ANALYTICAL AND PREPARATIVE METHODS BASED ON PRIMARY ANTIGEN-ANTIBODY BINDING
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Transcript of ANALYTICAL AND PREPARATIVE METHODS BASED ON PRIMARY ANTIGEN-ANTIBODY BINDING
ANALYTICAL AND PREPARATIVE ANALYTICAL AND PREPARATIVE METHODS BASED ON PRIMARY METHODS BASED ON PRIMARY
ANTIGEN-ANTIBODY BINDINGANTIGEN-ANTIBODY BINDING
IMMUNOAFFINITY CHROMATOGRAPHY
ELISA
SENSITIVITIES OF IMMUNOASSAYSSENSITIVITIES OF IMMUNOASSAYS
AFFINITY PURIFICATION OF ANTIBODIES AFFINITY PURIFICATION OF ANTIBODIES USING AN ANTIGEN-SORBENT COLUMNUSING AN ANTIGEN-SORBENT COLUMN
column polymer beads
covalently boundantigen
affinity purified antibody : monoclonal antibodies which can be ordered from catalogues are also purified using this technique
1) Addition of antibodies to be purified
2) Binding3) Washing4) Elution
STEPS OF PURIFICATIONSTEPS OF PURIFICATION
polimer bead
fixed antigen specific Abs on the surface of the bead
coloumn
PURIFICATION OF ANTIGENSPURIFICATION OF ANTIGENS
1) Loading the antigen mixture
2) Binding
3) Washing
4) Elution
Purified antigens
IMMUNOPRECIPITATIONIMMUNOPRECIPITATION
• isolation and concentration of a particular protein from a protein mixture
• detection of protein associations (e.g. members of receptor signalization)
ELISA plate
well
ELISAELISAEnzyme Linked ImmuneEnzyme Linked Immune Sorbent AssaySorbent Assay
enzyme linked immune sorbent
Antibody conjugated with enzyme
enzyme
Antigen/antibody adsorbed to solid
surface
ENZYME ACTIVITY IN ELISA IS ENZYME ACTIVITY IN ELISA IS DIRECTLY PROPORTIONAL TO THE DIRECTLY PROPORTIONAL TO THE
AMOUNT OF ANTIGEN PRESENTAMOUNT 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 INBASIC SETUPS IN ELISA ELISA / / IMMUNOHISTOCHEMISTRY IMMUNOHISTOCHEMISTRY / / FLOW CYTOMETRYFLOW CYTOMETRY
Direct method Indirect method
Antigen
Primary antibodies
Label
Label
Secondary antibodies
Enzyme/anti-enzyme system
PAP – peroxidase / anti-peroxidaseAPAAP – alkaline phosphatase / anti- alkaline phosphatase
Antigen
Primaryantibody
Secondaryantibody
Enzyme-specific antibody, same isotype as the primary antibody
Enzyme
BASIC SETUPS INBASIC SETUPS IN ELISA ELISA / / IMMUNOHISTOCHEMISTRY IMMUNOHISTOCHEMISTRY / / FLOW CYTOMETRYFLOW CYTOMETRY
Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity )
Basic ABC
Antigen
Biotinylated antibody
Avidin
Avidin-enzyme complexes
Biotin-enzyme complex
Avidin-biotin enzyme
complexes
BASIC SETUPS INBASIC SETUPS IN ELISA ELISA / / IMMUNOHISTOCHEMISTRY IMMUNOHISTOCHEMISTRY / / FLOW CYTOMETRYFLOW CYTOMETRY
SENSITIVITIES OF IMMUNOASSAYSSENSITIVITIES OF IMMUNOASSAYS
EXAMPLES EXAMPLES FOR FOR DIRECT, INDIRECT DIRECT, INDIRECT AND COMPETITIVE ELISAAND COMPETITIVE ELISAss
For antigens present at low concentration in complex biological samples
Removal of unbound material
Blocking free plastic surface with inert
protein
Removal of unbound protein
Addition of biotinylated antibody specific to a different epitope on
target protein
Removal of unbound material
Addition of avidin-conjugated enzyme
Addition of substrate
Coating with Ag-specific „capture”
antibody
Addition of antigen- containing solution
Removal of excess enzyme
STEPS OF COMBINED SANDWICH ELISA STEPS OF COMBINED SANDWICH ELISA
PRACTICAL USE OF IMMUNOASSAYSPRACTICAL 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
TUMORDIAGNOSTICSTUMORDIAGNOSTICS
