Different Methods of Hormone Estimation

Different Methods of Hormone Estimation

Introduction:
The problem in hormone estimation, that there are very small
amounts of hormone in a very complex mixture. In the preimmunoassay era hormone estimation was done by complex and
insensitive methods (chemical methods, whole animal or tissue
bioassay) and was insensitive, imprecise and inaccurate.
Immunoassay hormone estimation first described in 1960 and
have many advantages (simplicity, speed, precision, accuracy,
sensitivity).
Immunological techniques, like radioimmunoassays (RIA) and
enzyme immunoassays (EIA), are used because they are capable
of measuring small quantities of hormones. RIAs are highly
sensitive and have been the most common immunological
methods for hormone analysis used to date. However, an RIA
laboratory needs to be licensed for the use of radioisotopic
tracers, and gamma and beta scintillation detection equipment
are comparatively expensive. By contrast, EIAs do not utilize
radioactivity, equipment is less expensive and reagents are easy
to prepare, are highly stable and have a long shelf-life. Many EIAs
are now as sensitive as RIAs and so are gaining in popularity. The
purpose of this manual is to acquaint the reader with both RIA and
EIA techniques; however, the emphasis will be on EIA because of
its universal adaptability and potential for development as field
tests.
Principles of Immunoassay:
The essence of an immunoassay is the competition between
added labeled antigen (tracer) and unlabeled antigen (i.e.,
hormone in the sample) binding to an antibody. Highly sensitive
immunoassays rely on the use of a limited amount of antibody. If
the primary antibody is in excess, there is little or no competition
in binding between labeled and unlabeled antigen, thus no
discrimination in measuring concentration of the unknown. With a
limited amount of antibody, samples with higher concentrations of
hormone have a greater chance of competing against labeled
antigen for antibody binding than low concentration samples, and
this relationship is proportional to the amount of unlabeled
hormone added. Antibodies are produced against a specific
antigen by immunizing an animal and collecting immune serum.
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Different Methods of Hormone Estimation

A)Primary antibody (antisera):

Also called the first antibody refers to the hormone specific
antibody; the one that was produced from immunizing an animal
against the hormone to be measured. Primary antisera can be
produced against large proteins by direct injection of antigen
solubilized in a carrier that stimulates the immune response (i.e.,
Freunds adjuvant). Immunization against small proteins or
steroids (i.e., haptens) requires conjugation to immune
stimulating molecules (i.e., albumins, keyhole limpet antigen,
etc.). Primary antibodies are often produced in rabbits, guinea
pigs and monkeys.
B)Second antibodies:
Also called non-specific antibodies are those produced by
immunizing a different species like a goat or sheep against nonspecific IgGs of the species that produced the primary antisera
(e.g., goat anti-rabbit antisera).
C)Polyclonal antibodies:
They are produced by injecting an animal with a purified or
partially purified antigen and collecting the immune serum. The
antisera produced contains a variety of immunoglobulins against
the antigen (or antigens for partially purified immunogen) or
conjugate molecule (in the case of conjugated haptens). Antibody
production is limited to the life span of the immunized animal and
success of reimmunizations.
D)

Monoclonal antibodies:

They are produced by immunizing an animal (e.g., mouse) and

cloning individual antibody cells to produce a single variant
antibody against an antigenic determinant on the hormone.
Antibody production is indefinite as long as the cloned cells are
maintained (frozen in liquid nitrogen).
Principles of Radioimmunoassay:
- RIAs depend on the assumption that an antigen can be linked
to a radioactive molecule (e.g., 3H or 125I) and retain
immunological binding activity.
- Essential components of RIA:
Antibody: An immunoglobulin produced against a specific
antigen(s).

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Different Methods of Hormone Estimation

Tracer antigen: An antigen that is labeled in a manner that

permits its detection. The labeled antigen, or tracer, should
be structurally similar to the unlabeled antigen (or standard)
and generally is labeled with 125I or 3H.
Standard antigen: The standard hormone is the same
antigen that was injected into an animal to induce an
immune response for antibody production. Standards are
used in a series of different concentrations, against which
'unknown' concentrations of the antigen contained in
biological fluids can be estimated.
Separation method: The ability to separate bound Ab-Ag
complexes from free hormone.
- Steps of RIA:
1) Determination of antibody titer: Based on a binding
inhibition curve, the optimal working antibody dilution is
one that results in 30-40% binding of the total labeled
antigen. In general, increasing the antibody concentration
decreases assay sensitivity, whereas decreasing antibody
concentration will increase sensitivity.
2) Development of a standard curve: Incubation of a fixed
amount of tracer and antibody in the presence of different
concentrations of standard (unlabeled antigen). A graph is
generated which depicts the relationship between the
percentage of tracer bound (relative to the maximum
binding, or zero tube, see below) and relative mass of
standard added to each assay tube. There is an inverse
relationship between percentage binding of tracer to
antibody and hormone mass.
3) Optimizing the RIA: Performance of the RIA can be
affected by a variety of factors, including buffers, reagent
volumes, tracer or antibody concentration, incubation time
and temperature, and hormone mass.
4) Incubation time and temperature: The required time to
achieve maximum tracer binding, assessed by counting
binding in the zero tubes. Incubation temperatures range
from 4 to 37C depending on assay characteristics.
- Types of RIA:
A) Single antibody (charcoal-dextran separation) RIA:
This is a common assay system used for measurement of
steroid hormones. A hormone-specific antibody (or first
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Different Methods of Hormone Estimation

