Practical lessons Flashcards
Antigen
- Auto (self)
- Allo (other)
- Xeno (alien)
Epitope
Antigenic determinant
Two types:
- Conformational (recognized by BCR)
- Linear (recognized by TCR)
(and neoantigenic determinant - created by proteolysis)
Immune complex
Soluble antigen-antibody complex
- Overproduction: Hypersensitivity
=> ABO, Rh, hemolytic anemia, SLE, serum sickness
Serum electrophoresis
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
Turbidimetry
Measures protein levels in serum using light intensity
- More sensitive than nephelometry
Nephelometry
Measure protein levels in serum using light dispersion intensity (“scatter”)
- Less sensitive than turdbidimetry
Precipitation methods
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
Radial immunodiffusion
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)
Immune-electrophoresis
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
Immunofixation
To detect one antibody isotype
- Sample (any body fluid) is run by electrophoresis in six => separation proteins
- Add monospecific antibodies
- Precipitation
- Fix and stain
Application of immunoserology methods
- Blood group determination
a) Traditional slide/tile method
b) Quick bedside test
c) Micro column method in lab
Homing
Wandering in target tissue
- Tissue specific cytokines
Methods based on agglutination + fields of use
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
Labeling of diagnostic antibodies
1) Covalent conjugation to marker molecules
- Enzymes
- Radioisotopes
- Colloidal gold
- Fluorochrome
2) Biotinylated antibody
- Marked streptavidin can bind to it
ELISA
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)
ELISPOT
“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)
Western blot
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)
RIA
“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)
IRMA
“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
Immuno(histo)chemistry
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
Lateral flow test
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
Principles of flow cytometer and cytometry
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
Identification of cell populations by size and granularity
- Forward scatter: size
- Side scatter: granularity
Identification of cell populations by immunophenotyping
Identification of cells by their protein pattern
- Antigen expression (eg CD3+ is T cells)
- Proportion of detected cells
- Relative molecule expression
- Coexpression
Identification of cell populations by detection of soluble molecules
1) Cytokines:
- Microbead with anti-cytokine antibody 1
- Add fluorochrome-conjugated anti-cytokine antibody 2
- Use lateral flow cytometry
Identification of cell populations by cell cyle analysis
- Proprium iodide staining - stain DNA (differentiate necrotic, apoptotic and normal cells)
- In cancer => more cells in S phase
Role of flow cytometry in clinical practice
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)
Hemolytic test
Anti-erythrocyte Ab + patient serum => hemolysis
- Measure hemoglobin concentration
Measuring complement activation (CH50)
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
Detection of complement aberrations
- C3, C4 and factor B routinely tested
Which complement protein is present in highest conc. in our body?
C3
What does simultaneous reduction of C3 and C4 suggest?
Classical pathway (and MBL pathway) activation
In vitro complement activation
1) Hemolytic test
2) Complement fixation test
3) CH50 test
HANE (hereditary angioedema), “HAE” also used
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!!
How can we decide if anaphylactic reaction or HAE cause obstruction (in ex: laryngeal edema)?
- Epinephrine effect: anaphylactic reaction
- Epinephrine no effect: HAE
Detection of complement aberrations
- C3, C4 and factor B routinely tested
Hva mer??
Histogram fluorescence
- High intensity fluorescence = more antigens per(!) cell
- High no. (tall mountain) = more cells express the antigen
AIDS
- Western blot: HIV-positiv if (p31 or p24 AND gp160 or gp120 bands present simultaneously)
- AIDS: decreased CD4+ T cells in patients (FCM)
Methods based on antigen-antibody interaction
- ELISA
- RIA
- IRMA
- ELISPOT
- Immunohistochemistry
- Western blot
- ELFA
- Lateral flow test
Methods based on antigen-antibody interaction
- ELISA
- RIA
- IRMA
- ELISPOT
- Immunohistochemistry
- Western blot
- ELFA
- Lateral flow test
- Flow cytometry
Monoclonal vs polyclonal antibodies
- Monoclonal: specific to one epitope
- Polyclonal: recognize different epitopes
Hapten
Small molecule that elicit immune response only when attached to a carrier
Methods based on antigen-antibody interaction
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
Hapten
Small molecule that elicit immune response only when attached to a carrier
- Injected into mouse to create polyclonal antibodies
Sensitivity
- How sensitive the antibody is
- What % of sick samples are recognized as positive
Specificity
- The % of healthy samples tested as negative
Methods based on antigen-antibody interaction
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
Methods based on antigen-antibody interaction
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?
Antibody characteristics
- 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
NK cell and NKT cell CD
NK cell: CD56+ and CD3-
NKT cell: CD56+ and CD3+
T cell CD
CD3+
Th: +CD4+
Tc: +CD8+
γδT: +γδTCR+
B cell CD
CD19+
Consequenze of injecting a soluble ag subcutaneously?
- Spreads in the subcutaneous CT
- Gets degraded enzymatically after a while
- Endocytosis
- Ag presentation with MHC
- No costimulation: T cell anergy
- Low probably of contact with recirculating specific T or B cells
Immune response to foreign protein ag if introduced per os?
Generally, tolerance (similarly to food and normal bacterial flora derived proteins)
ADJUVANT
- definition
- content
- function
- in therapy
- examples
- 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
The significance of ag dose during immunization
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
Passive and active immunization (general)
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
Types of active vaccinations
- Whole virus vaccines
a. Live attenuated
b. Inactivated (killed, complete)
c. Live recombinant - 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
Features of effective vaccines
- 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)
T-dependent antigens
- Usually proteins
- T-B cooperation
- High affinity abs
- Memory
T-independent antigens
- Usually polysaccharides
- Only the B cell is activated
- Short term memory
Protective epitope
Very specific and unique epitope of pathogen. Involved in the pathogenesis.
Neutralizing ab can bind it -> the pathogen cannot penetrate cells -> no spreading
Phases of anticiral CD8+ T cells
- Clonal expansion upon ag exposure
- Contraction - after elimination of ag
- Establishment of memory
B cell response in infants/elderly
Consequence?
Solution?
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)
Types of B cells
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
Subunit conjugate vaccine
Epitope + toxoid
E.g. Tetanus toxoid protein + polysaccharide conjugate
E.g. HiB
Conventional strategy of vaccine production
Reproduction in the laboratory -> Antigens that are the basis for the vaccine are isolated one by one
“Reverse vaccinology” - In silico computational approach for vaccine production
- Starts from known genome of pathogen
- Only proteins can be used
- The selected sequence is cloned and expressed in a normal wet lab
