ENDOCRINE MANUAL FOR REPRODUCTIVE ASSESSMENT OF DOMESTIC ...

ENDOCRINE MANUAL FOR REPRODUCTIVE
ASSESSMENT OF DOMESTIC AND
NON-DOMESTIC SPECIES

Janine Brown, Ph.D.
Sue Walker, M.S.

Karen Steinman, B.S.

Conservation & Research Center
Smithsonian's National Zoological Park

1500 Remount Road
Front Royal, Virginia, 22630

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TABLE OF CONTENTS

Page

I. Introduction 5

II. Principles of Immunoassays 5

III. Principals of Radioimmunoassays 6
Essential components of an RIA 6
Steps to development of an RIA 6

IV. Types of Radioimmunoassays 8
Single antibody RIA 8
Double antibody RIA 8
Solid phase RIA 9

V. Principals of Enzymeimmunoassays 10
Essential components of an EIA 10
Steps to development of an EIA 10

VI. Types of Enzymeimmunoassays 12
Single antibody EIA 12
Double antibody (sandwich) EIA 13
Determining appropriate antibody dilution 15
Effect of antibody dilution on standard curve 15

VII. General Assay Terminology 16
Antibody characteristics 16
Assay characteristics 17
Types of assay variation 17

VIII. Monitoring Quality Control 17
Why is quality control necessary? 17
Proper use of the standard curve 17
Monitoring standard curve parameters 18
Monitoring assay quality 18
Example – LH EIA control monitor 20

IX. Laboratory Immunoassay Validation 21
Parallelism 21
Recovery/Accuracy check 22
Extraction efficiency 23

X. Biological Validation of Methods 25

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XII. Immunoassay Troubleshooting Endocrine Workbook
Color and curve problems
Loss of binding 26
Shifted standard curve
High non-specific binding 27
Poor precision and high CVs 27
27
XIII. Sample Collection/Storage Methods 28
Blood 28
Urine 28
Feces
Fecal dilutions 30
30
XIV. Hormone Extraction Methods 31
Blood 31
Urine 31
Feces
Conjugated steroid analysis 32
Fecal extraction sheet 32
32
XV. Assay Protocols 32
Creatinine assay protocol 33
LH EIA protocol 34
LH EIA reagent preparation
Cortisol EIA protocol 35
Cortisol EIA stock preparation 35
Sample sheet 37
38
XVI. Calculating EIA Results 40
Reading the plate 41
Calculating results from OD 42
Average blank value
Data matrix/table OD 43
Converting data from feces 43
Converting data from urine 43
Tips for reading EIA plates 43
44
XVII. Assay Reagents and Supplies 47
Steroid EIA recipes 48
LH EIA recipes 48
Creatinine assay recipes
Assay reagents for steroid and LH EIA 49
General supplies 49
General equipment 50
51
52
53
54

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XVIII. Review of Steroid Metabolism Conservation & Research Center
XIX. Review of Reproductive Physiology Endocrine Workbook
XX. Review of Adrenal Metabolism
55

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65

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I. INTRODUCTION

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 it’s universal adaptability and
potential for development as field tests.

II. PRINCIPLES OF IMMUNOASSAYS

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.

Antigen-antibody binding in RIA and EIA follows the Law of Mass Action. The distribution
between the bound and unbound phases is directly related to the total amount of antigen (Ag) in
the presence of a fixed amount of antibody (Ab), where k1 and k2 denote the association (forwards
reaction) and dissociation (reverse reaction) constants, respectively.

k2
Ag + Ab AgAb

k1
In the beginning, the rate of the reaction is greater in the forward direction (k1) until equilibrium is
achieved; at equilibrium there will be no further net change in the concentrations on either side of
the equation.

The affinity constant: K = k1/k2

High K implies that the reaction is favored in the forward direction; low K implies the reaction is
favored in the opposite direction. Given a constant amount of antibody of fixed K, the ratio of
bound to free antigen will be related to the total amount of antigen present.

Antibodies are produced against a specific antigen by immunizing an animal and collecting
immune serum.

• Primary antibody (or 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

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injection of antigen solubilized in a carrier that stimulates the immune response (i.e.,
Freund’s 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.
• Second antibodies, also called “non-specific antibodies” are those produced by
immunizing a different species like a goat or sheep against non-specific IgGs of the species
that produced the primary antisera (e.g., goat anti-rabbit antisera).
• Polyclonal antibodies 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 re-
immunizations.
• Monoclonal antibodies 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).

III. 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 AN RIA:

• Antibody: An immunoglobulin produced against a specific antigen(s).
• 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 TO DEVELOPMENT OF AN 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

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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.
1. 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 37˚C depending on assay characteristics.
5. Definitions:
• Total Counts (TC): Total amount of radioactivity added to each tube. Usually

~30,000 cpm.
• Non-specific Binding (NSB): Amount of radioactivity that remains after the separation

method has been employed in a tube containing all assay components except first
antibody. The amount of radioactivity remaining reflects the efficiency of the separation
method and defines the 'background' counts that will be present in every assay tube
independent of 'specific' binding of antigen to antibody.
• Total Binding (TB): Total amount of binding that occurs between labeled Ag and Ab in
the absence of competition from standard or unknown hormone. Also called “zeros”.
• Unknowns: Hormone concentrations in unknown samples determined by comparing
the specific binding (relative to the zero tubes) of the unknown tubes to the binding
obtained using standard hormone preparations of known mass (i.e., the standard curve).
• Buffers: Include phosphate, Tris or borate with pH ranges of 5.0 – 9.0 (generally pH
7.0 – 7.6) and molarities of 0.05 - 0.1 M. Phosphate buffers are most commonly used
and have the advantage of the pH not being affected by temperature (unlike Tris
buffers). Buffers generally are preserved by addition of sodium azide (0.1%) or
thimerosal/merthiolate (0.1%). Proteins like bovine serum albumin (BSA) often are
added to stabilize antisera and reduce non-specific binding. BSA can vary from batch
to batch and has a limited shelf-life (~1 year refrigerated).

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IV. TYPES OF RADIOIMMUNOASSAYS

SINGLE ANTIBODY (CHARCOAL-DEXTRAN SEPARATION) RIA

This is a common assay system used for measurement of steroid hormones. A hormone-specific
antibody (or first 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.

Example: Y First antibody O Unknown (sample steroid)
7 Charcoal-dextran
G 3H-labeled steroid

1. Incubate 2. Binding to antibody 3. Adsorption of free steroid

OOO+GGGG OOOOGGGGG O7G O7O O7G
OOO+GGGG YO YO GY YG GY YO YO YG YG YG

YYYYY

4. Centrifuge and decant supernatant containing Ab-Ag complexes into vial with scintillation
cocktail to enhance 3H signal.

5. Count radioactivity of liquid phase in a beta scintillation counter. The higher the radioactivity,
the lower the sample hormone concentration.

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 125I-labeled 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 Ab-Ag 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.

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Example: Y First antibody O Unknown (sample) G Labeled hormone Second antibody

(e.g., rabbit IgG) (e.g., goat anti-rabbit IgG)

1. Incubate 2. Binding to first Ab 3. Incubate with second Ab
OOGG
O O OG G G G OOOOGGGGG
O O OG G G G YO YO YG GY YG
OY YO GY GY GY
YYYYY

4. Centrifuge to separate free hormone from bound complexes.

5. Decant the liquid containing free hormone (supernatant) and count the pellet containing Ag-

Ab1Ab2 complexes in a gamma scintillation counter. The higher the radioactivity in the pellet,
the lower the sample hormone concentration.

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 technique is that centrifugation is
not required to separate bound complexes from free antigen.

Example: Y First antibody O Unknown (sample hormone) G 125I-labeled hormone (tracer)

1. Incubate with sample and tracer 2. Decant supernatant
containing free hormone

UU

3. Count tubes in a gamma scintillation counter. The higher the radioactivity, the lower the
sample hormone concentration.

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V. PRINCIPLES OF ENZYMEIMMUNOASSAY

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 THE EIA:

• 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 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 TO DEVELOPMENT OF AN EIA

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.

