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What Might Be The Reason For False Positive Results In The Lateral Flow Assay?

Posted on July 8, 2021 by lateralflow 

The lateral flow assay (LFA) is a well-established platform for point-of-care (POC) testing and the reason is its low cost and ease of use,  Conventional LFAs yield qualitative or semi-quantitative results and necessitate the use of specialized instruments for quantitative detection. The lateral flow assay (LFA) is a paper-based platform for detecting and quantifying analytes in complex mixtures. The sample is placed on a test device, and the results are displayed within 5–30 minutes. Because of the low development costs and ease of production of LFAs, their applications have expanded to a wide range of fields requiring rapid testing.

Test-strip-cutting-machine HOT SALE!

A liquid sample, such as blood, serum, plasma, urine, saliva, or solubilized solids, is added directly to the sample pad and wicked via the lateral flow device to be used. The sample pad neutralizes the sample and filters out undesirable particulates like red blood cells. The sample can then flow freely to the conjugate pad, which contains highly colored or fluorescent nanoparticles with an antibody on their surface. These dried nanoparticles are released and mix with the sample when the liquid reaches the conjugate pad. If the antibody recognizes any target analytes in the sample, they will bind to the antibody.

There has been an increase in demand for point-of-care multiple diagnostic assays with extensive testing lines that allow for the rapid and simultaneous detection of multiple analytes point-of-care samples in current history. Such assays (potentially a single LFA) should be simple to carry out without the assistance of a laboratory or individuals trained in chemical analysis. LFAs are excellent candidates because they are inexpensive to produce, simple to use, and, most importantly, widely accepted by users and regulatory authorities.

Even though lateral flow assay is a beneficial and the most used way for testing however there are some deficits and problems seen in the Lateral flow Assay and the results, specifically. This is an often asked question that why the positive false results in the lateral flow assay occur and there are different reasons for it depending upon the branch of science. However, the reason that the false positive results come out in the Lateral Flow Assay is discussed in detail in the following answer. When a test line is observable in the utter lack of the desired analyte, a false positive result can be caused by a variety of factors, including non-specific binding, cross-reactivity, or heterophilic antibodies.

To optimize the assay and eliminate false positive results, it is necessary to identify which of these factors or combinations of factors is causing a false positive result. Non-specific binding occurs when there is a non-specific interaction between the antibody-nanoparticle conjugate and the antibody at the test line, regardless of whether the target analyte is present or not. Non-specific binding can frequently be avoided by simply optimizing the conjugation procedure for a specific protein. Optimizing the antibody/protein loading (too little or too much antibody can result in non-specific binding), the antibody incubation time, and the reaction buffer are all aspects of conjugation optimization. Proteins, surfactants, or polymers can be used as blocking agents in a test strip component (e.g. conjugate diluent, sample pad pre-treatment, conjugate pad pre-treatment, running buffer, etc.). Cross-reactivity differs from non-specific binding in that it occurs when the antibody binds to an analyte in the sample that is NOT the target analyte. This is a more difficult problem to solve, and it usually necessitates the replacement of antibody systems that do not cross-react with unwanted analytes.

A sample containing heterophilic antibodies (endogenous antibodies that bind assay antibodies) may produce a false positive result. Several types of heterophilic antibodies can end up causing cross-linking between the antibody conjugated to the nanoparticle and the antibody at the test line, even when the target analyte is not present. If the false positive result shows the same signal intensity even after diluting the sample, rather than a linear decrease in signal intensity, it could be due to heterophilic antibodies. Human antibodies in a specimen that interact with assay antibodies to produce false-positive or false-negative results are referred to as heterophilic antibodies. The heterophilic antibody can develop in a patient as a result of exposure to specific animals or animal products, infection with bacterial or viral agents, or non-specifically. A false-positive result test can occur for several points of that some poor test technique by the screenee (e.g., thick smear on Haemoccult card) and invalid test reading. A delayed reading can also result in a false-positive result, especially when using a latex agglutination test. A false-positive result can also be caused by physiological blood loss in a subject who is not within the normal range.

False-positive results may happen as a result of homeostasis disruptions (e.g., incomplete excision repair, cell cycle arrest, inflammation) that can take place before cell membranes are compromised and necrosis/apoptosis is detectable by histopathology. These consequences can increase DNA migration at the site of contact as well as peripherally in other tissues as the tissue responds to cellular injury and the systemic inflammatory response progresses, depending on the dose concentration and administration frequency.


The lateral flow assay is simple, easy to access, and economical but due to different reasons that are above mentioned the results can come as not true, sometimes. LFAs' distinct and exceptional properties have aided in the detection of disease biomarkers and infectious agents in medicine, agriculture, food safety, and environmental safety. Although the method's principle has managed to remain unchanged for decades, LFA techniques have been continuously improved, resulting in increased sensitivity and reproducibility, as well as the simultaneous detection of multiple analytes. Importantly, these assays can now be performed outside of the laboratory, which offers significant benefits for use in developing countries and at the point-of-care, whether in the field or in more conventional clinical settings. Even though lateral flow assay can show false positive and vice versa results, other methods can be tried and tested for reassurance.


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