
  • Dichroic filter - accurate color filter
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23
Q

Identification of cell populations by size and granularity

A
  • Forward scatter: size

- Side scatter: granularity

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24
Q

Identification of cell populations by immunophenotyping

A

Identification of cells by their protein pattern

  • Antigen expression (eg CD3+ is T cells)
  • Proportion of detected cells
  • Relative molecule expression
  • Coexpression
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25
Q

Identification of cell populations by detection of soluble molecules

A

1) Cytokines:
- Microbead with anti-cytokine antibody 1
- Add fluorochrome-conjugated anti-cytokine antibody 2
- Use lateral flow cytometry

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26
Q

Identification of cell populations by cell cyle analysis

A
  • Proprium iodide staining - stain DNA (differentiate necrotic, apoptotic and normal cells)
  • In cancer => more cells in S phase
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27
Q

Role of flow cytometry in clinical practice

A

1) Hematological disorders
- Leukemia
- Minimal residual disease (MRD)
- Multidrug resistance (MDR)
- Monitoring bone marrow transplant
2) Immunodeficiencies
- AIDS (use combination of ELISA, western blot and FCM)

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28
Q

Hemolytic test

A

Anti-erythrocyte Ab + patient serum => hemolysis

- Measure hemoglobin concentration

29
Q

Measuring complement activation (CH50)

A

The denominator of serum dilution that lyses 50 % of sheep RBCs in a test tube

  • CH50 ref. value in serum; 142-279 CH50 unit
  • Normal CH50 value = all complement factors present (but does not show the level of them, unless highly altered)
  • CH50 changes if:
    a) Amount of complement factors elevated (more common - complement proteins are also acute phase proteins)
    b) -||- decreased (due to genetic mutations, hepatic dysfunction, starving, overconsumption in immunological disorders)
    c) Some factors missing
30
Q

Detection of complement aberrations

A
  • C3, C4 and factor B routinely tested
31
Q

Which complement protein is present in highest conc. in our body?

A

C3

32
Q

What does simultaneous reduction of C3 and C4 suggest?

A

Classical pathway (and MBL pathway) activation

33
Q

In vitro complement activation

A

1) Hemolytic test
2) Complement fixation test
3) CH50 test

34
Q

HANE (hereditary angioedema), “HAE” also used

A

Hereditary C1INH deficiency

  • C1 activity uninhibited => C4b and C2b incr. prod.
  • C4b and C2b rapidly consumed - C3 and C5 not activated!
  • Edema caused by the decreased action of C1INH in the bradikinin-kallikrein system!!
35
Q

How can we decide if anaphylactic reaction or HAE cause obstruction (in ex: laryngeal edema)?

A
  • Epinephrine effect: anaphylactic reaction

- Epinephrine no effect: HAE

36
Q

Detection of complement aberrations

A
  • C3, C4 and factor B routinely tested

Hva mer??

37
Q

Histogram fluorescence

A
  • High intensity fluorescence = more antigens per(!) cell

- High no. (tall mountain) = more cells express the antigen

38
Q

AIDS

A
  • Western blot: HIV-positiv if (p31 or p24 AND gp160 or gp120 bands present simultaneously)
  • AIDS: decreased CD4+ T cells in patients (FCM)
39
Q

Methods based on antigen-antibody interaction

A
  • ELISA
  • RIA
  • IRMA
  • ELISPOT
  • Immunohistochemistry
  • Western blot
  • ELFA
  • Lateral flow test
40
Q

Methods based on antigen-antibody interaction

A
  • ELISA
  • RIA
  • IRMA
  • ELISPOT
  • Immunohistochemistry
  • Western blot
  • ELFA
  • Lateral flow test
  • Flow cytometry
41
Q

Monoclonal vs polyclonal antibodies

A
  • Monoclonal: specific to one epitope

- Polyclonal: recognize different epitopes

42
Q

Hapten

A

Small molecule that elicit immune response only when attached to a carrier

43
Q

Methods based on antigen-antibody interaction

A 
Biological body fluid sample
- ELISA
- RIA
- IRMA
- Lateral flow
Isolated cells
- ELISPOT
Biological solid tissue section
- Immunohistochemistry
Isolated proteins
- Western blot
Other
- ELFA
- Flow cytometry
44
Q

Hapten

A

Small molecule that elicit immune response only when attached to a carrier
- Injected into mouse to create polyclonal antibodies

45
Q

Sensitivity

A
  • How sensitive the antibody is

- What % of sick samples are recognized as positive

46
Q

Specificity

A
  • The % of healthy samples tested as negative
47
Q

Methods based on antigen-antibody interaction

A 
Biological body fluid sample
- ELISA
- RIA
- IRMA
- Lateral flow
Isolated cells
- ELISPOT
Biological solid tissue section
- Immunohistochemistry
Isolated proteins
- Western blot
Other
- ELFA
- Flow cytometry
48
Q

Methods based on antigen-antibody interaction

A 
Biological body fluid sample
- ELISA
- RIA
- IRMA
- Lateral flow
Isolated cells
- ELISPOT
Biological solid tissue section
- Immunohistochemistry
Isolated proteins
- Western blot
Other
- ELFA
- Flow cytometry?
49
Q

Antibody characteristics

A
  • Mooclonal/polyclonal
  • Affinity (strength epitope-Ag binding site of Ab, Keq)/avidity (overall strength of Ag-Ab complex - IgM high avidity)
  • Specificitycross-reactivity (spec: ability to recognize and bind antigen, cross-r: can bind to other similar Ag’s with shared or similar epitope)
  • Ag-Ab: non-covalent and reversible
  • Ab is flexible
50
Q

NK cell and NKT cell CD

A

NK cell: CD56+ and CD3-

NKT cell: CD56+ and CD3+

51
Q

T cell CD

A

CD3+
Th: +CD4+
Tc: +CD8+
γδT: +γδTCR+

52
Q

B cell CD

A

CD19+

53
Q

Consequenze of injecting a soluble ag subcutaneously?