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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 antibody-antigen binding and substrate-enzyme conjugate binding can
decrease if the temperature is too high.
5. Definitions:
• Total Binding (TB): Maximum binding of enzyme conjugate (labeled antigen) to the

antibody in the absence of competition from unlabeled antigen (standard or unknown
sample). Also called “zeroes” and should have the highest optical densities. It is
assumed to be 100% binding of the enzyme conjugate. Most assay systems attempt to
reach optical densities of 0.8-1.0 in the zero wells.
• Unknowns: Unknown hormone concentrations in samples are determined by
comparing the specific binding of the samples to the binding obtained from standard
hormone concentrations of known mass.
• Non-specific binding (NSB): Amount of binding that occurs in the plate that is NOT
due to the antibody, but to other components of the assay. It also accounts for the
amount of interference the plastic bottom of the plate produces when the light from the
plate reader is refracted. The NSB wells should be less than 10% of the maximum
binding wells (zeroes). The NSB results are subtracted from the sample/standard
results (this is done by the microplate reader). NSB can be reduced by adding a protein
blocking buffer after antibody coating, adding detergent to the assay buffer or
increasing the number of washes.

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VI. TYPES OF ENZYMEIMMUNOASSAYS

SINGLE ANTIBODY EIA (e.g., cortisol)

A hormone-specific 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.

Example: HRP-labeled hormone
Substrate
Y First antibody

Free hormone

1) Antibody binding to solid phase (known as coating)

YYYYYYYY

2) Competition for antibody binding sites by labeled and sample hormone

YY Y Y YY YY

3) Wash away excess unbound hormone

YYYYY YYY

4) Substrate binding to HRP-labeled hormone

YYYYY YYY

YYYYYY YY YYY YYYYY

Zero standard High standard
Maximum color Minimum color

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SECOND ANTIBODY (SANDWICH) EIA (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 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.

Example:

Second antibody BSA

Y First antibody Biotin-labeled hormone Substrate

Free hormone Streptavidin-peroxidase (enzyme)

1) Second antibody binding to solid phase (coating)

2) BSA is added to block non-specific binding sites

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3) Addition of hormone specific (first) antibody, biotin-labeled hormone, and free hormone

YYY Y

4) Wash away unbound components

YYYY

5) Addition of streptavidin-peroxidase (enzyme)

YY YY

6) Addition of substrate

YYYY

YYYY YYYY

Zero High standard
Maximum color Minimum color

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HOW TO DETERMINE APPROPRIATE ANTIBODY DILUTION FOR RIA AND EIA

100

80

% Binding 60
(TB/TC) 40

Want Ab to bind ~30%
of labeled hormone

20

Use Ab at 1:120,000 dilution 1000
0

1 10 100

Ab Dilution (x 1000)

Appropriate first antibody concentration is determined by running a titration curve which involves
incubating serial dilutions of first antibody with a constant amount of tracer. Calculate the %
binding of the antibody to tracer and plot as a linear-log curve. The best antibody dilution (one
that provides good sensitivity with adequate detectibility) is ~30% (range 20-50%).

EFFECT OF ANTIBODY DILUTION ON STANDARD CURVE CHARACTERISTICS

120 Correct
100 too much

not enough

80

% B/TB 60

40

20

0
1 10 100 1000
Standard Concentration

Inappropriate first antibody concentration will result in a poor standard curve that is too ‘flat’ on
the top or bottom portion. Too much antibody also will reduce assay sensitivity.

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VII. GENERAL ASSAY TERMINOLOGY

ANTIBODY CHARACTERISTICS

Sensitivity: The ability to detect small quantities of antigen. It is the lowest concentration
of antigen that can be statistically distinguished from a sample with no antigen.

- objective determination - calculated as the value 2 SD from the mean
response of the blank or zero (Bo) tube.

- subjective determination - the value at 90% or 95% of maximum binding.

Specificity: The ability of the antibody to discriminate between antigens.

Crossreactivity: The ability of an antibody to have immunoreactivity with a more than one
Examples: antigen. Crossreactivity of an antibody with other hormones is determined
using binding inhibition curves. It is expressed as the standard concentration at
50% binding divided by the concentration of the competitive antigen at 50%
binding, expressed as a percentage.

• Hormone C is detected but the antibody is not as specific for it. The crossreactivity is less
than 100%.

• The antibody for progesterone crossreacts better with hormone D than the standard. The
crossreactivity is over 100%.

• Hormones A and B only bind the antibody at high concentrations and crossreactivity
cannot be calculated.

Crossreactivity of Different Antigens on a Progesterone EIA

100 Progesterone Standard
Hormone A
Hormone B
Hormone C
Hormone D

80

60
% B/TB

40

20

0 1 10 100 1000
0.1 Standard Concentration (ng)

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ASSAY CHARACTERISTICS

Accuracy: The degree to which the measured concentration corresponds to the true concentration
of a substance. This is related in part to the specificity of the assay.

Precision: Refers to the repeatability of a measured value or the consistency of results. It is a
measure of random error defined as the variation among replicate measurements of a
defined sample. It is expressed as the Coefficient of Variation (%CV) which is the
(Standard Deviation/Mean)*100.

TYPES OF ASSAY VARIATION

Intra-assay variation: The variation within assays determined by repeated analysis of samples,
typically low, medium and high, within a single assay.

Inter-assay variation: The variation between assays determined by repeated analysis of the same
sample in several assays. These samples are called controls and should bind
at 30% and 70%.

Note: A sample can be precise, but not accurate. This occurs when the measured value deviates
from the true value as a result of using a non-specific first antibody or to systematic error, like
improperly calibrated weighting or measuring equipment, improper technique (pipetting) or
sample problems.

VIII. MONITORING QUALITY CONTROL

WHY IS QUALITY CONTROL NECESSARY?

• Proper validation and standardization of an assay is only the first step towards establishing
a reliable endocrine monitoring program. Subsequent assessment of assay quality and
consistency is absolutely necessary to assure the biological relevance of results.

• For every assay system there is an inherent level of error which must be accepted.
• A quality control program indicates when that level of error becomes unacceptable.
• Quality control samples only have value if their analysis provides a reasonable confidence

in data for the whole assay, or reflects a true error in the method.
• It is an ongoing process; the value of quality control values increases with time.

PROPER USE OF THE STANDARD CURVE

• Assay range (the 20-80% rule): A subjective method of sample exclusion which
eliminates the use of values in which the binding is less than 20% or greater than 80% of
maximum binding. All outliers are re-analyzed after appropriate modification (i.e., taking
more or less sample to the assay). This rule is based on the assumption that most standard
curves are linear between 20 and 80% binding, and therefore the dose response is linear.
The binding cut-off limits may vary, however, and should be appropriate for each assay.

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MONITORING STANDARD CURVE PARAMETERS

• Total binding (%B/Bo): Indicates the maximum binding of label in the given assay
system. Given that the assay parameters are unchanged, the maximum binding should
remain relatively constant from assay to assay. A decrease in binding suggests one or
more assay factors are not optimized (see Troubleshooting).

• Non-specific binding (%NB/Bo): The amount of binding due to factors other than
specific antibody binding. It should remain constant from assay to assay. It also should be
kept to a minimum, generally less than 5% of the total counts added. An increase in NSB
again suggests one or more assay factors are not optimized (see Troubleshooting).

• Effective dose values (ED20, ED50, ED80): Gives a good estimate of the overall ‘shape’ of
the curve. The ED50 is especially useful for indicating changes in assay sensitivity. The
ED20 and ED80 can also indicate changes in the slope of the curve.

MONITORING ASSAY QUALITY

• How is quality control monitored? Assay consistency is monitored by analyzing
‘internal control samples’ which are treated as unknowns, but are run in every assay.
- how many to run? Usually 2 - 3 controls, assayed in duplicate or triplicate. Much of
this depends upon the variability of the assay.
- what concentrations to run? Controls should provide an estimate of variability over the
working range of the standard curve. Recommend controls be run at ~30% (high
concentration), 50% (medium) and 70% (low) of maximum binding.
- what samples can be used as controls? Almost any biological material containing the
relevant antigen, including serum pools, fecal extracts, purified steroids diluted in
buffer or other fluid (provided it does not interfere with the assay), pituitary extracts,
urine pools, purchased standards, etc.
i. prepare a large pool projected to last for several years.
ii. divide the material into small aliquots to avoid repeated freeze-thawing.
iii. keep the aliquots in a safe freezer (preferably one with a temperature alarm).
iv. store all materials, including samples, in a no frost-free freezer.
v. do not allow controls to run out before making up new controls.

• Assay coefficients of variation: (%CV = standard deviation/mean times 100)
- intra-assay CV - determines the within assay error (the error associated with running the
same sample in one assay). Most accurately determined by calculating the variation in
assaying multiple replicates of one sample throughout the assay (e.g., n = 10 replicates).
More typically, the average intra-assay CV is calculated from the internal controls
assayed at the beginning of the assay. A third method is to calculate the average CV of
all unknowns run in an assay. If more than one assay has been run for a particular study,
then the mean intra-assay CVs for those assays should be averaged.
- inter-assay CV - determines the between assay error (the error observed when the same
sample is run in different assays). Determined by calculating the variation in values for
samples run in every assay. Within a study can calculate the individual inter-assay CVs
for each internal control and then average those numbers.

