9-Measuring hormones

Question 1

Immunoassay

Answer 1

Depends on the fact specific antibodies can be made for hormones
Steroid hormones conjugated to an immunogenic peptide in order to elicit a response from rabbit
Animal produce=antiserum
Binding is not a permanent, reversible reaction
TSH, LH and FSH glycoprotein hormones have a very similar structure, so antibodies must be specific
Immunoassay is a “competitive binding assay” i.e. competition between labelled (i.e. “tagged”) and unlabelled forms of a hormone for a limiting amount of antibody
Both the labelled and unlabelled hormones are mixed and compete for limiting amounts of antibody

Question 2

Immunoassay method

Answer 2

The most common radioactive tag is Iodine 125, as it decays it releases radiation
Adding more unlabelled hormones will decrease the radioactivity
Iodine reacts with tyrosine residues in the hormone
I125 can destroy thyroid follicular cells
How much hormone displaced is related to how much unlabelled hormone is present
Originally have a known amount of labelled hormone= assay standards
Have to remove displaced radioactive hormone from the sample, otherwise, radioactivity will read the same
Use charcoal or cellulose to remove unbound labelled and unlabelled hormone
Charcoal forms a suspension, charcoal is porous
Bound hormones cannot enter pores, but inbound can
The solution is then centrifuged to remove the charcoal
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 the unbound hormone.
Not the most practical type of assay, can’t be exposed to too much radiation, hard to do on a large scale.

Question 3

Immunometric assay

Answer 3

Non-competitive
Uses 2 monoclonal antibodies that only bind to a specific epitope
Radioactivity rarely used
limited by the amount of antibodies originally bound to tube
So adding more hormone will stop having effects at one point
Horseradish peroxidase is usually used as the enzyme
Colour change measured by spectrophotometer

Question 4

Competitive ELISA

Answer 4

Labelled and unlabelled hormones compete for the bound antibody

Question 5

Pros of immune assays

Answer 5

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 a high degree of confidence that the measured value is repeatable);
Very convenient (cost-effective, kit-based, ease of operation, high sample throughput)

Question 6

Bioassay

Answer 6

Can tell you how much target cells are being affected
There are impaired proportions of bioactivity to response
Bioassays measure the magnitude and intensity of biological effect

Question 7

In vivo bioassays

Answer 7

Involve the administration of a test or standard hormone to an animal, with quantification of the response
Laborious; technically demanding; expensive; insensitive; poorly reproducible; subject to species-specificity limitations

Question 8

In vitro bioassays

Answer 8

Addition of a test or standard hormone to cell cultures or tissue fragments, with quantification of the response
Cost-effective; robust; reliable; sensitive; precise; reproducible; easier to avoid species specificity problems

Question 9

Receptor assays

Answer 9

Ligand binding assays
Isolate receptors from tissue by solubilisation and fractionation
Don’t have to find the best antibody for the job
Use labelled hormones along with unknown hormone
Same procedure as a competitive assay
Advantages and disadvantages of receptor assays:
Physiological – uses “natural” binding site for hormone
High affinity for hormone
High specificity for hormone
High sensitivity
Good precision
BUT – labour intensive, requires extensive tissue processing and is usually dependent on the availability of animal material