antibody) is added to a test tube with sample (or standards)

and a 3H-labeled tracer. The labeled and unlabeled antigens
compete for binding sites on the antibody during the
incubation phase. The unbound antigens are removed by
adsorption to dextran-coated charcoal. Only free antigen is
removed because the dextran coats the charcoal and blocks
the larger pores to prevent Ab-Ag adsorption. After
centrifugation the bound complexes are left in the
supernatant which is decanted into vials containing
scintillation cocktail for counting in a beta counter. The
method is inexpensive, but tends to be less sensitive than
other methods.
B) Double antibody RIA: This technique relies on the
precipitation of bound complexes with a second antibody
that is specific to the IgG of the species in which the first
antibody was made. In general, the first step is the
incubation of first antibody with labeled (usually an 125Ilabeled tracer) and unlabeled (sample) antigen. Tracer may
be added simultaneously with unlabeled antigen or after the
antibody and unknown antigen have incubated for a period
of time. Delayed tracer addition can increase assay
sensitivity. Normal serum from the same species as the
primary antibody, but not containing antigen-specific
antibodies, must be included to facilitate the formation of AbAg complexes. The Ab-Ag complexes are precipitated after
incubation with the second antibody and centrifugation.
Polyethylene glycol (PEG) can be added with the second
antibody to improve precipitation efficiency because PEG
decreases Ab-Ag complex solubility. After centrifugation, the
supernatant is discarded and radioactivity in the pellet is
counted in a gamma counter. The double antibody technique
has good sensitivity, but requires an additional incubation
period that can prolong assay time. It can be used for both
steroid and protein assays.
C) Solid-phase RIA: The hormone-specific antibody (first
antibody) is attached to the solid phase (i.e., adhered to the
wall of the plastic assay tube). Labeled ( 125I-labeled tracer)
and unlabeled (sample or standard) antigens are incubated
in the tube and unbound antigen is removed by decanting
the liquid phase. The radioactivity remaining in the tube is
counted in a gamma counter. This technique can be used for
protein and steroid hormones. The advantage to this
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Different Methods of Hormone Estimation

technique is that centrifugation is not required to separate

bound complexes from free antigen.
Principles of Enzyme immunoassay:
- EIA is also known as ELISA (Enzyme Linked ImmunoSorbent
Assay). EIAs depend on the assumption that an antigen can be
linked to an enzyme and retain both immunological and
enzymatic activity in the resultant conjugate. The soluble
antigen or antibody must also be linked to an insoluble phase in
a way in which the reactivity of the immunological component
is retained.
- Essential components of ELISA:
Solid Phase: The solid phase is the polystyrene microtiter
plate.
Antibody: An immunoglobulin produced against a specific
antigen. Polyclonal antibodies must be affinity purified for
EIA.
Coating buffer: The antibody is diluted with an alkaline
buffer, usually a carbonate/bicarbonate buffer of pH 9.6,
which causes it to passively adsorb to the well of the
microtiter plate.
Wash solution: Each incubation is terminated by a washing
step. The wash removes all unbound components from the
plate.
Enzyme conjugate (tracer): The enzyme conjugate is the
component of the assay that permits detection of antigen
concentration. For direct, single antibody EIAs, a common
enzyme conjugate is hormone conjugated to horseradish
peroxidase (HRP). For double antibody sandwich EIAs, the
enzyme conjugate complex is a biotin labeled hormone that
binds to peroxidase-labeled strepavidin.
Assay buffer: Phosphate or Tris buffers of pH 7.0 are
commonly used. Sodium azide cannot be used in buffers for
single antibody EIAs because the HRP is inhibited by azide.
Sample dilutions, standards and enzyme conjugate are made
up in assay buffer.
Standards or unlabeled antigen: The standard is usually
the same antigen that was used to make the antibody and
the same as the enzyme conjugate, or is structurally similar
so that it crossreacts with the first antibody. Standards are
used in a series of known concentrations against which
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Different Methods of Hormone Estimation