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• Causes of variation:
- intra-assay CV - generally is the result of the presence of unequal amounts of sample,
tracer or antibody (i.e., poor pipetting, or incomplete mixing of sample or reagents), but
also can be due to inconsistencies in counting or decanting.
- inter-assay CV - caused by system-related problems like reagent instability, procedural
variation or changes in standards (do not allow old standards to run out before making up
new standards!).

• What is an acceptable level of error?
- sample rejection - a level of error needs to be established that objectively determines
when a sample needs to be re-analyzed. However, the amount of error tolerated is
subjective and determined, in part, by how critically the data need to be interpreted. For
example, the error rate would be lower in a human endocrinology laboratory where
health status and potential treatments hinge on accurate measurements. Conversely,
more error might be tolerated in assays conducted to define hormonal trends (i.e., the
absolute values are not as important as the profile).
- rules of thumb for defining acceptable levels of error
i. fixed percentage of the CV - often designated as 10% such that all samples with a
CV >10% are re-analyzed.
ii. assay rejection criteria - often subjective, but can involve exclusions based on
control values falling within 2 SD of the mean of previous values, or a set
proportion of sample CVs being below 10%.

120 The same 5% error in binding
100 results in a greater %CV at each

80 end of the curve

% B/TB 60 10 100 1000 10000

40 Standard Concentration
20

0
1

• Proportional error: The CVs are higher and dose calculations are not proportional at the
extreme ends of the standard curve, due to the non-linearity of the curve (see above figure).

• External quality control procedures: Commonly used in clinical laboratories to ensure
that the quality of data are independent of the laboratory which generated it. Involves

analysis of samples provided by external sources.

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LH EIA Control Monitor

Controls Percent Binding For Standards

QCH QCH QCL QCL 2.0
3.9
7.8
15.6
31.3
62.5
125.0
250.0
500.0

Date Tech %B Dose %B Dose

4/22/02 sw 74.8 64.4 53.7 114.9 100.8 100.8 100.0 101.2 89.6 74.7 51.9 23.6 9.2

6/27/02 sw 69.8 57.4 48.0 106.6 93.7 94.6 92.6 88.7 82.2 67.5 42.2 16.9 6.4

6/28/02 sw 66.4 58.6 46.6 104.0 97.4 98.6 98.5 90.4 80.6 65.1 42.0 14.8 5.9

6/29/02 sw 70.5 57.1 47.8 106.6 97.2 100.5 99.0 92.2 83.9 67.1 42.7 16.1 6.2

6/30/02 sw 68.3 58.6 44.8 114.0 97.1 91.6 81.0 67.7 42.5 15.7 5.7

7/1/02 sw 63.0 68.5 44.5 114.1 95.1 98.0 99.0 90.9 80.7 66.0 42.2 15.7 5.2

7/1/02 sw 68.4 59.8 49.1 102.5 98.5 100.2 101.0 94.0 83.0 67.2 43.0 17.0 6.7

7/1/02 sw 73.4 44.8 50.7 94.3 93.6 96.6 91.5 88.7 79.8 64.7 42.0 16.3 6.0

7/1/02 sw 67.3 61.3 48.2 104.0 96.1 97.8 99.2 91.1 82.0 66.4 42.4 15.8 6.0

7/3/01 sw 71.6 50.0 47.0 102.8 98.8 100.3 96.7 92.6 83.4 63.4 41.6 16.7 6.3

7/3/01 sw 69.7 52.1 45.7 102.3 98.1 98.4 98.0 91.0 81.8 64.6 38.7 15.2 5.1

7/3/01 sw 71.9 54.4 46.3 108.7 97.1 95.2 93.9 94.7 83.1 66.8 41.4 16.4 6.1

7/3/01 sw 68.8 62.7 48.7 107.7 104.0 103.7 104.1 94.2 87.1 69.9 43.9 16.7 4.8

7/19/02 sw 64.8 53.4 42.0 104.6 95.9 96.6 96.5 87.4 77.7 60.8 36.3 13.3 3.1

7/19/02 sw 68.1 53.3 46.3 106.5 101.3 98.8 96.6 88.8 79.7 64.0 44.9 12.7 3.8

7/19/02 sw 69.6 47.4 43.0 102.3 103.1 102.4 101.1 91.5 78.6 64.0 35.4 13.1 4.0

7/19/02 sw 66.7 50.7 40.3 112.0 96.7 98.6 97.2 85.3 78.2 62.8 36.8 13.1 4.3

7/31/02 sw 68.6 59.6 49.8 105.8 94.6 94.2 96.3 88.1 81.0 67.9 44.8 20.6 12.8

7/31/02 sw 72.3 50.1 48.4 104.4 97.4 97.8 97.7 88.2 81.7 68.1 42.6 18.8 11.3

10/23/02 sw 73.4 54.0 51.4 103.2 100.0 100.0 102.0 91.7 85.2 70.4 45.3 17.7 5.6

10/23/02 sw 75.7 50.8 55.1 96.9 100.0 100.0 103.0 91.4 86.2 69.8 48.0 17.4 4.0

Mean 69.7 55.7 47.5 105.6 97.9 98.6 98.2 91.1 82.2 66.6 42.4 16.4 6.1
Count 21.0 21.0 21.0 21.0 21.0 20.0 20.0 21.0 21.0 21.0 21.0 21.0 21.0
Std Dev 3.2 5.9 3.6 5.2 2.8 2.5 3.2 3.3 3.0 3.1 3.7 2.5 2.4

CV 4.6 10.6 7.6 4.9 2.9 2.5 3.3 3.6 3.6 4.6 8.8 15.5 38.7
Mn
+1SD 72.9 61.6 51.1 110.8 100.8 101.1 101.4 94.4 85.2 69.7 46.1 18.9 8.5
Mn-1SD 66.4 49.7 43.9 100.4 95.1 96.2 94.9 87.8 79.2 63.5 38.7 13.8 3.7
Min 63.0 44.8 40.3 94.3 93.6 94.2 91.5 85.3 77.7 60.8 35.4 12.7 3.1
Max 75.7 68.5 55.1 114.9 104.0 103.7 104.1 101.2 89.6 74.7 51.9 23.6 12.8

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IX. LABORATORY IMMUNOASSAY VALIDATION

PARALLELISM

Definition: Parallelism is a way of determining if the assay is actually measuring what it should
be measuring. It can also tell what dilution of sample to use for the assay.

• Samples to test should reflect the range of normal concentrations expected (i.e., estrous
cycle and pregnancy, seasonal samples).

• Pool an equal amount of urine or fecal extract from each sample and dilute serially two-
fold in assay buffer (neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, etc.). For example add 200 ul of
buffer into tubes labeled from 1:2 to 1:8192 - then take 200 ul of the neat sample and mix
with the 200 ul in the “1:2” tube, vortex the “1:2” - then take 200 ul out of the 1:2 and
place in the “1:4” and so on. This will result in 200 ul in each tube except the last dilution
which will have 400 ul.

• Plot the % binding of samples by choosing an arbitrary concentration for the neat sample
and halving the concentration for each dilution.

• If the sample curve parallels the standard curve the sample hormone is immunologically
similar to the standard and can be measured proportionately.

100 Parallel
80 displacement
60
1:64
% B/TB 1:32

40 Use 1:16

1:8

20 1:4 Standard curve

0 1:2
0.1 neat

1 10 100 1000

Standard Concentration or Dilution

The best (most accurate) dilution to run samples is at ~50% binding (see above figure). The pool
shows parallelism with the standard curve and is therefore valid for use in the assay. Samples
should be run at a 1:16 dilution based on the binding inhibition observed at ~50%.

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120
100 No displacement

80 Non-parallel Low concentration
displacement Standard curve
% B/TB
1 10 100 1000
60
Standard Concentration or Dilution
40

20

0
0.1

In the graph above, samples that produced non-parallel or no displacement cannot be run in this
assay system because they do not demonstrate immunoactivity of endogenous antigen similar to
the assay standards. The sample with a low concentration of immunoactive antigen demonstrated
limited parallelism, but this type of sample could only be run ‘neat’ and there likely would be
problems with limited detection within the working range of the assay (i.e., <80% binding).

