elisa seminar final2

…By Dr. Deshkar DW

1798 - First demonstration of vaccination smallpox vaccination (Edward Jenner)

1890 - Demonstration of antibody activity against diphtheria and tetanus toxins.

1890 - Beginning of humoral theory of immunity. (Emil von Behring & Shibasaburo Kitasato)

1900 - Antibody formation theory (Paul Ehrlich)

1938 - Antigen-Antibody binding hypothesis (John Marrack)

1948 - antibody production in plasma B cells

1959 -1962 - Discovery of antibody structure

Enzyme Linked ImmunoSorbent Assay{ELISA}

History

• 1971 - Peter Perlmann and Eva Engvall at Stockholm University invented ELISA

• 1975 - Generation of the first monoclonal antibodies (George Kohler and Cesar Milstein)

• 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.

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

Enzyme Linked ImmunoSorbent Assay{ELISA}

History

• ELISA – an immunological test, using an enzyme as a label to determine presence of target protein.

• The enzyme linkage or labeling allows you to follow your target protein and if present (qualify) and at what amounts (quantify).

• An enzyme conjugate is an enzyme bound or joined with an antibody which binds with your target protein.

• This enzyme labeling is a safe and effective way to track your antibody.

Enzyme Linked ImmunoSorbent Assay{ELISA}

Components

Antigen

• The antigen is your target protein which comes from your sample extract.

• The antigen binds to the antibody.

• Any substance that stimulates an immune response.

Antibody

• An antibody is a protein made in response to an antigen.

• Each antibody binds only to its antigen.

Enzyme Conjugate

• An enzyme conjugate (EC) is an antibody joined with an enzyme.

• Enzyme labeling allows the researcher to follow the antibody.

• This joining of the enzyme to antibody is often called conjugation.

• Horse radish peroxidase (HRP) and Alkaline phosphatase (ALP) are the two most widely used enzymes employed in ELISA assay.

Components

Enzyme Function

• Enzymes are proteins that speed up the rate of a chemical reaction without being used up and usually react only to particular substrates.

• The rate of this reaction is proportional to the amount of enzyme present. In the case of non-competitive ELISA, the more binding you have of the enzyme conjugate to the antigen, the stronger your color development will be.

Components

Substrate

• ALP substrate - For most applications pNPP (p-Nitrophenyl-phosphate) is the

most widely used substrate.

• HRP substrates– Hydrogen peroxide. TMB (3,3’,5,5’-tetramethylbenzidine) OPD (o-phenylenediamine dihydrochloride)

ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt

Substrate Function• A substrate is a compound or substance that undergoes change. Substrates

bind to active sites on the surface of enzymes and are converted or changed. In ELISA the specific substrate used changes color.

Components

Four major advantages of ELISA

Data analysedstatistically

ELISA Enzyme Linked Immunosorbent Assay

Why ELISA?

Versatile Simple Sensitive Quantifiable

Many systems using different

combinations of reagents are

possible

Uses micro-titre plates and

allied equipment

Enzyme catalyst

amplification

Colour

Passive attachment to solid phase

High Capacity Ideal range

of sensitivity

for diagnosis

Read by eye

Easy separation of

bound and unbound

reactants by washing step

Rapid

Cheap

Kits feasible

Multi-channelspectrophotometers

Data can be stored

ELISA : Pros and Cons

Pros: 1.No radioactivity needed : safe and inexpensive

2.Less expensive equipment

3.Huge capacity through standard 96-microwell pattern: miniaturization and automation.

4.Equal or even better sensitivity than RIA

5.Versatility : same principle allows the use of different labels, incl. enzymes (colored end product or light) and fluorochromes.

Cons: Somewhat more difficult to develop, control and standardize than RIA.

General Procedure Of ELISA

Coating with Capture antibody

1. Coat the wells of a PVC micro-titer plate with the capture antibody2. Cover the plate with an adhesive plastic and incubate overnight at

4°C.3. Remove the coating solution and wash the plate twice by filling the

wells with 200 μl PBS.

Adding Samples4. Add 100 μl of appropriately diluted samples to each well, & Incubate

for 90 min at 37°C.5. Remove the samples and wash the plate twice by filling the wells with

200 μl PBS.

Incubation with Detection antibody6. Add 100 μl of detection antibody conjugated, diluted at the optimal

concentration in blocking buffer immediately before use.7. Cover the plate with an adhesive plastic and incubate for 1-2 h at

room temperature.8. Wash the plate four times with PBS.

Detection9. Dispense 100 μl (or 50 μl) of the substrate solution per well with a multi-

channel pipette or a multi-pipette.

A Microtiter plate (96-well) used for ELISA

General Procedure Of ELISA

Reading plates

•Select proper wave-length on machine.

• Carefully wipe bottom of plate to removeexcess moisture. Plates that are wet maydiffuse your light source, giving inaccuratereadings.

•Mix the plate. This will distribute colorevenly. Some machines have a ‘mix’ setting.

General Procedure Of ELISA

There is no need for phase separation. It is less sensitive than heterogenous ELISA.

Uses:

It is suitable for determination of lowmolecular weight substances. e.g drug monitoring.

PRINCIPAL:

Enzyme labeled Ag & unknown Ag compete together for Ab-binding sites .The enzyme labeled Ag that remain free is enzymatically active & can be determined in the presence of the bound form.

