ENZYME-LINKED IMMUNO SORBENT ASSAY

(ELISA) AND ITS TYPES

YESRA ARSHAD
INTRODUCTION

The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique which is used to measure the
concentration of an analyte (usually antibodies or antigens) in solution.
INTRODUCTION TO ELISA

 A 96 - well microtiter plate being used for ELISA.

 A test that uses antibodies and color change to identify a substance.
 ELISA is a popular format of a "wet-lab" type analytic biochemistry assay.
 ELISA involves at least one antibody with specificity for a particular antigen.
 ELISA involves detection of an "analyte" in a liquid sample by a method that continues to use liquid reagents
during the "analysis“.
PRINCIPLE

 The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding (antigen- antibody binding). The
enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab
binding.
 It combines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or
antigens coupled to an easily assayed enzyme.
 It involves the usage of an enzyme to detect the binding of an Antigen (Ag)and an Antibody (Ab).
 The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag : Ab
binding.
 Therefore, ELISA can be used to detect either the presence of antigens or antibodies in a sample depending upon
how the test is designed.
STEPS INVOLVED IN ELISA

1. Antigen Binding
 The sample antigen is binded or immobilized to the micro plate via adsorption to the surface.
 Binding is achieved by incubating the wells with a solution containing the antigen for 2hrs at RT or overnight at
4°C.
 The protein adheres due to hydrophobic interactions between the protein& the plastic.
 Coating is done using carbonate/bicarbonate buffer at a pH 9.6.
STEPS INVOLVED IN ELISA

2. Blocking
 All unbound sites on the solid support are blocked to prevent nonspecific binding of the antibodies.
 Blocking buffers like BSA, Non fat dry milk powder (casein) in PBS ( Phosphate buffered saline) or TBS ( Tris
buffered saline) at a pH 7.4 with minute percentage of Tween 20 are used to block the free sites.
 Protein in the blocking solution will attach to membranes in places where the target proteins have not attached.
 Excess blocking agent is removed by washing the plate membrane with washing buffer.
STEPS INVOLVED IN ELISA

3. Primary Antibody
 The 1° antibody is added and will be bound only if there is a recognized epitope within sample antigen.

4. Secondary Antibody
 An enzyme-linked 2° antibody is added with suitable dilutions which will bind to any available 1° antibody (i.e. it
is bound to the antigen).
 2° antibodies are linked to the enzyme through biconjugation.
 Enzymes generally used are Horse Radish Peroxidase (HRP) & Alkaline Phosphatase (AP).
 Plate is washed with buffer or a mild detergent to remove any unbound antibodies or proteins
STEPS INVOLVED IN ELISA

5. Detection/ Developing
 After the final wash step, the plate is developed by adding an enzymatic chromogenic substrate to give color.
 The entire plate is placed into a plate reader and the OD is determined for each well.
 Intensity of color reflects the amount of specific 2° antibody bound to the target.
 A spectrophotometer is designed to read each well in the 96 well plate. It is interfaced with a computer for data
management.
 Substrates used are pNPP ( p- nitro phenyl phosphate), BCIP (5-bromo,4- chloro,3-indoyl phosphate), NBT (nitro
blue tetrazolium), TMB (3,3´,5,5´- tetra methyl benzidine), DAB (3,3-diamino benzidine).
COMPONENTS OF ELISA

 Antibody: IgG fraction of serum.

 Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4 lysine residues.
 Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a catalyst to oxidize substrate in the
presence of Hydrogen peroxide to produce a blue color.
 Reaction stopped with dilute acid to cause complex to turn yellow.
REAGENTS USED

