Download 4th seminar (ELISA, functional)_2016

• Preparative and analytical methods based
on antibody-antigen interactions
(ELISA)
• Functional investigations of the cells of the
immune system
(2nd part)
The sensitivity of immunoassays
Measuring the functional activity of Band T-lymphocytes
Topics:
1. Polyclonal activation of T- and B-lymphocytes
lectin induced activation
α-IgM, α-CD3 or α-TCR induced activation
2. Investigation of the T- and B-cell response
activation markers
proliferative response 3H-thymidine incorporation
CFSE fluorescence dilution
cell cycle
antibody or cytokine production (ELISA)
3. Determination of the activated T- or B-cell number
ELISPOT
Intracellular cytokine staining
(review)
Phases of the humoral immune response
(review)
Phases of T cell response
(review)
Signalling of the B-cell antigen receptor complex
membrane
adaptor
protein
(NTAL)
(review)
Signalling of the T-cell antigen receptor complex
LAT (in the cell
membrane)
SLP-76(LCP2)
cytoplasmic
adaptor
All known steps can be examined
Investigations of the cellular
functions of the immune system
Immunodeficiencies mainly characterized by different
functional immunoassays
Lymphocyte activation by specific antigen is hardly detected,
because of the low number of the antigen specific cells
Lymphocyte function can be investigated by polyclonal T/B-lymphocyte
activator materials – polyclonal activators
Polyclonal activation of lymphocites by
LPS, lectins, PMA/ionomycin
B cell (mouse)
T cell
T cell
TLR4
(PMA activates protein kinase C)
BCR- or TCR-specific antibodies may also activate the lymphocytes
(Which antibody types can effectively activate the B- and T-cells?)
Polyclonal B cell activators
Activator
T cell dependency
Ig secretion
Human B cells
PWM (pokeweed mitogen)
no
yes
SpA (superantigen, staphylococcus protein A)
no
yes
EBV (transforming effect)
yes
yes
Anti-Ig
yes
In the presence of cytokines
Mouse B cells
LPS
no
yes
PWM (pokeweed mitogen, lektin)
yes
yes
PPD (purified protein derivate, mycobacterium) no
yes
Anti-Ig (antibodies)
In the presence of cytokines
no
Polyclonal T cell activators
Phytohaemagglutinin (PHA)
lectin
Phaseolus vulgaris
Concanavalin A (ConA)
lectin
Canavalia ensiformis
anti-CD3, anti-TCR
antibody
Pokeweed (PWM)
(Phytolacca americana) – formerly
used for colouring red wine
(toxic: triterpene saponin)
Chenopodiales
Phytolaccaceae
Phytohaemagglutinin (PHA)  Canavalia ensiformis – Jackbean or Sword bean
Concanavalin A (ConA)  Phaseolus vulgaris – common bean
A bab-félék nyersen, nem rendesen megfőzve a
lektin tartalmuk miatt mérgezőek!
(klasszikus ételmérgezési tünetek – rosszullét, hányás, hasmenés)
A bab beáztatása, és az áztatóvíz leöntése jelentős
mennyiségű lektintől szabadít meg, és a forralás
inaktiválja (irreverzibilisen denaturálja) a lektineket.
(Ezért nem célszerű a babféléket „sous-vide” –olni)
Biswas S, Kayastha AM.: J Biochem Mol Biol. 2002 Sep 30;35(5):472-5.
