L8 - Laboratory Measurements

Question 1

Why do we need to measure hormone levels? (4)

Answer 1

• Establishment of “normal ranges”
• Clinical diagnosis of endocrine malfunction and disease
• Effectiveness of treatment of patients
• Understanding of endocrine physiology

Question 2

The measurement or “assay” of hormones (e.g. in blood samples)

Answer 2

• Using a given test system, to compare the response produced by the hormone in the sample, with the responses produced by known concentrations of the same hormone

Question 3

The principle of immunoassay (3)

Answer 3

• SPECIFIC INTERACTION between antibody and antigen (i.e. hormone to be measured).
• Reaction is REVERSIBLE and will reach an equilibrium state.
• A competitive binding assay” i.e. competition between labelled (i.e. “tagged”) and unlabelled forms of a hormone for a limiting amount of antibody

Question 4

Physical separation of antibody-bound and “free” hormone

Answer 4

• At equilibrium, antibody-bound hormone (both labelled and unlabelled) must be separated from “free” (i.e. unbound) hormone using charcoal, cellulose etc.

• If the hormone level in a blood sample is low, then a higher level of labelled (e.g. radioactive) hormone will be bound to the antibody (i.e. remaining in solution within the incubation tube) following removal of unbound hormone.
o And vise versa

Question 5

Immunometric Assays

Answer 5

• These use two different antibodies (e.g. monoclonal antibodies), which bind to different regions (epitopes) of a single hormone molecule.

Question 6

The immunoradiometric assay (IRMA)

Answer 6

Known constant amount added.

After incubation, the amount of GH bound to the antibody is detected by adding an excess of a second labelled antibody to all tubes.

Any unbound antibody is removed, leaving the amount of triple complex to be deter¬ mined by quantifying the bound label (e.g. fluores¬ cence or radioactivity).

Question 7

Enzyme-linked Immunosorbent Assay (ELISA)

Answer 7

Look at pic

Question 8

Immunoassays: measurement of hormone mass

Answer 8

• Highly-sensitive (can measure less-than-physiological levels)
• Highly-specific (can distinguish between similar hormone structures, e.g. LH, FSH and TSH)
• Highly precise (give high degree of confidence that the measured value is repeatable)
• Very convenient (cost-effective, kit-based, ease of operation, high sample throughput).

Question 9

Biological Assays (Bioassays)

Answer 9

•	Measure the MAGNITUDE or INTENSITY of a BIOLOGICAL EFFECT
•	Typical bioassay end-points (effects):
o	Cell growth, 
o	Release of a metabolite, 
o	Uptake of a radioisotope

Question 10

The principal types of bioassay (2)

Answer 10

• In vivo bioassays: involve administration of a test or standard hormone to an animal, with quantification of the response
• In vitro bioassays: addition of a test or standard hormone to cell cultures or tissue fragments, with quantification of the response.

Question 11

Normal human thyroid follicular cells in monolayer culture

Answer 11

• Cells retain an intact adenylate cyclase response to TSH which generates cyclic AMP in proportion to the applied TSH dose

Question 12

Bioassays: limitations and advantages

Answer 12

• In vivo bioassays: laborious; technically demanding; expensive; insensitive; poorly reproducible; subject to species-specificity limitations.
• In vitro bioassays: cost-effective; robust; reliable; sensitive; precise; reproducible; easier to avoid species-specificity problems

Question 13

Assignment of potency to recombinant hormones

Answer 13

Calibration of R in terms of N (units of activity) and in terms of mass used (R =100% pure).

See pictures

Question 14

Advantages and disadvantages of receptor assays

Answer 14

• Physiological”, i.e. uses ‘natural’ binding site for hormone;
• High affinity for hormone;
High specificity for hormone;
• High sensitivity;
• Good precision;
• Good sample capacity
• But: labour-intensive; requires tissue processing; dependent on animal material.