HOMOGENOUS ELISA

HETEROGENOUS ELISA

PRINCIPAL:

• An essential component of these assay is the separation of the bound enzyme labeled component from free labeled one after the immunological incubation.

• The assay can be :a) Competitive.b) Non Competitive

TYPES OF ELISA

• There are three main methods that form the basis to all ELISAs. 1. DIRECT ELISA 2. INDIRECT ELISA 3. SANDWICH ELISA

All these systems (1-3) can be used in assays called:4. COMPETITION OR INHIBITION ELISA

DIRECT ELISA

The direct ELISA uses the method of directly labeling the

antibody itself. Microwell plates are coated with a sample

containing the target antigen, and the binding of labeled

antibody is quantitated by a colorimetric,

chemiluminescent, or fluorescent end-point.

DIRECT ELISA

DIRECT ELISA

DIRECT ELISA

Advantages of Direct Detection

• Quick methodology since only one antibody is used. • Cross-reactivity of secondary antibody is

eliminated.

Disadvantages of Direct Detection

• Immunoreactivity of the primary antibody may be reduced as a result of labeling.

• Labeling of every primary antibody is time-consuming and expensive.

• No flexibility in choice of primary antibody label from one experiment to another.

• Little signal amplification.

DIRECT ELISA

The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody.

Since the labeled secondary antibody is directed against

all antibodies of a given species (e.g.anti-mouse), it can be

used with a wide variety of primary antibodies (e.g. all

mouse monoclonal antibodies).

INDIRECT ELISA

INDIRECT ELISA

INDIRECT ELISA

Advantages of indirect detection

• Wide variety of labeled secondary antibodies are available commercially.

• Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.

• Immunoreactivity of the primary antibody is not affected by labeling.

• Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.

INDIRECT ELISA

Disadvantages of indirect detection

• Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal.

• An extra incubation step is required in the procedure.

INDIRECT ELISA

SANDWICH ELISA

1. Plate is coated with a capture antibody

2. Sample is added, and any antigen present binds to capture antibody

3. Detecting antibody is added, and binds to antigen

4. Enzyme-linked secondary antibody is added, and binds to detecting antibody

5. Substrate is added, and is converted by enzyme to detectable form.

SANDWICH ELISA

SANDWICH ELISA TYPES

– Sandwich Direct – Sandwich Indirect

Sandwich Direct

A. The detecting antibody is labelled (conjugate)

B. The capture and detecting antibodies can be from the same sample

C. The detecting antibody can be from different species

Sandwich Indirect

A. The detecting antibody is NOT labelled

B. The detecting antibody is not prepared in same species as capture antibody

C. The detecting antibody is detected using an anti-species conjugate

SANDWICH DIRECT

SANDWICH DIRECT

SANDWICH INDIRECT

SANDWICH INDIRECT

COMPETITIVE ELISA

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.

COMPETITIVE ELISA

ELISA Reverse method & device (ELISA-R m&d)

A newer technique uses an solid phase made up of an

immunosorbent polystyrene rod with 4-12 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.

ADVANTAGES

• The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and different antigens for multi-target assays.

• The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples.

• One ogive is left unsensitized to measure the non-specific reactions of the sample.

• The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating ready-to-use lab-kits and on-site kits.

PRECAUTIONS

Negative control with strong signal

The excessive background signal can be caused by

inadequate rinsing of plates, reagents not sufficiently

diluted, inadequate blocking of plates or non-specific

binding of enzyme conjugate. The appearance of color in

negative control wells may also indicate cross-reactivity of

secondary antibody with components in the antigen

sample.

PRECAUTIONS contd…..

• Negative control with strong signal

The excessive background signal can be caused by

inadequate rinsing of plates, reagents not sufficiently

diluted, inadequate blocking of plates or non-specific

binding of enzyme conjugate.

The appearance of color in negative control wells may also

indicate cross-reactivity of secondary antibody with

components in the antigen sample.

ELISA with weak signal

• Wash buffer not adequately drained after every wash step.

• Inadequate incubation times.

• Detection reagents too dilute. Perform checkerboard titrations.

• Enzyme conjugate defective or inhibited by contaminant.

• Substrate defective or contaminated.

• Microwell plates poorly coated.

• Loss of capture antibody during blocking/washing. Decrease or eliminate use of Tween-20.

PRECAUTIONS contd…..

APPLICATIONS

• Screening donated blood for evidence of viral contamination by – HIV-1 and HIV-2 (presence of anti-HIV

antibodies) – hepatitis C (presence of antibodies) – hepatitis B (testing for both antibodies and

a viral antigen)

• Measuring hormone levels – HCG (as a test for pregnancy) – LH (determining the time of ovulation) – TSH, T3 and T4 (for thyroid function)

APPLICATIONS contd….

• Detecting infections – sexually-transmitted agents like HIV, syphilis

and chlamydia – hepatitis B and C – Toxoplasma gondii

• Detecting allergens in food and house dust • Measuring "rheumatoid factors" and other

autoantibody in autoimmune diseases like lupus erythematosus

• Measuring toxins in contaminated food• • Detecting illicit drugs, e.g.,

– cocaine – opiates