ELISA

Total slides : 115 1April 7, 2023

ELISAImmunochemistryIsfahan University of Medical Science, School of Pharmacy

Department of Clinical Biochemistry

Created by:

A.N. Emami Razavi

Enzyme Linked Immunosurbent Assay

ELISAELISA


ELISAImmunochemistry

Outlines

What is ELISA History Application Mechanism & Reagents Types of ELISA Methods Quality control HIV test

What is ELISA?

ELISAELISA



Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.



Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.



Enzyme-linked immunosorbent assay

Name suggests three componentsAntibody

Allows for specific detection of analyte of interest

Solid phase (sorbent)Allows one to wash away all the material that is not

specifically captured

Enzymatic amplificationAllows you to turn a little capture into a visible color

change that can be quantified using an absorbance plate reader

History

ELISAELISA



Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal which indicates whether a specific antigen or antibody is present in the sample. Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960.



The American physicist Rosalyn S.Yalow (born 1921) made her most outstanding contribution to modern medicine in developing radioimmunoassay (RIA), for which she received a Nobel Prize in physiology/medicine (1977).

The American physicist Rosalyn S.Yalow (born 1921) made her most outstanding contribution to modern medicine in developing radioimmunoassay (RIA), for which she received a Nobel Prize in physiology/medicine (1977).



Dr. Solomon A. Berson, M.D. '45 (1919-1972) and Dr. Rosalyn Sussman Yalow (1921- ) co-developed the radioimmunoassay (RIA) in 1959.

Dr. Solomon A. Berson, M.D. '45 (1919-1972) and Dr. Rosalyn Sussman Yalow (1921- ) co-developed the radioimmunoassay (RIA) in 1959.



Because radioactivity poses a health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a non-radioactive signal in place of the radioactive signal. When certain enzymes (such as peroxidase) react with appropriate substrates (such as ABTS or 3,3’,5,5’-Tetramethylbenzidine), they can result in changes in color, which can be used as a signal. However, the signal has to be associated with the presence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G.B. Pierce.



Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container, i.e. the immunosorbent has to be prepared. A technique to accomplish this was published by Wide and Porath in 1966

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



Eva Engvall is one of the scientists who invented the ELISA test in 1971. She is shown at her home near Buellton, Calif. Engvall is currently a professor at the Burnham Institute in La Jolla, Calif.

Eva Engvall is one of the scientists who invented the ELISA test in 1971. She is shown at her home near Buellton, Calif. Engvall is currently a professor at the Burnham Institute in La Jolla, Calif.

Applications

ELISAELISA



Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs.



What is it used for?

Measure antibody levels (allergies, vaccines) Detect viruses (hepatitis, HIV, venereal

diseases) Detect hormonal changes (pregnancy) Detect circulatory inflammatory markers

(cytokines)



Advantages of ELISA

Sensitive: nanogram levels or lower Reproducible Minimal reagents Qualitative & Quantitative

Qualitative Eg. HIV testing quantitative assays Eg. Drug Monitoring

Wells can be coated with Antigens OR Antibodies Suitable for automation high speed NO radiation hazards



Sensitivity

Relative sensitivities of tests (approx)

Usual operating range [Ab] or [Ag]

precipitationimmunoelectrophoresisdouble/radial diffusion

10 g/ml - 1 mg/ml

immunofluorescence 0.1 - 10 g/ml

ELISA (colour) 0.1 - 10 ng/ml (chemiluminescence) 0.01 - 10 ng/ml

radioimmunoassay 0.01 - 10 ng/ml



Enzymes with Chromogenic Substrates

High molar extinction coefficient (i.e., strong color change)

Strong binding between enzyme and substrate (low KM)

Linear relationship between color intensity and [enzyme]



Limitations

Results may not be absolute

False positive possible

False negative possible Antibody must be available



Materials needed

Testing sample Antibody (1st, 2nd) / Antigen Polystyrene microtiter plate Blocking buffer Washing buffer Substrate Enzyme

Mechanism & Reagents

ELISAELISA



Mechanism

The basic mechanism involved in these test utilizes absorption of antigen to a solid surface which is placed in contact with a dilution of serum. The reaction which detects and quantifies the binding of antibody uses an antibody labeled with an enzyme followed by the addition of an appropriate substrate on which the enzyme can act to produce a colour reaction. Two distinct test mechanisms are “noncompetitive " and "competitive".