RECOVERY/ACCURACY CHECK

The degree to which the measured concentration corresponds to the true concentration of a
substance. Tests for potential interference caused by substances contained within the biological
sample that are independent of specific antigen-antibody binding.

• Make a sample pool. It should have a low concentration of hormone (e.g., for
progestagens use samples from the follicular phase).

• Spike aliquots of pooled sample (i.e., 100 ul) with an equal amount (i.e., 100 ul) from each
standard.

• Analyze the spiked samples as unknowns in the assay.

• The sample pool also needs to be analyzed without added standard to determine the
amount of endogenous (or background) hormone present. That will have to be subtracted
out from each spiked sample.

Results:

Amount Expected = (Concentration of standard spiked with / 2)
Amount Observed = (Concentration observed from assay results – minus background)
% Recovery = (Amount Observed/Amount Expected)*100

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Example:

Standard Amount added Amount Minus % Recovery
concentration - Amount observed background 87.46
expected (pg/well) 81.78
(pg/well) (pg/well) (pg/well) 78.42
500.00 250.00 235.48 218.64 82.18
250.00 125.00 119.07 102.23 84.86
125.00 62.50 65.85 49.01 84.64
62.50 31.25 42.52 25.68 80.51
31.25 15.63 30.10 13.26 87.18
15.62 7.81 23.45 6.61
7.80 3.90 19.98 3.14
3.90 1.95 18.54 1.70
Endogenous/
background 16.84
0.00

Plot Amount Expected vs Amount Observed - conduct a linear regression (y=mx+b) analysis.
Slopes > or < 1 suggests an over or under estimation of hormone mass, respectively.

250 Elephant Urine PdG Recovery

200 y = 0.8679x + 15.343
R2 = 0.9986
Amount 150
Observed 100

50

0
0 100 200 300

Amount Expected

EXTRACTION EFFICIENCY

Definition: A way of defining how efficient or effective the extraction method is at ‘extracting’ or
pulling out metabolites of interest (steroid or protein hormones) from a specific matrix (urine,
fecal, blood or saliva). Monitoring extraction efficiency also permits monitoring how consistent
the extraction process is from sample to sample.

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• Similar to recovery but samples are spiked before extraction.
• Spike each sample with:

Labeled hormone: ~3000 cpm/100 ul of 3H or 14C radiolabeled steroid
or

Unlabeled (cold) hormone: (mass is dependant upon sensitivity of assay system) – add
enough mass to read at about 50% binding
• Extract sample as per protocol.
After extraction:
• Determine “Amount Observed”:
Labeled hormone: aliquot 50 ul from the final 1.0 ml extract to scintillation vial and
count radioactivity.
or
Unlabeled hormone: analyze by RIA or EIA to determine concentration.
• Determine “Amount Expected”:
Labeled hormone: aliquot 100 ul of original stock of radiolabeled steroid to scintillation
vial and count radioactivity.
or
Unlabeled hormone: dilute standard stock the sample the sample was spiked with to
determine concentration.
Results:
Amount Expected = (expected minus background)
Amount Observed = (observed minus background)
% Extraction efficiency = (Amount Observed/Amount Expected)*100

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X. BIOLOGICAL VALIDATION OF METHODS

It is crucial to demonstrate that hormonal measures accurately reflect the physiological events of
interest. From a practical standpoint, it is unnecessary to determine the specific molecular
structure of the hormones being monitored in each species. However, it is critical to prove that
fluctuations in the hormonal metabolite being measured provide physiologically relevant
information. For example, an ovarian cycle might be validated by comparing two independent
measures of the same hormone in matched samples (i.e., fecal vs. urinary estrogen), or by
comparing temporal hormonal excretion patterns with external signs of reproductive status (e.g.,
sex skin swelling, copulatory behavior). Similarly, showing a predicted rise and fall in a
metabolite coincident with pregnancy onset and parturition, respectively, is solid evidence of a
hormone’s utility for assessing pregnancy status. Administering a drug known to stimulate
hormonal production is also useful for demonstrating a cause-and-effect relationship between its
exogenous administration and the subsequent excretion of the target hormone. Typically,
hormone challenges include gonadotropin releasing hormone (GnRH) to study pituitary hormones
(LH, FSH) and subsequent androgen production (in males), or adrenocorticotropic hormone
(ACTH) and cortisol secretion. This approach also clarifies the excretory lag-time between
stimulation of an endocrine gland and the appearance of its hormonal metabolites in excreta.

General protocol for ACTH or GnRH challenges:

• GnRH is typically administered at a dose of 1 ug/kg body weight; ACTH (gel form
preferred) is given at doses of 2-5 IU/kg. Drugs can be administered i.v. (preferable), i.m.
or s.c.

• If blood collection is possible, samples should be collected as follows: -20 min, -10 min
and 0 min, with 0 = time of hormone injection; 15 min, 30 min, 45 min, 60 min, 90 min, 2
hr, 3 hr, 4 hr, 6 hr and 8 hr post-injection. It is important that at least one pre-injection
sample be collected, and that several are collected for at least 6 hr after injection.

• For urinary hormone validation, several pre-injection samples should be collected (can
start collecting the week before the test) and every urine sample for at least two days post-
injection.

• For fecal hormone validation, several pre-injection samples should be collected and every
fecal sample for at least five days post-injection.

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XI. HPLC FOR ANALYSIS OF METABOLITES

High performance (or pressure) liquid chromatography is a separation technique used to identify
specific metabolites or components within a given sample. HPLC separates a sample into
fractions, each of which is analyzed for crossreactivity in a given assay. The separation is
performed when two phases are brought in contact, one phase being stationary (the column) and
the other being mobile (a liquid). The sample mixture is forced through the column under
pressure and undergoes a series of interactions between the stationary and mobile phases.
Interactions exploit differences in the physical or chemical properties of the components within
the sample. These differences govern the rate of migration of the individual components under the
influence the mobile phase moving through a column. Separated components emerge in the order
of increasing interaction with the stationary phase. The least retarded component elutes first, the
most strongly retained material elutes last. Selection of the appropriate HPLC method is governed
by sample properties such as molecular weight, solubility and polarity.

The most common method for the separation of steroids is reverse phase chromatography and is
based on polarity [a non-polar stationary phase (a column packed with hydrocarbons) and a polar
mobile phase (eg., methanol, water, acetonitrile and ethanol)]. The sample components are
attracted to the surface of the adsorbent (the solid phase) with differing strength. Non-polar
samples are attracted to the stationary phase and are retained, the more polar compounds elute
first. As the gradient of the mobile phase is adjusted over time components within the sample are
separated. As the polarity of the mobile phase increases the non-polar samples begin to elude
from the column.

HPLC of Fecal Estrone Metabolites

7000 9000

6000 Estrone 8000
5000 (Fraction 63)
Estrone-3- 7000
4000 Sulfate 6000
Radioactivity (Fraction 16) 5000 Immuno-
(cpm/fraction) 3 0 0 0
activity
2000 4000 (pg/fraction)
1000 3000
2000

1000

00

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76

HPLC fraction Known Antigens
Immunoreactivity on EIA
Unknown metabolite

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XII. IMMUNOASSAY TROUBLE SHOOTING

Some of the problems listed below are for EIA only. Those relevant to both EIA and RIA are
designated by an asterisk “*”.

COLOR AND CURVE PROBLEMS

Color develops too quickly (or covers entire plate)
• Conjugate is too concentrated – use higher dilution
• Curve will be flat on top
• This could also be the effect of too much first antibody
•

Color develops too slowly (or not at all)
• Conjugate is too weak – retitrate to lower dilution (add more)
• Curve will be flat on bottom
• This could also be the effect of too little first antibody
• Also, hydrogen peroxide could be bad (re-make fresh), diluted wrong or not added.
• Also see Loss of Binding Section
•

Patchy color development
• See Poor Precision and High CV’s section

LOSS OF BINDING

Decomposition of the antibody*
• Some antibodies are more stable than others. Best to store antibody frozen at a set dilution
in a no frost-free freezer, or preferably an ultra-low freezer (especially for long-term
storage of stocks). Store antibody upright (to decrease surface area exposed) in o-ring
vials to ensure a tight seal.
• Antibody solutions should be prepared carefully (e.g., do not expose to excessive heat or
shake vigorously).
• Most problems with loss of binding are NOT due to the antibody unless it is because not
enough antibody was added.

Poor label (or conjugate) characteristics*
• This is the most common factor causing low binding.
• Making fresh label will often correct low binding and improve color development.
• Care must be taken with these reagents; they are the signal suppliers of the assay.
i. Always store label at recommended temperatures.
ii. Never store working label dilution for long periods of time.
iii. It should always be made up just before use.
iv. Never leave label on the bench at RT for excessive amounts of time.

Ineffective separation
• Wash plates the recommended number of times and maintain consistency across the plate.
Improper washing will lead to high CV’s and loss of binding.

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Reduced quality of assay reagents*
• It is important that buffers are made properly and often. Check buffer pH. Be sure that
proteins like BSA are stored properly and not too old.

Calculation error*
• Take care that all reagent calculations are correct. This is an easy place to make a simple
mistake! Keep records of all calculations for each assay.

SHIFTED STANDARD CURVE

Parallel shift*
• Shift to the left - too much standard
• Shift to the right – too little standard
Causes:
- Inaccurate weighing of standards – check balance calibration
- Inaccurate diluting of standards – check pipette calibration
- Contamination of standard stock - make new standard stock
- Changes in standard stock – due to evaporation, adsorption to storage vessel, etc.

Non-parallel shift*
• Contaminated buffers – contamination with cross-reacting analytes or other compounds
that interfere with binding.

HIGH NON-SPECIFIC BINDING*

Decomposition of label*
• Non-specific binding often increases as labels age or become damaged

Adherence of label to well or tube*
• Can be due to label decomposition or problems with buffer proteins that no longer prevent
sticking

Incomplete removal of label*
• This can be caused by poor washing of the plate or decanting of assay tubes

Non-specific attachment of antibody
• Unsuitable blocking buffer used (needs to be re-made) or omission of blocking buffer
(forgot to add it).

POOR PRECISION AND HIGH CVs*

Human error*
• Inconsistent pipetting, washing etc. The key is to be consistent. The reproducibility of any
assay relies in part on the accuracy of the workers involved.
• Avoid bubbles in pipet tips.

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• Reading of the well occurs through a thickness of liquid, any change in volume in a well
will result in an alteration of optical density an increase in variation. Thus, it is important
to add solutions to the wells evenly and accurately to achieve the same volume in each well
and limit the effect of volume changes (EIA only).

Incomplete mixing of samples or other reagents
• All frozen samples must be thoroughly, but gently, mixed after thawing. Never shake
samples or assay reagents vigorously.

Equipment failure*
• Check that all wells are being washed evenly.
• Pipets are fundamentally important to the accuracy of the assay. They should be checked
on a regular bases for precision and accuracy of delivery volumes*.

Poor handling of the plates
• Clean the bottom of plates with a kimwipe before inserting into the reader. Film, dirt or
fingerprints on the bottom of the plate could interfere with optical density readings
resulting in poor CV’s.
• Avoid stacking plates. Keep separated.
• Always keep plates sealed with plate covers during incubation steps.
• Always use the same procedure for addition of reagents.

Using the wrong plates
• Always use the recommended plate for a particular assay.
• Never use a tissue culture grade plate for EIA, it will result in high variability.

Water*
• This can be a major problem for the daily functioning of assays within a laboratory as well
as for standardization of assays across laboratories even when identical reagents are used.
The reasons for why water affects the assay have not been extensively examined, and no
single factor has emerged as being most important.

Laboratory Glassware*
• All glass and plastic should be cleaned and rinsed well in distilled water. This avoids the
introduction of contaminants or adverse pH conditions into the assay. An enzymatic type
detergent (such as Terg-A-Zyme) should be used to wash dishes. If precision is an issue, a
simple first step in troubling shooting is to wash and replace all plastic and glassware.

Timing of steps*
• Individual steps should be timed accurately. For example for a 1 hour incubation step, no
more than 5 minutes either way should be tolerated.

Temperature*
• Temperature can have an impact on assay development. Make a note of temperature and
its variation during the year, this may explain variation in results at different times.

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XIII. SAMPLE COLLECTION/STORAGE METHODS

It is very important that all samples be properly labeled with the animal’s name or
studbook/identification number, facility, and date.

BLOOD:

• When collecting multiple samples per day, label the time. This is especially important for
hormone challenges, like ACTH and GnRH.

• After blood has been collected and centrifuged, pour serum into the labeled storage tube.
Leave room in the tube for expansion of sample during freezing (e.g., put 4 ml into a 5-ml
vial). Place samples upright into storage box and freeze at -20°C. Store samples in the
box from left to right and front to back (see box below). Place the first sample in space #1
and repeat for each new sample added. This way, when processing samples, they are
already organized and in order by date and time.

• Remember to keep all samples frozen until analysis.
• Label the outside of the storage box with the name of the animal, species (if necessary), the

location of the animal, the matrix (urine, serum, fecal extract), and the dates of the first and
last sample in the box. For example: Shanthi, Asian Elephant, National Zoo, urine, 1 Oct
00 – 20 Nov 00.

Sample storage box:

91 95 100

81 85 90

71 75 80

61 65 70

51 55 60

41 45 50

31 35 40

21 25 30

11 15 20

1 2 3 4 5 6 7 8 9 10

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URINE:

• Label collection tube with animal’s name, date, and time collected. If necessary, record if
the sample may have been contaminated with feces, water, etc. Whenever possible,
centrifuge sample and decant supernatant to remove debris.

• Pour urine sample into labeled tube and place in storage box. Leave room in the tube for
expansion of sample during freezing (e.g., put 4 ml into a 5-ml vial). Store sample in
freezer at -20°C. Store samples as described previously.

• If storing samples at RT or in the refrigerator (for up to a month) instead of freezing, add
10% ethanol to each tube as a preservative.

• Thoroughly clean syringes or cups used to collect urine samples to prevent cross-
contamination.

FECES:

• Place feces in collection bag or tube labeled with animal’s name, date, and time (if
relevant) and store at -20°C.

• For fecal extracts, label storage tube with animal’s name, date, time, and the extraction
medium. For example: Shanthi, 1 Oct 00, 0800h, neat extract MeOH.

• If making dilutions from the extract and the dilutions will be stored as well, label the
storage tube with the animal’s name, date, time, and the dilution. Remember, dilutions are
always made in assay buffer.

• Place fecal extract tubes in storage boxes as described previously and store at
-20°C. Fecal extracts are in alcohol so they will not freeze.

FECAL DILUTIONS

For fecal extracts and urine samples, dilutions often have to be made, especially for extracts stored
in alcohols.

• The appropriate dilution to use is determined from the parallelism curve.
• Dilutions are always made with assay buffer. Dilutions are always stated as 1 to a number.

For example: 1:100 (1 + 99) means one part sample to 100 total parts volume.

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XIV. HORMONE EXTRACTION METHODS

Definition: Extraction is a method by which components of interest in a sample are isolated,
removed or concentrated, making it free of interference from other particulates found within the
matrix (fecal, urine, blood or saliva). This generally refers to steroids.

For most assay systems, free steroids must be extracted from the sample matrix. A few systems
have been developed to analyze unextracted samples (e.g., commercial RIA kits). The extraction
method used is dependent on the properties of the component of interest. For more specific
extractions (eg., pulling in a specific group of steroids, or removing proteins or lipids from a
sample) varying solvents and/or ratios of two or more solvents can be used.

BLOOD SERUM OR PLASMA

• Ether extraction is most commonly used. Diethyl ether is used for progestagens; ethyl
ether is used for estrogens and androgens. Standard protocol involves extracting with a
10:1 volume of ether:sample by vortexing for 1 minute followed by snap freezing in liquid
nitrogen or dry ice/acetone. The ether supernatant is dried and the sample reconstituted in
assay buffer, vortexed for 1 min and stored frozen (-20˚C) until analyzed.

URINE

• Ether can be used for gonadal steroids similar to blood serum/plasma extractions.
• For corticoids, dichloromethane is very effective. For that method, urine (0.5 ml) is

combined with 1 ml dichloromethane in a 12 x 75 mm polypropylene tube, capped tightly,
shaken gently for 10 min and centrifuged (1500 x g) for 5 min. The lower
dichloromethane fraction is removed by aspiration using a Pasteur pipet and placed into a
glass 12 x 75 mm tube. Six hundred µl are then transferred to another glass 12 x 75 mm
tube using a positive displacement pipet and dried under air. Samples are reconstituted in
assay buffer, vortexed for 1 min and stored frozen (-20˚C) until analyzed.

