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ELISA
Enzyme Linked Immune Sorbent Assay
in basic science and clinical practice
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
STEPS OF BASIC INDIRECT ELISA
Detection of antigen specific antibody
Adsorption of antigen (coating)
Removal of excess antibody
Removal of excess antigen
Saturation of uncovered
surface area with proteins
Addition of Ag-specific
antibodies
Addition of Secondary
Ab conjugated with
enzyme
Addition of
chromogenic substrate
Removal of excess protein
EXAMPLES FOR DIRECT AND
INDIRECT ELISAs
TESTING VIRAL INFECTION
Viral antigens cannot be detected efficiently in lot of case (latency), but you can
efficiently detect the antibodies that were produced 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.
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).
DETERMINATION OF ANTIBODY ISOTYPE
Sometimes the antigen specific antibodies indicate the presence of the antigen in the body (see the
”viral infection testing” part )
The isotypes of these antibodies additionally could reveal whether aan ongoing immune response is
a primary (IgM dominance) or a or a secondary/memory response (IgG dominance). To determine
the isotype of reacting antibodies, one should use 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
STEPS OF COMBINED SANDWICH ELISA
For antigens present at low concentration in complex biological samples
Coating with Ag-specific
„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
Detection of tumor antigens, cytokines, hormones
doping/drug assay: EPO (erythropoietin), steroids
hCG (human chorionic gonadotropin) – pregnancy test
sandwich assays
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)
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. The majority of patients with
bladder cancer release large quantities of NMP22 into their urine,.
Thyroid Cancer :
Increased Serum Thyroglobulin (sTG) can be detected by immunoassay
Hepatocarcinoma
Alpha Fetoprotein (AFP) is a 68 kDa protein, Its normal concentration in serum is below 9 ng/mL;
higher concentrations are associated with hepatomas and ovarian cancer.
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.
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)
Today’s Practical: Pregnancy test based on
the principle of capture ELISA
You may have met such diagnostic tools or you certainly are are going to
meet them during your career
Test is based on detection of human chorionic gonadotropin in serum or urine
(pregnancy test)
The principles of these tools are similar to the capture ELISA
assay we discussed earlier.
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.)
ITT ÉRDEMES BEFEJEZNI, DE HA GONDOLJÁTOK A KÖVETKEZŐ KÉT DIA AZ
EIA TESZT KVANTITATÍV KIERTÉKELÉSÉRŐL SZÓL. AZ IDŐBE VALÓSZÍNŰLEG BELEFÉR
Tamás, mennyire tanítottátok meg nekik a számításokat?
DETERMINATION OF THE CONCENTRATION
A quantitative property of an indicator refers to the concentration:
 color (absorbance, optical density)
 fluorescence
Quantified concentration can be obtained by comparison with
signals obtained with a serial dilution of a known standard sample
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