Lateral-flow assays are essential tools in diagnostics, offering rapid and accessible testing solutions across a variety of applications.
However, assembling a functional and reliable assay requires precise techniques, careful material handling and an understanding of flow dynamics.
This step-by-step guide highlights the full process of creating a generic hCG lateral-flow assay, offering practical advice and insider tips to optimize assembly techniques and material integration insider tips to optimize assembly techniques, select appropriate materials, and integrate them effectively across the test strip.
Download this guide to explore:
- Best practices for membrane preparation, striping and assembly
- Key strategies for minimizing defects and improving reproducibility
- Insights into material overlaps, sample handling and quality control
A step-by-step guide
Creating a
lateral-flow
assay
A step-by-step guide for
creating a lateral-flow
assay
If you’re curious about the practical aspects of the assembly and striping process for
a lateral-flow assay, this guide is for you. Please note that this information is a guide
for creating a generic hCG test format. Different ways of assembling the system and/
or applying reagents exist. You should thoroughly investigate all patents and methods
that could be used in the creation of your diagnostic test to optimize your process.
Getting started
Bring all your components and laminate cards together so that you can begin to
assemble your assay in an orderly and organized manner.
Step 1
Lay out your laminate. Some device manufacturers create
small jigs that allow you to align all the components of
your test uniformly and prevent the cards from slipping.
You can mount pins on the jig to keep the laminate
securely in place and to mark specific sites where material
will be placed or overlapped.
Top tip: A jig is not always necessary but is of great
assistance in building a uniform test.
2Creating a lateral-flow assay
Step 2
Once the laminate is laid out, uncover the adhesive section
of the plastic so that it is facing up and not in contact with
anything else.
You’ll lay down the diagnostic membrane of your choice in the
next step, but first you should examine the membrane to be
certain that it’s free from scratches, particles, or other defects
that may alter your test results.
Don’t handle the membrane with your bare hands!
Hold the membrane up to the light using forceps, tweezers, or
gloved hands so that you can look at it at an angle along the
surface.
Step 3
Hold each end of the membrane so that the membrane is taut.
Your gloved fingertips (or forceps) should touch only the last
centimeter of each end of the membrane. Lay the membrane
down in a straight line on the adhesive.
Use a wooden brayer to carefully press the membrane down
onto the adhesive so that there are no air bubbles. A brayer
is a small tool with rotating head similar to a wallpaper roller.
Before using, thoroughly wash the brayer’s roller in alcohol
and allow it to dry. Then gently use the brayer to press the
membrane onto the adhesive.
3Creating a lateral-flow assay
Step 4
Add the absorbent pad. The absorbent pad is placed at the far
end of the test in the direction of flow, after the test area, and
should overlap the membrane by a few millimeters. The exact
amount for overlap depends upon your assay chemistry, but a
typical amount is 1.8 to 2.0 mm.
The absorbent pad should be handled in the same manner
as the membrane. It can be rolled with the brayer to ensure
evenness and to remove air bubbles.
Step 5
The next component to add to the laminate is the conjugate
pad. This material is added to the opposite end of the test
as the absorbent pad (i.e., at the origin of sample flow) and
should overlap the membrane by 1 to 2 mm. Again, handle the
conjugate pad the same way as the other components and roll
to secure.
Normally, the conjugate release pad is pretreated with a
combination of reagents to create a stable environment for
the protein and ensure good resolubilization by the sample.
Another option is using pretreated conjugate release media.
4Creating a lateral-flow assay
Step 6
Next, place the sample wick on top of the conjugate release
pad. The sample wick is the beginning point for the test. Ensure
a small overlap between the sample wick and conjugate pad.
The sample wick may also be pretreated. Sample wicks often
contain a cocktail of detergent and buffer to condition the
sample prior to entering the experimental zones.
Step 7
The next step is to stripe the test. Normally, you stripe 3 lines. • The first line in a one-step hCG system is a gold coupled
mouse anti-hCG antibody. The conjugate should be striped
down in a buffer that allows rapid resolubilization by the
sample. Rapid resolubilization is necessary to allow the
antibody and analyte to bind to one another to create a
complex that will then attach at the readout (test) line.
• The next line is the test line, which is where the antibodyanalyte complex binds if present in the sample. This line is
located on the membrane. In our generic hCG system, this
line is another mouse-anti-hCG antibody.
• The final line is also located on the membrane and is the
control line. At this line, a goat-anti-mouse antibody is bound
irreversibly to the membrane. Any conjugate material not
bound to the original test line will bind here to signal that the
sample front has passed the test line and an accurate test
reading can be made.
5Creating a lateral-flow assay
Step 8
Select the reagents to make up the various buffers and
impregnating agents in the test. Buffers suitable to
optimize are:
• Buffer for striping capture antibodies on the reaction
membrane: 10 mM phosphate, pH 7.2
• Buffer for conjugate application: 20 mM phosphate,
10% sucrose, 0.2% Tween 20, 1% PVP K-30
Step 9
Once you’ve completed the processes of assembly and striping,
you can cut the laminated cards into appropriately sized strips.
You can cut the strips by hand or use an automated “guillotine”
device that cuts the completed cards into strips of uniform
width. The strips are then ready to be placed into a cassette
that will protect the test and provide ease of handling for the
end user.
6Creating a lateral-flow assay
Useful content • Lateral flow assay troubleshooting guide &
how to switch diagnostic membranes
• Exploring the future of Lateral Flow Assays
Development
• 10 top tips for LFA development • Troubleshooting lateral-flow tests • Top 5 tips for testing stability in lateral-flow
assays
• Interactive brochure: Every step of the assay • Blog: Custom lateral flow assay projects • Blog: How depth filters work in blood
separation
Lateral-flow assay development
• Blog: Blood separation challenges (and how
to solve them!)
• Infographic: Considerations for lateral flow
membrane selection
• Sample pads • Blood separators • Conjugate release pads • Nitrocellulose membrane selection • Unbacked membranes for lateral flow
immunoassays
• Absorption pads
Other point-of-care testing • Blog: Membranes 101: A guide to membranes for POC diagnostics • Infographic: Navigating flow-through immunoassays • Infographic: Did you know…track-etched membranes • Flow-through immunoassays • Dipstick colorimetric assays
7Creating a lateral-flow assay
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