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Human Reproduction 316
Week 10 Monday 2005 Neville Bruce
Reproductive Endocrinology Practical Aspects of Measurement
Measurement (why and where)
 Clinic:
Diagnosis
Programming treatment
Effects of manipulation
 Personal: Pregnancy tests, monitoring cycle for contraception or conception
 Research: Human: Individual, Population or group trends
Animal
 Public Health Future possibilities
Screening; eg onset of puberty, decisions on what this should confer (age of consent?)
Analysis of fertility; Is our life style affecting our fertility and hormones?
Other??. Selecting out for warriors? Athletes?

Measurement (how)
Chemical and physical, generally purify and measure (weight, colour etc)
Bioassays; Egypt, Germany, Aschiem Zondek, mouse ovary and uterus, toad clutch!
Competitive Binding Assays; Immunoassays. The big break through.
Measurement (what) What is the question being addressed?
blood or plasma
urine
saliva
Blood concentration = production rate/metabolic clearance rate
Hormone Assay
Background
A hormone assay essentially detects and measures the concentration of a hormone in a given sample: eg
cortisol, ng/mL (weight of the hormone in a 1 mL solution volume).
Hormone concentrations are generally expressed as weight per unit volume, (eg ug/mL, ng/mL, pg/mL or
ug/L (L = litre) or ng/dL (dL is deciliter , 100mL) or a molecular weight equivalents per litre eg nMol/L
(nanomol/litre). The latter enables comparison of relative number of molecules between different
hormones etc. To convert from nMol/L to ng/mL multiply by molecular weight and divide by 1000(to
get from L to ml). We will generally use ng/mL or pg/mL.
Useful units (need to understand well).
liter (L), milliliter (mL) = approximately 1 gram if water , microliter (uL or L, the latter is correct but
often computer bugged so uLl is mostly used here)
gram (g) miligram (mg = 1 uL if water), microgram (ug), nanogram (ng), picogram (pg)
Measurement Steps
1) Collect samples:
1) Prepare samples: this may include:
a) extraction; To remove nonspecific and cross reactants and/or concentrate samples
b) dilution to suit sensitivity of curve
2) Run assay with standards and unknowns
3) Compute results by comparing values of unknowns against the standard curve.
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Competitive protein binding assay. Immunoassay
Principles. General principles same for all assays but there are many variations on the theme.
(ELISA example The hormone to be measured or hormone sample Hs and its specific antibody Ab are
mixed together at carefully regulated quantities so that after equilibrium is reached there will be some
free hormone Hs, some free antibody, Ab and some bound hormone-antibody complex HAb. We
generally aim to have around 50% bound. A standard amount of an identical hormone that has been
labelled Hl is then added. The Hlwill compete with the Hs for binding to the Ab. High concentrations of
Hs will mean that little Hl will bind to the Ab. Low concentrations of Hs will mean that a lot of Hl will
bind to the Ab. After the reactions are complete, the amount of Hl-Ab is measured and compared against
a standard curve to estimate the concentration of Hs in the sample (see diagram below).
The big advantages of competitive binding assays are:
1) their high level of specificity (only measure the hormone of interest, not similar hormones or cross
reacting molecules)
2) their high level of sensitivity (can measure exceedingly small amounts of hormone down to pg levels)
3) can be adapted to measuring a large number of samples, efficiently and economically.
Competitive binding assays revolutionized understanding of endocrinology
Second
antibodyenzyme
step:
The second
antibody,
conjugated to
an enzyme
binds directly to
the first
antibody
(incubation 1.5
h). All then
washed out
other than the
Ab2-e bonded
to the first
antibody on
walls of well.
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Antibody HAb
Enzyme Linked Immuno Sorbent Assay (ELISA)
As used to measure salivary cortisol
Hormone, labeled Hl
Hormone sample/standard Hs
Antibody – hormone bound
Colour agent,
Low concentration
standards / samples
High concentration
standards / samples
Plate step: fixed amount of
hormone antibody
is bonded
to walls of each well (incubation, 2h
or overnight 4 ºC). Remaining
(unbonded) is then washed out.
Addition of labelled hormone
and
either standards or unknown sample
hormone
The mix is allowed to incubate for 1
hour and any unbound hormone
washed away
After incubation the mix of labelled
and tagged (sample or standard) will
bind to the antibody in proportion to
amount of each in the well. All still
unbound hormone is then washed
out.
Substrate colour step: Colour is
added to the wells and when solution
is bluish, reaction is stopped with
H2SO4 and colour read (absorbance)
on microplate reader.
Dense colour represents small
amount of hormone in standard or
sample, light colour represents very
little hormone