Lysozyme: Strongly associates with proteins having low
isoelectric points (pI). Immunoglobulins have a pI of
around 5 and lysozyme may form a bridge between the
solid–phase IgG and the signal antibody.
Endogenous hormone-binding proteins: These are
present in varying concentrations in all serum and plasma
samples and may markedly influence assay performance.
SHBG (sex hormone binding globulin) interferes in
immunoassay of testosterone and estradiol.
TBG, (thyroxine binding globulin) and NEFA (non
esterified fatty acid) interfere with the estimation of free
Abnormal forms of endogenous binding proteins: These
are present in the plasma of some patients. They are
present in familial dysalbuminemic hyperthyroxinemia
(FDH) in which albumin molecules bind to thyroxine (T4).
Individuals with FDH can be diagnosed as thyrotoxic, in
Heterophilic antibodies: They may arise as a consequence
of intimate contact, either intentional or unintentional,
with animals. The most familiar effect of heterophilic
antibodies is observed in two-site sandwich reagent-excess
assays, in which a ‘bridge’ is formed between the two
antibodies forming the sandwich. Assays that are affected
by heterophilic antibodies include for CEA, CA 125, hCG,
TSH, T3, T4, free T4, Prolactin, HBsAg and Digoxin.
Fibrinogen from incompletely clotted samples interferes
with sampling procedures on automated immunoassay
instruments and may produce spurious results.
Paraproteinemia causes interferences in many assays by
Non-specific interference may arise from excessive
concentrations of other constituents of plasma. Free fatty
acids affect some assays for free T4 by displacement of T4
from endogenous binding proteins.
The “Hook Effect” is characterized by the production
of artefactually low results from samples that have
extraordinarily high concentrations of antigen (analyte),
far exceeding the concentration of the upper standard in
The Hook effect is most commonly found in single-step
immunometric assays, a popular format, chosen for its
specificity and speed, particularly with high-throughput
immunoassay analyzers. The assays most affected are
those that have analyte concentration that may range over
several orders of magnitude. For example, α Feto protein
(AFP), CA 125, hCG, PSA, TSH, prolactin and Ferritin are
The incidence of Hook effect can be reduced (but not
eliminated) by careful assay design – incorporating a wash
step prior to addition of the second antibody, thereby
avoiding simultaneous saturation of both antibodies.
Despite attempts to eliminate or reduce the Hook
effect by careful assay design the only reliable method of
routinely eliminating the effect is to test the samples that
are likely to be affected by Hook effect in undiluted and
also at a suitable dilution. Such samples should be diluted
using either the assay diluent or serum from a normal
subject until a stable quantitative response is achieved.
It is one of the most important requirements of
immunoassays. Interference occurs in all situations in
which the antibody is not absolutely specific for the analyte.
Consequently, assessment of specificity is a vital step in the
optimization of every new immunoassay. Poor specificity
results in interference from compounds of similar
molecular structure or which carry similar immunoreactive
epitopes. In determining the overall specificity of an assay,
a major factor is the cross reactivity of the antibody.
Some the major specificity problem areas are related
to measurement of steroids and structurally related
compounds. All commonly used testosterone assays, cross
react in varying degrees with 5α-dihydrotestosterone, and
all cortisol assays cross react with prednisolone.
Assessment of the specificity of immunometric assays
employed, each having unique specificity for a different
epitope on the antigen. It is usual practice to employ at
least one monoclonal antibody, which can be selected by
epitope mapping to react only with predetermined sites on
the antigen molecule. Use of two monoclonal antibodies
can introduce extreme specificity.
What is the difference between an antigen and
The word “antigen” is conventionally used to describe
as antibody generators, i.e. that can generate antibody
against itself. Also, anything that is foreign to the body is
also known as antigen. This definiton of foreignness has
become irrelevant with autoantigens. Antigen can be
defined as those that bind with the antibody. They need
not be foreign in nature. Some antigens also require a
carrier/helper to bind with the antibody.
Immunogens, as the name goes are those that can
elicit an immune response. It may be either T-cell or
B-cell response. All immunogens can be antigens. But all
antigens need not be immunogens.
1. What are the different types of epitopes?
of peptides. Conformational epitopes are formed when
the protein chain is folded. Disulfide bonds are important
for maintaining the conformational integrity.
Sometimes, the value of an analyte obtained by laboratory
testing will be very low in spite of suspicion that it will
be high. This false low values derived in spite of it being
very high is known as Hook effect. This is due to very
high concentration in the blood. The levels are so high
that they actually mask the binding sites available in the
immunoassay system, leading to very low values. (Imagine
one hundred persons fighting to sit in 5 chairs. Even
though there were hundred the actual number of people
who sat were only 5). This is observed in parameters like
PSA,hCG, CEA, etc. The solution for this is to dilute the
and fluorescence? Which is better?
Fluorescence is a phenomenon where molecule absorsbs
light in one wavelength and emits in another wavelength.
In this, there is a source of excitation. Chemiluminescence
is the production of light by a chemical reaction. The main
difference is that there is no radiation is absorbed. The
energy required to emit light comes from the energetics
of chemical reaction. Definitely chemiluminescence is a
better technology for use in immunoassays.
4. What is meant by apoptosis?
In an organism such as a human, the number of new cells
created must be balanced by an equal number of cells
dying. Sometimes cell death occurs as a result of injury;
most often, however, it is a planned, natural process called
apoptosis. Apoptosis is sometimes called cellular suicide
because it is a cell’s own gene products that carry out its
death. While it kills a cell, apoptosis is beneficial to the host
as a whole - it is important, for example, in development,
5. How does secondary response differ from primary
It differs mainly in three ways:
¾ It involves an amplified population of memory cells
¾ Higher levels of antibodies are formed than primary
6. What are primary and secondary lymphoid organs?
Primary lymphoid organs are the bone marrow and
thymus. These organs function as sites for B-cell and T-cell
maturation, respectively. Secondary lymphoid organs
are spleen, lymph nodes and various mucous associated
lymphoid tissues. All these trap antigens and provide sites
lymphocytes can interact with antigen.
7. What is the difference between active and passive
Active immunity Passive immunity
Produced actively by the host Received passively by the host
Induced by infection Conferred by introduction of
Durable and effective protection Protection transient and less
Immunity effective only Immunity effective immediately
Immunological memory present No immunological memory
Negative phase may occur No negative phase
Not applicable in immunodeficient host
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