QUANTITATIVITY OF PCR AND ELISA vs LFAs AND EQUIVALENCE VALUES
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QUANTITATIVITY OF PCR AND ELISA vs LFAs AND EQUIVALENCE VALUES
Here are our most frequently asked questions about equivalence values. If you do not find an answer to a question you may have, please contact us via the chat bubble in the bottom right corner.
PCR and ELISA are both assays that, when invented, were performed manually (without a machine), and where the results were read visually by the naked eye or under a microscope.
Before diving into details, let’s first define and explain frequently used terms that relate to assays and diagnostic testing in general.
An important epidemiological distinction is that of “infected” vs “infectious” individuals.
Negative individuals may be “naive” (having never been infected in the first place) or previously infected, but where the pathogen is no longer present or undetectable (but antibodies against them may persist.)
Infectious individuals are infected with actively replicating or replicating competent pathogens, and the pathogen is detectable.
Infected (but non-infectious) individuals had an infection that was cleared and where replication competent pathogens are not being shed, excreted, or exhaled. Pathogens, parts of pathogens, viral fragments, etc. that are “replication incompetent” are often detected by PCR although such particles do not possess threat of infection to others.
All life, no matter how complex, on the most fundamental level have one thing in common; deriving metabolic advantage from a proton gradient.
All organisms, from the simplest virus to animals, are made up of proteins, which in turn are made up of peptides, which are made up of amino acids. Simple sugars make up polysaccharides, which organisms synthesize and use in metabolism.
Proteins and sugars make up complex molecules. On a microscopic level, these consist of a complex arrangement of micromechanical structures with a myriad of unique electrostatic field signatures based on the individual atoms and the states that they are composed of. Every microscopic subsection of these molecules, if they can be uniquely identified, could be considered to be an antigen.
The ideal antigen is a particular part of a particular molecule that can be uniquely recognized.
What “recognizes” the antigens are antibodies, and they can do so from a distance, because it is not a visual or chemical reaction that takes place when they bind, but rather a match in the electrostatic signature of the antigen and the opposite electrostatic signature of the antibody that cause them to “bind”. They “click” together much like if you fixed a bunch of magnets in a complicated arrangement in 3D space, and then you made an opposite arrangement of reversed polarized magnets that would “bind” if the arrangement matched.
Here one should comprehend that in this analogy, the arrangement could still “bind” together if one or more of the magnets were shifted just slightly out of its ideal position, although the binding would be “sub-optimal” and less “attractive” overall. If you kept shifting the magnets around, you would eventually end up with an arrangement that would not bind at all.
The analogy of antigen-antibody optimal vs sub-optimal binding is what happens as mutations in organisms produce a shift in the antigen structures that cause the binding to be less optimal. The level of binding capability of an antibody to an antigen is called “affinity”.
The specific region of an antigen that an antibody can bind to is called an “epitope.”
Natural infections in immuno-competent “naive” individuals (someone with a healthy immune system that has not previously been exposed to a particular pathogen), typically produce a number of different antibodies against many different epitopes.
Artificial infections (vaccines) that use live or attenuated virus or bacteria plus an adjuvant to generate an immune response, typically stimulate the immune system to generate many different types of antibodies, similar to a natural infection.
Protein sub-unit vaccines however, generate a very specific and narrow immune response only against the specific epitope that is present in the vaccine. Such vaccines are called “monovalent”.
To overcome this narrow response or target multiple pathogens with a single vaccine, more that one sub-unit may be added to the vaccine, making it “multivalent”.
Current mRNA vaccines (aka gene-therapy) also generate a very specific and narrow immune response against the epitope on the antigen that is produced when the mRNA is transcribed into proteins in the ribosomes of infected cells.
We know historically from the treatment of bacterial and viral infections that a single anti-bacterial or anti-viral drug when used over time causes the pathogen to develop anti-bacterial or anti-viral resistance, as the organism naturally evolves and mutations select for variants that escape the destructive mechanism of the drug. Therefore, modern treatments of infections often contain “a cocktail” of different drugs, each with different destructive properties, that work together to be less susceptible to single target resistance adaptations as the pathogen inevitably evolves.
Analogous to this, an immune response that consists of a plethora of antibodies rather than just a single epitope binding antibody, has a much greater chance to fight off a current infection as well as future infection, because the chances that the pathogen simultaneously will evolve to escape all of the target regions of antibodies at the same time is astronomical in the evolutionary arms race between hosts and pathogens.
For this reason, natural infection (presuming that the host survives), or vaccination with a sterilizing multi-epitope target vaccine, inevitably produces a better and longer lasting immune response than single epitope non-sterilizing vaccines.
LFA antibody tests provide an inexpensive, quick, and convenient way to determine if and what kind of antibody response an individual has mounted against a particular pathogen, and studying how the various antibody levels are weaning over time, is a great tool in our immunological understanding of a disease, and to determine the effectiveness over time of a particular vaccine vs natural infection.
What we have also discovered is that the nasal secretions and the composition of the mucus membranes vary between peoples of various ethnicities. Thus, its particularly important to test LFAs across a wide variety of people with different ethnical backgrounds and correct for variations in the composition of excretion. This is typically done by increasing the amount of detergent used in the reagent buffer, but not so much as to affect the sensitivity or limit of detection (LOD) of the test kit.
Real-Time (RT) PCR assays are qualitative diagnostic tests that, when not invalid, produce either a positive or negative result. The number of cycles a PCR assay runs for is typically decided by a country’s health government, such as the CDC in the USA. The higher the number of cycles the PCR runs to, the higher the chance to catch weak positive results, but this also means that false positive results will occur more frequently.
Distinguishing false positives from weak positives at high cycle values is not trivial, and has caused a lot of controversy in the recent SARS-CoV2 epidemic.
The “Cycle threshold” or Ct value of an RT-PCR assay refers to the number of cycles needed to amplify viral DNA/RNA to reach a detectable level.
If a signal indicating the presence of the target sequence is detected before the number of cycles hits the recommended upper value, the test is considered to be positive and the number of cycles is recorded as the Ct value for that specimen.
While the PCR assay itself is not technically quantitative on a cycle by cycle basis, counting the number of cycles until a target is detected can be interpreted quantitatively.
PCR test are often referred to as Nucleic Acid Amplification Tests (NAAT).
PCR, when used correctly, is a great diagnostic tool for detecting very low concentrations of a target sequence, potentially down to just one copy or DNA/RNA in the sample, which makes it very sensitive. Also the PCR method is great at amplifying (making copies) of a target sequence, as the amount doubles for every cycle.
This however also comes with some drawbacks. The ultra sensitive nature of PCR makes it extremely susceptible to process errors, contamination from other neighboring samples, or from the environment, since just one single fragment of contamination will be amplified along with the specimen.
Because every cycle doubles the amount of target if present, the final Ct of a sample depends substantially on how many target particles were in the sample to begin with, which can vary greatly between same samples taken from the same patient or by different collection methods or people.
PCR Ct values are therefore not a good measure of viral load, because variations in Ct values will vary much more depending on the microscopic details of how well a sample is collected from a patient rather than from how infectious or infected a person is.
In summary the most common PCR drawbacks are:
Real-Time (RT) PCR assays are qualitative diagnostic tests that, when not invalid, produce either a positive or negative result. The number of cycles a PCR assay runs for is typically decided by a country’s health government, such as the CDC in the USA. The higher the number of cycles the PCR runs to, the higher the chance to catch weak positive results, but this also means that false positive results will occur more frequently.
Distinguishing false positives from weak positives at high cycle values is not trivial, and has caused a lot of controversy in the recent SARS-CoV2 epidemic.
The “Cycle threshold” or Ct value of an RT-PCR assay refers to the number of cycles needed to amplify viral DNA/RNA to reach a detectable level.
If a signal indicating the presence of the target sequence is detected before the number of cycles hits the recommended upper value, the test is considered to be positive and the number of cycles is recorded as the Ct value for that specimen.
While the PCR assay itself is not technically quantitative on a cycle by cycle basis, counting the number of cycles until a target is detected can be interpreted quantitatively.
