A Guide To Rapid Antigen & Lateral Flow Tests

A Guide To Rapid Antigen & Lateral Flow Tests

A comprehensive guide to rapid and lateral flow testing, including the advantages of this method and how it works

Rapid or lateral flow testing, also known as rapid antigen testing, is a method for determining the presence of certain infectious organisms in a sample. They have been in common use for testing for and detecting COVID-19 during the recent pandemic but the technology has existed for some time and has a number of other uses. 

Commonly the technology is used in self-testing or at-home testing. It is different from a PCR test, even though the two share similar or overlapping use cases.

What is a rapid antigen test?  

A rapid antigen test is a diagnostic test that detects the presence of a specific viral antigen in a sample. This test is often used to detect the presence of a viral infection, such as COVID-19, in a patient. It is a quick and easy way to get results, as the test can be performed on-site in a matter of minutes.

The test involves taking a sample, such as a swab from the nose or throat, and then using a special device to detect the presence of the viral antigen. Rapid antigen tests are often used in settings such as clinics, nursing homes, or schools, where fast results are important for identifying and controlling outbreaks. 

What is a lateral flow test and is it different from a rapid antigen test?

A lateral flow test is a type of rapid diagnostic test that is used to detect the presence of a particular substance, such as a virus or antibody, in a person's body. It works by using a paper strip or membrane that contains specific markers or antibodies that react with the substance being tested for.

A lateral flow test is different from a rapid antigen test in that it can be used to detect a wider range of substances, including proteins, DNA, and antibodies, while rapid antigen tests are usually used to detect a specific virus or bacteria. Additionally, the results of a lateral flow test are usually read by the naked eye, while an antigen test may require a specialised reader or machine to interpret the results.

Overall, both lateral flow tests and rapid antigen tests are valuable tools for quickly diagnosing and identifying infectious diseases in a variety of settings, from hospitals and clinics to schools and workplaces.

If the test detects viral proteins are present it will be indicated by a colour strip on the test results showing alongside a control line. 

Common names for a lateral flow test or devices

Lateral flow tests are often referred to as:

  • Lateral flow tests (LFT)
  • Lateral flow devices (LFD)
  • Lateral flow assays (LFA)
  • Lateral flow immunoassays (LFIA)
  • In-vitro diagnostic devices (IVD)

However, they are more commonly referred to as rapid, quick or RAT tests by the public.

How does a lateral flow test work?

Once a sample is introduced, it will travel through the test device, starting from the conjugate pad, then onto the nitrocellulose membrane and finally onto the absorbent pad. In sandwich assays, a positive outcome is indicated by a coloured line appearing at the test line position whereas in competitive assays, a positive result is indicated by the lack of a coloured line at the test line position.

This process, known as lateral flow assay, is a rapid and cost-effective method for detecting various analytes in biological samples. It is widely used in medical diagnosis, food safety testing, environmental monitoring, and more.

The simplicity of the test device makes it suitable for use in remote and resource-limited settings. With advances in technology, lateral flow assays have become increasingly sensitive and specific, providing accurate and reliable results within minutes.

What is a sandwich assay and what is a competitive assay? 

A sandwich assay, also known as a capture assay, is a type of immunoassay used to detect and quantify the presence of a specific antigen in a sample. In a sandwich assay, two antibodies are used to sandwich the antigen of interest between them, forming an antibody-antigen-antibody complex.

One antibody is immobilised on a solid surface, such as a microplate, while the other antibody is labelled with a detection system, such as an enzyme or fluorescent molecule. The amount of signal generated is directly proportional to the amount of antigen present in the sample.

On the other hand, a competitive assay, also known as an inhibition assay, is another type of immunoassay used to detect and quantify the presence of a specific antigen in a sample. In a competitive assay, a labelled antigen competes with the unlabelled antigen in the sample for binding to a limited amount of immobilised antibody.

The amount of signal generated is inversely proportional to the amount of antigen present in the sample, since higher amounts of unlabelled antigen in the sample will result in less labelled antigen binding to the immobilised antibody, and therefore less signal.

Competitive assays are often used when high sensitivity is required, or when the antigen of interest is small and difficult to work with.

