203.

ELISA Biosensor Test Format

Learning outcome.

Students will be able to learn and explain the use of ELISA based test format in Biosensor.

1. Introduction
2. Biosensor is a mini testing device that is used in diagnostics for estimating and detecting
different analytes.
3. For testing a molecule a number of lab designed assay methods are successfully used in
diagnostic Biosensors.
4. Enzyme linked immuno sorbent assay (ELISA) for proteins.

Enzyme linked immunosorbent Assays

Enzyme linked immunosorbent Assays (ELISA) based are one of the formats applied in biosensor
measurements.

Micro plate are used in ELISA for the immobilzation of immunogenic molecules.

In biosensor the immunogenic molecules are immobilized on the surface of active transducer.

Types

Enzyme linked immunosorbent Assays (ELISA) assay based are one of the formats applied in biosensor
measurements.

In principle, four categories of test formats are available:

Direct Detection

Sandwich assay

Competitive assay

Direct Assay

In direct assay a label-free transduction principle is used.

It is a one-step process without any other reagents.

It allows real-time monitoring of binding events to the biosensor surface.

The signal response changes as soon as the analyte binds to the biorecognition element
immobilized on the transducer surface.
Sandwich Assays

In a sandwich there are two biorecognition elements.

Capture antibody that captures the target molecule.

Detection antibody that detects the capture antibody.

Response is obtained when the detection antibody binds with the capture antibody.

Competitive Assay

In competitive Assay there are two types of Analytes.

In a competitive assay analyte and a known concentration of labeled analyte (derivative).

The compettive assay could of two types

 Surface competitive assay

 Solution competitive Assay
Advantages.
Low detection limits
High analyte selectivity
High throughput of samples
Reduced sample preparation
Versatility for target analytes
Cost effectiveness
Aadaptability to field use.

Disadvantages.
Low detection limits
High analyte selectivity
High throughput of samples
Reduced sample preparation
Versatility for target analytes
Cost effectiveness
Aadaptability to field use.

Related Notes

Biosensor test formats; a) direct detection, label-free: quantifiable signal response ob-
tained by binding of analyte; b) sandwich assay, label optional: quantifiable signal response ob-
tained by binding of secondary biorecognition element subsequent to analyte binding; c)
com-petitive assay, label required: quantifiable signal response obtained by binding of labeled
analyte derivative competing with analyte for the surface bound binding sites; d) binding
inhibition as-say, label optional: after a pre-incubation step, in which equilibrium between
analyte molecules and unbound biorecognition elements is achieved, the remaining free
biorecognition elements will be detected via immobilized analyte (derivative) allowing
calculation of the original analyte concentration n a sandwich assay the analyte-related
signal response is obtained when a second biorecognition element binds to the analyte
after the analyte has bound to the biosensor surface. This requires analyte molecules
which are large enough for two independent biorecognition elements to bind. Whether the
second biorecognition element has to be labeled or not depends on the transduction prin- ciple.
It is also possible to use a third biorecognition element binding to the second one (“indirect
sandwich”). The use of more than three biorecognition ele-ments is not common . In a
competitive assay analyte and a known concentration of labeled analyte (derivative)
compete for the surface bound ana-lyte-specific binding sites provided by the biorecognition
element. This test format including the corresponding transduction principles requires labels. In
the binding inhibition assay a pre-incubation step, in which analyte and a known
concentration of unbound biorecognition element equilibrate, precedes the actual
measurement. Again, whether the biorecognition element has to be labeled or not depends on
the underlying transduction principle. After equilibrium, the remaining free biorecognition
elements’ binding sites are detected with a biosensor providing immobilized analyte (derivative)
allowing to derive the original analyte concentra-tion in the sample . This method is particularly
suitable for label-free detection of small molecules, as the signal response is achieved by
binding of the corre-sponding biorecognition element. The latter is usually a protein of
higher mass and size and hence should create a higher signal response.