ELISAFrom A ～Z

1-Introduction

ELISA (Enzyme-linked immuno-sorbent assay) is one of immunoassay method used to detection of

1-Antibodies2-Proteins3-Peptides

4-Biomolecules

ELISA

What is immunoassay?

The term “immunoassay” is a combined term of “immuno”(= immunological, practically immunochemical antigen-antibody-reaction) and “assay” (= determination of the purity of a substance or the amount of any constituent of a mixture.

1 .Antigen/antibody of interest is absorbed on to plastic surface (‘sorbent’).

2. Antigen is recognised by specific antibody (‘immuno’).

3. This antibody is recognised by second antibody (‘immuno’) which has enzyme attached (‘enzyme-linked’).

4. Substrate reacts with enzyme to produce product, usually coloured .

Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960 .

In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands independently published papers that synthesized this knowledge into methods to perform EIA/ELISA.

History of Elisa

Rosalyn Sussman 1960Eva Engvall 1971

History of Elisa

1-Antibody (antiserum)

2-Antigen

Important components in immunoassay

3-Labeling materials

1-Antibody (antiserum)Antibody: proteins produced by the

immune system which help defend against antigens

SYMBOL FOR ANTIBODY

The variable regions are though to be the place for recognition and binding with the antigen.

Any molecule that induces production of antibodies when introduced in the body is called antigen. ORAny “thing”, foreign to the immune system.

e.g. bacteria, viruses, (or their parts), pollen, etc. SYMBOL FOR ANTIGEN

2-Antigen

3-Labeling materials In immunoassay, it is necessary to use any marker to know the antigen-antibody binding. For such purpose, we label either antigen or antibody with some materials that do not interefere with the binding.

e.g:horseradishperoxidase enzyme substrate: trimethylbenzidine

ELISA READER

ELISA KIT

Components of Kit

Pre-Coated, Stabilized 96-well Microtiter Plate.

Sample DiluentStandards and controlsConjugated Detection Antibody10X Wash SolutionSubstrateStop Solution

3-Basic principal of

ELISA

ELISA.wmv.MP4

Advantages of ELISA

Reagents are relatively cheap & have a long shelf life

ELISA is highly specific and sensitiveNo radiation hazards occur during

labelling or disposal of waste.Easy to perform and quick procedures Equipment can be inexpensive and

widely available.ELISA can be used to a variety of

infections.

Disadvantages of ELISA

Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes.

Enzyme activity may be affected by plasma constituents.

Kits are commercially available, but not cheapVery specific to a particular antigen. Won’t

recognize any other antigenFalse positives/negatives possible, especially

with mutated/altered antigen

4-Types Of

Elisa

1-Direct ELISA2-Indirect ELISA

3-Sandwich ELISA4-Competitive ELISA

5-Ogives ELISA

Types Of Elisa

1-Direct ELISA1-Direct ELISA

The direct detection method uses a labeled primary antibody that reacts directly with the antigen. Direct detection can be performed with antigen that is directly immobilized on the assay plate . Direct detection is not widely used in ELISA but is quite common for immunohistochemical staining of tissues and cells.

2-Indirect ELISA

The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody.The secondary antibody has specificity for the primary antibody

Direct and Indirect ELISA

3-Sandwich ELISAThe sandwich measures the amount of antigen between two layers of antibodies.

Sandwich are especially useful if the concentration of antigens is low or they are contained in a mix of high concentrations of contaminating protein

To utilize this assay, one antibody (capture) is bound to a microtiter plate well. Antigen is then added and bound to the antibody. Unbound products are then removed, and 2ry antibody is added (detection), then add the 3rd labeled antibody to complete the sandwich

Major advantages of this technique are that the antigen does not need to be purified prior to use, due to its high specificity .

In this Unlabeled antibody is incubated in the presence of its antigen. These bound antibody/antigen complexes are then added to an antigen coated well. The plate is washed unbound antibody is removed. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.

4-competitive ELISA

A newer technique uses an solid phase made up of an immuno-sorbent polystyrene rod with 8-12 protruding ogives.

The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous ) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents

5-multiple and portable ELISA

Ogives pins

and rack

The method

APPLICATIONS :

1-HIV-1 and HIV-2 (presence of anti-HIV antibodies). hepatitis C (presence of antibodies) .

2-hepatitis B (testing for both antibodies and a viral antigen) .

3-Measuring hormone levels HCG (as a test for pregnancy) .

4-LH (determining the time of ovulation). TSH, T3 and T4 (for thyroid function) .

ELISA step by step

Reading the kit insert before working

1 .Coating of Wells with Antibody100 μL of antibody diluted in buffer is added to each well .

Cover the plate and incubate at 4 °C overnight.

2 .Washingwash manually 3 times as follows:Empty the plate by inversion over a sink. Tap the inverted plate against some layers of soft paper tissue to remove residual liquid. Wash the plate by filling the wells by immersion in buffer B. Leave on the table for 3 minutes. Empty the plate as described above and repeat washing two more times.

2-A concentrated solution of non-interacting protein, such as bovine serum albumin (BSA) or casein, is added to all plate wells.This step is known as blocking,because the serum proteins block nonspecific adsorption of other proteins to the plate.

3 .Incubation with Test Samples.100 μL of test sample or standard diluted in buffer is added

per well.Cover the plate and incubate at room temperature for 2 hours.

