Introduction Immunochromatographic assays also called lateral flow dipstick immunoassay or simply strip tests Are simple devices intended to detect the presence or absence of a target analyte ID: 565575
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Slide1
ImmunochromatographySlide2
Introduction
Immunochromatographic assays, also called lateral flow dipstick immunoassay or simply strip
tests
Are simple devices intended to detect the presence (or absence) of a target
analyte
in sample (matrix) without the need for specialized and costly equipment.
A widely spread and well known application is the home pregnancy test.Slide3
The
liquid sample is dropped on the sample pad, the antigen in the sample forms an
immunocomplex with the antibody labeled with colloidal gold.
Its complex moves along with the liquid sample, and makes a contact with the antibody immobilized on the membrane, followed by forming an immunocomplxes with the immobilized antibody, resulting in generating a colored red purple linePrinciple of Immunochromatography Kit:Slide4
Appearance
of red purple line on the membrane indicates the presence of antigen in interest in the sample. Since the liquid of the sample migrates through the membrane very fast, it makes it possible to detect the presence or absence of antigen within 15 minutes
.
An Immunochromatographic unit is completed by attaching the sample pad at the end of the membrane.Principle of Immunochromatography Kit:Slide5Slide6Slide7
What do you need to know about your sample before?
Variability of target molecule concentration (defines sample volume to be applied)
Variability of sample composition, e.g. pH (sample composition may have to be adjusted by sample pad pretreatment).
Sample viscosity (limits density of the pad material)
Unspecific interactions of your target with the pad material (defines pad blocking requirements).
Unspecific interactions of the target with test line reagents (may require additional adjustments).
Need of retention of particles contained in the sample (e.g. red blood cells).
Sample PadSlide8Slide9
Typically, it is the physically smallest part in the lateral flow test.
Fulfills a diversity of functions:
Absorbs the volume in which the detector conjugate is added to the pad.Does not interact with the conjugate.
Maintains he conjugate integrity upon dryingMaintains the conjugate in the dry state (can easily be more than a year at RT).Releases the conjugate easily and completely upon contact with the sample liquid.Allows for interaction between the detector reagents in the conjugate and the target in the sample.Conjugate pad:Slide10
Typically, this is a “large pore sized “ nitrocellulose (NC) membrane.
The membrane are available in a very broad range of sample flow characteristics.
All NC membranes contain a surfactant, usually an anionic surfactant, that makes them hydrophilic.
The Analytical Membrane:Slide11Slide12
Its task is to soak the sample liquid and all reagents that have not been absorbed at the test and control lines.
It must prevent the backflow of this liquid into the drying membrane as long as possible.
Select a cotton paper with an absorption capacity that is much higher than the sample volume.
The WickSlide13
Step 1: Sample placement
To
perform the test, a sample is placed on the sample pad at the end of the strip.
The sample may be used alone as commonly done with urine or serum or whole blood or plasma compatible tests, or it may be mixed with a buffer specific to the test
How they work:Slide14
Step 2: Molecules
solubilize
with the addition of the sample, the detector molecules are solubilized.
When solubilized the detector molecules mix with and bind to the analyte in the sample (if analytes is present).Slide15
Step 3: Capillary action
Then
capillary action draws the fluid mixture up the sample pad and into the membrane.
The sample/detector molecule mix continues to move up the membrane until it reaches the analyte capture molecule.
In
these lines a second (and third) antibody or antigen, immobilized as a thin stripe in the nitrocellulose will then capture the complexes if it is positive for the target analytes.
The
control line should always show as a visible line, otherwise the test is invalid and must be repeated. If the test is positive, a colored (typically pink or purple ) line develops along with the control line Slide16
Step 4: Excess absorbed
Excess
buffer along with any reagent not captured at the test of control line will then moves into the absorbent wicking pad.Slide17Slide18
Can detect antigen or antibody
Commercially available.
Easy to perform
Limited / no instrumentation.Single use, rapid test.User friendly format Very short time to get test result.Long-term stability over a wide range of climates
Relatively inexpensive to make.
Advantages:Slide19
Results are qualitative.
Rapid tests can be less sensitive and less accurate.
Disadvantages:Slide20
Strips
Cassette
Multi-devices
Midstream (hCG and LH only).Type of Lateral test (Format):Slide21Slide22
It can be applied for multiple test platforms for liver, sexually transmitted diseases, cardiac markers, as well as women’s and men’s health (hCG, VDRL, CMV, PSA, H. Pylori, Troponin I, TORCH, HIV, HBs Ag, HBV, HCV, RA, CRP, ASO, SLE, IM, salmonella IgM & IgG, …)
Applications of Immunochromatographic assays: