Molecular detection methods in particular are indispensable in any diagnostic laboratory. At the same time, rapid test formats are moving into the focus of public awareness. Due to their simplicity, these rapid tests can be used as a point of need tool. The following article focuses on one of the most exciting topics in diagnostics today. It is about the connection of complex molecular biological detection techniques combined with the simplicity of universal lateral flow assays. The Milenia HybriDetect is a universal platform which allows individual and innovative Lateral Flow development. The first part of this series of articles deals with the most important basics: What is Lateral Flow? What is the Milenia HybriDetect and how is it possible to detect anything with it?
What is a Lateral Flow Assay?
Lateral flow assays (LFAs) are characterized as simple, robust and easy-to-handle rapid tests. This diagnostic tool can be used without special equipment or trained personnel. These charcteristics define LFAs as rapid tests, that are explicitly suitable for laboratory-independent analysis. Lateral flow rapid tests are often described as a combination of chromotography and immunostaining. A lateral flow device (LFD) is composed of several overlapping layers, containing specific catcher and detector elements, that are able to „recognize“ certain analytes, including proteins, drugs, hormones, metabolites and others. In particular, specific antibodies play an important role in this context, which gives the test format the name lateral flow immunoassay (LFIA). These LFIAs can be impressivly robust and allow analysis of a wide variety of samples, such as blood, serum, saliva, swabs, urine and many more.
The most famous Lateral Flow Test
The most famous of all LFIAs is the pregnancy test, capable to detect the typical pregnancy hormone hCG directly from urine. After applying the sample to the test strip, lines can be seen with the naked eye on the LFD after 10 to 15 minutes. This short description of a lateral flow assay emphasizes the most important characteristics of this diagnostic platform. It‘s a simple, easy-to-handle, robust, rapid, point-of-need, and equipment-free test system. The following figure describes the mechanism of a “classic” Lateral Flow Immunoassay (example: pregnancy test).
Figure 1. Principle of “classic” Lateral Flow Immunoassay – the pregnancy test
What is Milenia HybriDetect?
Milenia HybriDetect is a universal lateral flow development platform for researchers all over the world to design their individual rapid test. It is a versatile tool to detect DNA-amplification products, DNA, RNA, proteins, peptides, antibodies, metabolites, cells, etc…
Over the past decade the combination of DNA-amplification and lateral flow has developed into an important methodological niche. The use of universal lateral flow devices is an essential component for the simplification of complex methods. For this reason the Milenia HybriDetect is the perfect fit for a sensitive and specific nucleic acid detection where it is needed. Particularly in the context of the current pandemic, it becomes clear how important a precise and sensitive point-of-care diagnosis is.
Video 1. Lateral Flow Assay using the Milenia HybriDetect 2T (timelapse of approx. 2 min of LFA)
One Lateral Flow Development Platform – Two Devices
Currently, there are two different test strips belonging to the Milenia HybriDetect „family“ available. Both strips contain a control line confirming the overall functionality of the immunoassay. Furthermore, the Milenia HybriDetect LFD contains one test line, whereas the Milenia HybriDetect 2T contains an additional test line for multiplex applications (Video 1). Both strip-types are manufactured as „dipsticks“, which are basically „naked“ strips and are not incorporated in a plastic housing. This is not only environmentally friendly, but it also enables user friedly dip applications, in which the strips can be positioned directly in small reaction tubes, such as 96-well plates or PCR tubes.
How to perform a Lateral Flow Assay
The simplicity is one of the most important advantages of LFAs, beside the robustness and timesaving character. After application of the sample directly on the strip, the HybriDetect is placed in a sufficient volume of running buffer initiateing the lateral flow. After a few seconds the user can see how the goldconjugate particles migrate over the entire analytical membrane. Normally it takes 2 to 5 minutes until clear signals appear. Alternatively, the Milenia HybriDetect dipsticks can be placed directly into the sample or sample-containig running buffer. The presence (or absence) of the test line gives information about a positive or negative test result. Furthermore, the appearance of the control is an important feature, indicating the overall functionality of the LFA. This is particullary important for the validity of the analysis.
The following video shows how to perform a LFA using the Milenia HybriDetect 2T in just one minute.
Video 2. How to perform a Lateral Flow Assay using the Milenia HybriDetect 2T.
How Milenia HybriDetect works – the detection principle
Milenia HybriDetect is a universal lateral flow development platform that allows the detection of multiple analytes. The decisive requirement for the successful detection is the „incorporation“ or „attachment“ of defined labels:
- Milenia HybriDetect: Biotin and FITC/FAM (test line)
- Milenia HybriDetect 2T: Biotin and FITC/FAM (T1), Digoxygenin and FITC/FAM (T2)
If these labels, like bitoin, FITC / FAM or digoxygenin are somehow „attached“ to the analyte, the test strip(s) will be able to detect it. Biotinylated and FITC-/FAM-labeled analytes are detectable on the (bottom) test line of the Milenia HybriDetect test strips. Additionally, digoxygenin and FITC-/FAM-labeled analytes are detectable on the test line 2 of the HybriDetect2T.
