Enzyme-Linked Immnoabsorbent Assay (ELISA) Methods for Detecting Antigen!
ELISA method is 10 to 1000 folds sensitive than the older methods like agglutination and immunoelectrophoresis. A variety of modifications of ELISA are available, some of which are described here.
Table 1.0: Advantages of enzyme immunoassays
1. Amplification effect of enzymes results in development of more sensitive assays
2. Reagents have a long shelf life and are comparatively cheap
3. A wide variety of assay configurations can be developed
4. Equipment is less costly and available widely
5. No radiation hazards
6. Can be performed in small laboratories also
Enzyme-linked immnoabsorbent method can be used to detect either
antigen or antibody. The ELISA method has many advantages over other
methods (Table 1.0).
ELISA method is 10 to 1000 folds sensitive than the older methods like agglutination and immunoelectrophoresis. A variety of modifications of ELISA are available, some of which are described here.
Table 1.0: Advantages of enzyme immunoassays
1. Amplification effect of enzymes results in development of more sensitive assays
2. Reagents have a long shelf life and are comparatively cheap
3. A wide variety of assay configurations can be developed
4. Equipment is less costly and available widely
5. No radiation hazards
6. Can be performed in small laboratories also
ELISA to Assay Antibodies:
Known antigen is coated on to a microliter plate wells (small plastic
plate, treated to maximize protein binding; contains 96 wells with a
volume of 200 µl).
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Human test serum is added to the well and incubated.
i. If the serum contains antibodies against the coated antigen, the antibodies bind to the antigens.
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The wells are washed to remove unbound serum components.
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Enzyme conjugated anti-human immunoglobulin (known as conjugate) is added to the wells and incubated.
ii. The conjugate binds to the antigen-bound antibodies in the well.
iii. If the test serum doesn’t contain antibodies against the antigen, the conjugate remains in the solution.
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The wells are washed to remove unbound conjugate.
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A suitable substrate is added to the wells and incubated.
iv. The enzyme of the conjugate in the antigen-antibody-
conjugates complex acts upon the substrate and split the substrate to
produce a coloured product. Development of color indicates that
antibody specific to the antigen coated on the wells is present in the
test serum.
v. Non-development of color indicates that the test serum doesn’t have antibodies against the antigen coated on the wells.
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A stop solution (usually, IN sulphuric acid) is added to stop the
reaction. Then the plate is kept in an ELISA reader and the optical
density (OD) of the wells is measured. The OD corresponds to the amount
of antibody in the test serum.
By using known concentrations of antibodies, a graph can be
constructed. The antibody concentrations are plotted on the X axis and
the corresponding ODs are plotted on the Y axis and a curve is drawn. By
interpolating the OD value of the test serum, the concentration of
antibodies in a test serum is determined.
ELISA to Assay Antigen:
A known monoclonal antibody (referred to as primary antibody) to an antigen is coated onto the microtiter wells.
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The sample supposed to contain the antigen is added to the well and incubated.
i. If the sample contains the corresponding antigen, the antigen
binds to the antibody on the well and forms an antigen-antibody complex.
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The wells are washed to remove unbound materials.
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A known enzyme conjugated mAb (called as conjugate) against the antigen is added to the wells and the plate is incubated.
i. If the well contains antigen-antibody complex, the conjugate binds to the antigen in the complex.
ii. If there is no antigen-antibody complex, the conjugate remains in solution.
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The wells are washed to remove the unbound conjugate. A suitable substrate is added and incubated.
i. If there is conjugate in the well, the enzyme of the conjugate splits the substrate and produces a colored product.
iii. Absence of development of color indicates that there is no
antigen in the sample (against the antibody coated on the wells).
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A stop solution is added to stop the reaction. ODs of the
individual wells are read in the ELISA reader. As explained earlier a
graph (consisting of different concentrations of antigens on the X axis
and the corresponding ODs on the Y axis) is constructed and the curve is
drawn. The concentration of the antigen in the test sample is
determined by interpolation of its OD.
Antibody Capture ELISA Method:
Wells of the micro titer plate are coated with anti-human IgM antibodies.
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Patient’s serum is added and incubated. The IgM antibodies in the serum bind to the anti-human IgM antibodies on the wells.
The wells are washed and then a known antigen is added and incubated.
i. If IgM antibodies against the antigen are present in the well,
the antigen will bind to the IgM antibody and remain in the well.
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The wells are washed to remove unbound antigen
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An enzyme conjugated mAb to the antigen is added and incubated.
i. If antigen is present in the well (anti IgM-IgM-antigen complex), the conjugate will bind to the antigen.
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The wells are washed to remove unbound conjugate and substrate is added.
i. Development of color indicates that IgM antibodies against the antigen are present in the patient’s serum.
ii. Non-development of color indicates that the patient’s serum doesn’t contain IgM antibodies against the antigen.