Reagents Antigens may be produced using any of the standard

techniques. They may be purified by precipitation and ultra-centrifugation or by column chromatography before being absorbed onto the surface. An alternative method which allows the use of relatively impure antigens is to bind specific antibody to the surface and allow it to absorb the antigen from the preparation. Since the latter approach usually adds an extra stage to the test this is not the preferred approach for commercial test kits. In most cases the antigen is delivered pre-absorbed onto plates, though they need to be carefully dried and packed to maintain their stability at refrigerator temperature for a reasonable period.



The labeled anti-globulin used in the standard test is included in the test kits as is the appropriate substrates and stop-solutions for use with it. All of these reagents may be purchased separately from a number of sources however their titration to a standard level of activity for a specific purpose is rarely worthwhile for a commercial laboratory carrying out routine serology.

Reagents



Finally, and most importantly, standard negative sera and positive sera of known potency are required. These are even more important than those used in other serological tests since it is common practice to express the results of the test sera by comparing them with those of the control sera (serum-to-positive or serum-to-negative ratios). The objective of this is to remove some of the variation due to operator, environment and plate effects.

Reagents

Types of ELISA

ELISAELISA



Noncompetitive binding assay or Sandwich method

Antigen measuring system [Titrewells coated with antibodies ; Enzyme labelled antibodies]

Antibody measuring system [Titrewells coated with antigens ; Enzyme labelled antiantibodies]

Competitive binding assay Titrewells coated with antibodies ; Enzyme labelled

antigens



Noncompetitive or Sandwich Assay Antigen measuring system

Titre wells coated with suitable antibody Add patient sample containing the antigen Incubate: till antigen antibody reaction is complete Wash remove unbound antigen Add Antibody labelled with Enzyme Incubate till antigen binds labelled antibody Wash remove unbound labelled antibody Add substrate ; incubate Enzyme + Substrate Product measure colour Colour proportional to antigen in patient sample



Noncompetitive or Sandwich Assay Antibody measuring system

Titre wells coated with suitable antigen Add patient sample containing the antibody Incubate: till antigen antibody reaction is complete Wash remove unbound antibody Add Antiantibody labelled with Enzyme Incubate till labelled antiantibodies binds antigen-

antibody complex Wash remove unbound labelled antiantibody Add substrate ; incubate Enzyme + Substrate Product measure colour Colour proportional to antibody in patient sample



Competitive binding assay

Titrewells coated with antibodies Known quantities of patient sample containing

antigen + antigen labelled with enzyme Incubate: till antigen antibody reaction is complete Wash remove unbound antigens Add substrate ; incubate Enzyme + Substrate Product measure colour Colour inversely related to antigen in patient sample



Enzyme labels

Enzyme labels should have high specific reactivity Should be easily coupled to ligands & the labelled

complex must be stable The reactivity should be retained after linking of the

enzyme to the antigen/antibody The chosen enzymes should not be normally present

in the patient samples Examples of enzyme labels

Horse radish peroxidase, Alkaline phosphatase, Glucose oxidase



Enzyme-Mediated Detection

Enzyme Horseradish Peroxidase

Substrate Abbrev. Color

Diaminobenzidine DAB Brown

Enzyme Alkaline Phosphatase (AP)

Substrate Abbrev. ColorBromochloroindolylphosphateNitro Blue Tetrazolium

BCIP (AP substrate) NBT (Enhance color)

Purple

Methods

ELISAELISA



BASIC FORMAT

Solid phase = 96 / 384-well microplate



96 Well 350µL Polypropylene Plates









96 Well 1mL Polypropylene Plates



96 Well 2mL Polypropylene Plates



384 Well Polystyrene Assay Plates



384 Well Polypropylene Microtiter Plates



Analyte = antibody Analyte = antigen

Incubate, wash

1. Coat solid phase withantigen when analysing antibody

antibody when analysing antigen



2. Block free binding sites. Incubate. Wash.




3. Add sample. Incubate. Wash




4. Add conjugate. Incubate. Wash.


E E E E



5. Add substrate

6. Incubate, stop, measure colour change

Colourless

ENZYME

OD

CONCENTRATION



COATING THE PLATE

Dilute the Ag in PBS containing 0.02% NaN3 (5-10 ug/ml) and despense 50 ul into each well of 96-well microtiter plate using a multipipette/dispenser. (Micro plates vary in their ability to bind Ag. So each resercher should test for the plates of choice for their specific Ag). Seal plates with adhesive plate sealer and incubate overnight at 4° C or 2 to 3 h at 37° C (coated plates can be prepared and stored in the refrigerator for several weeks).



