Hormones Analysis

Hormone measurement
• The Problem
– very small amounts of hormone in a very complex mixture
• Pre-immunoassay
– complex and insensitive methods (chemical methods, whole
animal or tissue bioassay)
– insensitive
– imprecise
– inaccurate
• Immunoassay
– first described in 1960
– very rapid expansion since early 1970s
– advantages (simplicity, speed, precision, accuracy, sensitivity)
Definitions
• Potency estimate: the concentration of the analyte.
• Sensitivity: the minimum amount of the analyte
which can be accurately detected.
• Specificity: the ability of IA to uniquely measure
the analyte of interest.
• Accuracy: agreement between the true answer and
the answer obtained in the IA.
• Precision: expressed as inta- interassay variation,
calculated as CV.
Systemic versus random errors
• Systemic: errors deflect repeated

measures from true or accurate value.
• Random: are those which primarily
affects precision. Random errors can
not be eliminated but can be
minimized.
Opening definitions
• Is standardisation of Immunoassay
different from standardisation of any other
types of assay system?
• What do we mean by Standardisation,

Calibration?
Do the ‘analytes’ exist?
• Cortisol, Testosterone, Thyroxine can be
weighed out, but...
• Free Thyroxine [ie non-protein bound]

• Urinary Free Cortisol
[i.e. cortisol that is not conjugated and is in urine]
• Protein/polypeptide hormones
• TSH, hCG, LH and FSH etc.

• Standardisation
Calibration using a measurement standard
• Calibration
set of operations that establish under specified
conditions. The relationship between values of
quantities indicated by a measuring instrument or
measuring system, or values represented by a
material measure or a reference material, and
corresponding values realised by standards
In broad terms ...
• Standardise = make readings comparable

• Calibrate = make readings correct
• We want to do both!!
Where is immunoassay used
• Medical laboratory
– hormones, drugs, tumour markers, specific proteins, viral antigens, etc
• Point of Care Testing
– Drugs, cardiac markers, anticoagulants etc
• Over the Counter
– Pregnancy tests, fertility tests etc
• Agriculture, veterinary, pharmaceuticals, research etc

Limited reagent immunoassay
+ +
50% bound
Solid phase antibody Antigen Bound antigen Free antigen
+ +
25% bound
+ +
12.5% bound
Include labelled antigen (fixed amount) to indicate the distribution of bound and free analyte
Count bound fraction after separation and washing .
Limited reagent immunoassay standard curve
Label
activity
bound to
solid
phase
Concentration of antigen
2-site immunometric assay
+ +
Solid phase antibody Antigen Labelled antibody (excess)

(excess)
Separate and count activity bound to solid phase

2-site immunometric assay standard curve
Label
activity
bound to
solid
phase
Concentration of antigen
Basic requirements for
immunoassay
• Standards
• Specific antibodies
• Labelled antigen or antibody
• Separation system
• Quality control
Types of label
– Radioactive (125I, 32P etc)
– Fluorescence (Direct, time-resolved)
– Enzyme (colorimetry, fluorimetry, enhanced
chemiluminescence)
– Luminescence (bioluminescence, phosphorescence)
– Microparticle
– Streptavidin/avidin-biotin
– Amplification
Ideal immunoassay label
• Detectability.
• Reactivity.
• Nonspecific binding.
• Stability.
Advantages of 2-site
immunometric assays
• Increased sensitivity
• Increased precision
• Better specificity
• Greater assay range
• Shorter assay times
Disadvantages of 2 site
immumetric assays
• Need for large quantities of pure antibody
(monoclonal antibodies usually employed)
• 2 antibody binding sites required (limit range of
analysis)
• High dose “hook” effect
• Need for multiple washing steps
• Non specific interference due to heterophyllic
antibodies
Advantages of isotopic labels
• Simple coupling reactions
• Label properties do not alter on coupling
• No background signal
• Efficient/convenient detection systems
• No additional cost to detect signal
• Very useful for research assays
Non isotopic labels - advantages
• No radioactivity
– safety aspects
– disposal
• Extended life of label
• Speed of detection
• Ease of automation
• Theoretical increase in sensitivity
• Possibility of homogeneous assays
• Simple/safe label preparation
Non isotopic labels - disadvantages
• Safety aspects of labels/substrates
• Serum/buffer effects
• Extra manipulations in detection
• Inefficient detection in some cases
• No recounting possible in some systems
• Limitation of separation system
• “Dedicated” instruments
• Commercial pressures
Specialised Immunoassays
• Free hormone assays
• Homogeneous (non separation )