Thermal stability of Phaseolus vulgaris leucoagglutinin: a differential scanning calorimetry study
Receptor crosslinking
(immediate)
phosphorilation steps
- Western blot
- Bead array
(seconds-minutes)
Antigen receptors (TCR, BCR), and different
other receptors (e.g. cytokine receptors)ic Ca2+ increase
- FACS, microscopy
Gene activation
- RT-PCR
Cytokine synthesis - IC cytometry
Cytokine secretion
- ELISA, ELISPOT
- DNA content
- IN antigens
Cell-cycle/apoptosis
Lymphocyte activation
Cell division
- H-thymidine, CFSE, MTT
and the examination
possibilities
The examination often requires specific
3
Ag-Ab reactions
A jelenlevő vagy termelt „antigének”
(pl. fehérjék, citokinek, ellenanyagok, ….)
vizsgálata
ELISA
Enzyme Linked Immune Sorbent Assay
ELISA plate
well
Different plastic surfaces for different materials
enzyme linked
immune sorbent
enzyme
Antibody conjugated with
enzyme
Antigen/antibody
adsorbed to solid
surface
ENZYME ACTIVITY IN ELISA IS
DIRECTLY PROPORTIONAL TO THE
AMOUNT OF ANTIGEN PRESENT
Enzyme activity is measured by the color
reaction due to conversion of substrate
Similar principle applies to many other antibody-based
detection methods
Miért enzimet használunk jelzésként?
Jelölésekről/jelzésekről az előző szemináriumban esett már említés:
• radioaktív izotópok  RIA főleg régen használták (ELISA elődje)
• fluoreszcens festék
• ….
egy jelzés  egy jel
(fluoreszcencia: fakulás (fading) )
enzim előnye: sok szubsztrát átalakítás (turnover szám)  jel felerősítése
ÉRZÉKENYSÉG NÖVEKEDÉSE!
BASIC SETUPS IN
ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Direct method
Indirect method
(simplicity)
(more label  sensitivity)
(“modularity”)
Label
Label
Secondary
antibodies
Primary
antibodies
Antigen
surface
BASIC SETUPS IN
ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Enzyme/anti-enzyme system
(generally applied in microscopic immunohistochemistry)
PAP – peroxidase / anti-peroxidase
APAAP – alkaline phosphatase / anti- alkaline phosphatase
Enzyme
Enzyme-specific
antibody, same (iso)type
as the primary antibody
Primary
antibody
Antigen
Secondary
antibody
Az ELISA, immunhisztokémia, citometria során
használt módszerek alap jellegzetességei
Biotin-avidin rendszerrel kombinált indirekt módszerek
Az avidin nagy affinitással képes a biotint kötni
Egyszerű
Avidin-enzim komplexek
„ABC”
Avidin Biotin
enzimComplexek
Jel felerősítése
(arányok!!! mint a
precipitációnál)
Biotin-enzim komplex
Avidin
Biotinált ellenanyag
Antigén
(szabad)
BASIC SETUPS IN
ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Indirect systems combined with biotin-avidin signal
amplification (Avidin binds biotin with very high affinity )
Basic
Avidin-enzyme complexes
ABC
Avidin-biotin
enzyme
complexes
Biotin-enzyme complex
Avidin
Biotinylated antibody
Antigen
Protein biotinylation is easy
Modified biotins with different reactive chemical
functional groups can be bound to diverse materials
M.Wilchek, EA. Bayer et al.
Biotin
ELISA systems can be used not only for the
detection of a material, but its exact concentration
can also be measured by the help of a known
concentration standard
The different direct or indirect, biotin-avidine
methods can be used in combination
See the next examples!
EXAMPLES FOR DIRECT, INDIRECT
AND COMPETITIVE ELISAs
STEPS OF COMBINED SANDWICH ELISA
Goal: examination of antigens present at low concentration in complex
biological and other samples (body fluids, natural waters, foods)
e.g. hormons, cytokines, etc.
Coating with Agspecific „capture”
antibody
Blocking free plastic
surface with inert
protein
Removal of excess enzyme
Removal of unbound material
Removal of unbound protein
Removal of unbound material
Addition of antigencontaining solution
Addition of biotinylated
antibody specific to a
different epitope on
target protein
Addition of avidinconjugated enzyme
Addition of substrate
Antigen specific antibody pair is a prerequisite
TUMORDIAGNOSTICS
Tumor specific (TSA) and tumor associated (TAA) antigen
recognizing antibodies can be used in diagnostics
The antigens can be detected by sandwich techniques
Tumor Ag specific
detecting/reporter
antibody (suplemented)
Tumor antigen (patient serum)
PROSTATE CANCER:
Tumor Ag specific
capture antibody
precoated plate
(as a part of the kit)
Prostate-Specific Antigen (PSA) derives its name from its first known site of origin, the
prostate gland. Serum concentrations of PSA are elevated in patients with prostate
cancer, benign prostatic hypertrophy (BPH) and prostatitis. In addition, PSA serum levels
appear to correlate with the volume and clinical stage of prostate cancer.