PCR test are often referred to as Nucleic Acid Amplification Tests (NAAT).
PCR, when used correctly, is a great diagnostic tool for detecting very low concentrations of a target sequence, potentially down to just one copy or DNA/RNA in the sample, which makes it very sensitive. Also the PCR method is great at amplifying (making copies) of a target sequence, as the amount doubles for every cycle.
This however also comes with some drawbacks. The ultra sensitive nature of PCR makes it extremely susceptible to process errors, contamination from other neighboring samples, or from the environment, since just one single fragment of contamination will be amplified along with the specimen.
Because every cycle doubles the amount of target if present, the final Ct of a sample depends substantially on how many target particles were in the sample to begin with, which can vary greatly between same samples taken from the same patient or by different collection methods or people.
PCR Ct values are therefore not a good measure of viral load, because variations in Ct values will vary much more depending on the microscopic details of how well a sample is collected from a patient rather than from how infectious or infected a person is.
In summary the most common PCR drawbacks are:
ELISA is typically used in diagnostics labs to detect antibodies from plasma or serum. Performing this assay involves these steps:
Since the process of mechanically washing and electronically reading the specimens requires two different sets of instrumentation, these tasks are typically performed in the lab by two separate machines. Both of these machines are not typically used outside of a clinical lab environment and not suitable for use in the field or in an outdoor environment.
An absorbance reader is a lab bench instrument that is used to automate the reading or multi-well microtiter plates.
A dispenser and plate washer is a lab bench instrument that is used to automate dispensing and washing of each well in multi-well microtiter plates.
ELISA while being a popular and ubiquitous diagnostic test in serology labs, does have some drawbacks:
An absorbance reader is a lab bench instrument that is used to automate the reading or multi-well microtiter plates.
ELISA is typically used in diagnostics labs to detect antibodies from plasma or serum. Performing this assay involves these steps:
Since the process of mechanically washing and electronically reading the specimens requires two different sets of instrumentation, these tasks are typically performed in the lab by two separate machines. Both of these machines are not typically used outside of a clinical lab environment and not suitable for use in the field or in an outdoor environment.
A dispenser and plate washer is a lab bench instrument that is used to automate dispensing and washing of each well in multi-well microtiter plates.
ELISA while being a popular and ubiquitous diagnostic test in serology labs, does have some drawbacks:
LFA is a versatile diagnostic assay that is well established, and unlike PCR, can be used in the field and outside of a sterile lab environment. LFAs consist of laminated nitrocellulose paper strips imprinted with an antigen/antibody pair that react to produce a control line when a liquid is absorbed and flows through the paper. The control line is always located furthest away from the analyte pad, and its purpose is to indicate that a sufficient amount of liquid has been used for the flow to reach the far end of the paper. The control line becomes visible when colored nanoparticles are released as an antibody and antigen pair binds. The control line is also an indication that the assay is not damaged by radiation, age, chemicals or other damaging factors.
Unlike PCR, LFAs (or RDTs) have the unique advantage that they can be used both for antigen antibody testing. Antigen tests are typically done from swabs, whereas antibody tests are done from capillary blood (finger-prick), venous blood(blood draw), or serum (centrifuged venous blood).
Antibody tests in diagnostic labs typically use Enzyme Linked Immunosorbent Assays (ELISA). These types of assays also require a sterile lab environment to be processed correctly and to avoid environmental and cross contamination between samples.
A substantial advantage with LFAs is that they can be used as alternatives both to PCR and ELISA assays.
Some of the key advantages of LFAs over PCR are:
During a clinical trial of LFAs in the USA in Dec 2020, while comparing 3 vendor’s test kits against PCR, a disagreement between PCR and all 3 test kits was found, where the PCR showed a positive result and all 3 LFAs showed a negative result. Retesting with PCR gave a negative result, thus this study concluded that 1 in 60 PCR tests are false positives.
While LFAs are inexpensive and practical, they do have several drawbacks that needs to be considered:
Note that most issued with LFAs are related to human errors and readings by human eyes.
Rapid tests come in a variety of shapes and sizes. Some popular types are seen above in this picture.