A
  1. Spreads in the subcutaneous CT
  2. Gets degraded enzymatically after a while
  3. Endocytosis
  4. Ag presentation with MHC
  5. No costimulation: T cell anergy
  • Low probably of contact with recirculating specific T or B cells
54
Q

Immune response to foreign protein ag if introduced per os?

A

Generally, tolerance (similarly to food and normal bacterial flora derived proteins)

55
Q

ADJUVANT

  • definition
  • content
  • function
  • in therapy
  • examples
A
  • Enhancer of the effect, enhances immune response to given ag by inducing costimulation
  • Danger signal (e.g. PAMP, DAMP)
  • Exert function via PRRs
  • Can be co-injected with vaccine to enhance the chance of encounter of the ag with the specific lymphocyte -> Greater effect
  • neutral liposomes, mineral salts, cationic liposomes, saponins, PRR/TLR angonists, microspheres
56
Q

The significance of ag dose during immunization

A

High dose tolerance (the absence of an expected immunological response after repeated injections of large amounts of an antigen)

  • > T cell anergy - absence of costim. molecules
  • > Deletion of T cell (CD95 death receptor on T cell with CD95L on APC)

Low dose tolerance (a temporary and incomplete immunosuppression induced by the administration of subimmunogenic doses of soluble antigen)
-> Treg activation - T cell suppression through inhibitory receptors and cytokines

57
Q

Passive and active immunization (general)

A

PASSIVE

  • Injecting antibodies (IgG) and cells
  • For therapy
  • Immediate action: neutralization/opsonization/complement activation (Innate immune response)
  • No memory
  • Enzymatically degraded in 1-2 weeks

ACTIVE

  • Injecting antigen
  • Increases the specific ag recognition
  • Development of specific immunological memory
  • For prevention
  • Attenuated/dead pathogen or its subunits
  • Abs, T-cells, memory cells (adaptive immune response)
  • In repeated infection: activation of memory cells and differentiation into effector cells
58
Q

Types of active vaccinations

A
  1. Whole virus vaccines
    a. Live attenuated
    b. Inactivated (killed, complete)
    c. Live recombinant
  2. Subunit vaccine (only some parts of agent) - purified or recombinant ag protein
    a. Synthetic peptide DNA (RNA)

Results: Ab production, effector T cells, memory cells

59
Q

Features of effective vaccines

A
  • Safe (not cause illness/death)
  • Protective (from live pathogen)
  • Sustained protection (last for years)
  • Induce neutralizing abs (to prevent infection as certain pathogens, e.g. polio, infect cells that cannot be replaced, e.g. neurons)
  • Induce protective T cells (as certain pathogens, e.g. i.c. are more effectively eliminated by cell-mediated response)
  • Be practical (biologically stable, easy administration, few side effects, cheap)
60
Q

T-dependent antigens

A
  • Usually proteins
  • T-B cooperation
  • High affinity abs
  • Memory
61
Q

T-independent antigens

A
  • Usually polysaccharides
  • Only the B cell is activated
  • Short term memory
62
Q

Protective epitope

A

Very specific and unique epitope of pathogen. Involved in the pathogenesis.

Neutralizing ab can bind it -> the pathogen cannot penetrate cells -> no spreading

63
Q

Phases of anticiral CD8+ T cells

A
  1. Clonal expansion upon ag exposure
  2. Contraction - after elimination of ag
  3. Establishment of memory
64
Q

B cell response in infants/elderly

Consequence?
Solution?

A

Infants -> Low memory B cell pool
Elderly -> Low naive B cell pool

= Both have inability to respond to new ags (but for different reasons)

Consequence: Vaccine efficacy is limited
Solution: optimal adjuvants, higher ag dose, repeated injection (boost)

65
Q

Types of B cells

A

Follicular

  • In spleen/other lymphoid organs
  • TD response to protein ag
  • Germinal center reaction
  • Long lived plasma cells that produce isotype switched, high affinity Igs: IgG, IgA, IgE

Marginal zone

  • In spleen/other lymphoid organs
  • TI response to polysaccharides, lipids, etc
  • Short-lived plasma cells that produce mainly IgM

B1

  • Mucosal tissues, peritoneal cavity
  • TI response to polysaccharides, lipids, etc
  • Short-lived plasma cells that produce mainly IgM
66
Q

Subunit conjugate vaccine

A

Epitope + toxoid
E.g. Tetanus toxoid protein + polysaccharide conjugate
E.g. HiB

67
Q

Conventional strategy of vaccine production

A

Reproduction in the laboratory -> Antigens that are the basis for the vaccine are isolated one by one

68
Q

“Reverse vaccinology” - In silico computational approach for vaccine production

A
  • Starts from known genome of pathogen
  • Only proteins can be used
  • The selected sequence is cloned and expressed in a normal wet lab

Immunology Jenny (16 decks)

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