WASHING THE PLATE

Wash the plate three time with PBS using the Nunc-Immuno wash device (this device simultaneously delivers and aspirates fluid)



BLOCKING THE PLATE

block any residual binding capacity by adding 50 ul of a blocking buffer (PBS containing 5% BSA, 0.02% NaN3) to each well. Incubate the plate for 1 h at room temperature.



SAMPLE

Dilute in buffer-Tween 20 Include known positive and negative samples Standards……. recombinant protein

International standard antibodyDouble-dilute from 10 pg/ml - 10 ng/ml

100 l/well, duplicates 2 - 4 hours 20/37oC or overnight 4oC 3 - 6 washes with buffer-Tween 20



CONJUGATE

For assays of (human) antibodies use anti-(human) Ig-enzyme

For assays of antigens use enzyme-conjugated antibody



Conjugating antibodies to Alkaline Phosphatase

Centrifuge 5 mg of alkaline phosphatase suspension (supplied as a suspension in 65% (NH4)SO4) in a microfuge for 5 to 10 min. resuspend the enzyme pellet in a total volume of 1 mg of the antibody to be labeled.

Dialyzed overnight against 0.1 M sodium phosphate buffer, pH 6.8 to remove any contaminating free amino groups.

In a fume hood, slowly add 50 ug 1% glutaraldehyde solution while stirring for 5 min. Incubate at room temperatyre for 2 to 3 h, add 100 ul of 1 M ethanolamine, pH 7, and then incubate for additional 2 h at room temperature.

Dialyze overnight against PBS and remove any insoluble materials by centrifugation at 40,000 x g for 30 min.

Store the conjugate (supernatant) in the presence of 50% glycerole,1mM MgCl2 1mM ZnCl2, and 0.02% NaN3 at 4ºC



AMPLIFICATION

E

Directly conjugated developingantibody may give weak signal



amplify with

unlabelled (rabbit) anti-(human) Ig followed by

anti-(rabbit) Ig-enzyme

EE



or

Biotin-labelled anti-Ig followed by

streptavidin-enzyme

E-S B S-E

ES



SUBSTRATES

See Sigma catalogue for list of conjugates and substrates

Orthophenylene diamine Tetramethyl hydrochloride (OPD) benzidine (TMP)

Horse radish peroxidase (HRP) Orange, 490 nm Yellow, 450 nm Spectrophotometer



Paranitrophenyl phosphate (PNP) Methyl umbelliferol phosphate

Alkaline phosphatase

Yellow, 405 nm Methyl umbelliferone Spectrophotometer

365 nm 445 nm Fluorimeter









Micro-titre plate with a standard Elisa test seen from below. The first 2 wells at top right are the negative controls. The following 2 are positive controls. The remaining sera are field test sera. Usually at least 2 wells with a known laboratory positive control are included on each plate.



INDIRECT ELISA TO DETECT SPECIFIC ANTIBODIES

• screening hybridoma supernatants• detecting clinically important antibodies - autoantibodies - anti-pathogens - anti-allergens

1. Antigen




2. Sample (human) antibody

1. Antigen




E E E

3. Anti-(human) Ig-enzyme


1. Antigen




E E E4. Substrate

3. Anti-(human) Ig-enzyme


1. Antigen



ANTIGEN-CAPTURE ELISA TO DETECT SPECIFIC ANTIBODIES

Useful when pure antigen not available or antigen coats poorly

1. Specific antibody




2. Impure antigen eg tissue homogenate





3. Wash pure antigen

2. Impure antigen





4. Sample (human antibody)


2. Impure antigen





E E5. Anti-human Ig-enzyme



2. Impure antigen





6. Substrate

5. Anti-human Ig-enzyme



2. Impure antigen




ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS

eg. hormones drugs tumour antigens cytokines

1. Anti-analyte




1. Anti-analyte

2. Sample



1. Anti-analyte

2. Sample

3. Anti-analyte-enzyme

E E




1. Anti-analyte

2. Sample

3. Or: anti-analyte-biotin followed by streptavidin-enzyme

E-S B S-E

ES

E S E-S B S-E




1. Anti-analyte

2. Sample

4. Substrate

3. Or: anti-analyte-biotin followed by streptavidin-enzyme




COMPETITION ELISA TO DETECT ANTIGENS

1. Analyte

(antigen-coated plate)



2. Anti-analyte-E + sample

1. Analyte

Low [analyte] High [analyte]

E E E

E EE

EE

E

E




3. Wash


1. Analyte


E E E

E




3. Wash


1. Analyte

4. Substrate






1. Anti-analyte

(antibody-coated plate)