Is a rapid antigen test the same as a lateral flow test?

In general, yes – rapid antigen tests use lateral flow technology to test for the presence of antibodies. These have been popularised during the recent pandemic and are commonly used in COVID-19 rapid antigen tests but have been used prior to COVID in many other clinical and non-clinical settings.

Rapid antigen tests or IVDs are a type of test used to detect the presence of foreign antibodies (e.g. SARS-CoV-2) and other diseases in test subjects. It works by detecting antibodies that your body produces when it encounters certain types of viruses or bacteria.

What is a lateral flow immunoassay?

A lateral flow immunoassay (LFA) is a laboratory test that utilises antibodies to determine if there are molecules called antigens present in pathogens. A variety of antigens, like those found in viruses and bacteria, may be detected using an LFA. The LFA is a useful, sensitive, and accurate method of detecting the existence of antigens in a sample and has been especially effective during the COVID-19 outbreak.

Nonetheless, the technology has been in use for other purposes prior, including quick and non-invasive detection of Influenza, HIV or STIs. Additionally, LFAs have applications outside of medical diagnostics, such as in environmental science, chemical and pharmaceutical analyses, and even food and agriculture. 

What is the advantage of these types of tests?  

These types of tests offer many advantages. Firstly, they are quick and easy to administer, so a large number of people can be tested in a short amount of time. Secondly, they are cost-effective, making them accessible to a wider range of people.

Additionally, they are non-invasive and do not require a blood sample or other invasive procedure, which can be uncomfortable or even painful for some individuals.

Lastly, these tests can detect potential health problems at an early stage, enabling treatment to begin sooner and potentially preventing more serious health complications.

Overall, the advantage of these tests are speed and ease of use. They have a very low complexity level and offer results quickly.

How accurate are rapid flow tests?

Or how do we test and measure their accuracy? This link may help shed some light on the subject. Rapid antigen tests have become an important tool in the fight against COVID-19. However, their accuracy has been a topic of debate.

To measure the accuracy of a rapid antigen test, we can compare it to a gold standard test, such as a PCR test. Sensitivity and specificity are two important measures of a test's accuracy. Sensitivity refers to how well a test can correctly identify individuals who have the virus, while specificity refers to how well a test can correctly identify individuals who do not have the virus.

Recent data has shown that some rapid antigen tests have high sensitivity and specificity, with accuracy rates comparable to PCR tests. However, the accuracy of rapid antigen tests can be affected by various factors, such as the timing of the test, the performance of the test kit, and the prevalence of the virus in the population being tested.

It's important to note that rapid antigen tests should not be used as the only measure to diagnose COVID-19. They should be used in conjunction with other testing methods, and especially in symptomatic cases, seeking proper medical attention is essential. To reduce the spread of COVID-19, public health measures should also be taken, such as mask-wearing and social distancing.

Overall, rapid antigen tests can provide a fast and efficient way to detect COVID-19, but their accuracy should always be evaluated in the context of the specific circumstances and population being tested.

Overall rapid testing has proven to be very accurate as outlined in this longitudinal study of COVID diagnostic tests, however reports also indicate that there are contributing factors around the frequency and timing of testing that impact the results. As the study suggests, different variants of a virus can produce varying test results.

Further research by medRxiv highlights that some variants may produce false negatives or positives, leading to incorrect diagnoses. The findings highlight the importance of using accurate and reliable testing methods, especially in the context of a pandemic where prompt and accurate diagnoses can help control the spread of the virus.

As detailed in an investigative journal by Jama Internal Medicine, continuing to test over a period of time was important to the accuracy of results of over-the-counter rapid antigen tests, versus PCR tests which most often led to positive results after a single test.

Do rapid and lateral flow tests work for asymptomatic people?

As the previous report found, the tests can detect results in asymptomatic subjects, however, the best results were achieved by testing frequently over a number of days during which symptoms were displayed. 

Where the tests have been used among asymptomatic people in real environments settings, the reported performance has indeed been lower and was determined by factors including rate or testing and viral load.

How can lateral flow and PCR tests work together?