Human prostatic acid phosphatase (PAP) in human serum can be used similarly in
quantitative measurement.
Bladder cancer:
NMP22 is a Nuclear Matrix Protein found in human epithelial cells. In the urine of healthy
individuals, the protein is present at low levels. The majority of patients with bladder cancer
release large quantities of NMP22 into their urine, that can be detected by immunoassay
Thyroid Cancer :
Increased Serum Thyroglobulin (sTG) can be detected by immunoassay
Alpha Fetoprotein (AFP) is a 68 kDa protein, which is produced primarily during fetal life by
the fetal liver and yolk sac. AFP also appears in the maternal serum, presumably by
transplacental transfer . After birth, serum AFP levels decline rapidly during the first year of life
and low basal levels are then apparently maintained throughout childhood and adult life. Its
normal concentration in serum is below 9 ng/mL; higher concentrations are associated with
hepatoma and ovarian, testicular and presacral teratocarcinomas, and other cancers.
Carcinoembryonic Antigen (CEA) is a glycoprotein involved in cell adhesion. It is
normally produced during fetal development. serum from individuals with colorectal and other
carcinomas had higher levels of CEA than healthy individuals and can be used to monitor the
response to colon cancer treatment.
Her-2/neu protein is a 185 kD trans-membrane glycoprotein associated with tyrosine
kinase activity. Approximately 20-30% cases of breast cancer show an amplification
and/or over-expression of Her-2/neu in tumor cells. Since the introduction of Herceptin
as a targeted therapy for breast cancer, the clinical testing of Her-2/neu in breast
carcinoma has become very important in patient care. It can be shown by
immunohistochemistric or immunofluorescent methods from biopsy.
Detection of antigen specific antibodies
• Tracking the effectiveness of an immunisation
• Findig cell microcultures which produce antigen specific antibodies
(monoclonal antibody production / hybridome technics)
• Detection of antigen specific antibodies in clinical samples
(infections, autoimmune process – see the forthcoming examples)
(simple) ELISA
STEPS OF BASIC INDIRECT ELISA
Goal: Detection of antigen specific antibody
(This type will be performed at the practice)
Adsorption of antigen
(coating)
Saturation of uncovered
surface area with
proteins
Removal of excess antibody
Removal of excess antigen
Removal of excess protein
Addition of Agspecific antibodies
Addition of
Secondary Ab
conjugated with
enzyme
Addition of
chromogenic
substrate
e.g. pure protein antigens, or
large synthetic peptides
(at least µg/ml concentration)
TESTING VIRAL INFECTION
Viral antigens cannot be detected efficiently in lot of case (latency), but you can
efficiently detect the antibodies that were produced by the body in response to
the viral infection. These antibodies can serve as diagnostic markers.
term: SEROPOSITIVITY
Human Ig specific labeled antibodies indicate the
presence of the virus specific antibodies.
The serum of the infected person with virus specific
antibodies. The antibodies could bind the virus antigens.
viral antigen precoated test plate
EXAMPLES:
• Epstein-Barr Virus (EBV) test kit → ELISA method for the qualitative detection of IgG
antibody to Epstein-Barr Virus nuclear antigen-1 (EBNA-1) in human serum
• HIV assay kit → enzyme-linked immunosorbent assay for the detection of antibody to HIV-1 in
serum, plasma or dried blood spots
•Toxoplasmosis (Toxoplazma gondii – parasitic protozoa) → chromatographic immunoassay
for the qualitative detection of human IgM antibodies against Toxoplasma in serum or plasma
AUTOIMMUNITY
Autoantibodies from different autoimmune diseases can be detected similarly.