Tumor specific (TSA) and tumor associated (TAA) antigen recognizing antibodies can be used in diagnostics
The antigens can be detected by sandwich techniques
Tumor antigen (patient serum)
Tumor Ag specific detecting/reporter
antibody (suplemented)
Tumor Ag specificcapture antibody precoated plate
(as a part of the kit)PROSTATE CANCER:
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 THE ROLE OF THE HER-2/NEU PROTEIN IN THE PATHOGENESIS OF BREAST CANCER AND THE HERCEPTIN THERAPYOF BREAST CANCER AND THE HERCEPTIN THERAPY
STEPS OF BASIC INDIRECT ELISA STEPS OF BASIC INDIRECT ELISA Detection of antigen or specific antibody
Adsorption of antigen (coating)
Removal of excess antigen
Saturation of uncovered surface area with
proteins
Removal of excess protein
Addition of Ag-specific antibodies
Addition of Secondary Ab
conjugated with enzyme
Removal of excess antibody
Addition of chromogenic
substrate
TESTING VIRAL INFECTIONTESTING 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.
viral antigen precoated test plate
The serum of the infected person with virus specific antibodies. The antibodies could bind the virus antigens.
Human Ig specific labeled antibodies indicate the presence of the virus specific antibodies.
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
AUTOIMMUNITYAUTOIMMUNITYAutoantibodies 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 DETERMINATIONSERUM ANTIBODY ISOTYPE DETERMINATIONSometimes 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.
Antigen
IgM IgG
α-IgM α-IgG
Memory response
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!))
antibody capture antibody
antibody from the serum
isotype specific antibody
COMPETITIVE ELISACOMPETITIVE ELISA
Highly sensitive method used to detect and quantitate small amounts of antigen 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
Maybe you 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
nitrocellulose membrane(signal detection pad)
glass fiber membrane with visually labeled detection antibodies
front view side view
hCG capture antibody lane
control antibody lane(detection antibody capture)
absorbtion pad (cellulose)
sample application pad
urine
hCG capture antibody lane
control antibody lane
detection antibodies
hCG
control lane (C)
test lane (T)
hCG positive hCG negative
detection antibody capture antibodies
hCG lane( bound hCG)
control lanedetection antibody
capture antibodycontrol lane
test lane
hCG positive hCG negative
Competitive system
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)
• paint-filled latex beads
e.g.: aflatoxins (mycotoxins of Aspergillus spp.)
The assay can be used in semi-quantitative manner
Other uses of immunechromatographic test strips:
detection of toxins in food
ELISA PLATES - RESULTSELISA PLATES - RESULTS
ELISA plate with different concentration samples after the
addition of the chromogen substrate
ELISA plate with different concentration samples after
stopping the enzyme reaction
DETERMINATION OF THE CONCENTRATIONDETERMINATION 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 !!!PARTIAL TRUTH !!!
concentration
10
00
50
0
25
0
12
5
62
31
16
7.8
3.9
1.9
0.9
7
0.4
9
0.2
4
0.1
2
0.0
30
0.0
61
0.0
07
0.0
15
0.0
04
0
The sample with unknown concentration
ODThe serial dilution of the standard
According to OD: it could be anyone
?
You should also dilute the unknown sample
This region could indicate the concentration
This region could indicate the concentration