Over the recent COVID pandemic, the use of rapid lateral flow tests has been effective in the scale and speed at which they could be rolled out as part of a testing regime, which was supported by the 'gold' standard of PCR testing for confirmation and more complex cases.

The ability to test often, at home or outside of a clinical setting and receive results within minutes was highly useful, and when supported or confirmed by PCR or lab testing it was an important part of the later pandemic management in Australia. 

What is the difference between rapid antigen tests, lateral flow tests, and PCR tests?

PCR tests or Polymerase Chain Reaction tests use a different approach to process samples and detect a virus. With a PCR test, a sample is placed into a solution containing reagents that cause DNA and RNA molecules to bind with each other. This causes the formation of an insoluble solid mass that is then analysed in order to determine if they contain viral particles.

These tests should not be confused with rapid antibody tests, which use blood samples to detect antibodies generated in response to infection.

PCR tests Polymerase Chain Reaction tests detect the virus’s RNA in a sample by amplifying very small amounts of genetic material to use for detection. This allows for more accurate and sensitive detection of the virus than other methods, such as antigen tests. PCR tests are commonly used to diagnose COVID-19, as they can detect the virus even in asymptomatic individuals.

The process involves taking a sample, such as a nasal or throat swab, and then extracting RNA from the virus. The RNA is then copied and amplified through a series of cycles until it can be detected. Results can typically be delivered within hours to a few days, depending on the laboratory’s capacity. PCR tests have played a critical role in identifying and tracking COVID-19 infections and helping to control their spread.


Using a rapid antigen test 

Who can use the lateral flow test kit?

Laterla flow test kits are designed to be self-administered at home or in non-clinical settings—they do not require any clinical expertise to use. The tests are simple and quick to perform, and come packaged with instructions for use.

The test is designed to detect antigen proteins that are found on the surface of the virus. The antigen proteins bind to the antibodies in the sample and a coloured line appears on the test strip if the virus is present. These tests are often used to quickly identify people who may be infected with the virus, without the need for laboratory testing.

The accuracy of these tests can vary depending on the sample type and the amount of virus present in the sample. As such, it is important to understand the limitations of these tests and how to use them correctly.

Finally, it is important to note that these tests are not a replacement for traditional laboratory testing and should be used alongside other diagnostic methods to ensure the most accurate results.

Who can administer the test?

Most rapid antigen tests are designed to be self-administered. If you are intending to use these for a child or someone else, then do refer to the instruction for use guidelines from the test manufacturer. 

How much does a test kit cost?

In Australia rapid antigen tests cost on average between 5 and 15 AUD. 

How do I take the test?

All tests are required to come with instructions. All Biolink tests come with an instructions for use leaflet inside the packaging, and illustrated instructions on the packaging itself. You should always check the expiry date on the test, to ensure the test is valid for use, and carefully follow the manufacturer instructions.

General steps when using a rapid antigen or lateral flow test

Rapid lateral flow tests may require either an oral (saliva) or a nose swab. Biolink's nasal tests require a lower nasal swab but it is advised that you consult the specific instructions for use for each individual brand and test, as procedure steps may differ slightly.

The test you have might be different to the ones you have done in the past, even if it’s the same brand, so it's important to read the instructions carefully before you do any test to receive the most accurate results.

Before taking the swab

Wash your hands with soap and water or use a hand sanitiser. On a clear, clean and flat surface, lay out all the items in the test kit if your test does not come with a pre-filled tube like in the Ecotest version. Follow the instructions for use leaflet. 

Performing the swab

The user should perform the actions as per the instructions slowly and carefully for either a nasal or oral sample. Each test should be performed as instructed to ensure the best possible results / outcome from the sample collection.

Completing the test

Once the swab has been mixed in with the buffer solution, swirl it around in a circular motion for 10 to 15 seconds. Make sure to keep the swab submerged in the liquid solution while swirling.

Next, remove the swab from the collection device and discard it in the designated waste container, as directed in the test kit instructions. If you are using an Ecotest or Biolink pen, place the test device back in the buffer cap base, and push firmly. Wait the required time before reading results.

