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

protein such as BSA, casein etc..

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)

(See the past case study about myeloma multiplex)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 + 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 colors”)•colored latex beads

competitive immunochromatographic test strips for detecting aflatoxin

The assay can be used in semi-quantitative manner

ELISA PLATES - RESULTSELISA PLATES - RESULTS

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