Figure 2. Milenia HybriDetect – Composition and general detection principle (AuNP – Goldnanoparticle, rb – rabbit, gt – goat, DIG – digoxygenin, A1 – Analyte 1, A2 – Analyte 2)
But how is it possible to „incorporate“ or „attach“ the required labels?
This is exactly the point where it gets exciting, because there are an infinite number of answers to this simple question. The explanation of four basic, representative scenarios should give an idea of the wide versatility of the Milenia HybriDetect development platform. All scenarios are described by using the test strip with only one test line for a better understanding.
Scenario I – detection of amplicons
The HybriDetect is frequently used for the quick and easy detection of reaction products from DNA amplification reactions. Therefore, many developers use modified primers for the incorporation of the relevant labels, biotin and FITC/FAM. Consequently, the reaction products contain these labels for the successful detection on the LFD. Accordingly, the appearance of the test line correlates with the presence of the biotin- and FITC-/FAM-labeled amplification product. This reprensentative example is used in several DNA-amplification methods, such as PCR and multiple isothermal amplification techniques (e.g. LAMP-LFA). Furthermore, this approach allows amplification free detection of abundand nucleic acid (e.g. ribosomal RNAs) due to specific hybridization of labeled probes.
Figure 3. Labeled primers are incorporated in an amplification product, which is detectable on the test line (T) of the Milenia HybriDetect
Scenario II – detection of reporter degradation
Reporters are defined as components that contain both relevant labels (biotin and FITC/FAM). During the detection procedure, this reporter is somehow degraded. The reporter status can be assessed with the help of the Milenia HybriDetect.
A defined amount of reporter is used in the LFA, The reporter retains the majority if not all of the mobile gold conjugate particles at the bottom line. As a result the top line, which was originally defined as immunoassay control line, does not appear (negative result, intact reporter). If the reporter is degraded, labels are seperated from each other. As a consequence, less of the mobile gold conjugate can be retained on the bottom line. This results in an increasing intensity of the top line (positive result, degraded reporter).
Figure 4. Principle reporter degradation. Dual labeled reporter is degraded during detection reaction. Increasing degradation correlates with increasing C-line intensity.
The development of reporter-based assays is playing an increasing role in molecular biological applications. Innovative test formats such as CRISPR/Cas-based assays (e.g. SHERLOCK or DETECTR), benefit from the ability of the Milenia HybriDetect, which allows this kind of detection strategy.
Scenario III – non competetive (immuno-) assays
In the following example, a comparable strategy with completely different biological components is used. Milenia HybriDetect is suitable as a universal platform for the development of individual antibody-based rapid tests. Therefore, specific antibodies adressing different epitopes have to be labeled with Biotin and FITC/FAM. These primary antibodies are able to bind the analyte of interest. Secondary antibodies, which are part of the LFD, are responsible for the appearance of the test line. The presence of the test line correlates with the successful detection of the desired analyte.
Figure 5. Non competetive (immuno-) assay. Labeled antibodies are able to specifically bind the analyte of interest. This immunological “sandwich” is detectable on the Milenia HybriDetect.
Already established Immunoassays (e.g. ELISAs) can be easily transferred into a universal lateral flow rapid test format by using this labeling- and detection- strategy. Moreover, a mixture of a polyclonal antibodies allows the development of a simple rapid test.
Scenario IV – competetive (immuno-) assays
The appearance of the test line is consciously forced in competetive LFAs. The weakening or absence of this signal correlates with the presence of the analyte. Accordingly, the weakening of the intensity of the test line defines a positive test result. No change in the intensitiy of the test correlates with a negative test result. This approach is initially counter-intuitive, but can be very effective for certain questions. The following figure illustrates the principle of competetive assays using the Milenia HybriDetect.
Figure 6. Principle of a competetive (immuno-) assay. Labeled analyte competes with non-labeled analyte (in sample) for binding of the labeled antibody. Decreasing T-line intensity correlates with an increasing amount of the analyte of interest.
Scenario V to ∞
Each part of the detection strategies described here can be varied or combined. The simple and universal character of the Milenia HybriDetect platform allows extremely versatile and individual Lateral Flow development.
„Only your own creativity is limiting at this point…“
The next part of this blog series focuses on the most frequently used methods combined with a universal Lateral Flow readout …