The LFA strip, although it can be used by itself, is commonly placed inside a plastic cassette as seen in this picture.
The outer plastic shell protects the Lateral Flow Assay from being touched directly and allows for easier handling. Windows in the plastic allows parts of the assay to be exposed for reading and helps guide the analyte onto the correct location on the test strip.
Assaya is inventing more environmentally friendly solutions, and working with manufacturers, to make tests that use much less plastic and help transitioning the world of RAPID diagnostic into a greener one with less waste and easier to recycle materials.
The LFA is the core of the Rapid Diagnostic Test (RDT) and contains the nitrocellulose paper where the actual chemical reactions take place. For prototyping, a simple automatic cutter can be used.
In this video RAPID test that looks an negative test to the human eye, is actually a weak positive test using n-protein diluted down to 5.0 ng/ml. Machine reading confirms this, as the iaX analyzes the specimen to have an equivalent Cycle threshold value to PCR of 33, which is quite close to the PCR limit of 35 for a negative test.
When investigating the OpenDB entry for this test, and zoom in on the pictures of the test-line, we can see a slight red haze, which is an indication that there was indeed some n-protein in the specimen, but that it was way to weak or diluted to be observed visually by the human eye. The iaX however, were able to detect this and calculate an equivalent Cycle threshold (eCt) to PCR of 33 cycles.
Rapid tests come in a variety of shapes and sizes. Some popular types are seen above in this picture.
LFA is a versatile diagnostic assay that is well established, and unlike PCR, can be used in the field and outside of a sterile lab environment. LFAs consist of laminated nitrocellulose paper strips imprinted with an antigen/antibody pair that react to produce a control line when a liquid is absorbed and flows through the paper. The control line is always located furthest away from the analyte pad, and its purpose is to indicate that a sufficient amount of liquid has been used for the flow to reach the far end of the paper. The control line becomes visible when colored nanoparticles are released as an antibody and antigen pair binds. The control line is also an indication that the assay is not damaged by radiation, age, chemicals or other damaging factors.
The LFA strip, although it can be used by itself, is commonly placed inside a plastic cassette as seen in this picture.
The outer plastic shell protects the Lateral Flow Assay from being touched directly and allows for easier handling. Windows in the plastic allows parts of the assay to be exposed for reading and helps guide the analyte onto the correct location on the test strip.
The LFA is the core of the Rapid Diagnostic Test (RDT) and contains the nitrocellulose paper where the actual chemical reactions take place. For prototyping, a simple automatic cutter can be used.
Assaya is inventing more environmentally friendly solutions, and working with manufacturers, to make tests that use much less plastic and help transitioning the world of RAPID diagnostic into a greener one with less waste and easier to recycle materials.
Unlike PCR, LFAs (or RDTs) have the unique advantage that they can be used both for antigen antibody testing. Antigen tests are typically done from swabs, whereas antibody tests are done from capillary blood (finger-prick), venous blood(blood draw), or serum (centrifuged venous blood).
Antibody tests in diagnostic labs typically use Enzyme Linked Immunosorbent Assays (ELISA). These types of assays also require a sterile lab environment to be processed correctly and to avoid environmental and cross contamination between samples.
A substantial advantage with LFAs is that they can be used as alternatives both to PCR and ELISA assays.
Some of the key advantages of LFAs over PCR are:
In this video RAPID test that looks an negative test to the human eye, is actually a weak positive test using n-protein diluted down to 5.0 ng/ml. Machine reading confirms this, as the iaX analyzes the specimen to have an equivalent Cycle threshold value to PCR of 33, which is quite close to the PCR limit of 35 for a negative test.
When investigating the OpenDB entry for this test, and zoom in on the pictures of the test-line, we can see a slight red haze, which is an indication that there was indeed some n-protein in the specimen, but that it was way to weak or diluted to be observed visually by the human eye. The iaX however, were able to detect this and calculate an equivalent Cycle threshold (eCt) to PCR of 33 cycles.
While LFAs are inexpensive and practical, they do have several drawbacks that needs to be considered:
Note that most issued with LFAs are related to human errors and readings by human eyes.
