Lecture 7 - Hormone and receptor binding assays Flashcards
what are the steps for validation and assay methods?
- must have a standard of known activity
- dose response relationship (standard curve)
- sufficient replicates to demonstrate reproducibility
- must be sensitive
- simple, low cost, suitable for routine use
- type of measurement used must relate to hormone activity
- use of quality control samples in each batch of samples
- assess intra- and inter-assay accuracy and precision (within and between) - limit of detection
- with signal to noise ratio of 3 (how much signal before detected) - limit of quantification
- lowest concentration that can be measured with acceptable accuracy and precision
Why is parallelism of standard curves important?
A comparison of standard curves should show parallel curves
- parallelism needs to be validated for each tissue type (e.g. plasma will be different than muscle) to demonstrate that the response in the assay is not dependent on the sample matrix
What is the principle behind a biological assay (bioassay)?
= GOLD STANDARD
Measures a biological/physiological response (whole animal/tissue response)
- direct relation between hormone and response
- response needs to be quantifiable = positive/negative (yes/no) or dose response (intensity of response proportional to amount of hormone)
What are the advantages/disadvantages of bioassays?
Advantages:
- directly measures effect of hormone response through biological response
Disadvantages:
- activity and concentration aren’t necessarily related
- different forms of hormone may have different potencies (e.g. glycosylated vs. non-glycosylated; purification/extraction can also alter potency)
- not completely reproducible (variability between animals)
- expensive
- ethical considerations (use of animals)
Describe chemical assays (advantages/disadvantages)
- Liquid chromatography = purification (HPLC) –> ion exchange, adsorption (normal phase), hydrophobic interaction (reverse phase) – separated based on differences within structure
- gas chromatography = separates compounds by adsorption or partitioning between the gaseous mobile gas phase and the lipid/solid stationary phase
- electrophoresis - native or non-reducing gels separate intact protein molecules based on share, while gels containing SDS separate molecules based on molecular weight and not charge
Based on some aspect of the structure of the particular hormone
- sometimes it is possible to take advantage of unique structure of hormone to design simple/specific measure (using thyroxine to measure total protein bound iodine)
- solubility/stability related to structure
Disadvantage:
- chemical assays that test for functional groups on the hormone molecule that may not be involved with activity can give false results compared to biological activity
Describe the principle and methods of competitive binding assays for measurement of hormones
Based on specific binding of a hormone by a protein (usually specific antibody)
- a fixed amount of labeled hormone and fixed amount of binding protein are used, along with variable amount of unlabelled hormone
- equilibrium is established between amount of unlabelled hormone and labelled hormone that is complexed with binding protein
- the proportion of labelled vs. unlabeled hormone bound to the antibody at equilibrium depends on the amount of unlabelled hormone present in the assay
- the amount of label decreases with increasing amount of unlabelled hormone
- we can only measure amount of labelled hormone bound to protein, we use this to determine how much unlabeled hormone is present
- prepare standard curve to determine useful concentrations of binding protein (fixed amount of binding protein, with same amount of radioactive hormone, adding various amounts of unlabelled hormone)
- y axis = amount of labelled hormone
- x axis = concentration of unlabeled hormone
The hormone concentration in a test sample is determined by passing the sample to the binding protein and labelled hormone mixture
- the amount of labelled hormone bound is measured and the hormone concentration can be determined from the standard curve
Competitive binding requirements
- must have hormone standard that is able to bind receptor
- must have receptors in native (active) form
- must be able to detect interaction between hormone and receptor (some sort of tag/label)
- always include control to assess non-specific binding (we are interested in specific binding!)
Describe Scatchard analysis for hormone receptor binding. What important info does it give? How is it different from competitive binding assay?
Used to determine hormone-receptor binding
- similar to competitive binding assay but is used to measure the characteristics of receptors instead of the amount of hormone
- specific (low capacity, high affinity) binding of hormone receptors must be determined separately from non-specific (high capacity, low affinity) binding of hormone to other components of system
Saturation curve generated by incubating labelled hormone with a receptor preparation
- bound hormone separated from free hormone and the amount of hormone binding is measured
Total binding = specific + non-specific
Explain how you determine total binding, non-specific binding and specific binding for receptor binding assays?
The total binding is the amount of radioactivity bound to the receptor preparation and included hormone that is bound to receptors as well as non specific binding of hormone
The non-specific binding is determined by including a large amount of unlabelled hormone to saturate all the specific receptor sites
- When radioactive hormone is added, there are no specific receptors available for binding, so the binding is now due to the large number of available non-specific sites
The specific binding is determined as the difference between non-specific and total binding
Why is the Sandwich ELISA so names? Why is it more effective when contaminants are present in sample?
Used to measure hormone concentrations
- a capture antibody is bound to a solid support and incubated with unlabelled hormone
- this is then followed by second incubation with a second indicator antibody against the horses that is conjugated to an enzyme
- the activity of the enzyme is then measured
- the amount of hormone bound to the capture antibody is estimated from the enzyme activity on the bound indicator antibody
- attachment of antibody support
- addition of sample and radio-labelled hormone
- wash to remove unbound labelled hormone and count radioactivity
Useful for detecting low levels of hormone of when many contaminants are present
You just discovered a new hormone with a highly positively charged structure. Unfortunately, no antibody against it is available, and its receptor is unknown. However, you really need to purify and quantify it. What technique would you use?
Chromatography colomn - ion exchange = separates based on charge
