• Membrane flow rate too fast
• System flow rate too fast
• Inactivation of detection line antibodies (antigens)
• Insufficient detection line antibodies (multiple reasons)
• Insufficient gold label
• Inactivation of gold-labeled antibodies
• Gold label not reaching the detection line (multiple reasons)
• Inactivation of the analyte
• Unoptimized pH
• Unoptimized salt concentration
• Insufficient sample volume
• Substances inhibiting antigen-antibody reaction

False positive and false negative results are inaccuracies that may occur in diagnostic tests, impacting their overall accuracy. In a nutshell:
• False positive: This happens when a test incorrectly indicates the presence of a target analyte (e.g., a specific protein, antigen, or antibody) in the sample when it's actually absent. This can lead to unnecessary treatments or interventions for the tested individual. Factors contributing to false positives include cross-reactivity with other molecules, test component contamination, and incorrect interpretation of results.
• False negative: This occurs when a test incorrectly indicates the absence of a target analyte in the sample when it's actually present. This can result in delayed treatment or intervention for the tested individual. Factors contributing to false negatives include the target analyte's concentration being below the test's detection limit, improper sample collection or preparation, and degradation of test components over time.
It's important to note that no diagnostic test is perfect, and all tests have some degree of false positive and false negative results. LFAs are known for their ease of use, rapid results, and low cost. However, they may have lower sensitivity and specificity compared to other diagnostic methods, which could result in higher rates of false positive and false negative results. To ensure accurate interpretation, it's essential to consider the test's limitations and the specific context in which it's used. In some cases, confirmatory testing using a more accurate method may be necessary.

False positive test results can occur due to a variety of factors:
1. Gold standard particle issues: Ineffective protein coating or loss of coating during handling or storage can cause gold standard particles to bind non-specifically, leading to false positives. Excess gold standard particles can also contribute to this issue.

2. Capture antibody-related factors: Non-specific binding reactions of the capture antibody can occur due to hydrophobic forces, non-specific immune reactions, antibody additives, large positive charges, and high concentrations of sulfur-containing amino acids.

3. Chemical reagent problems: Chemical reagents used during the production process can be flushed out, resulting in a more hydrophobic membrane that causes background staining or false positive signals. Using excess protein or surfactant for membrane closure can also result in false positives.

4. Sample-related issues: Some samples contain substances that bind non-specifically to gold standard particles or capture antibodies, producing non-specific results. Factors such as hydrophobic bacterial fragments, high concentrations of sulfur or SH groups, high positive charges, or a large number of molecules that block the membrane can all interfere with the test and cause false positives.

To minimize the risk of false positive results, ensure proper handling and storage of gold standard particles, use high-quality and well-coated particles, optimize the production process for the nitrocellulose membrane, and carefully select and use chemical reagents and additives. Additionally, consider the properties of the sample being tested and any potential interference it may cause.

1. Marker destruction:
• Loss of antibodies and competition between them
• Damage caused by hydrophobic effects on antibody surfaces
• Antibody hydrolysis due to insufficient drying or storage in non-dry conditions

2. Ineffective capture antibodies:
• Failure to bind to capture antibodies
• Hydrophobic effects damaging the antibodies
• Excessive salt in labeled antibodies

3. Poor release of markers:
• Hydrophobicity of the marking pad
• Hydrophobicity of the membrane
• Insufficient sugar in markers due to storage in humid conditions

4. Poor membrane performance:
• Sticky samples, requiring filtration or smaller pore size
• Excessively fast flow rates
• Blocked membrane due to hydrophobic effects

LFA applications are numerous and diverse, making it impossible to cover all of them here, but the following is a selection of the most popular use cases:

Pregnancy Tests:
One of the most famous examples of LFAs might be the home pregnancy test, which has been around since the 1980s and detects hCG in urine produced by the placenta during pregnancy. Over the years, sensitivity has improved, and current tests usually provide a positive result within 10 or 11 days after conception.

At-home COVID-19 Tests:
Many people have likely experienced at-home COVID-19 antigen tests. Most devices used at home aim to detect the presence of the virus itself (i.e., potential infection) rather than antibodies indicating exposure or vaccination. They target antigens associated with viral exposure and accessible parts of the virus, such as spike, envelope, membrane, or nucleocapsid proteins.

