Introduction

As the name suggests, an immunoassay is a test which is used to either show the occurrence or measure the concentration of an analyte by the use of either an antibody or an antigen.
Any kind of molecule, regardless of its size or type can be used for an immunoassay as long as the suitable antibodies required for the assay are developed.

There are many types and formats of immunoassays. Some immunoassays require multiple steps while some involve simply mixing the reagents and the analyte and then making a measurement.

History

1950s: Rosalyn Yalow and Solomon Berson develop the first immunoassays.
1977: Rosalyn Yalow is awarded the Nobel Prize for her work on immunoassays.
1983: Professor Anthony Campbell substitutes radioactive iodine used in immunoassay with an acridinium ester which generates its own light, a phenomenon known as chemiluminescence.

Various Labels used in immunoassays:

There are certain molecules or groups used in immunoassays known as labels that help in the detection of antibody/antigen.
E.g. enzymes, radioactive isotopes, etc.

Examples of Immunoassays

Examples of Immunoassays:

1) Home pregnancy testing kits- Test for human chorionic gonadotropin (HCG)

2) Glucometer/ measuring insulin in blood.

Enzyme Linked Immunosorbent Assay (ELISA)

Introduction and types:

ELISA is a plate based immunoassay technique which has been created in order to detect or measure the concentration of substances such as proteins, antibodies, etc. in biological fluids.
It was first described by Weiland in 1978.
There are 4 main types of ELISA based on how the samples and antibodies are used and bound.

Direct ELISA- Used for antibody measurement.

Steps:

Addition of antigen to the microtiter plate and its adhesion to plastic.
Addition of inert protein such as casein to fully coat the plastic surfaces of the plate.

Addition of the primary antibody with an attached enzyme is added which binds to the test antigen coating the well.

Addition of the substrate for this enzyme which changes colour on reacting with the enzyme.

Sandwich ELISA- Used for antigen/analyte measurement
Steps:
Preparation of surface on which a known quantity of capture antibody is bound.
Blocking of the non-specific binding sites on the surface.
Application of the antigen-containing sample to the plate and its capture by the antibody.
Washing of the plate to remove unbound antigen.

Addition of another antibody known as specific antibody and its binding to antigen.

Application of secondary enzyme linked antibodies/ detection antibodies which bind specifically to the antibody’s Fc region.

Washing of the plate to remove the unbound antibody-enzyme conjugates.

Addition of a chemical which is used by the enzyme to get converted into a colour/ fluorescent/electrochemical signal.

Measurement of absorbance/fluorescence/electrochemical signal to determine the occurrence/concentration of antigen.

Competitive ELISA- Another technique for the measurement of the analyte/antigen

Steps:

Incubation of unlabelled antibody in the presence of its antigen/epitope.
Addition of bound antigen-antibody complexes to an antigen coated well.

Washing of the plate to remove unbound antibodies.

Addition of the secondary antibody specific to the primary antibody and its coupling to the enzyme.

Addition of substrate and eliciting a chromogenic or fluorescent signal by the remaining enzymes.

Arresting the reaction for the prevention of eventual saturation of the signal.

Reverse ELISA- Another technique used for detection of antibodies, traditional wells not used.

Steps:
Incubation of unlabelled antibody in the presence of its antigen/sample.
Provision of a sufficient incubation period to enable the antibodies to bind to the antigens.

Passing of the sample through the scavenger container which can either be a test tube or a specifically designed flow through channel whose surface has antigens called Scavenger antigens attached to it.

Passing of the sample containing the antibodies (tagged and bound both) through a detector which may be a flow cytometer or any other detector that brightens the tags and records the subsequent response.

Basic principle of ELISA:
ELISA makes use of both the specificity of antibodies and the sensitivity of enzyme assays by making use of antibodies/antigens attached to an enzyme (that can be assayed easily).

It provides a measurement of the amount of both antigen as well as antibody.

ELISA can either detect antigens recognized by antibodies or it can be used to check for the presence of antibodies that recognize an antigen.

Applications of ELISA:

ELISA can be used to check the antibody concentrations in serum in the case of diseases like HIV, west Nile Virus, etc.

In the food industry, it is used for detecting likely allergens such as milk, nuts, etc.

ELISA is also used as a serological blood test for celiac disease.

It is also used in toxicology for a quick, preliminary screening of drugs of certain classes.

In medical labs, ELISA is used as an in-vitro diagnosis technique.

Radioimmunoassay (RIA)

Introduction:

Radioimmunoassay (RIA) is a type of immunoassay that makes use of radioactive isotopes to form an antigen-antibody complex in a stepwise way.

This immunoassay technique albeit extremely sensitive, specific and requiring specialised equipment is one of the least expensive techniques to check the levels of antigens in the blood.

Since radioactive isotopes are used in this technique, special care and safety measures are needed.

Steps:

Making a radioactively labelled antigen by labelling it (the antigen) with the radioisotope 125-I and then subsequently attaching it to tyrosine.

Mixing the radiolabelled antigen with a known quantity of antibody specific for the antigen and the later specific binding of the antigen and the antibody to each other.

Addition of the sample of serum from the patient having an unknown amount of the same antigen (not radiolabelled) resulting in a competition between the unlabelled and labelled antigens for the antibody binding sites.

Increase of the amount of unlabelled antigen and the subsequent displacement and separation of bound and unbound antigens.

Measurement of radioactivity of the unbound antigens with the help of a gamma counter.

Applications:
Narcotic drugs like heroin and morphine can be detected in the hair by using RIA.

Drugs like hydromorphone and hydrocodone that are used in combination with antitussive and analgesic-antipyretic mixture can be detected in the plasma using RIA.

Flunisolide, a corticosteroid used for the treatment of asthma and other respiratory diseases can be detected in plasma with the help of RIA.

RIA is used in the differentiation between iron deficiency and the inability of the body to use iron by measuring the levels of ferritin in the serum.

In order to rule out digoxin toxicity rapidly and accurately by measuring its levels in the serum, RIA is used.

It is also used in determination of a patient’s thyroid status and also to check the effectiveness of I-131 doses after I-131 treatment in patients.

Immunoradiometric Assay (IRMA)

It is an immunoassay that uses radiolabelled antibodies instead of radiolabelled antigens.
IRMA also differs from RIA as the analyte to be analysed immediately combines with the radiolabelled antigens instead of other antigens being displaced.

It was first introduced by Miles and Hales in 1968.

Principle/Steps:

Labelling of antibodies with the help of radioisotopes which bind the antigens present in the sample.

Attachment of the radioactively labelled antibodies to the unbound epitopes of the antigen and the subsequent formation of the antigen-antibody complex after addition of a sample having the desired analyte.

Removal of unbound labelled antibodies using an antigen bound to a solid surface.

Measurement of the concentration of the remaining radioactively labelled antibodies using a gamma counter.

Applications of IRMA:

IRMA is used for measurement of hormones such as TSH (Thyroid Stimulating Hormone), ACTH (Adrenocorticotropin) and PTH (Parathyroid Hormone).

It is also used to measure the concentration of HCG (Human Chorionic Gonadotropin) during early pregnancy.

IRMA is also used in the monitoring of the condition of cancer patients by measuring the levels of the urokinase receptor in their serum.

Two site IRMA is used for the detection of Plasmodium falciparum antigen in the blood, thus helping in the diagnosis of malaria.

An IRMA of Factor IX, a protein is used in the prenatal diagnosis of Haemophilia B.