Developing a point-of-care (POC) immunoassay can be both time-consuming and costly, with potential challenges in various aspects of its development and optimization.
From challenges with cross-reactivities to issues with inconsistent results, these problems are well-known and proven techniques exist to address and troubleshoot them.
Download this infographic to find answers to the following questions and more:
- Why am I encountering false positives in sandwich tests or false negatives in competitive tests with artificial samples?
- Why do I see variability between tests with artificial samples?
- Why are there cosmetic issues with my tests?
Do you experience false positive
results in artificial samples in your
sandwich tests or false negative
results in your competitive tests?
Troubleshooting
lateral-flow tests
Possible reasons and solutions for faulty results
Do you experience challenges with
human anti-animal antibodies (HAAA)?
Do you experience inconsistent results
from your tests?
Do you experience challenges with
cross-reactivities?
Do you experience issues with
samples from patients that suffer
from autoimmune diseases?
Do you experience challenges with
samples from specific regions or
ethnic origins?
Do you experience false negative
results in artificial samples in your
sandwich test or false positive
results in your competitive tests?
Do you experience test-to-test
variability with artificial samples?
Possible reasons Possible solutions
Antibody activity destroyed by membrane surfactant Use another membrane and/or replace antibody
Interfering substances in pad pretreatment buffers Analyze every chemical separately for test compatibility
Capture antibody (only in sandwich tests) or detector
antibody have insufficient on-rates
Use slower membrane and/or optimize T-line position
Conjugate is released too quickly and runs in front
of analyte
Optimize sugar concentration in conjugate dispensing
buffer
Faulty cassette Check and correct cassette design
Possible reasons Possible solutions
Hydrophilic interaction between conjugate and test line
(T-line)
Increase NaCl concentration in pads or run test at
different pH
Hydrophobic interaction between conjugate and T-line Add (nonionic) surfactants
Backflow from wick Increase wick absorption
Mechanical retention of conjugate particle Replace polyclonal antibody (Ab) with monoclonal Ab in
conjugate, improve conjugate blocking and/or dispensing
buffer to prevent aggregation
Specific interaction between conjugated antibody and
T-line antigen
Solutions can be very specific, very often the use of F(ab)2
fragments in the conjugate helps a lot
Possible reasons Possible solutions
Anti-rodent antibodies may be present due to frequent
contact of patients with rodents in the environment or
due to previous antibody therapies.
Add heteroblockers to the sample pad pretreatment
buffer.