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Stop solution is added and the ODs of wells are individually
measured in the ELISA reader. The quantity of IgM antibodies against the
antigen can be determined by drawing a graph as described earlier.
A similar procedure can be adopted to detect IgG antibodies against an antigen.
The enzymes commonly used in the ELISA techniques are horseradish
peroxidase and alkaline phosphatase (Table 1.1). These enzymes are
covalently coupled to mAbs. A variety of substrates are acted upon by
these enzymes to produce colored products. Since the last step of the
ELISA method is enzymatic, the ELISA method is extremely sensitive
(i.e., very low concentrations of antigen or antibody are detectable).
Sensitivity of ELISA assays are enhanced by using additional
reagents (For example, biotin/avidinimmunoassay). Recently, the use of
horseradish peroxidase substrates that produce chemiluminescent products
has further enhanced the sensitivity. Measurement of the rate of the
reaction, rather than the extent of the reaction at a single fixed
instant, gives more accurate quantitative ELISA results.
Table 1.1: Characteristics of enzymes used in enzyme immunoassays:
| Characteristics | Horseradish peroxidase | β-Galactosidase | Alkaline phosphatase |
| Source | Horseradish | Escherichia coli | Bovine intestine |
| MW (daltons) | Ca.40,000 | Ca.530,000 | 140,000 |
| Specific activity | 250U/mg | 600 U/mg | 1000 U/mg |
| Turnover rate | 10,000 | 318,000 | 250,000 |
| Enzyme measurement | Colorimetry, fluorometry, luminometry | Colorimetry, fluorometry, luminometry | Colorimetry, fluorometry, luminometry |
| Enzyme labeling method | Periodate oxidation | Dimaleimide method, cross-linking reagent | Glutaraldehyde method, cross-linking |
Biotin-Avidin Enhanced ELISA:
Instead of labeling the mAb with an enzyme, the MAb can be labeled with biotin (Vitamin B12).
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Then enzyme labeled avidin (a protein component of the egg white) is added.
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Avidin binds to biotin with very high sensitivity and affinity.
Subsequently the substrate is added. The enzyme (in the
mAb-biotin-avidin-enzyme) complex acts upon the substrate and produce a
colored product.
Biotin-avidin enhancement is also used in Immuno- fluorescent assays.
Micro Enzyme Immunoassays:
Instead of coating the micro titer plate wells with antigens or
antibodies, small beads (1 mm in diameter) are coated with antigen or
antibody and ELISA assays are performed. The microparticles offer larger
surface area for antigen or antibody coating so that higher
concentrations of antigen or antibody can be coated, which helps in
shortening the time required for binding reactions.
Fluorescent enzyme immunoassay is identical to other EIAs except
that they use fluorescent substrates. In the fluorescent EIA, the
fluorophore is generated by an enzyme reaction. Following excitation of
the fluorophore at its optimal wavelength, light at a characteristic
wavelength is emitted. The emitted fluorescent light is measured.
Time-resolved Fluoroimmunoassay:
This method needs special instrumentation and special fluorescent
labels to increase the assay sensitivity. The fluorescent label used in
this assay exhibit a delayed fluorescence with a time period of 100 ns
or more between excitation and emission. Most substances responsible for
the background fluorescence have a short decay period. Therefore, the
measurement of the delayed fluorescence signal will reduce the effects
of background fluorescence.
This purpose is achieved with the use of special time- resolved
fluorometer that produces a fast light pulse that excites the
fluorophore. Fluorescence is measured a little while after excitation.
Thus, the effect of non specific background, which generally decays in
less than 10 ns can be eliminated.
The fluorophores exhibiting delayed fluorescence are:
i. Pyrene derivatives with a decay time of almost 100 ns.
ii. Rare earth metal chelate labels [such as europium (Eu3+), samarium (Sm3+), and terbium (Tb3+)] that have a very long decay time of about 50 to 100 µs.
Chemiluminescent Immunoassay:
Chemiluminescent immunoassays (CL-EIAs) use chemiluminescent
substrates that react with various enzymes. The chemiluminescent
substrate-enzyme reaction generates light, similar to bioluminescence.
The chemiluminescent EIA systems are definite improvement over RIAs in
terms of sensitivity, time efficiency, and procedural simplicity.
Chemiluminescence immunoassays use chemiluminescence generating molecules as labels.
i. Luminol derivatives
ii. Acridinium esters
iii. Nitrophenyl oxalate derivatives
iv. Ruthenium tri-bipridyl with tripropylamine for electrochemiluminescence
v. Basically, the assay methods don’t differ from RIAs/ FIAs.
This page was last modified on May 17, 2014.