During a clinical trial of LFAs in the USA in Dec 2020, while comparing 3 vendor’s test kits against PCR, a disagreement between PCR and all 3 test kits was found, where the PCR showed a positive result and all 3 LFAs showed a negative result. Retesting with PCR gave a negative result, thus this study concluded that 1 in 60 PCR tests are false positives.
The assay comparisons so far indicate that each assay type has pros and cons, and there is no “one-size-fits-all”.
Notice however one major advantage of PCR & ELISA over LFAs …
They are Machine Interpreted.
So what happens then if we try to Machine Interpret LFAs?
They become QLFAs – Quantitative Lateral Flow Assay
As you will see, they adopt many of the advantages of both PCR and ELISA without inheriting the drawbacks.
Using the Assaya iaX-2101 intelligent analyzer eXpress, any LAF can be turned into a Quantitative Lateral Flow Assay (QLFA). The iaX is fast, can be used in the field, outdoors, at events, or anywhere else LFAs need to be read fast and accurately, while minimizing human errors.
The analyzer was designed specifically to accommodate any existing LFA on the market today, and already supports hundreds of tests from over 40 different manufacturers.
To consistently and properly be able to determine an accurate Quantitative reading, the iaX uses ultra durable LEDs with multiple wavelengths across the entire range from deep UV to IR, and each test type and test cassette is calibrated to be able to be optimally read in a few seconds using a macro-view fixed-focus camera.
The iaX first reads the barcode of the test to uniquely identify the test and its calibrated test profile, and then quickly analyzes the test cassette for anomalies, and then interprets the exact intensity of each of the control and test lines on the test cassette to calculate the values.
The Assaya analyzers use the UTID specification for test barcode interpretation, which is a free and OpenSource system for identifying vendors and test cassette profiles.
EVs are test line values read by the iaX that are interpreted into quantitative values that are calibrated according to their equivalent values of traditional PCR and ELISA tests.
The units of EVs are also equivalent to those of other assays. As an example, PCR which typically gives a Cycle threshold (Ct) value is interpreted by the iaX into an eCt (equivalent Cycle threshold) value.
This method of interpreting test and control lines and the system and algorithms implemented in the iaX to interpret them are patent pending.
Assaya’s founder Clas Sivertsen designing the iaX-2101 Universal QLFA reader in Feb 2021. Machine reading LFAs are important to achieve accurate, consistent, and quantitative results, reduce human intervention, errors, and improve throughout for mass testing.
Using the Assaya iaX-2101 intelligent analyzer eXpress, any LAF can be turned into a Quantitative Lateral Flow Assay (QLFA). The iaX is fast, can be used in the field, outdoors, at events, or anywhere else LFAs need to be read fast and accurately, while minimizing human errors.
The analyzer was designed specifically to accommodate any existing LFA on the market today, and already supports hundreds of tests from over 40 different manufacturers.
Assaya’s founder Clas Sivertsen designing the iaX-2101 Universal QLFA reader in Feb 2021. Machine reading LFAs are important to achieve accurate, consistent, and quantitative results, reduce human intervention, errors, and improve throughout for mass testing.
The iaX first reads the barcode of the test to uniquely identify the test and its calibrated test profile, and then quickly analyzes the test cassette for anomalies, and then interprets the exact intensity of each of the control and test lines on the test cassette to calculate the values.
To consistently and properly be able to determine an accurate Quantitative reading, the iaX uses ultra durable LEDs with multiple wavelengths across the entire range from deep UV to IR, and each test type and test cassette is calibrated to be able to be optimally read in a few seconds using a macro-view fixed-focus camera.
The Assaya analyzers use the UTID specification for test barcode interpretation, which is a free and OpenSource system for identifying vendors and test cassette profiles.
EVs are test line values read by the iaX that are interpreted into quantitative values that are calibrated according to their equivalent values of traditional PCR and ELISA tests.
The units of EVs are also equivalent to those of other assays. As an example, PCR which typically gives a Cycle threshold (Ct) value is interpreted by the iaX into an eCt (equivalent Cycle threshold) value.