In 60% of SLE patients autoantibodies can be detected against double strand DNA. Anti-ENA
antibodies can be seen in many systemic AIDs. (See Ouchterlony method in the previous
practice)
Type I (autoimmune) diabetes can be detected by the presence of antibodies against islet-cell
specific antigens.
Glutamic acid decarboxylase (GAD65), specific antibodies have been found in 70-90%
of prediabetic and Type 1 diabetic patients (including approximately 7-10% of adult onset
diabetics with Type 1 diabetes)
IA-2 (a tyrosine phosphatase-like protein) specific Ab. are found in 50-75% of Type 1
diabetic patients at and prior to disease onset, are generally more prevalent in younger
patients, and are associated with rapid progression to overt disease. These
autoantibodies have been detected in some ICA positive/GAD Ab negative patients, and
therefore can be considered independent markers of disease.
Insulin: anti-insulin ABs are found predominantly, though not exclusively, in young
children (<5 years) developing Type 1 diabetes. In insulin-naive (untreated) patients, the
prevalence of autoantibodies to insulin is almost 100% in very young individuals and
almost absent in patients with adult onset of Type 1 diabetes. (It should be noted that
insulin autoantibodies are indistinguishable from insulin antibodies that commonly
develop with insulin therapy).
SERUM ANTIBODY ISOTYPE DETERMINATION
Sometimes the antigen specific antibodies could refer the presence of the antigen in the
body (see the ”viral infection testing” part )
The isotypes of these antibodies additionally could refer the fresh/persistent (IgM
dominance) or repeated/memory immune response (IgG dominance) against the parasite.
The possibilities can be discriminated by the use of isotype specific secondary antibodies.
α-IgM
α-IgG
Memory response
IgM
IgG
Antigen
Increased IgE level can be seen in atopic allergy cases, and
some autoimmune process, and in the case of some parasite
infection
Abnormal levels of serum immunoglobulin isotypes can
be seen in the case of some immunodeficiencyies (e.g. hyper
IgM syndrome, multiplex myeloma (case study!))
isotype specific
antibody
antibody from the serum
antibody capture antibody
COMPETITIVE ELISA
Goal: detection of small antigens (haptens) in complex biological samples
when the sandwich ELISA can’t be applied (no need for antibody pairs)
Antigen in solution and on the solid surface compete for the binding site of labeled
specific antibody.
- Coating with antigen, blocking
- Addition of experimental sample that contains or lacks antigen
- Addition of labeled antibody  binding
- Washing
- Addition of enzyme substrate
RIA - RadioImmunoAssay
(Inconvenient because of the usage of radioactive isotopes)
Usage:
• Some small antigen (e.g. haptens) can’t be conjugated by large labels
without the destruction of the epitope
• As a competitive method  one type of antigen specific antibody is
enough to perform
Generally 125I or 131I is the label (to tyrosine side chain in the proteins)
EXAMPLE:
cAMP RIA:
125I 
labelled cAMP:
measured cAMP:
125I
bound
labelled or “cold” cAMP
Ag specific
capture antibody
ELISA types (summary)
capture/sandwich
simple
e.g. detection of antigen
specific antibodies
Ag specific
detector/reporter detector/reporter
antibody
antibody
Ag specific
antigen
antibody
Ag specific
antigen
capture antibody
soluble Ag are present in
the sample
competitive
detection of antigens
no soluble Ag
Ag specific
antibody
antigen
detection of haptens or detection of an antigen with only one type of antigen specific antibodies
ELISA methodical errors
Aspecific adhesion of the materials
The antibodies (as proteins generally) could
also bind to the surface aspecifically
- directly
- indirectly
the color is the same
Aspecific adhesion of the proteins
can be reduced by:
•blocking of the surface
•applying detergent
Hook effect -
It could happen in ”one step”, ”without washing” sandwich tests. Large antigen
concentration could give invalid small value. Unwashed soluble antigens compete with the captured
surface bound ones. It can be avoided by efficient washing steps, or with the dilution of the sample.