unknown concentrations of antigen in the sample can be

measured and calculated.
Substrate: The substrate reacts with the bound enzyme
conjugate and changes color. It consists of three
components: buffer, chromagen, and catalyst. The buffer has
an acidic pH and is either citric acid or phosphate citrate
buffer. The chromagen is the color changer and is usually
azino-bis-3-ethyl benzthiazoline-6-sulfonic acid (ABTS) or
tetramethylbenzadine (TMB). ABTS turns a green color and
TMB turns blue. The catalyst is what causes the reaction, via
oxidation-reduction, and is hydrogen peroxide or sodium
perborate.
Stop solution: Sulfuric acid solution that stops the
substrate reaction and allows the plate to be read at any
time. It is used primarily in the double antibody EIA. It causes
the blue substrate to turn yellow.
- Steps of ELISA:
1) Determination of antibody titer. An appropriate antibody
titer is one that results in adequate color change while
retaining good sensitivity. In general, increasing the antibody
concentration increases the enzymatic color change, but
decreases
assay
sensitivity.
Decreasing
antibody
concentration (more dilute) increases sensitivity, but the
color change is less.
2) Determination of enzyme conjugate dilution. Increased
enzyme conjugate concentration results in a stronger color
change but decreased assay sensitivity, whereas decreased
conjugate concentration increases assay sensitivity but
reduces color intensity. An appropriate combination of
antibody and enzyme conjugate results in adequate color
intensity with high assay sensitivity.
3) Development of standard curve. Incubation of a fixed
amount of enzyme conjugate and antibody in the presence
of different concentrations of standard (unlabeled antigen). A
graph is generated that depicts the relationship between the
percentage of bound enzyme conjugate (relative to the
maximum binding of the enzyme conjugate, zero well) to the
concentration/mass of the standard added. The relationship
of the percent binding and the standard mass is inversely
proportional.
4) Time and temperature of incubation. Incubation time
can be decreased with increased temperature but antibodyPage 6|8

Different Methods of Hormone Estimation

antigen binding and substrate-enzyme conjugate binding can

decrease if the temperature is too high.
- Types of ELISA:
1) Single antibody ELISA (e.g., cortisol). A hormonespecific antibody (or first antibody) is passively adsorbed
(i.e., coated) to a polystyrene microtiter plate. Unabsorbed
antibody is washed away. Known (standards) and unknown
(samples) concentrations of hormone (unlabeled antigen)
and the hormone-specific enzyme conjugate (HRP) (the
labeled antigen) are added to the well. The labeled and
unlabeled antigens compete for binding sites on the antibody
during the incubation phase. The unbound components are
washed away. The substrate is added and reacts with the
bound enzyme conjugate and changes color. The more color
change in the well, the more enzyme conjugate is bound,
meaning less hormone. The relationship of color to hormone
concentration is inversely proportional. The zero wells
contain only enzyme conjugate so there is no competition for
antibody binding. The zero wells represent the maximum
binding of the labeled antigen and, hence, have the most
color change.
2) Second antibody ELISA (e.g., luteinizing hormone). In
this antibody system a second antibody that recognizes the
first antibody is used to coat the microtiter plate. Following
incubation, unbound second antibody is decanted and a
blocking buffer, usually containing a protein such as BSA is
added to reduce non-specific binding. After incubation, plates
are washed and any unbound components removed. The first
antibody and unlabeled antigens (sample or standards) are
added to the wells. The first antibody binds to the second
antibody and the free hormone then binds to the first
antibody. Reagents are allowed to incubate followed by
incubation with the biotin-labeled antigen. Plates are
washed, removing any unbound biotin-labeled antigen, and
streptavidin-peroxidase is added. The distinguishing feature
of the avidin-biotin system is the extremely high affinity of
the avidin (from egg white or a bacteria -Streptomyces
avidinii) for biotin (a water-soluble B vitamin). The speed and
the strength of binding between these two molecules is used
to provide an amplification of the enzyme signal. This
binding also is not influenced as much by extreme
temperatures or pH levels. The peroxidase enzyme is
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Different Methods of Hormone Estimation

incorporated into the streptavidin and after binding to the

biotin, forms the enzyme conjugate complex. Following plate
washing substrate (TMB) is added. The chromagen within the
substrate reacts with the bound enzyme conjugate and
changes color. Similar to the direct single antibody
competitive EIA, the more color change in the well the more
enzyme conjugate bound, meaning less hormone. The
relationship of color to hormone concentration is inversely
proportional.

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