elisa- lite

ELISA

Presenter: Dr Pranav SoporyJunior Resident

Dept. of PharmacologyAll India Institute of Medical Sciences

New DelhiMob: 9999-491-690

Email: [email protected]

Contents

Antigen-Antibody Reactions

Introduction to ELISA

Non-competitive ELISA

Reporting

AntibodyProduced by:

B cells Plasma Cells AntibodyChromosome:

2 Heavy Chains - Chrm. 142 Light Chains - Chrm. 2 (ϰ)

Chrm. 22 (λ)Disulfide bonds

Fab region:Fragment antigen-bindingVariable regionParatope binds to Epitope (Ag)

Fc region:Fragment crystallizable regionInteracts with - cell surface (Fc receptor)

- complement systemActivates the immune system

Fab region

Fc region

Antigen – Antibody Reaction• General Features

1. Specific, but specificity is not absolute!

2. Requires

a. Specific pH

b. Correct temperature

c. Electrolyte balance

3. Bonds involved

a. Van der waal

b. Hydrogen

c. Ionic

4. Combination: firm and reversible

Types of Ag-Ab reactions

Sensitive for Ag

Soluble Ag+

Ab+

Electrolyte=

PRECIPITATION

Particulate Ag+

Ab+

Electrolyte=

AGGLUTINATION

Sensitive for Ab

ELISA• Enzyme Linked Immunosorbent Assay

1. Plate based Immunoassay

2. Works on the principle of Ag-Ab binding

3. Based on enzymatic-color reaction

4. Detects and quantifies substances such as peptides, proteins, hormones and antibodies.

5. Types of ELISA

a. Qualitative: Positive or negative results

b. Quantitative: Optical density interpolated into a standard curve, which is

typically a serial dilution of the target

Immunoassay HistoryBefore 1970s:

Radioimmunoassay: Used radioactively-labeled antigens or antibodies.

Was the only test available.

Radioactivity provides signal indicating presence of Ag or Ab.

Radioactivity poses a health risk to the technician.

1971: Peter Perlmann and Eva Engvall at Stockholm University invented ELISA

1986: Generation of the first monoclonal antibodies (George Kohler and Cesar Milstein)

Specimen sample for ELISA

Serum HIV

CSF Neurocysticercosis

Sputum Brucellosis

Urine Legionnaire’s disease

Stool Giardiasis

Microtiter Well

Characteristics:

1. Marked on top: Numerically

2. Marked on the side: Alphabetically

3. Generally: 96 wells

Indirect ELISA

Enzyme cleaves substrate to produce color

Add Substrate (chromogen) for the Enzyme

Wash – To remove unbound secondary Ab

Add secondary Ab (Enzyme Linked)

Wash – To remove unbound Antibodies

Add Serum (contains primary Ab)

Microtiter well coated with Antigen

Indirect ELISA• The indirect ELISA detects the presence of antibody in a sample

• Advantages:

1. Wide variety of Enzyme linked secondary antibodies available

2. Versatile: many primary antibodies can be made in one species and the same

labeled secondary antibody can be used for detection.

3. Increased sensitivity: each primary antibody contains several epitopes that can be

bound by the labeled secondary antibody, allowing for signal amplification.

• Disadvantages:

1. Cross-reactivity: might occur with the secondary antibody, resulting in nonspecific signal.

2. An extra incubation step is required in the procedure. (compared to Direct ELISA)

ELISA: materials required

1. Testing Sample

2. Antibody (1st and 2nd)/ Antigen

3. Microtiter well

4. Blocking Buffer

5. Washing Buffer

6. Substrate

Nomenclature

EL: The second antibody is attached to an enzyme (‘enzyme-linked’)

I: Antigen is recognized by specific antibody (‘immuno’)

S: Antigen of interest is absorbed on to plastic surface (‘sorbent’)

A: Substrate reacts with enzyme to produce a product, usually colored,

which can be assessed (‘assay’)

Direct ELISA




Add Serum (contains primary Ab that is Enzyme Linked)

Microtiter well coated with Antigen

Direct ELISA• The direct ELISA detects the presence of antigen in a sample

• Advantages:

1. Quick methodology since only one antigen is used

2. Cross reactivity of second antibody is eliminated

• Disadvantages:

1. Immunoreactivity of primary antibody is reduced because its enzyme linked

2. Lesser signal amplification than Indirect ELISA

Sandwich ELISA



Add Enzyme linked Antibody


Add serum (contains Ag to be detected)

Microtiter well coated with Capture Antibody

Sandwich ELISA

• The sandwich ELISA detects the presence of antigen in a sample

• Advantages:

1. High specificity because the antigen is specifically captured and detected.

2. Suitable for crude/impure samples as the antigen does not require purification

prior to measurement.

3. Flexible and sensitive, both direct or indirect detection methods can be used.

BuffersBlocking protein

• a.k.a. “Detergent”• Prevents other proteins from adsorbing to the

plate.• Non-reactive protein like

1. Bovine Serum Albumin

Washing

• Necessary to remove non-bound reagents• E.g.

• 1. Phosphate-buffered solution (PBS)• 2. Tris – buffered saline (TBS)

Enzymes used in ELISA

Enzyme Substrate Chromogen

Horseradish Peroxidase (M/C)

H2O2 Tetra methyl benzidine (TMB)

Alkaline Phosphatase Nitrophenyl Phosphate (NPP)

Diethandamine + MgCl2

Results

• Absorbance from Microtiter wells detected via Microplate readers

Final plate of ELISA Microplate reader

Results

Source

Lens

Filter

MTP

Detector Calculation

REPORT

22

Calculation

• Data graphed between Optical density Vs Log concentration.• Known conc. of Ag are used to produce a standard curve.• This data is used to measure the conc. of unknown samples by

comparison to the linear portion of the standard curve.

Conc. of substance (pg/ml)

Opti

cal D

ensit

y

ELISA

Advantages• Long shelf life• Easy to perform• Quick• Widely available

Disadvantages

• Enzyme activity maybe affected by plasma constituents• Expensive

Limitations

• Results may not be absolute• Ab may be not available• False positives/negatives

(mutated antigen)

Thank You

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