FECES

• Boiling in ethanol is the easiest and most common extraction method.
• Weigh out feces in 16x125 mm glass tubes (~0.2 g dry, 0.5 g wet). Record exact weight of

each sample. Add 5.0 ml of 90% ETOH (4.5 ml ETOH, 0.5 ml distilled water) and vortex
briefly. If monitoring extraction efficiency add appropriate amount of tracer to each tube.
Boil tubes in a water bath (90˚C) for 20 minutes adding ETOH to keep from boiling dry.
Bring the volume of extract up to approximately pre-boil levels with ETOH and centrifuge
at 1500 rpm for 20 minutes. Pour off extracts into a second set of labeled 16x125 mm
tubes. Add 90% ETOH to the remaining fecal pellets and vortex for 30 seconds.
Centrifuge at 1500 rpm for 15 minutes. Combine the first and second extracts. Dry down
extract and reconstitute in 1 ml of MeOH, vortex briefly. If possible, sonicate for 15
minutes. Store the MeOH extract at –20˚C and/or make appropriate dilution in buffer
(usually 1:10 in 12x75 mm tubes) for frozen storage.

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• Feces can be dried using a lyophilizer, savant rotary evaporator or conventional oven
(~50˚C for several hours or until dry; i.e., weight no longer changes). Solar ovens are not
recommended due to potential steroid alteration by UV light. Drying times will vary for
each technique and even among individual samples.

CONJUGATED STEROID ANALYSIS
• Steroid conjugates (sulfates or glucuronides) can be assayed directly using antibodies that
crossreact with conjugates.
• Conjugated steroids can be assessed indirectly after enzyme hydrolysis (e.g., sulfatase,
glucuronidase or ‘snail juice’, Helix pomatia, which contains both glucuronidase and
sulfatase activity) and assaying the resulting free steroids. Extract urine, serum or fecal
extracts (resuspended in 0.5 ml buffer) with 10 volumes of diethyl ether to separate water-
soluble from ether-soluble forms. Hydrolyze residual aqueous samples diluted 1:1 in 0.5
ml in 0.1 M acetate buffer (pH 5.0) with 50 µl ß-glucuronidase/aryl sulfatase (20,000
Fishman U/40,000 Roy U, respectively; Boehringer Mannheim Corp., Indianapolis, IN) at
37˚C for 24 hours. Samples are then extracted with 10 volumes of diethyl ether to separate
enzyme-hydrolyzable (organic phase) from non-hydrolyzable (aqueous phase) forms.

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Fecal Extraction Sheet ID: Zoo:

Species: Dat e: Sex:

Sample DATE WEIGHT Sample DATE WEIGHT Sample DATE WEIGHT
1 50 99
2 51 34
3 52 10 0
4 53 10 1
5 54 10 2
6 55 10 3
7 56 10 4
8 57 10 5
9 58 10 6
59 10 7
10 60 10 8
11 61 10 9
12 62 11 0
13 63 11 1
14 64 11 2
15 65 11 3
16 66 11 4
17 67 11 5
18 68 11 6
19 69 11 7
20 70 11 8
21 71 11 9
22 72 12 0
23 73 12 1
24 74 12 2
25 75 12 3
26 76 12 4
27 77 12 5
28 78 12 6
29 79 12 7
30 80 12 8
31 81 12 9
32 82 13 0
33 83 13 1
34 84 13 2
35 85 13 3
36 86 13 4
37 87 13 5
38 88 13 6
39 89 13 7
40 90 13 8
41 91 13 9
42 92 14 0
43 93 14 1
44 94 14 2
45 95 14 3
46 96 14 4
47 97 14 5
48 98 14 6
49 14 7

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XV. ASSAY PROTOCOLS

CREATININE ASSAY

Definition: Creatinine is a breakdown product of muscle protein that is excreted in urine at a
constant rate. It is a way to measure how concentrated a urine sample is and whether or not a
certain sample is viable. For instance, if it contains too much water and thus mass within the
sample is too dilute to measure, it is not a viable sample.

• Thaw all samples to be analyzed.
• Creatinine can degrade so samples should be thawed once or kept cold. It is best to run

creatinine assay after the first thaw of the sample, but as long as the sample is kept cold
after thawing it can be refrozen and run at a later time.
• Creatinine is less stable than other hormones, which can usually survive repeated freezing
and thawing (although it should be avoided as much as possible).

PROTOCOL FOR CREATININE ASSAY - per plate

1. Standards
• Standard values used are 100, 50, 25, 12.5, and 6.25 ug/ml
• Dilute top standard stock (Sigma – 925-11; 10 mg/dl or 100 ug/ml, 4˚C) serially 2-fold
using 200 ul stock plus 200 ul assay buffer or tap water and mix well
• Use same buffer or tap water as the zero standard (Note: water quality is not critical)

2. Samples
• Dilute samples to appropriate amount (1:5 - 1:100 in buffer or water)

3. Plate loading
• Use Dynatech plates (Immulon 4 Flat Bottom Plates – Cat # 0110103855)
• Add 50 ul per well of standard and sample in duplicate according to plate map
• Speed of addition is unimportant as this is not a binding assay
• Start at A1 and go down each set of columns
• Pipet all solutions in this order
• Avoid splashing or touching the samples in the wells
• Add 50 ul of tap water to each well using the repeater pipet
• Add 50 ul per well 0.75 N NaOH
• Add 50 ul per well 0.4 N picric acid using the repeater pipet
• Shake plate briefly to mix (by tapping) and incubate at RT for 30 minutes

4. Plate reading
• Wipe bottom of plate to make sure it is clean
• Read at 490 nm

5. Assay dilution/value cutoffs
• Avoid running samples at <1:5 or >1:100 dilution
• Any sample with a creatinine value of <0.1 mg/ml is too dilute and should not be used

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Creatinine Assay Template

ASSAY SPECIES
DATE ANIMAL ID
DILUTION

1 2 3 4 5 6 7 8 9 10 11 12
A 0 50
B 0 50
C 6.25 100
D 6.25 100
E 12.5
F 12.5
G 25
H 25

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PROTOCOL FOR LH EIA - per plate

DAY 1 • use NUNC Maxisorb plates
1. Plate coating • For each plate add 250 ul of anti-mouse IgG (second Ab) to 25 ml of

DAY 2 coating buffer
2. Plate buffering • add 250 ul per well of coating antibody solution using a repeater pipet
• do not coat column 1 (except NSB’s)
DAY 3 • start at A2 and go down each column (see plate map)
3. Standards • pipet all solutions in this order
• tap plates gently to ensure that coating solution covers well bottom
• label, cover with acetate plate sealer and leave overnight at room

temperature

• dump contents of plate with one good shake into sink
• add 300 ul per well of blocking buffer
• do not coat column 1 (except NSB’s)
• start at A2 and go down each column
• cover with acetate plate sealer and store overnight at RT
• NOTE: plate can be used after 3 hours, or saved for 2-3 weeks at room

temperature if sealed well or for 4 weeks if frozen in ziplock bag

• standards: 500, 250, 125, 62.5, 31.2, 15.6 and 7.8 pg/well
• dilute standard stock (10 ng/ml or 500 pg/well, -20˚C) serially 2-fold

using 200 ul stock plus 200 ul assay buffer

4. First Ab • make first Ab working dilution (1:600,000 or run titer for best dilution)
by adding 16.7 ul of first Ab (1:1000, 4˚C) in 10 ml assay buffer

5. Plate washing • wash the plate 3 times with wash solution to remove blocking buffer
• do not allow plates to dry – load immediately

6. Plate loading • add 50 ul per well assay buffer into NSB wells
• add 50 ul per well standards, controls and samples
DAY 4 • using the repeater pipet and immediately add 100 ul per well of first Ab
7. Label
(except NSB)
• avoid splashing or touching the samples in the wells
• cover the plates and incubate overnight at RT

• biotinylated label (BL) working dilution is 1:200, 000 (or run titer)
• add 50 ul stock of BL (1:1000) to 10 ml assay buffer
• using the repeater pipet, add 100 ul per well of BL
• cover the plates and incubate for 4 hr at RT

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8. Streptavidin • prepare immediately before use
• combine 1 ul streptavidin-peroxidase conjugate and 24 ml assay buffer
• wash the plate 4 times with wash solution
• add 200 ul streptavidin to all wells using repeater pipet
• cover and incubate with shaking for 40 minutes at RT

9. Substrate • prepare TMB substrate solution immediately before use
• take 1 phosphate-citrate capsule (Sigma P-4922) and break open by
twisting
• dump contents into 100 ml H2O – Let dissolve (10 min) in the dark (light

sensitive)
• take 2 TMB (Sigma T-3405) tablets in 20 ml phosphate-citrate buffer

per plate
• wash the plate 4 times with wash solution
• add 200 ul substrate solution to all wells using repeater
• cover and incubate with shaking for ~ 40 minutes at RT (depending on

color intensity)

