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ANALYTICAL AND PREPARATIVE
METHODS BASED ON PRIMARY
ANTIGEN-ANTIBODY BINDING
IMMUNOAFFINITY CHROMATOGRAPHY
ELISA
SENSITIVITIES OF IMMUNOASSAYS
AFFINITY PURIFICATION OF ANTIBODIES
USING AN ANTIGEN-SORBENT COLUMN
column
”affinity purified antibody”: monoclonal
antibodies which can be ordered from
catalogues are also purified using this
technique
polymer beads
covalently bound
antigen
STEPS OF PURIFICATION
1)
2)
3)
4)
Addition of
antibodies to
be purified
Binding
Washing
Elution
PURIFICATION OF ANTIGENS
1) Loading the antigen mixture
coloumn
2) Binding
3) Washing
polimer bead
4) Elution
fixed antigen specific Abs on the surface of the bead
Purified antigens
IMMUNOPRECIPITATION
• 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
ELISA
Enzyme Linked Immune Sorbent Assay
ELISA plate
well
Different plastic coatings 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
BASIC SETUPS IN
ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Direct method
Indirect method
Label
Label
Secondary
antibodies
Primary
antibodies
Antigen
BASIC SETUPS IN
ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Enzyme/anti-enzyme system
PAP – peroxidase / anti-peroxidase
APAAP – alkaline phosphatase / anti- alkaline phosphatase
Enzyme
Enzyme-specific
antibody, same isotype
as the primary antibody
Primary
antibody
Antigen
Secondary
antibody
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
M.Wilchek, EA. Bayer et al.
Biotin
Modified biotins with different reactive
chemical functional groups can be bound
to diverse materials
SENSITIVITIES OF IMMUNOASSAYS
EXAMPLES FOR DIRECT, INDIRECT
AND COMPETITIVE ELISAs
STEPS OF COMBINED SANDWICH ELISA
For antigens present at low concentration in complex biological samples
Coating with Agspecific „capture”
antibody
Blocking free plastic
surface with inert
protein
Addition of antigencontaining solution
Addition of biotinylated
antibody specific to a
different epitope on
target protein
Addition of avidinconjugated enzyme
Addition of substrate
Removal of excess enzyme
Removal of unbound material
Removal of unbound protein
Removal of unbound material
PRACTICAL USE OF IMMUNOASSAYS
hCG (human chorionic gonadotropin) – pregnancy test
Detection of tumor antigens, cytokines, hormones
doping/drug assay: EPO (erythropoietin), steroids
sandwich assays or competitive tests
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.
THE ROLE OF THE HER-2/NEU PROTEIN IN THE PATHOGENESIS
OF BREAST CANCER AND THE HERCEPTIN THERAPY
STEPS OF BASIC INDIRECT ELISA
Detection of antigen specific antibody
Adsorption of antigen
(coating)
Saturation of uncovered
surface area with
proteins
Addition of Agspecific antibodies
Addition of
Secondary Ab
conjugated with
enzyme
Addition of
chromogenic
substrate
Removal of excess antibody
Removal of excess antigen
Removal of excess protein
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.
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
Highly sensitive method used to detect and quantitate small antigens
(haptens) in complex biological samples. 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
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.
ELISA PLATES - RESULTS
(Chromogenic substrates can have different colors)
You could have already met such kind of diagnostic tools or you are
going to meet them during your career
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
The assay can be used in
semi-quantitative manner
Other uses of immunechromatographic
test strips:
detection of toxins in food
e.g.: aflatoxins (mycotoxins of
Aspergillus spp.)
DETERMINATION OF THE CONCENTRATION
A quantitative property of an indicator refers to the concentration:
 color (absorbance, optical density)
 fluorescence
 cell number (e.g. in determination of growth factor concentration)
Quantified concentration can be obtained by comparison
with known concentration sample (standard)
The principle of comparison:
equal absorbances  equal concentrations
PARTIAL TRUTH !!!
The serial dilution of the standard
OD
The sample with unknown
concentration
You should also dilute the
unknown sample
Only this region indicates
valid concentrations
All these concentrations could be choosen?
concentration
0.004
0.007
0.015
0.030
0.061
0.12
0.24
0.49
0.97
1.9
3.9
7.8
16
31
62
125
250
500
1000
?
0