4 .Wash as described in step 2.

5 .Incubation with enzyme- Conjugated Antibody.100 μL of enzyme-conjugated antibody diluted in buffer is

added to each well.Cover the plate and incubate at room temperature for 1 hour.

The enzyme-conjugated antibody should be directed against the antigen to be determined.

The conjugatec antibody must be specific for the antigen of interest

6 .Wash as described in step 2.

7 .Colour Development 100 μL of chromogenic substrate is added to each well.

Cover the plate and incubate for 15 minutes, or until a suitable colour has developed. The plate should preferably be protected against light during this incubation.

8 .Stopping the Colour DevelopmentStop the reaction by adding 100 μL 0.5 M H2SO4 to each well.

9 .Reading of ResultsRead results directly through the bottom of the microwell plate using an automated or semiautomatedphotometer (ELISA-reader). The subtraction of theabsorbance at a reference wavelength (between 620 and 650 nm) is recommended.

The result

Fundamental techniques for performing ELISA

1 .How to use a tip-exchange type pipette?

5. St r uct ur e of ant ibody-coat ed micr opl at e and

t r ea t ment

Fundamental techniques for performing ELISA

6 .How to wash a microplate

Fundamental techniques for performing ELISA

7-HOW TO TREAT WITH THE REAGENTS?

Use reservoir for each reagent

Label the reservoi

Don’t use the same reservoir for multiple

regents

Don’t return the reagents to the stock

8 .Shaking of the well-plate for mixing

Place the plate on the flat and smooth surface of a laboratory table, hold the plate and move the plate roundly to draw circles rapidly for approx. 10 seconds while lightly pressing the plate on the surface. Repeat 3 times.

Important points in performing ELISA and improvement of assay

performance

1-Sample treatment.3-Stability of assay samples.

2-Infleunce of humidity and air stream.

Important points in performing ELISA and improvement of assay performance

1 .Sampling and treatments of samples

Serum or plasma

In general, we recommend using

Use of fluoride must be avoided because fluoride ion is a potent inhibitor of peroxidase .

sampling

serum

When getting plasmaheparin is most often used as an anti-coagulant

An important phenomenon with frozen plasma is that an insoluble substance (fibrin) will be formed when thawed. In this case, the sample must be mixed and centrifuged, then the insoluble cluster flowing in the plasma should be taken out by a thin wire needle sharply bent at an end. If such fibrin remains in the sample, it may clog the tip of a pipette and influences assay variability

TAKE CARE

Hemolysis and Lipemia

Serum or plasma, when fresh, shows pH near neutral, however, it very quickly goes to alkaline more than pH 8 by losing CO2.

In alkaline pH, the antigen-antibody reaction is interfered. resulting in cancellation of the assay or giving inaccurate assay values. So, in such case, before hand dilution of serum or heparinized plasma with assay buffer will be helpful.

pH Of the sample

Sample storage temperature is better to be lower than -35 C. Ultra-low temperature such as -80 C is recommended for a long-term storage.

Repeated freezing and thawing is also harmful to the protein, and may cause inactivation.

Storage temperature and freezing-thawing.

When samples are taken out from the freezer and thawed, never forget to mix these samples because the solution after thawing is not homogeneous, and the bottom area containsmore solute

2-Stability of assay samples.

In assay, the problem of sample stability, i.e. how long the substance to be measured can keep its immunoreactivity, in serum or plasma, is very important. Blood samples also contain enzymes to destroy peptides or proteins, and stability against those enzymes differs from substance to substance.

Freezer of –20C is not trustable for the constancy of temperature but use of a freezer of –35 C or lower

temperature is recommended .

Avoid sunlight

Avoid air fans

3-infeluence of humidity and air stream

During all the incubation process, the well-plate should be covered using the attached plate cover. Plate cover is effective only under the most suitable condition, i.e. room temperature, humidity more than 50%, and air stream of less than 0.2m/sec.

N.B It is recommend to get

a small semi-transparent plastic box, and put moistened paper towel on the bottom.

Trouble shooting in ELISA

1-Poor or no coloration after the last step 1 )The standard or samples might not be added.

2 )Reagents necessary for coloration might not be added.

3 )Wrong reagents related to coloration might have been added.

4 )Influence of the temperature under which the kits had been stored.

5 )Excessive hard washing of the well plate.

2(The standard curve obtained was not smooth.

There might be some mistake in the serial dilution of the original standard solution. 3)Flat

standard curve .

Standard solutions are not added.

4-Big variation between two wells in duplicated assay was observed. 1 )Scratching the bottom of the well by aspirator tip during aspiration of washing buffer.

3)Assay might be started while the well-plate was still cooler than room temperature.

2 )Scratching the bottom of the well by pipette tip during addition of standards, samples, or reagents.

4 )Air stream, warmer or cooler than room temperature

5 )Air stream from air conditioner or other instrumentsmight dry wells.

6 )Insufficient removal of washing buffer from the wellsmight dilute reagent solution added in the following step of the procedure.

7)Big variation would be obtained if the sample is nothomogeneous.

Standard curve Of ELISA

Shapes of standard curves depending on scales in X-and Y-axes.

Standard curve of ELISA prepared by plotting standard concentration on X-axis and absorbance on Y-axis, both in normal scale, looks like a linear line except for lower concentration area.

Standard curve Of ELISA