10. Stop reaction • add 50 ul of sulfuric acid stop solution to each well (blue will turn yellow)
• 0 wells should read ~1.0 OD

11. Plate reading • read at 620 nm (Reference Filter), 450 nm (Measuring Filter)

LH EIA REAGENT PREPARATION

1. Second antibody • dissolve whole vial of anti-mouse IgG (whole molecule affinity purified,
2. First antibody Sigma M8645; 1 mg) in 5 ml of coating buffer
3. Biotinylated label • aliquot into 1 ml fractions (each 1 ml aliquot will coat 4 plates (50
ug/plate) and freeze
• stock; LH 518-B7, order from Jan Roser 1 mg/vial lyophilized
• freeze stock upon arrival
• reconstitute stock in 50 mm Tris, pH 9.0 or 0.03M NH4HCo3
• aliquot and freeze at -20oC (stable for at least 1 year )
• stored as a 1:1000 dilution in RIA buffer
• EZ-Link Sulfo-NHS-LC-Biotinylation Kit (PIERCE cat#21430)
• weigh out 2 mg of ovine LH (reagent grade - NIADDK-oLH-26)
• make a 2 mg/ml oLH stock by adding 1 ml of PBS
• make a 10 ug/ul biotin solution just before conjugation by adding 2 mg of
biotin (in kit) to 200 ul of dH2O
• add 86 ul (calculation from kit based on a MW 26,000) of the biotin to 1
ml of the 2 mg/ml oLH stock
• incubate the biotin/oLH solution for 30 min at RT
• run 30 ml PBS down desalting column
• layer the 1 ml biotin/oLH solution onto the column, allow solution to flow
into column

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4. Streptavidin • layer 20 ml PBS and collect 20, 1 ml fractions (can be stored frozen)
5. Standards • run fractions on plate at 1:50,000 to see where label eluded
• combine fractions that contain label and titrate out to determine working

concentration
• dilute lyophilized streptavidin (Boehringer 1 089 153; 500 U) in 1 ml

sterile water at a concentration of 500 U conjugate/ml
• reconstituted solution is stable for up to 6 months at 4˚C (do not freeze)
• bovine LH (NIH-bLH-B10, AFP-5551B)
• weigh out ~1 mg of the ovine LH reconstitute in assay buffer for a 1

mg/ml (1,000,000 ng/ml) stock
• dilute 1,000,000 ng/ml stock 1:1000 by adding 10 ul of 1,000,000 ng/ml

stock to 9990 ul assay buffer – gives 10 ml of a 1000 ng/ml stock
• dilute 1000 ng/ml stock 1:100 by adding 500 ul of 1000 ng/ml stock to

49.5 ml of assay buffer – gives 50 ml of a 10 ng/ml stock
• aliquot 1 ml aliquots and store at 4˚C

LH EIA Template

ASSAY SPECIES
DATE ANIMAL ID
DILUTION

1 2 3 4 5 6 7 8 9 10 11 12

Blank 0 62.5 C1 15.6
A

31.2
B

7.8 125 C2 62.5
C

125
D

15.6 250 250
E

500
F

31.2 500 00
G

7.8 0
H

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Day 1 PROTOCOL FOR CORTISOL EIA - per plate
1. Plate coating
• use NUNC Maxisorb plates
Day 2 • add 50 ul antibody stock (1:85, -20°C) to 5 ml coating buffer (working
2. Standards
dilution, 1:8500)
• add 50 ul per well antibody solution using the repeater pipet
• do not coat column 1
• start at A2 and go down each column (see plate map)
• pipet all solutions in this order
• tap plates gently to ensure that coating solution covers well bottom
• label, cover with acetate plate sealer and leave overnight at 4°C

• standards: 1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8 and 3.9 pg
cortisol/well

• dilute standard stock (20 ng/ml or 1000 pg/well, -20˚C) serially 2-fold
using 200 ul stock plus 200 ul assay buffer

3. Samples/controls • dilute urine or fecal extracts in EIA buffer to the appropriate dilution
• dilute controls to the appropriate dilution, if necessary

4. HRP label • HRP-labeled cortisol working dilution is 1:20,000
• add 25 ul stock (1:100, 4˚C) to 5 ml EIA buffer

5. Plate washing • wash plates five times with wash solution using Dynex plate washer
• blot the plates on paper towel to remove excess wash solution
• allow plates to dry inverted on a paper towel

6. Plate loading • add 50 ul standard, sample, or control per well in duplicate as quickly
and accurately as possible, according to plate set-up (<10 minutes)

• using the repeater, immediately add 50 ul per well of diluted Cort-HRP
• avoid splashing or touching the samples in the wells
• cover the plates, label with the time and incubate at RT for 1 hour

7. Plate washing • wash the plates 5 times with wash solution and blot dry
8. Substrate • plates are fairly stable and can be left until all plates are washed
• prepare ABTS substrate immediately before use
• combine 40 ul 0.5 M H2O2, 125 ul 40 mM ABTS and 12.5 ml citrate

buffer
• add 100 ul ABTS substrate to all wells using repeater pipet
• cover and incubate at RT with shaking for 10 minutes to 1 hour

depending on color intensity (0 wells should read ~1.0 OD)

9. Plate reading • read at 405 nm (Measuring Filter), 620 nm (Reference Filter)

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CORTISOL STOCK PREPARATION

1. Antibody • dilute cortisol R4866 (Coralie Munro, University of California, Davis,
USA) at a dilution of 1:85 by adding 24 ul of stock to 2000 ul of coating
buffer

• aliquot 100 ul into O-ring vials and freeze
• antibody stock is stored in the freezer

2. HRP conjugate • dilute cortisol-horseradish peroxidase (Coralie Munro, University of
California, Davis, USA) 1:100 by adding 50 ul of stock to 4.95 ml EIA

assay buffer and store at 4˚C
• refreeze remaining stock

3. Standards • weigh out 1 mg cortisol (Sigma Diagnostics), add 1 ml ETOH for a 1
mg/ml primary stock solution

• take 1 ml primary stock (1 mg/ml) and add to 99 ml ETOH assay buffer
for a 10 ug/ml (10,000 ng/ml) working stock in alcohol

• dilute a further by adding 100 ul working stock to 49.9 ml EIA buffer to
prepare 20 ng/ml or 1000 pg/well standard

• aliquot and store all stocks at -20˚C

Cortisol EIA Template

ASSAY SPECIES
DATE ANIMAL ID
DILUTION

1 2 3 4 5 6 7 8 9 10 11 12

Blank 0 31.2 500 3 1.2
A

6 2.5
B

3.9 62.5 1000 12 5
C

25 0
D

7. 8 125 C1 0 500
E

3.9 1000
F

15.6 250 C2 7.8 0

G

15.6 0

H

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Sample Sheet

Species: Date: Assay: Dilution: Diluent: Zoo:

Assay Sample # ID/Date Assay Sample # ID/Date Assay Sample # ID/Date

1 1 1
2 2 2
3 3 3
4 4 4
5 5 5
6 6 6
7 7 7
8 8 8
9 9 9
10 10 10
11 11 11
12 12 12
13 13 13
14 14 14
15 15 15
16 16 16
17 17 17
18 18 18
19 19 19
20 20 20
21 21 21
22 22 22
23 23 23
24 24 24
25 25 25
26 26 26

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XVI. CALCULATING EIA RESULTS

READING THE PLATE

• Since the product of EIA is color, it can be read in two ways: 1) by eye inspection; or 2) by
using a spectrophotometer.

• Reading plates by eye is usually only used for positive/negative type results.
• The product of the substrate catalysis by enzyme is measured by transmitting light of a

specific wavelength through the product and measuring the amount of light adsorption by a
plate reader (i.e., optical density).
• Since different products can be produced in EIAs, the correct filter for the detection of the
wavelength must be used.

CALCULATING RESULTS FROM OPTICAL DENSITY (OD)

• There are no set rules on how to set up a plate; it is a matter of choice.
• We run plates by moving down columns (standard curve in duplicate – zeros; standards

S2-S10; controls, C1-C2; samples, T1-T26; standard curve in singlet to account for any
time lag in loading the plate).

