Assays and Hormones Flashcards
Immunoassay
- Immunoassays rely on antigens (hormone) and antibodies (that bind to hormones).
- Antigen - any substance that can elicit an antibody response (E2, P4, hCG, FSH)
- Antigens can be labeled for use in immunoassays (e.g. radioactive iodine 131-I attached chemically to steroid hormone via a carrier molecule such as a tyrosine residue)
- Antibody - immunoglobulin protein defined by its ability to bind antigen (Polyclonal vs monoclonal)
- Antibodies can be labeled as well (radioactive vs non-radioactive: e.g. chemical attached causing a color change/light reaction)
Polyclonal antibodies
Derived from animal SERUM
1. Inject Ag into host animal
2. Collect serum from animal ~ 3 months later
3. Combine fixed amount of Ag with serial dilutions of serum to isolate antibodies used for assay
*Polyclonal abs will be LESS specific since it is polyclonal (serum abs may bind to your Ag as well as other Ags)
Monoclonal antibodies
Derived in cell culture
1. Inject Ag into host animal
2. Harvest SPLEEN from animal
3. Serially isolate B-CELLS until the cell that produces abs to your Ag has been identified
4. Fuse B-cell with immortalized myeloma cell to form “hybridoma” > creates clonal cell line that continuously produces abs
*Monoclonal abs are much more SPECIFIC since they are created from individual spleen B-cells isolated to target Ag specifically
Types of immunoassays:
- RIA
- IRMA (including ELISA)
RIA - Radio-immunoassay
IRMA - Immunoradiometricassay
- ELISA - Enzyme-linked immunosorbant assay
Both rely on interaction between Ag and ab for detecting levels of Ag (hormone)
RIA
- Process
- Pros/Cons
RIA: radio-immunoassay (indirect measure)
“ANTIGEN excess” assay
- Start with KNOWN amount of Ab
- Add KNOWN amount of labeled Ag
- In assay: add UNknown amount of UN-labeled Ag (what you are trying to measure)
The UNknown amount of UN-labeled Ag competes with known labeled Ag for binding at Ab sites.
- Measure how much labeled, bound Ag is present by separating bound Ag using a 2nd ab or magnetic particles»_space; can then determine how much UN-known, UN-labeled Ag is present in assay (if labeled, bound Ag is high, then you know there is a low amount of UN-labeled Ag present in sample and vice versa)
- INDIRECT measurement (you are actually measuring the inverse of the sample) > inverse S-shaped curve.
- Accuracy of assay is highest in the linear portion of the S-curve, where slope is consistent (this is 20-80% of range of assay; if estrogen is too high, may require dilution to be on the curve > then multiply by dilution factor to determine total amount)
Pros of RIA: good for small steroid hormones, multiple hormones can be assayed from the same serum sample
Cons of RIA: time consuming, $$, cross-reactivity, auto-abs can skew results
IRMA
IRMA: immunoradiometricassay
“ANTIBODY excess”
- Excess Ab
- UNknown amount of Ag
- Sandwich Ab (2nd) to bind to Ag-Ab complex
(In IRMA, radiolabeled; in ELISA, enzyme-linked label in the solid phase)
DIRECT measurement of Ag amount
Pros: very sensitive, ideal with monoclonal abs, better for large, protein hormones
Cons: not as good for assay of small, steroid hormones, interference from other species-specific abs, hook effect
Hook effect
When there is a very high amount of Ag
Measured Ag overwhelms the amount of Ab available»_space; excess free Ag binds to radiolabeled Ab (saturated up)»_space; the actual measured amount of “sandwich” Ab-Ag-labeled Ab is low
Graph looks like a “hook”
Occurs with prolactin
Which assay is best for small, steroid hormones?
Which assay is best for large, protein hormones?
Small steroid - RIA
Large protein - IRMA (ELISA)
What is sensitivity in the context of assay validation?
Specificity? How do you obtain specificity?
Sensitivity: the smallest amount of hormone being measured that can be distinguished from zero (what is the lowest amount of hormone that can be measured in your assay?)
Specificity: ability to differentiate the hormone you are testing from other similar hormones (how much cross-reactivity?) Use mass spec (gold standard) to obtain specificity
Assay validation:
- Accuracy
- Precision
Accuracy: ability of assay to provide a value that agrees with the actual/true value (to assess: compare values obtained from your assay to known standards)
Precision: assay variability when multiple measurements are taken on the same sample (intra-assay variability = same sample, same day vs inter-assay variability = same sample, different days/assays)
Calculate coefficient of variance
CV (%) = standard deviation (SD) /mean x 100
Measures variation of values that the assay produces
Measure of precision
Mass spectrometry
- Gold standard of measuring hormones
- Measurement of hormones based on mass and charge
- Direct measure of hormone quantity
- Good for measuring testosterone in females (lower threshold values)
What is the gold standard for measuring free testosterone?
Equilibrium dialysis method
What would you expect on an estradiol assay for:
- Ethinyl estradiol
- 17 beta estradiol (Estrace, transdermal patch)
- Premarin
- Ethinyl E2: will not show up on assay due to ethinyl group
- 17 beta E2: WILL show up/be accurate on assay
- Premarin: inaccurate, only some will be detectable on assay (because derived from pregnant horses)
What is the type of assay used to measure prolactin?
IRMA (ELISA)
- Remember “hook effect”
- Consider macroprolactin
