Immunoassay- A Powerful Analytical Technique

Immunoassay is a biochemical test that measures the concentration of an analyte in a biological liquid using the reaction of an antibody or antibodies to its antigen. The assay takes advantage of the specific binding of an antibody, which are special proteins made in the body in response to invading microorganisms or other foreign substances, to its antigen. The qualities of immunoassay that makes it one of the powerful analytical techniques are its specificity, sensitivity and versatility.

This technique can measure both the presence of antigen (direct) or antibodies (indirect) in a given specimen. For instance, when seeking to detect the presence of an infection the concentration of antibody specific to that particular pathogen is measured. For measuring hormones such as LH, FSH and Insulin, the hormone acts as the antigen.

Detecting the quantity of antibody or antigen can be achieved by labeling either the antigen or antibody. The label may consist of an enzyme: Enzyme immunoassay (ELISA), radioisotopes such as I-125: Radioimmunoassay (RIA), magnetic labels: Magnetic immunoassay (MIA) or fluorescence. Other techniques include agglutination, nephelometry, turbidimetry and Western Blot. Calbiotech offers the former two-immunoassay kits (ELISA & RIA) with a superior quality. The majority of the Calbiotech kits are in ELISA format.









Figure 1. Simplified scheme of Calbiotech ELISA kits.

In addition to colorimetric (ELISA) assays, Calbiotech also offers chemiluminescent (lumELISA) assays, which employs special type of substrate, known as luminol that emits chemically produced lights. For better sensitivity, dynamic range, linearity and shorter assay time, Chemiluminescent kits are the options.

Commonly the designs of a Calbiotech ELISA kit fall within one of the following types. To keep the assays easy to understand, the brief explanations are accompanied with pictorially representations.

1. Sandwich assay Type

• Antibodies immobilized on the polystyrene micro-titer plate (capture antibody) will bind the target analyte present in samples.
• The second antibody (detection antibody), tagged to an enzyme, will recognize a different epitope on the analyte and binds the target on a different position.
• The unbound analyte and/or detection antibodies will be washed out
• Substrate (TMB) is added. The remaining bound enzyme catalyzes the oxidation reaction of TMB to produce color (Blue).
• The enzymatic reaction is stopped by adding diluted acid to yield a yellow color.
• The yellow color can be read spectrophotometrically at 450nm.


Figure 2. Direct immunometric assay (ELISA) detecting antigen.

The Calbiotech chemiluminescent (lumELISA) version of the typical sandwich assay follows similar protocol to the regular ELISA kits except that luminol substrate is used and the enzymatic reaction of the substrate is not stopped by a diluted acid solution. Once the luminol substrate is added, the photons should be counted immediately using luminometers.


Figure 3. Direct immuno-metric assay (lumELISA) detecting antigen

The typical dose-response curve looks like the following:


Figure 4. Dose-response curve showing direct relationship between signal developed and analyte concentration.

Assays are also designed to measure antibodies, produced by the body against a certain pathogens, in biological samples. In this type of assays, the antibodies are considered as the analyte. Because the analyte is surrounded on two sides, the assay procedure can be categorized as sandwich assay.

In this assay type:

• The pathogen itself is immobilized on the polystyrene micro-titer plate and the target antibody (if present) will bind itself to the antigen
• After un-reacted analyte is washed out , a secondary antibody tagged to the an enzyme is added and binds to the target antibody
• The unbound enzyme tagged secondary antibody is washed out
• Substrate is added and the remaining bound enzyme will catalyze its oxidation reaction to produce color (Blue)
• The oxidation reaction is stopped by adding a diluted acid to yield yellow color.
• The yellow color can be read spectrophotometrically at 450nm.


Figure5. Indirect immunometric assay (ELISA) detecting antibody

In all of the above immunometric assays, the intensity of the color developed is directly proportional to the concentration of the analyte.

Generally sandwich assay designs are suitable if the target molecules are large enough (proteins and antibodies) to bind two separate antibodies at the same time. Competitive assay designs are for small target molecules.

2. Competitive Assay Type

• Capture antibody is immobilized on the polystyrene micro-titer plate
• Both antigens (the analyte and the conjugated antigen) will compete to the same site in the capture antibody
• The plate is washed out to remove unbound free and conjugated antigens.
• Substrate(TMB) is added and the remaining enzyme catalyzes its oxidation reaction to produce color (blue)
• The oxidation reaction is stopped by adding diluted acid to yield yellow color
• The yellow color can be read spectrophotometrically at 450nm


Figure 6. Competitive assay detecting antigen.

In competitive assays, the color developed is inversely proportional to the analyte concentration in the biological sample.


Figure 7. Dose-response curve showing indirect relationship between the signal generated and the analyte concentration.

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