S= Standard C= Control T= Test Sample

1 2 3 4 5 6 7 8 9 10 11 12
A Blank Zero S5 S9 T1 T5 T9 T13 T17 T21 T25 S5
B Blank Zero S5 S9 T1 T5 T9 T13 T17 T21 T25 S6
C S2 S6 S10 T2 T6 T10 T14 T18 T22 T26 S7
D S2 S6 S10 T2 T6 T10 T14 T18 T22 T26 S8
E S3 S7 C1 T3 T7 T11 T15 T19 T23 Zero S9
F S3 S7 C1 T3 T7 T11 T15 T19 T23 S2 S10
G S4 S8 C2 T4 T8 T12 T16 T20 T24 S3 Zero
H S4 S8 C2 T4 T8 T12 T16 T20 T24 S4 Zero

AVERAGE BLANK VALUE:

• This is the average OD of both blank wells (background or non-specific binding)
• This average is then removed from every other well on the plate

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DATA MATRIX/TABLE : OD

• These are the final OD readings from the plate reader after the background has been
removed.

• The printout will identify which OD value corresponds to which well.

Example: “Well A3” OD equals 0.722 on print out

1 2 3 4 5 6 7 8 9 10 11 12

A Blank 0.722

B Blank

C

D

E

F

G

H

Results:

• To determine % specific binding, calculate the average OD of the “zero” wells

1 2 3 4 5 6 7 8 9 10 11 12
A .831
B .928
C
D
E .864
F
G .860
H .877

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Example: (Well A2, B2, 11E, 12G, 12H)/5
From printout - (0.831 + 0.928 + 0.864 + 0.860 + 0.877)/5 = 0.872

• “0.872” is the denominator by which all other OD values are divided

1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E .857
F
G
H

Example: OD from “Well E2” equals (0.857/0.872) * 100 % = 98.2%

• Calculate the % binding for each of the standard wells.

1 2 3 4 5 6 7 8 9 10 11 12

A 82 26 81

B 82 25 68

C 105 70 15 52

D 103 68 14 35

E 98 55 24

F 99 52 94 15

G 92 39 93

H 92 38 94

• Take the average % binding of the standards that have been run in triplicate.

Example: ‘Standard 10’ which is 1000 pg/well is found in wells C4, D4 and F12
From print out – (15 + 14 + 16)/3 = 15%

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• Now plot the standard curve.

120

100

80

%B/TB 60

40

20

0

1 10 100 1000 10000

Standard Concentration (logrithmic scale)

• Calculate the % binding of all controls and test samples

1 2 3 4 5 6 7 8 9 10 11 12
A 79 80 73 68 67 70 121
B 74 78 64 59 63 63 112
C 75 79 71 71 65 108 115
D 76 78 71 69 63 109 108
E
F 44 77 76 73 64 65 106
G 43 73 73 71 56 62 106
H 81 78 70 40 69 76 108
81 77 67 43 69 76 109

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• Calculate dose for each control and test sample by extrapolating data from standard curve

120
100

80 1st
% B/TB 60

40 2nd
20

0
1 10 100 1000 10000

Standard Concentration (logrithmic scale)

Example:
• Test sample one or “T1” was placed into “Well A5” and “Well A2”.
• The percent binding was calculated to be 79% and 74%, respectively.
• The average % binding would be 76%.
• The concentration at 76% is 42.85 pg/well.

• Results are converted to concentration/ml of sample.

Example:
• 42.85 pg/well is equal to 42.85 pg/50 ul
• The 50 is much was loaded into the well, for the cortisol assay this is 50 ul/well.

Thus, 42.85 pg/50 ul = 0.857 pg/ul
0.857 pg/ul * 1000 (1 ml) = 857 pg/ml
857 pg/ml/1000 (pg to ng) = 0.857 ng/ml

CONVERTING DATA FROM FECES

Example:
• Need weight of feces originally weighed out for extraction (e.g., 0.5 g)
• Final volume from the extraction (e.g., 1 ml)
• From the assay calculate the mass (e.g., pg/well to ng/ml….say 120 ng/ml)
• Dilution factors need to be calculated into the data (e.g., samples ran at a 1:10 – then 120 *
10 = 1200 ng/ml)

Thus, 1200 ng/0.5 g = x ng/1g of feces = 240 ng/g of feces

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CONVERTING DATA FROM URINE
Example:

• From the assay calculate the mass (e.g., pg/well to ng/ml….say 20 ng/ml)
• Dilution factors need to be calculated into the data (e.g., samples ran at a 1:10 – then 10 *

20 = 200 ng/ml)
• Then divide the sample value by the creatinine value (e.g., 0.182 mg/ml creatinine)
Thus, 200 ng/ml divided by 0.182 mg/ml = 1098.9 ng/mg creatinine
TIPS FOR READING EIA PLATES
• Clean bottom of plate before reading
• Make sure the right filter is used to read the plate
• Get print out of OD’s
• Remove average background OD’s from all other OD values
• Calculate average ‘zero’ OD value
• Use this to calculate % binding of all other wells - (OD of each well/ ‘zero’ OD average

value) *100%
• Plot the standard curve – concentration (x axis - log scale) vs % binding (y axis - linear

scale)
• Average % binding’s for each sample (controls and test samples)
• Extrapolate concentrations based on % binding
• Convert data to a per ml or per gram mass concentration (Example: pg/well to ng/ml)

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XVII. ASSAY REAGENTS AND SUPPLIES

1. STEROID EIA ASSAY RECIPES (full and half recipes)

COATING BUFFER Full Half (Sigma, S2127)
Na2CO3 (Anhydrous) 1.59 g .795g (Sigma, S8875)
NaHCO3 2.93 g 1.465g
H2O 1000 ml 500 ml
pH to 9.6, store at 4˚C

ASSAY BUFFER (EIA BUFFER)

Stock A 0.2M NaH2PO4 27.8 g 13.9g (Sigma, S9638)
Stock B 0.2M Na2HPO4 28.4 g 14.2g (Sigma, S0876)
H2O 1000 ml 500 ml

Stock A 195 ml 97.5 ml

Stock B 305 ml 152.5 ml

NaCl 8.7 g 4.35g (Sigma, S9625)

BSA 1.0 g 0.5g (Sigma, A7906)

H2O 500 ml 250 ml
pH to 7.0, store at 4oC

“Dilution buffer” for fecal extraction in EIA buffer minus the addition of BSA.

WASH SOLUTION CONCENTRATE

NaCl 87.66 g 43.83g (Sigma, S9625)

Tween 20 5.0 ml 2.5 ml (Sigma, P1379)

H2O 1000 ml 500 ml
store at 4˚C

Dilute 10-fold for working wash solution (100 ml/50 ml/25 ml wash solution

plus 900 ml/450 ml/225 ml H2O), store at RT

SUBSTRATE BUFFER 9.61 g 4.805g (Sigma, C0759)
Citric acid (anhydrous) 1000 ml 500 ml
H2O
pH to 4.0, store at 4˚C

ABTS (40 mM)

ABTS 0.55 g (Sigma, A1888)

pH to 6.0. ABTS is light sensitive - use brown glass or foil for storage
store at 4oC

HYDROGEN PEROXIDE (0.5M)

H2O2 (30% Solution) 500 ul (Sigma, H1009)
H2O 8 ml 49
store at 4˚C

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2. LEUTINIZING HORMONE EIA ASSAY RECIPES

COATING BUFFER Full Half

Na2CO3 1.59g 0.795g (Sigma S7795)
NaHCO3 2.936g 1.468g (Sigma S6014)
H2O 1000 ml 500 ml
pH to 9.6 with 1M HCL

(100 ml 37% HCL (JT Baker 9535-02) 900 ml dH2O)
store at 4˚C

BLOCKING BUFFER

Tris 2.42g 1.21g (Sigma T1503)
8.96g (Sigma S9625)
NaCl 19.92g (Sigma A7906)
5g (Sigma S8032)
BSA 10g 0.5g
500 ml
Sodium azide 1g

dH2O 1000 ml

pH to 7.5 with 1M HCL, store at 4˚C

ASSAY BUFFER 2.42g 1.21g (Sigma T1503)
Tris 17.9g 8.95g (Sigma S9625)
NaCl 1.0 g 0.5 g (Sigma A7906)
BSA 1 ml 0.5 ml (Sigma P1754)
Tween 80 1000 ml 500 ml
H2O
pH to 9.6 with 1M HCL

WASH SOLUTION O.5 ml 0.2 ml (Sigma P1379)
Tween 20 17.9g 8.95g (Sigma S9625)
NaCl 1000 ml 500 ml
H2O
store at RT

TMB SOLUTION

1 phosphate-citrate capsule 100 ml (Sigma P4922)
(Sigma T3405)
2 TMB tablets/plate 20 ml phosphate-citrate

buffer/plate

Make up immediately before use. Keep in the dark.

STOP REAGENT 27 ml
473 ml
H2SO4 (95-97% sulfuric acid FW=98.08)
H2O
store at RT

50