methodical errors of the ELISA
The soluble antigen can compete with the surface bound one:
• In the case of very high antigen concentration
• In the case of ineffective/missing washing steps (e.g. “one step” or
“no-wash” procedures)
Hook effect - false negative or decreased results
appropriate ELISA
Soluble antigen could be present in the case
of very high antigen concentration or without
washing the test plates. This results false
negative or diminished positive readout.
0
The competitive ELISA avoids this phenomenon
The competitive tests are more reliable in the clinical diagnostics
ELISA PLATES - RESULTS
(Chromogenic substrates can have different colors)
Diagnostic quick tests:
e.g. detection of human chorionic gonadotropin in serum or urine
(pregnancy test)
The principles of these tools are similar to the ELISA
assay you have met before.
hCG Rapid One-Step Immunochromatographic Assay strip
front view
side view
absorbtion pad (cellulose)
control antibody lane
(detection antibody capture)
hCG capture antibody lane
nitrocellulose membrane
(signal detection pad)
glass fiber membrane with visually labeled detection antibodies
sample application pad
urine
detection antibody
capture antibodies
control
antibody lane
hCG capture
antibody lane
hCG positive hCG negative
control lane (C)
test lane (T)
detection antibodies
similar to sandwich ELISA
hCG
Competitive system
hCG positive
control lane
detection antibody
capture antibody
(
hCG lane
bound hCG)
hCG negative
control lane
test lane
similar to competitive ELISA
You don’t need to use additional
enzymatic incubation steps, because the
labels on the detection antibodies can be
observed by naked eye:
• colloidal gold („surface plasmon” resonance)
• colored latex beads
Other uses of immunechromatographic
test strips:
detection of toxins in food
e.g.: aflatoxins
(mycotoxins of Aspergillus spp.)
ELISPOT
Enzyme Linked Immuno-Spot
the principles are similar to ELISA
capable to determine the number of cells that
produce antibodies, cytokines, chemokines,
granzymes and other soluble effector molecules
the sensitivity allows the determination 1 activated
cell among 300 000 other, so it can reveal activated
effector cells not only after policlonal-, but after
antigen specific activation
the first steps should be done in aseptic conditions
ELISPOT
The process
- coating with antigen specific capture antibodies
- blocking
- administration of the cells (activation, incubation)
- washing
- administration of biotin conjugated
antigen specific secondary antibody
- avidin-enzyme conjugate
upper view of a well on
an ELISPOT plate with
- administration of the chromogenic
the generated spots
substrate which forms insoluble
precipitate  spot (AEC 3-amino-9-ethylcarbazol)
A spot showing
the place of the
cytokine
producing cell
It can be evaluated by microscopy (slow, manual process)
or you can use “ELISPOT plate reader” (fast + standardizable spot
number and size determination)
ELISPOT
• Spot number represent the number of the effector
•
molecule (e.g. cytokine) producing cells
A spot size represent the amount of the effector molecule
which is produced by one cell
• The overall/cumulative spot area calculated by the
software summarise this two parameter together, which
represents the overall effector molecule production
Cellular ELISA
Identification of cell surface or intracellular antigens
• Different cell surface molecules
• Intracellular molecules
• Signalling
ratio of the phosphorylated / nonphosphorylated signalling molecules
(two color ELISA - 2 different enzyme and
substrate or two different
fluorescent/luminescent dyes)
• Adherent cell can simply grow on the surface of the plate
• Non-adherent cells can be “fixed” on the surface
e.g. poly-L-lysine precoated plates
Western blot
It can detect the presence or even different modifications
(e.g. phosphorylation state) of specific proteins
The cells’ activation stage can be „frozen” at different times, so
the events of the activation can be monitored in parallel
samples.