Reporting your result lateral flow test results

Depending on the test type, and where you are doing it, there can be different ways or requirements for reporting your test results. You should seek advice based on your relevant government authority.

Performing the test on someone else

If you are performing a test on someone else, it will help to talk them through the procedure and remain calm. If your test requires you to take a throat swab, use a light or lamp to improve visibility. Ask them to open their mouth to say “ahhhhhhh” loudly so you can see clearly. Stop doing the test if they become distressed. It's important to use a separate test kit for each person and each result—do not use the same test twice.

What do I do if my PCR is positive and my rapid test is negative or vice versa?

If your PCR (polymerase chain reaction) test is positive for a virus such as COVID-19 and your rapid test is negative, or vice versa, there are a few things you should do. First, it's important to understand that both tests have different sensitivities and specificities, which means they have varying levels of accuracy.

If your PCR test is positive and your rapid test is negative, it's possible that the rapid test missed the virus or that you are in the early stages of infection and the virus has not yet multiplied enough to show up on the rapid test. In this case, it's important to follow the guidance of public health officials and isolate yourself to prevent the spread of the virus to others. You should also consult with a healthcare provider to determine the best course of action and to receive care if needed.

If your rapid test is positive and your PCR test is negative, it's possible that the rapid test produced a false positive result. In this case, you should also consult with a healthcare provider to determine the next steps and to receive an accurate diagnosis. It's also important to continue following the guidelines of public health officials to prevent the spread of the virus.

Overall, if you receive conflicting results from your PCR and rapid tests, it's important to seek guidance from a healthcare provider. They can help you understand your results and provide the necessary care and support during this challenging time.

If you have a PCR test that is positive and your rapid test is negative, you should remain cautious and act based on the lab test results, as they are considered the gold standard for (COVID) testing. As mentioned previously, there are contributing factors that affect the results of over-the-counter or self-test antigen tests such as viral load and time of testing. 

What happens if I have a negative rapid antigen test result?

A PCR or NAACT is the gold standard of testing for COVID-19 and where possible, should be used to confirm a result from a rapid antigen test result for any type of test.

Depending on your circumstances and advice from your relevant government authority you can choose to confirm the rapid test with a PCR for a more conclusive result.

Can lateral flow or rapid antigen results be faked?

There have been reports in the media of people using other liquid substances to change or impact the result of a lateral flow test, causing them to show positive results. This article from the BBC suggests that people have been using other liquids (soft drinks like cola, cordial) to cheat, falsify or spoof positive results.

There is a possibility that certain drinks may contain antibodies that a lateral flow test uses to generate its results, leading to an a false positive result—likely due to the acidity levels, which may likely have an effect on the very sensitive test. 

Do rapid tests detect all known COVID variants?

According to the World Health Organisation and the Therapeutic Goods Administration of Australia, there are five main variants of COVID that are considered to be of concern: 

  • Alpha – first found in the United Kingdom
  • Beta – first found in South Africa
  • Delta – first found in India
  • Gamma – first found in Brazil
  • Omicron – first found in South Africa

To find out which tests can detect which variants, you can consult the TGA website, which publishes a list of Rapid Antigen Tests and their ability to detect variants.

Rapid antigen tests & other respiratory diseases

Do rapid tests detect the common cold?

Rapid tests are not primary diagnostic tools for detecting the common cold. This is mainly because the common cold can be caused by a variety of viruses, and there are no definitive diagnostic tests that can accurately identify all of them. Moreover, the symptoms of the common cold are generally mild and subside on their own after a few days without requiring extensive medical attention.

Therefore, diagnostic tests are not usually necessary, and healthcare providers primarily focus on managing the symptoms. However, if the symptoms worsen or persist, further diagnostic tests may be used to rule out other bacterial or viral infections.

Do rapid tests detect influenza?  

Yes, rapid tests can detect influenza. Rapid influenza diagnostic tests (RIDTs) are immunoassays that detect influenza viral antigens in respiratory specimens. RIDTs can often provide results within 15 to 30 minutes, making them a quick and convenient tool for diagnosing influenza infections.

However, it is important to note that RIDTs have limitations and may not detect all strains or types of influenza viruses with 100% accuracy. Therefore, confirmatory testing or further evaluation may still be necessary in certain cases.

