Speed of the reaction: The solid phase is coated with avidin
and the capture antibody is biotinylated,this minimizes
the need for other coating methods and facilitates the use
of antibodies with high affinities.
Temperature stability and other problems: Non-bound
avidin is very thermostable for the folded-unfolded
transition,Tm = 85°C (pH 7–9). When biotin is bound,the
protein acquires greater thermostability Tm = 132°C.
Thus, the avidin-biotin system is more resistant to high
temperature. This greater thermostability of avidin-biotin
system overcomes/reduces the problems faced during:
The sensitivity of any analytical technique is defined as the
minimal concentration that can be reliably estimated.
In any immunometric assay the signal measured at the
end of the assay consists of two types of signals:
1. The signal seen due to the presence of analyte–what
2. The signal due to non-specific adsorption of labeled
antibody–commonly called background absorbance.
Technically sensitivity can be defined as the minimal
concentration of analyte that is statistically unlikely to form
part of the range of signals seen in the absence of analyte.
1. Third Generation Ultrasensitive assay designs are
based on maximizing the Signal Noise Ratio (S/N).
Streptavidin-biotin based assays offer four fold increase
in signal generation, thus making the background noise
negligible. Chemiluminescent assays are also based on
the principle of generating very high signal in presence of
analyte in order to make the background noise negligible.
Primary Calibrators and Matrix Effect
The aim of standardization of laboratory is to improve
the accuracy, i.e. the results should be as close to the true
Immunoassays do not actually measure the analyte.
A prerequisite for standardization is that the standard/
calibrator and analyte are identical. In other words, the
calibrator should contain the analyte in a form identical to
Calibrators should ideally be prepared by using a base
material identical to that in the test samples. For clinical
applications Human Serum is the preferred base matrix.
The matrix of a calibrator needs to behave in a similar
For assay of hormones that are bound to serum protein,
it is hard to use any other matrix other than Human Serum.
Calibration of direct assays for protein bound hormones
is complicated because of interference by the binding
proteins. The effect of the binding proteins is hard to
eliminate completely. Therefore, serum based calibrators
Few immunoassays are totally free from interference from
the ill-defined composition of biological fluids under test
(matrix). Different samples containing the same amount of
analyte may give different results due to this Matrix effect.
Assays having higher sensitivity are able to better identify
and amplify the analyte thereby reducing the matrix effect,
and improving the assay accuracy.
Most of the diagnostic immunoassay kits are not
exhausted overnight. Repeated usage and the store/use/
store cycles, exposes the immunoassay system to multiple
thermal shocks. This impacts the analytical performance
of the immunoassays due to the lowering of sensitivity.
This shift in sensitivity affects the ultrasensitive assays
lesser since the percentage change in sensitivity would
be proportionally smaller as compared to assays having
Due to their higher sensitivity and amplification ability,
ultrasensitive assays enable test run on the smaller volume
samples, such as capillary blood from children. This not
only assures better testing confidence but also minimizes
Ultrasensitive assays have opened up new opportunities
in the diagnosis of diseases or clinical conditions which
were previously unrecognized or the test for which were
unavailable. A good example is the development of Third
Generation Thyrotropin (TSH) assays/Ultrasensitive TSH
assays which for the first time opened up the possibility of
differentiating between the euthyroid and hyperthyroid
As compared to low sensitivity assays, ultrasensitive
assays also offer an increase in signal ratio as well as
improvement in rate of change of the measured signal which
in turn offers the immunoassay users greater accuracy from
In conclusion: Ultrasensitive assays are more robust,
more accurate, versatile systems that improve reliability
of results and provide confidence to the clinicians on the
Epitope: Part of an antigen to which antibody binds.
Epitype: Part of an epitope where the antibody binds.
¾ Conformational or discontinuous: T3, TSH
REPRESENTATIVE ELISA/CLIA TECHNIQUES
ELISA/CLIA Analyte Determination Principles
Thyroid stimulating Immunoenzymometric/Sandwich
Total triiodothyronine Competitive
Free-triiodothyronine Competitive
Total thyroxine (T4) Competitive
Free-thyroxine (FT4) Competitive
Anti-thyroglobulin Immunoenzymometric/Sandwich
Anti-thyroperoxidase Immunoenzymometric/Sandwich
Lutropin (LH) Immunoenzymometric/Sandwich
Follitropin (FSH) Immunoenzymometric/Sandwich
Prolactin (PRL) Immunoenzymometric/Sandwich
Prolactin (PRL) Immunoenzymometric/Sandwich
Sequential (Streptavidin Biotin)
Beta-hCG Immunoenzymometric/Sandwich
Insulin Immunoenzymometric/Sandwich
C-Peptide Immunoenzymometric/Sandwich
AFP Immunoenzymometric/Sandwich
CEA Immunoenzymometric/Sandwich
PSA Immunoenzymometric/Sandwich
CA-125 Immunoenzymometric/Sandwich
Free beta hCG Immunoenzymometric/Sandwich
Free PSA Immunoenzymometric/Sandwich
CA 19-9 Immunoenzymometric/Sandwich
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