2. Analyte-E + sample

1. Anti-analyte





3. Wash


1. Anti-analyte

E E E E E E E





3. Wash


1. Analyte

4. Substrate





CYTOKINES

12 1

2

HEALTH

CANCER VIRUSES

MYCOBACTERIAASTHMA, ALLERGY

LUPUS

RHEUMATOID ARTHRITISMULTIPLE SCLEROSIS DIABETES

IL2IL12IFNTNF

IL4 IL5 IL6 IL10

Type 1 Type 2



Detection of cytokines by ELISA

Plasma or supernatant of cultured mononuclear cells

Coat plate with anti-CK (Pharmingen) 0.5 g/ml in bicarbonate buffer, 4o overnight

Wash x 2 with PBS-T

Block with PBS + 10% FCS, 2 hours RT

Wash x2 with PBS-T



Add standards (recombinant CK 10 pg-10 ng /ml), controls and samples 4o overnight

Wash x3 with PBS-T

Add biotinylated anti-CK (Pharmingen) 0.5 g/ml

RT 60 minutes

Wash x6 with PBS-T

Add streptavidin-peroxidase

RT 30 min



Wash x8 with PBS-T

Add OPD substrate

15 min RT, dark

Stop with N H2SO4

Read A490

2

1

0

[rCK]

HIV Test

ELISAELISA



HIV

The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.



HIV

An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. The test is performed in a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.



An ELISA plate



The ELISA Method Partially purified, inactivated HIV antigens pre-

coated onto an ELISA plate Patient serum which contains antibodies. If the

patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate.

Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies.

Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody.



Negative ELISA Test

Positive ELISA Test



ELISA data from three patients

Above is ELISA data from three patients. Numbers are expressed as optical density at 450 nm. The cutoff value indicating a positive result is 0.500. Optical densities of 0.300 to 0.499 are indeterminate and need to be retested. Values below 0.300 are considered to be negative. In most cases, a patient will be retested if the serum gives a positive result. If the ELISA retests are positive, the patient will then be retested by western blotting analysis.

Positive Control Negative Control Patient A Patient B Patient C Assay Control

1.689 0.153 O.055 0.412 1.999 0.123



Sources of Error for HIV EIA Tests



Sources of False Negative Results

Early Seroconverters

the window between infection and an antibody response to the virus

Operator Error Fail to add serum or reagent to the correct well Reagent diluted in wrong diluvent or in wrong dilution

Equipment Error



Source of False Positive Results

MULTIPLE PREGNANCY

MULTIPLE TRANSFUSION

AUTO IMMUNE DISORDER

CHRONIC HEPATITIS,

CHRONIC ALCOHOLIC

HBV VACCINATION

ANTIBODY TO POLYSTERENE



Trouble shooting EIA Kit integrity

Controls (kit and in-house)

Equipment

Cross contamination

Sample quality

Personnel training

Correct validation and interpretation of results



Kit integrity

Was cold chain maintained during kit transport?

Has the kit expired?

Has the kit been stored properly in your lab?



Controls

Kit Controls should be run on each EIA plate. Indicates whether the kit components are functioning. Used to validate EIA run, calculate cut off

In-house controls Kit controls are designed to be quite robust and do not reflect subtle

changes in testing. In-house controls should be calibrated to test as a low positive (above

cut-off, below maximum)



Equipment : Pipettes

Single and Multi channel Pipettes should be calibrated on a monthly basis.



Equipment : Microplate Washers

Daily Prime the washer with wash solution before running sample plates

Set the washer to wash the recommended number of times (with correct volume)

Check for accurate dispensing and complete aspiration in each plate well, if not clean the washer head

Listen for changes in the sound the washer makes, this can indicate a vacuum leak

At the end of the day prime the washer with DI water



Equipment : Microplate Washers Weekly

If a washer is not used during the week rinse it out with DI water to reduce microbial growth.

Monthly Run a 10% solution of ethanol through the washer to disinfect.

This can also be done if the washer exhibits signs of contamination (high background).

Thoroughly rinse the washer after alcohol is used.



Equipment : Micro-plate Reader Daily

Each time a reader is turned on it runs a self test, it will then report any errors.

Weekly Run a control plate weekly. Variations in positive or negative specimens

could be a sign of a bad diode or a spill on a diode.