Reagent Composition
 Coating Buffer » » 0.01M Phosphate Buffer + 0.15 M NaCl (PBS)
 Diluting/Washing Buffer » » 0.01 M Phosphate Buffer + 0.50 M NaCl + 0.1% Tween 20
 Blocking Buffer » » Bovine Serum Albumin (BSA)
 Enzyme » » Horse-redish peroxidase (HRPO)
 Chromogenic Substrate » » Trimethyl benzidine (TMB)
 Stop Solution » » 0.5 M H₂SO₄
 Solid phases used in ELISA include cross-linked dextran or polyacrylamide beeds, filter paper(cellulose) disc or
polypropylene tubes and disposable polystyrene microtitre plates.
 Antigen or antibodies attached to solid phase by passive absorption.
 Conjugate used must contain a highly reactive antibody coupled to an enzyme with a highly turnover
number(alkaline phosphatase and horseradish peroxide are commonly used enzymes).
TYPES OF ELISA

 Non Competitive ELISA

 Direct ELISA
 Indirect ELISA
 Sandwich ELISA

 Competitive ELISA
 Reverse/Multiple/portable ELISA
DIRECT ELISA

 It uses a primary labeled anti-body that react directly with the antigen.
 It can be performed with the antigen that is directly immobilized on assay plate.
 Not widely used but common for immuno-histochemical staining of cells & tissues.
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.
 Little signal amplification.
INDIRECT ELISA

 It utilizes a primary un-labeled antibody in conjunction with a labeled secondary antibody.

 Secondary antibody has specificity for primary antibody.
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.
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.
SANDWICH ELISA

 Antigens like Tumor markers, hormones, serum proteins may be determined.

 Antigens in the sample bind with the capture antibody & become immobilized.
 The antibody of the enzyme conjugate bind with the immobilized antigen to form a sandwich of Ab-Ag-Ab/
enzyme bound to microwell.
SANDWICH ELISA
SANDWICH ELISA

ADVANTAGES
 A major disadvantage of the indirect ELISA is the method of antigen immobilization is not specific.
 Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may
competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.
 Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen
from complicated mixtures before the measurement.
 Simplifies the assay.
 Increases the specificity and the sensitivity of the assay.
COMPETITIVE ELISA

 Antibody coated microwell.

 Serum antigen & labeled antigen added together .... Competition
 Ab-Ag enzyme complex bound is inversely related to the conc. of antigen present in sample.
 Increased serum antigen results in reduced binding of Ag- enzyme conjugate with the antibody producing less
enzyme activity & (yellow) color formation.
 Used to determine small molecules like T ₃ , T ₄ & Progesterone.
COMPETITIVE ELISA
COMPETITIVE ELISA
REVERSE ELISA

 A new technique uses a 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 chromigeneous) are carried out by dipping the ogives in microwells of
standard microplated prefilled with reagents.
Advantages of this technique:
 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, food and environmental samples.
 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
ELISA RESULTS

 ELISA data is typically graphed with Optical density Vs Log concentration to produce a sigmoidal curve.
 Known concentrations of antigen are used to produce a standard curve and then this data is used to measure the
concentration of unknown samples by comparison to the linear portion of the standard curve.
 This can be done directly on the graph or with curve fitting software which is typically found on ELISA plate
readers.
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.
 Positive control with no signal Microwell plates not coated properly. 
 Reagents applied in wrong order or step omitted. 
 Secondary antibody not matched to the species of primary antibody. 
 Enzyme conjugate defective or inhibited by contaminant. 
 ELISA with weak signal  Wash buffer not adequately drained after every wash step.  Inadequate incubation times.  Enzyme
conjugate defective or inhibited by contaminant.  Substrate defective or contaminated.  Microwell plates poorly coated.  Loss of
capture antibody during blocking/washing.
 SENSITIVITY ELISAs are one of the most sensitive immunoassays available. The typical detection range for an
ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the
antibody – antigen interaction. In addition, some substrates such as those yielding enhanced chemiluminescent or
fluorescent signal, can be used to improve results. As mentioned earlier, indirect detection will produce higher
levels of signal and should therefore be more sensitive. However, it can also cause higher background signal thus
reducing net specific signal levels.
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)
§  Detecting infections
§  Sexually-transmitted agents like HIV, syphilis and chlamydia
§  Hepatitis B and C
§  Toxoplasma gondii
§  Detecting illicit drugs.
§  Detecting allergens in food and house dust
Thank you