at least 105-106 cells required
WESTERN BLOT
Steps:
Anode(+)
1) sample preparation
(cells, tissues)
2) gel electrophoresis
3) blotting
4) labeling
Cathode(-)
5) detection
Usage: identification of defined
components from protein mixtures by
antigen specific antibodies
WESTERN BLOT
The use of antibodies in molecular biology is widespread
It is probably most often encountered in Western analysis
SDS-PAGE gel  resolved into single protein bands (overlap possible)
Presence of a protein is determined by hybridizing the proteins, transferred
or applied to a membrane, with the relevant antibody
Antibody recognizes
epitope in specific protein
Protein
Standard sample
SDS-PAGE
Membrane
Western blot
e.g. the phosphotyrosine and ZAP-70 blot of activated T-cells
non-activated
ConA
PHA
After removing (elution) of the anti P-Y
antibodies, the membrane can be
incubated with anti ZAP-70 antibodies
also, so the phosphorylation state of
the ZAP-70 can be evaluated
Y
P-Y blot
P-Y P-Y
The usage of the WB in HIV diagnostics:
Western-blot:
Western-blot of the HIV-1 carrier CEM cell line (T-cell line) lysate.
The blot membrane with the HIV antigens is reacted with the patient’s serum.
The serum of an infected individual contains anti-HIV antibodies which react with
the antigens on the blot membrane and produces specific WB lane patterns.
days of seroconversion
WESTERN BLOT
Detection methods:
Colorimetric (peroxidase enzyme – densitometry /
spectrophotometry)
Chemiluminescent (ECL)
Radioactive (isotope – X-ray)
Fluorescent (staining – CCD camera)
ENHANCED CHEMILUMINESCENCE (ECL)
•
•
Immobilized
proteins
•
•
Primary
Fabantibody
Fc
Horseradish
peroxidase
H2O2
Epitope on
protein
surface
Secondary
antibody
Membrane
In the presence of H2O2, horseradish peroxidase (HRP)
oxidizes diacylhydrazides such as luminol
Directly after oxidation, luminol is in an exited state, and
emits a photon to return to the ground state
This photon can be detected with a film or a camera
Light emission can be enhanced by ~1000-fold with
phenolic compounds such as 6-hydroxybenzothiazole
(enhancer)
H2O
luminol
luminol
h
enhancer
Detection
IMMUNOPRECIPITATION
DO NOT MIX this term with the precipitation
reaction of the soluble immuncomplexes
• isolation and
concentration of a
particular protein
from a protein
mixture
• detection of protein
associations (e.g.
members of
receptor
signalization)
CHROMATIN
IMMUNOPRECIPITATION
(ChIP)
Identification of molecules (mainly
transcription factors) binding to a
specific site of the DNA
Provides information about the
link between signaling pathways
and gene activation
IMMUNOHISTOCHEMISTRY
Labeled antibodies added to fixed tissue sections detect
the distribution of the chosen antigen within the tissue or
within the cells of a particular tissue
• Immunofluorescence
•Fluorescent dye coupled to antibody
FITC – fluorescein isothiocyanate (green)
PE – phycoerythrin (orange)
• Immunoenzyme method
• enzyme-coupled antibody
P – peroxidase
AP – alkaline phosphatase
(Substrates converted into an insoluble compound)
IMMUNOHISTOCHEMISTRY
Fixation
Tissue
sample
Freezing
Sectioning
Section before
staining
IMMUNOHISTOCHEMISTRY
Enzyme
X
Avidin
Biotin
Secondary antibody
Primary antibody
Slide
Cells
Tissue
sample
Classical histochemistry
Acute bronchopneumonia (hematoxylin-eozin staining)
Only few cell types could be identified
Immunohistochemistry
(CD68+ macrophages and lymphocytes, granuloma)
Detection of actin microfilaments (TRITC)
A fixed and permeabilized skin fibroblast
Mitochondria
F-actin
Nucleus
Antinuclear (ANA) autoantibodies from the serum of a SLE
patient can be visualized in cell culture (Hep-2) by indirect
fluorescent labeling (immunofluorescence)
Intracellular cytokine detection by
immunofluorescence
cytokine specific antibody with fluorescent labelling
- the cell membrane should be permeabilized (detergent)
- the cells should be fixed previously avoiding the
decomposition of the cells (e.g. aldehyde fixation)
- optionally the cells could be
labelled by some cell type specific
antibody in the beginning (e.g. CD4)
cytokines
The result:
You can determine which cell type has produced
the cytokines!
