Immunoassay Flashcards
What is biotin
Water soluble vitamin B7
Cofactor in carboxylase reactions
Dietary sources of biotin
Egg yolk
Soybeans
Yeast
Liver
Kidney
Nuts
Cereals
Vitamin supplements
What is biotin prescribed for?
- Multiple sclerosis
- Hair, nail, skin supplements
- Biotinidase deficiency
- Mitochondrial disorders
Normal daily requirement of biotin
35-70mcg daily
Biotin content of regular multivitamin
30mcg
Dose of high dose biotin supplements
> 1mg/day = 30× usual multivitamin dose
Up to 10mg/day in hair/nail/skin supplements
Describe relationship between biotin ingestion and blood concentration
Peak 1-2hr after ingestion
Type of bond between biotin and streptavidin
Non-covalent, but strong, stable and specific
Type of bond between biotin and biological targets
Covalent, but does not interfere with target’s activity
Direction of biotin interference with sandwich assay?
Falsely low
Platforms using bitoin-stretavidin
Roche Elecsys
Ortho Clinical Diagnostics Vitros
Siemens Centaur
Beckman Access
Beckman DXI
Siemens Immulite
Direction of biotin interference with competitive assays?
Falsely high
How does biotin interference cause interference in immunoassays?
In both sandwich and competitive assays, biotin binds the solid phase. Analyte binding to solid phase, either by capture antibody or detection antibody is blocked. Analyte is washed away. In sandwich immunoassays, the direct relationship between analyte concentration and light signal means that when analyte is washed away, you get a falsely low result. In competitive assays, where the relationship between analyte concentration and light signal is inversely proportional, you get a falsely high result.
Examples of immunoassays prone to biotin interference
Roche TSH, FT4 and FT3
COrisol, ACTH
FSH, LH, E2, P4
HCG
Trop T
Testosterone
Total B12
Ways to detect and mitigate biotin interference
Serial dilution study
Biotin depletion protocols - eg addition of streptavidin-agarose beads to remove biotin before running on the analyser
Measurement of biotin
Repeat measurement using an alternate method unaffected by biotin
How long should biotin be withheld before repeating affected analyses?
At least 3 days
Draw an antibody/immunoglobulin
Constant region
Variable region - Ag binding site here
What is the difference between monoclonial and polyclonal antibodies.
Monoclonal antibodies are produced by a single clone of plasma cells and all bind to the same epitope of an antigen. Polyclonal antibodies are produced by many plasma cell clones and bind to a wide variety of epitopes on the antigen
Define “affinity” of an antibody
How tightly it binds to an antigen
Define “avidity” of an antibody
Number of potential binding sites. How many different epitopes it can bind on the antigen
What is a heterogeneous immunoassay?
Analyte-antibody complex is separated from remaining sample prior to analysis
What is a homogeneous immunoassay?
Analyte-antibody complex is NOT separated from the rest of the sample prior to analysis
What are three methods for separating antibody-analyte complexes from sample in heterogeneous immunoassays?
- Precipitation of antibody-analyte complexes
- Cross-linking
- Binding to solid-phase followed by a wash step
Classification of competitive assays
Sequential (sample added and incubation occurs before labelled analogue added) vs simultaneous (sample and labelled analogue added together at once and incubated)
Immunoassay detection methods
Fluorescence
Chemiluminescence
Radioactivity
Enzyme-based
Fluorescence detection methods for immunoassay?
- Dye-labelled antibody - fluorescent signal proportional to concentration of analyte
- Fluorescence polarisation (FPIA) - fluorescent labelled analogue, not analyte. When analogue is unbound by antibody it is free to rotate and light remains unpolarised. When analogue is bound to antibody, rotation slowed by mass of antibody and there is an increase in polarised light. Change in polarisation is directly related to amount of analyte in sample
Enzymatic detection methods for immunoassay?
- Enzyme labels
- ELISA
- EMIT
- CEDIA
Advantage of enzymatic immunoassays?
Amplification is inherent to the enzymatic process - more sensitive assay
What are some common enzymatic labels?
ALP
Peroxidase
G6PD
Beta-galactosidase
What are the products of enzyme-labelled immunoassays?
Colour product
Fluorescent product
Photon (chemiluminscent substrate)
Intermediate substrate for a second enzymatic reaction
With the aid of a diagram, describe an enzyme-linked immunosorbent assay (ELISA)?
Sandwich format with capture antibody bound to solid phase and enzyme-labelled detection antibody.
Describe EMIT (enzyme multiplied immunoassay technology)
Enzyme-labelled analogue competes with analyte for binding to antibody. Antibody-bound enzyme is unable to convert substrate to product. The higher the analyte concentration, the more enzyme labelled analogue will remain free to convert substrate to product. Analyte concentrations are therefore propotional to enzyme activity.
Describe CEDIA (cloned enzyme donor IA)
Genetically-engineered fragments of beta-galactosidase compete with analogue for binding to antibody, Only free enzyme fragments can combine to produce an active enzyme. The higher the analyte concentration the fewer bound enzyme fragments there are and the greater the activity of whole enzyme.
Describe turbidimetric detection in immunoassays
Turbidimetry measures the reduction in light transmission as light scattering increases due to the formation of large immunocomplexes by antigen-antibody cross-linking occurs
Describe nephelometric detection in immunoassays
Nephelometry is the measurement of scattered light at a specific angle of observation. As immunocomplexes form, light scattering increases and detected light at the specified angle increases.
Types of point of care immunoassay?
- Lateral flow
- Optical IA
Describe lateral flow immunoassay
Fluid sample is drawn through a porous membrane. Analyte binds labelled antibody and is captured by a capture antibody with a colour development step or the use of coloured microparticles
Describe optical immunoassay
Optical immunoassays use a change in light reflection when the thickness of a thin antibody film becomes thicker due to antigen binding
What are the applications for POC immunoassay?
Emergency department
In home tests
Important feature(s) of POC immunoassay devices?
They have a built in quality monitor to verify appropriate storage and device operation
Why do different immunoassays produce different analyte concentrations?
Different manufacturers use different antibodies with different affinities, avidities and binding epitopes. This can be circumvented using the same method for serial measurements but makes it difficult to establish universal reference intervals.
Investigations for interference for heterophile antibodies
- Check on another assay
- Serial dilution
- Removal of heterophile by HBT/normal mouse serum/immobilised protein A column/PEG