immunoassays
Measurement of free hormones
• ? Free hormones closely reflect the true (active)
hormonal state in the body
• In theory, these are an optimal test of hormonal
function
• Measurement presents a challenge
– very low concentrations
– avoiding disturbing equilibrium between bound and
free hormone during measurement
– sera from some patients (non-thyroidal illness) contain
interfering substances that can invalidate measurements
Measurement
of free hormones
• Reference methods
– Initial separation by equilibrium dialysis or
ultrafiltration and measurement of analyte in
the separated fraction by immunoassay
– Only minimal dilution of sample possible
– Expensive, time consuming, unsuitable for high
volume work
– Commercial methods now available
Methods of free hormone
estimation
• Index methods (calculation)
• 2 step immunoassay
• Analogue immunoassay
• Labelled antibody assays
Index Method
• Correct total hormone concentration for
abnormal binding protein concentration
• Measure total hormone (free and bound)
• Measure binding protein (i.e. TBG, SHBG)
• Apply formulae to estimate free hormone
• Simple, rapid, inexpensive
• Variable performance
• Not accurate at very high or low binding
protein concentrations
2 Step Immunoassay
• Extract free hormone from serum by adding
solid phase antibody
• Wash
• Add label which binds to remaining
unoccupied antibody binding sites
Analogue Immunoassay
• Label = analogues which bind to antibody
but not to binding proteins (disputed)
• Mix sample, antibody and analogue label
– binding of label to antibody is inversely
proportional to free hormone concentration in
the sample
Labelled Antibody Assay
• Label = antibody to hormone (usually
monoclonal) labelled with 125I or non-
isotopic label
• Solid phase = derivative of antigen attached
to coated tube or magnetic particles etc
Problems with current
free hormone assays
• Reference methods not practical
• Lack of information from manufacturers on kit
performance in binding abnormalities
• Frequent changes in kit formulation and methodology
• Large range in concentrations measured in EQAS schemes
when comparing different methods
• Patients with same abnormality (ie. non- thyroidal illness)
can have normal results with one kit and abnormal results
with another kit supposedly based on the same analytical
principle
Homogeneous immunoassays
– No separation step therefore simple and easy to
automate
– Lack sensitivity
• Fluorescence polarisation
• Turbidimetry/nephelometry/latex agglutination
Reference Range
• A guide to the levels expected in normal
people
• “Normal population”
– laboratory staff
– hospital out patients
– occupational health
– medical students
• Must take age/sex etc into account
• Must take a sufficient sample (≈ 100)
• Age
– T3 in elderly people
• Sex
– Testosterone in males/females
• Time of day
– Cortisol
• Time of month
– Oestradiol/progesterone (females)
• Diet
– Insulin
• Illness
– Sick euthyroid
Hormone assay in the future
• Dominated by immunoassay techniques
• ? GCMS
• Increased sensitivity
• Better automation
– computers
– robotics
– non isotopic labels
• Near patient testing devices
Immunoassay automation
• Non isotopic labels
• Microprocessor power
• Improved robotics
• Better antibodies → faster reaction times
Immunoassay Analysers
• Immunological reagents
• Precision usually good
• Wide variations in sensitivity, specificity
and accuracy between analysers
• Careful definition of assay requirements
• ?Whether any one analyser will satisfy all
requirements
Types of automated system
• Work simplified batch systems
• Automated batch systems
• Total automation (black box) - random
access
• Portable (bedside biochemistry) instruments
Advantages of automation
• Increased precision
• Work simplification
• Versatility
• Less contact with samples
• Rapid turnaround time
Disadvantages of automation
• Lack of reagent choice
• Total reliance on manufacturer
• Lack of range of analytes
• Little use for “research” assay
• Increased cost

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Hormone measurement

• Systemic: errors deflect repeated

• What do we mean by Standardisation,

• Free Thyroxine [ie non-protein bound]

• TSH, hCG, LH and FSH etc.

• Standardise = make readings comparable

• Agriculture, veterinary, pharmaceuticals, research etc

Solid phase antibody Antigen Labelled antibody (excess)

Separate and count activity bound to solid phase

• Free hormone assays

• Homogeneous (non separation )

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