Techniques Using ELISA
The use of enzymes as immunochemical labels for use in competitive binding
was reported in 1971.
The most widely used names are ELISA.
E- Enzyme
L- Linked
I- Immuno
S- Sorbant
A- Assay;
EIA- Enzyme Immuno-Assay; and EMIT- Enzyme multiplied Immuno Assay technique.
The EIA are capable of detecting extremely small quantities of immune
reactants. The advantages of EIA are:
1. Sensitive assays can be developed by the amplification effect of enzymes.
2. Reagents are relatively cheap and can have a long shelf life.
3. Multiple simultaneous assays can be developed.
4. A wide variety of assay configuration can be developed.
5. No radiation hazards occur during labeling or disposal of wastes.
6. Rapid simple EIA adaptable to automation can be developed.
7. Homogeneous EIA can be developed for haptens and proteins.
EIA can be classified by:
1. Which reactant is determined i.e. antigen or antibody
2. Which reactant is labeled.
3. Competitive or non competitive assay
4. Method of separation of bound and free reactants.
There are several important criteria in the selection of particular enzyme
label, including:
1. Turnover numbers-number of substrate molecules converted to product
per enzymes site/ unit of time.
2. Purity
3. Sensitivity
4. Ease and speed of detection
5. Absence of interesting factors in test fluid
6. Potential reactive groups
7. Stability
8. Suitability for homogenous EIA
There are two major types of EIA - heterogenous and homogenous. In the
heterogenous system, the antigen-antibody reaction does not effect the activity
of the enzyme label. In homogenous EIA, the antigen-antibody interaction
modulates the activity of the enzyme eliminates the need for a separation step.
The term ELISA identifies a heterogenous enzyme assay differentiating it from
enzyme linked antibody conjugates used in microscope immuno-histo-chemical
staining reactions. ELISA may be developed in different configurations, and one
of the reactants is immobilized onto a solid phase matrix.
Hetrogenous EIA:
The principles involved in this method involves measurement of enzyme activity.
The choice of a separation phase in heterogenous EIA is limited by the large
size of enzyme label. Acceptable methods include use of solid phase
antigen or antibody, or use of beads such as agarose or polyacrylamide. Some
investigators have utilized magnetic beads for an ingenious separation system.
The commonly employed heterogenous EIA are:
1. Competitive EIA for antigen
2. Immuno-enzymo-metric assay
3. Double immuno-enzymo-metric assay for antigen determination.
Competitive EIA for antigen
It is a competitive EIA between the unlabeled antigen and enzyme labeled antigen
which are competing for a limited amount of specific antibody binding sites.
The amount of enzyme labeled antigen bound by antibody is inversely
proportional to the concentration of the unlabeled antigen.
The disadvantage of this method is that significant amounts of pure antigen must
be isolated to label with enzyme.
Immuno-enzymo-metric assay:
In this EIA, the reactants are in stoichiometric excess. The enzyme labeled
antibody is first reacted with antigen, and excess solid phase antigens is then
added to remove unreacted enzyme labelled antibody. The enzyme activity
bound to the solid phase is inversely proportional to the concentration of free
antigen.
This configuration may be used for small hapten molecules which cannot be easily
assayed by two site immunoenymometric assay. As long as the enzyme labeled
antibody is specific, the antigen does not have to be isolated in pure form.
Two site Immuno-enzymo-metric assay for antigen:
A solid phase antibody is first incubated with the antigen to be measured.
After washing, enzyme labeled antibody is then added. The enzyme
activity bound to the solid phase is proportional to the concentration of the
antigen present.
This method is useful for measuring complex antigens and can be used only for
antigens which are able to bind atleast two antibodies. Monoclonal
antibodies reactive at different antigenic sites, one labeled and second
unlabelled solid phase, may be incubated with the unknown antigen
simultaneously.
Sandwich assay for antibody detection:
The solid phase antigen is incubated with a sample containing the antibody to be
detected. After the reaction and appropriate washing, the enzyme labeled
second antibody is added. The second antibody is reactive against the
first antibody. The amount of enzyme activity found bound to the solid
phase is directly proportional to the amount of antigen specific antibody.
Double antibody immuno-enzymo-metric assay for antigen determination:
This method employs a solid phase antigen. Free antigen prevents antigen
specific antibody from binding to the solid phase. The solid phase is then
washed and reacted with enzymes labeled second antibody reactive against the
first antibody. The amount of enzyme labeled second antibody bound to the
solid phase is inversely proportional to the quantity of the free antigen in the
sample.
The advantage of this method is that one enzyme labeled antibody can measure
many different antigens.
Homogenous Enzyme Immuno Assay:
The term homogenous immuno assay may be applied to any antigen antibody reaction
system in which the measurement of the degree of the immune reaction is carried
out without a separation of the free and the antibody bound components. Homogenous
enzyme immuno assays have been used for assay of drugs and hormones in the
clinical laboratory. At present homogenous EIA are generally less
sensitive than heterogenous EIA. The hetrogenous EIA has equaled the
sensitivity of RIA in many applications whereas the homogenous EIA may be of one
or two order of magnitude less sensitive than RIA. Homogenous EIA may
require complex immuno chemical reagents. However the systems are rapid,
simple and adaptable to automation. Currenty the chief application of
homogenous EIA is in the determination of low molecular weight analyets such as
hormones and drugs. More recently, homogenous EIA methods have been
developed for complex higher molecular weight protein antigen such as IgG.
there are various types of homogenous EIA. In each of these assays, the
antigen antibody interaction modulates the activity of their enzymes or enzyme
labeled in the presence of substrate. The modulation of the enzyme
activity reflects the degree of immuno chemicals reaction.
Radioimmuno Assay:
The antibody is attached to the wells of a micro titre plate. The antigen
in serum when added binds to antibody. The radio-labeled antibody is then
added which binds to the antigen. The radio activity is recorded by a
gamma-counter.