The sensitivity could reach that of the Western blot.
(e.g. with chilled CCD camera mounted microscope:
You need only one cell for detection)
Bioassay
Cytokine dependent or cytokine sensitive cell lines can serve as “bio
indicators” of that given cytokine. Those cytokines can be detected by
high sensitivity with these indicator cells:
e.g. CTLL2 cells
IL-2 present
no IL-2
TNF present
no TNF
e.g. Wehi 164 cells
Problem: cross reactivity of different cytokines
The survival/proliferation of the cells can be measured by a
colorimetric assay (MTT assay)
MTT assay:
cytokine concentration
IL-2
CTLL2
TNF
Wehi 164
There could be some cross reaction between cytokines. e.g. the CTLL-2 cell line can be
stimulated by IL-4 also
Detection of intracellular (cytoplasmic) Ca2+ concentration
An inrease in cytoplasmic Ca2+
levels can be detected by
fluorescent indicator dyes.
/Fluo-3 or Indo-1/
for example – ic Ca2+ signal in a single cell
B cell
antigen presentation by B cell to T cell
(click)
T cell
Investigation of gene activation
Activation of T cells can be monitored by the detection
of the transcribed mRNA of the activated genes.
e.g. activation of cytokine genes
method: RT-PCR, QRT-PCR
cells  RNA isolation
RNA  (reverse transcriptase)  cDNA
cDNA  (PCR)  determination of the length and quantity
RT-PCR: agarose gel (densitometry)
QRT-PCR: fluorescent method
(TaqMan probe (FRET) or dsNA intercalating fluorochrome  SYBR green)
The size of the cycling cells are increased –
called blast transformation
Cell-cycle
Possibility of the examination
Stimuli
(e.g. antigen)
resting lymphocyte
(G0)
effector cell
- transcription (RT-PCR)
- protein synthesis
memory cell
(Immunoassay)
changes in the RNA- and protein
synthesis, in the cell membrane
and in the transports
cell division
change in the
number of the cells
(MTT, CFSE, special histone
modifications)
DNA-synthesis
DNA quantification
(fluorescent DNS intercalating agents,
3H-thymidine)
The cell cycle can be examined by fluorescent dye
G2
G0
M
that intercalates stoechiometrically
into the double stranded DNA
(e.g. propidium iodide, PI)
DNA analysis
G1
cell number
s
G0G1
G2 M
s
0
200
400
600
800
1000
4N
2N
DNA content
Distribution of a normal cycling cell-population by
DNA content (flow cytometry)
Methods for determinating the
B/T cell proliferation
3H-labeled
thymidine incorporation – measures the increasing DNA
content by β decomposition, and does not answer the numbers of cell
division, and the dividing cell number
thymidine-analog bromodeoxyuridin (BrdU) can be administered to
experimental animals, or cell cultures, and the proliferating cells can be
detected by labelling with BrdU specific antibody (microscopy, FACS)
Carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescent
stain can be used to tracking the cell divisions:
Tracking the cell divisions
“Cell tracer” or “Cell tracker” dyes enter the cell, and
trapped there. The apolar CFSE can bind covalently to the
cellular proteins. Later the stain can only be diluted by the
cell divisions: distributed equally between the two daughter
cells – the fluorescence intensity decreases to the half also.
cell divisions:
7 6 5 4 3 2 1 0