Do rapid tests detect RSV?  

Yes, rapid tests are available that can detect respiratory syncytial virus (RSV) in a short amount of time. These tests work by identifying specific proteins or genetic material associated with the virus. Rapid RSV tests are often used in healthcare settings to quickly diagnose and treat patients with respiratory illnesses.

However, it's important to note that rapid tests may not be as accurate as laboratory-based tests and false negatives can occur. If there is suspicion of RSV infection, it may be necessary to follow up with additional testing or consult with a healthcare provider.

What about a combination rapid test that would involve using multiple testing methods to diagnose a condition?

Ordinarily, to diagnose a condition, it would involve a combination of blood tests, urine tests, and imaging tests to provide a comprehensive diagnosis.

The advantage of a combination rapid test is that it can provide a highly accurate diagnosis by gathering information from multiple sources, in a single testing device. It can save time, cost, and reduce the need for multiple follow-up appointments.

However, a combination rapid test cannot yield as much information, and may not be as accurate as comprehensive medical tests (such as blood, urine, and imaging tests).

Ultimately, the decision to use a combination rapid test should be based on the unique needs of each patient and the judgement of their healthcare provider.

Do rapid tests detect Strep A?   

Yes, rapid tests can detect Streptococcus pyogenes, also known as Strep A. These tests are commonly used in doctors' offices and clinics to quickly diagnose strep throat in patients presenting symptoms such as sore throat, fever, and difficulty swallowing.

The rapid tests involve taking a swab from the back of the throat, and results can be obtained within minutes. If the test comes back positive for Strep A, the patient can be started on antibiotics right away to help clear the infection.

Other applications for rapid antigen tests

Do rapid antigen tests detect HIV?

No, rapid antigen tests do not detect HIV. Rapid antigen tests are designed to detect specific proteins on the surface of a virus, while HIV is a virus that contains genetic material in its core. HIV detection requires specific tests that target the genetic material or antibodies produced by the body in response to the virus. Rapid HIV tests are available for quick and early detection of infection.

Do rapid antigen tests detect HPV?

No, rapid antigen tests do not detect HPV. Rapid antigen tests are designed to detect specific viral antigens of certain infections, such as COVID-19, influenza, or Strep A. HPV detection requires specialised tests, such as the HPV DNA test or a pap smear, which examine samples for the presence of HPV DNA or abnormal cells that may indicate HPV infection.

Do rapid antigen tests detect STIs?

No, rapid antigen tests are not designed to detect sexually transmitted infections (STIs). They are mainly used to diagnose infectious diseases in humans caused by viruses such as influenza or COVID-19.

Do rapid antigen tests detect diseases in livestock or pets?

Rapid antigen tests are occasionally used to detect diseases in livestock and pets, such as avian influenza in birds and bovine viral diarrhea virus in cattle. These tests are quick and easy to administer, making them a valuable tool for veterinarians and farmers in diagnosing and controlling disease outbreaks. However, it's important to note that not all diseases can be detected using rapid antigen tests, and confirmatory testing may be necessary for accurate diagnosis.

Are rapid antigen tests used for drug or alcohol detection?

No, rapid antigen tests are not used for drug or alcohol detection. They are primarily used for detecting certain microbial infections, such as the flu or COVID-19.

Is a pregnancy test a rapid antigen test?  

A pregnancy test is not a rapid antigen test. It is a different type of test that detects the hormone human chorionic gonadotropin (hCG) in a person's urine to determine if they are pregnant.

While the use of rapid antigen and lateral flow tests have come into common and popular usage during the COVID-19 pandemic, the technology is not new. It was modified to detect the novel coronavirus but has been in use in clinical and non-clinical settings for many years.

The devices or cassettes can be modified or set up to detect many different viruses, and continued work is being done to modify them to be more effective in the detection of multiple viruses from a single sample/test. Currently, tests are being developed for use in detecting InfluenzaHIV, and other diseases such as STI's, Zika, HPV.

Ana Martins

Ana is Biolink's Senior Medical Product Engineer
Back to blog