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

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Include labelled antigen (fixed amount) to indicate the distribution of bound and free analyteCount bound fraction after separation and washing .

50% bound

Solid phase antibody Antigen Bound antigen Free antigen

25% bound

12.5% bound

Limited reagent immunoassay standard curve

Labelactivitybound tosolidphase

Concentration of antigen

2-site immunometric assay

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Solid phase antibody(excess)

Antigen Labelled antibody (excess)

Separate and count activity bound to solid phase

2-site immunometric assay standard curve

Labelactivitybound tosolidphase

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