Cross contamination

Can be caused by:

Reusing pipette tips (contaminated with + plasma)

Splashes from one well to another during removal of plate covers



Sample Quality

Properly collected (no haemolysis)

Transport conditions

Storage conditions

Number of freeze/thaw cycles

Age of sample



Validation and Interpretation of Results

Positive and Negative controls must fall within a certain range.

Controls are used to calculate a cut-off.

Samples below cut-off are negative, those above are positive



Test your self

1. What does ELISA measure?

2. What if serum were left out?

3. Omission of the wash step

4. Which patient is HIV positive?

Use these problems to test your understanding of this topic.



Problem 1

What is the ELISA test intended to measure?Antibody to HIV only

Antigen to HIV only

Presence of free, circulating virus in the patient

Antibodies directed against HLA molecules

BB

AA

CC

DD



Problem 2

What would happen if serum were omitted from the ELISA, but all other steps remained the same and were performed properly?

Anti-human Ig-conjugate would not bind and be washed away.

Anti-human Ig-conjugate would bind non-specifically to the ELISA plate.

The O.D. values would be nearly the same as the assay control.

Both A and C.

BB

AA

CC

DD



Problem 3

What would happen if the anti-human Ig-conjugate were not washed free of the well before the substrate was added? The ELISA would not develop when the substrate was

added. The ELISA would develop normally.

All wells would show uniform over development due to unbound and excess anti-human Ig enzyme conjugate.

Both A and B.

BB

AA

CC

DD



Problem 4

From the ELISA data, which patient is seropositive for HIV?

Patient A Patients A and B

Patient B Patient C

Positive Control Negative Control Patient A Patient B Patient C Assay Control

1.689 0.153 O.055 0.412 1.999 0.123

BB

AA CC

DD

Thank youThank you

Questions Questions



Antibody to HIV only

Antibody from patient serum made in response to various HIV proteins binds to antigen.

AA

Correct!



Antigen to HIV only

HIV antigens are already coated onto the plate. Antibody from the patient binds to the HIV antigens.

BB

Incorrect!



Presence of free, circulating virus in the patient

Serum antibody is detected, not circulating virus in the serum.

CC

Incorrect!



Antibodies directed against HLA molecules

Although there may be anti-HLA antibodies present in the serum, the test is not designed to measure them.

DD

Incorrect!



Anti-human Ig-conjugate would not bind and be washed away.

but there is another correct answer

AA

Correct!



Anti-human Ig-conjugate would bind non-specifically to the ELISA plate.

The anti-human Ig conjugate is specific for human Ig and has very little nonspecific binding activity.

BB

Incorrect!



The O.D. values would be nearly the same as the assay control.

but there is another correct answer.

CC

Correct!



Both A and C.

Since no serum was added, the anti-human Ig conjugate would not be able to bind to human Ig and the O.D. values would be the same as the assay control.

DD

Correct!



The ELISA would not develop when the substrate was added.

The substrate would overdevelop because an excess of enzyme coupled to the anti-human Ig was present.

AA

Incorrect!



The ELISA would develop normally.


BB

Incorrect!



All wells would show uniform over development due to unbound and excess anti-human Ig enzyme conjugate.

Since the enzyme which acts on the substrate is in excess, it would turn all the wells a uniform color whether they were truly positive or not.

CC

Correct!



Both A and B.


DD

Incorrect!



Patient A

Patient A has an O.D. of 0.055 and would be considered negative.

AA

Incorrect!



Patient B

Patient B would be considered indeterminate and would need to be retested.

BB

Incorrect!



Patients B and C

Patient A has an O.D. of 0.055 and would be considered negative and patient B would be considered indeterminate and would need to be retested.

CC

Incorrect!



Patient C

Serum from patient C showed an O.D. of 1.999 which is even higher than the positive control O.D. for the assay.

DD

Correct!







Extinction coefficient

The extinction coefficient for a particular substance is a measure of how well it scatters and absorbs electromagnetic radiation (EM waves). If the EM wave can pass through very easily, the material has a low extinction coefficient. Conversely, if the radiation hardly penetrates the material, but rather quickly becomes "extinct" within it, the extinction coefficient is high.

A material can behave differently for different wavelengths of electromagnetic radiation. Glass is transparent to visible light, but many types of glass are opaque to ultra-violet wavelengths. In general, the extinction coefficient for any material is a function of the incident wavelength. The extinction coefficient is used widely in ultraviolet-visible spectroscopy.

ELISA

Education

rosalyn sussman

molar absorption

absorbs electromagnetic

low extinction

linked immunosorbent

detect specific

antibody sandwich

wash pure