Human Pathogens:
While SARS-CoV-2 might be the first human pathogen that comes to mind, LFAs are also useful for detecting a range of targets, including Plasmodium parasites causing malaria, Mycobacterium tuberculosis (the causative agent of tuberculosis), Hepatitis B Virus, and Human Immunodeficiency Virus (HIV).

Animal Pathogens:
Not just limited to human medicine, LFAs also benefit veterinary applications. In addition to pregnancy detection in animals, they can detect various veterinary pathogens, making them particularly useful for practitioners, farmers, breeders, or owners working outside of clinics. Examples include African Swine Fever and bovine enteric pathogens. LFAs played a crucial role in eradicating rinderpest, and farm testing also helps prevent pathogens in food production animals from reaching consumers. However, there has been criticism of the quality of detection for some diseases, such as rabies.

Food Contaminants:
Food contaminants come in various forms, from pathogens and pesticides to heavy metals and toxins. Aflatoxins, secondary metabolites produced by Aspergillus and Penicillium that are present in cereal-based foods, nuts, and spices, are one such example. Numerous LFAs have been developed for these toxins, covering a group of chemically related compounds. Due to their small size, LFAs for aflatoxins are usually indirect. Multiplex tests for aflatoxins and other mycotoxins have also been developed. Rapid diagnosis of Salmonella, a common foodborne pathogen, helps speed up treatment, identify contamination sources, and prevent further spread. LFA technology offers a means to determine effective binders for test strips using phage display technology and can even distinguish between live and dead Salmonella enterica.

Environmental Pollutants:
Detecting environmental pollutants, including pesticides, Bisphenol A (BPA), and heavy metals, is vital for protecting humans, animals, and the environment. Portable detection solutions enable analysts to identify problems on-site and take quick action. LFAs have been developed to detect mercury, chromium, and cadmium ions. BPS, a concerning endocrine-disrupting compound, can also be detected with this technology, showing good results compared to lab-based tests. Organophosphate pesticides can be detected with indirect LFAs, and a test for simultaneous detection of clothianidin and thiamethoxam in water has been developed.

Allergens:
Individuals with allergies must carefully ensure they avoid their triggers, and food manufacturers must ensure products declared as "free from" are truly so. LFAs offer a quick and simple method for ensuring safety and have been developed for a range of common allergens, including gluten, casein, soy, mustard.

Therapeutic drugs that require close monitoring:
For some therapeutic drugs, there is a fine line between ineffective doses, optimal doses, and levels that exceed safety (where serious adverse reactions may occur). To ensure that patients stay within the optimal dose range, close monitoring is needed, and diagnostic aids like LFTs can provide this monitoring. Digoxin is a cardiac glycoside used to treat tachycardia and is one such drug. Scientists have developed an LFT that can be read by a smartphone app to help patients self-monitor their medication.

Abused drugs:
Urine analysis (which LFA is well-suited for) remains one of the most common methods for illicit drug testing in the UK, providing rapid results for workplace testing or law enforcement scenarios. A detection method for cocaine has also been developed, which differs from many abused drug detection methods due to the small molecular weight of cocaine, adopting a competitive format and using biomimetic materials for non-competitive formats. LFAs have even been developed that can detect Δ9-tetrahydrocannabinol, cocaine, opiates, and amphetamines in sweat from fingerprints.

1. The advantages of LFTs include:
2. Affordability
3. Rapid results (usually within minutes)
4. Convenience and portability, especially for visually read methods
5. Ease of use
6. Stability at room temperature, making them useful for point-of-care, home testing, or remote areas with limited refrigeration
7. Long shelf-life
8. No power source required for most tests
9. A variety of digital readers available for different environments
10. Small sample volume requirement
11. Readers that can improve result interpretation and record accuracy
12. Capability for multiplex detection

1. The disadvantages of LFTs include:
2. Particulate or sticky samples may cause device blockage or instability
3. Pretreatment may be necessary for some sample types
4. Lower accuracy compared to laboratory-based tests (lower sensitivity and specificity)
5. Proper handling of potentially infectious samples is required after testing, which can be challenging outside of care settings
6. Generally not suitable for high-throughput analysis
7. Results need to be read within a limited time to avoid fading or overdevelopment
8. Typically qualitative (yes/no), hindering interpretation of infection stage or risk (except digital reading devices)
9. Desktop or handheld readers add complexity and cost for users
10. Reproducibility may vary between batches
11. Cross-reactivity can be challenging, especially for multiplex assays