This method of interpreting test and control lines and the system and algorithms implemented in the iaX to interpret them are patent pending.
QLFAs are Machine Read LFAs, which greatly reduces human eye fatigue, human processing errors, and the lab-bound complexity of processing ELISA and PCR assays.
QLFAs meet all the requirements for mass RAPID testing at Point of Test and Clinical labs.
When developing EVs for a new test kit, first make sure your UTID is printed as laser marking or label on each test cassette. The number of test cassettes you need vary depending on process variation and desired accuracy across variations in test-line intensity (manufacturing consistency of the test kit), but a good place to start is to have minimum 2 negative and 6 positive samples that have been developed from positive controls. The 2 negative tests should be one with and one without matrix.
Be sure to distribute the concentrations across the entire valid spectrum as much as possible, for example: 0ng/ml, 1ng/ml, 2ng/ml, 3ng/ml, 5ng/ml, 8ng/ml, 10 ng/ml.
Note that the more tests you have the better, although a good starting point is 2+6.
After each test has developed to the correct time, insert each test into the iaX one at a time and wait for the status light to light before you pull the test out and insert the next. Once all tests have been read, you can proceed to the net step.
Next, in the Assaya OpenBio Portal, locate the test that was just read and enter manually the desired target EVs for each test as notes, and make sure to include the desired unit.
Click the button to export the dataset as CSV and open it in a spreadsheet like Excel, Open Office, Numbers, Google Sheets, etc.
Assaya technical support teams around the world assist test kit vendors with the test kit on-boarding.
Many kinds of EVs can be developed. A popular EV is equivalent Cycle threshold (eCT ) values to add quantitative value to LFAs similar to that of PCR assays.
There are many useful units that can be developed into EVs for tests like antibodies, water quality, vitamins, pH, Pregnancy, and many more.
The EV system is flexible enough to accommodate any values and units, such as:
Below image shows a simplified comparison between theCycle threshold values commonly reported from PCR tests calibrated and comparted to the equivalent Cycle thresholds used in Quantitative LFAs.
Many kinds of EVs can be developed. A popular EV is equivalent Cycle threshold (eCT ) values to add quantitative value to LFAs similar to that of PCR assays.
There are many useful units that can be developed into EVs for tests like antibodies, water quality, vitamins, pH, Pregnancy, and many more.
The EV system is flexible enough to accommodate any values and units, such as:
Below image shows a simplified comparison between the Cycle threshold values commonly reported from PCR tests calibrated and comparted to the equivalent Cycle thresholds used in Quantitative LFAs.
When developing EVs for a new test kit, first make sure your UTID is printed as laser marking or label on each test cassette. The number of test cassettes you need vary depending on process variation and desired accuracy across variations in test-line intensity (manufacturing consistency of the test kit), but a good place to start is to have minimum 2 negative and 6 positive samples that have been developed from positive controls. The 2 negative tests should be one with and one without matrix.
Be sure to distribute the concentrations across the entire valid spectrum as much as possible, for example: 0ng/ml, 1ng/ml, 2ng/ml, 3ng/ml, 5ng/ml, 8ng/ml, 10 ng/ml.
Note that the more tests you have the better, although a good starting point is 2+6.
After each test has developed to the correct time, insert each test into the iaX one at a time and wait for the status light to light before you pull the test out and insert the next. Once all tests have been read, you can proceed to the net step.
Next, in the Assaya OpenBio Portal, locate the test that was just read and enter manually the desired target EVs for each test as notes, and make sure to include the desired unit.
Click the button to export the dataset as CSV and open it in a spreadsheet like Excel, Open Office, Numbers, Google Sheets, etc.
Assaya technical support teams around the world assist test kit vendors with the test kit on-boarding.
Assaya provides affordable and equitable human error reducing diagnostics at scale, with real-time reporting. Our mission is to provide the world with reliable, augmented, practical, and inexpensive diagnostics (RAPID) that can be deployed anywhere, anytime, to identify biomarkers and dangerous pathogens in near real-time. We help people everywhere shift from illness to wellness, and prevent the spread of disease, in a sustainable and ethically responsible manner.
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