ELISA plate

well

ELISAEnzyme Linked Immune Sorbent Assay

in basic science and clinical practice

Different plastic coatings for different materials

enzyme linked immune sorbent

Antibody conjugated with enzyme

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

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 IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY

Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity )

Basic ABC

Antigen

Biotinylated antibodyAvidin

Avidin-enzyme complexes

Biotin-enzyme complex

Avidin-biotin enzyme complexes

BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY

STEPS OF BASIC INDIRECT ELISA Detection of antigen specific antibody

Adsorption of antigen (coating)

Removal of excess antigenSaturation of uncovered

surface area with proteinsRemoval of excess protein

Addition of Ag-specific antibodies

Addition of Secondary Ab conjugated with

enzyme

Removal of excess antibody

Addition of chromogenic substrate

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.

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

AUTOIMMUNITYAutoantibodies 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 ISOTYPESometimes 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.

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

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

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 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)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

HepatocarcinomaAlpha 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.

Aspecific adhesion of the materials

Aspecific adhesion of the proteins can be reduced by:

• blocking of the surface• applying detergent

ELISA methodical errors

The antibodies (as proteins generally) could also bind to the surface aspecifically

- directly

- indirectly

the color is the same

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

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

similar to sandwich ELISA

hCG lane( bound hCG)

control lanedetection antibody

capture antibodycontrol lane

test lane

hCG positive hCG negative

Competitive system

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

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

ITT ÉRDEMES BEFEJEZNI, DE HA GONDOLJÁTOK A KÖVETKEZŐ KÉT DIA AZEIA 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 !!!

concentration

1000 50

0

250

125 62 31 16 7.8

3.9

1.9

0.97

0.49

0.24

0.12

0.03

0

0.06

1

0.00

7

0.01

5

0.00

4

0

The sample with unknown concentration

ODThe serial dilution of the standard

All these concentrations could be choosen?

?

You should also dilute the unknown sample

Only this region indicates valid concentrations

Only this region indicates valid concentrations