Lateral Flow Assay

Lateral flow assays (LFAs) are a simple, paper-based detection device. Their low-cost and portable designs allow for rapid detection (5-30 mins) of a specific analyte in a sample mixture. 

When a liquid sample (blood, saliva, urine, etc.) is placed on an LFA, the sample travels across the strip through different regions in which specific molecules have been immobilized. The interactions between the immobilized molecules and the analyte present in the sample produces a visible result that can be seen by the user [2]. If there is no analyte present in the sample, these interactions do not take place, and thus no visible result is presented.

The visible results produced by different LFAs can be either qualitative (“yes or no” result, such as in a pregnancy test) or quantitative (concentration of analyte indicated by varying signal intensities) [2].  Since all of the necessary chemistry involved in this process takes place on the strip itself, there is no need for training or previous qualifications of the user. This makes LFAs a very useful point-of-care testing technology. In this activity, we will be utilizing LFAs to produce quantitative results.  

LFAs are widely used in medical diagnostics as they are a very useful point-of-care technology. For instance, the home pregnancy test is an LFA that detects the hormone hCG in urine. 

Lateral Flow Components: 

An LFA is composed of a sample pad, conjugate pad, nitrocellulose membrane with test and control lines, and absorbent pad. When constructing the LFA, an overlap of 1-2 mm between each component most occur.

Sample Pad

• • The sample pad accepts, neutralizes, and then evenly distributes a liquid sample, sending it towards the conjugate pad.  

Conjugate Pad

• • The conjugate pad immediately follows the sample pad and is impregnated with antibodies that are specific to the analyte being targeted by the LFA [2]. These antibodies are conjugated to colored particles that will eventually be responsible for producing the visible results of the test.  

Nitrocellulose Membrane 

• • Most commonly made from nitrocellulose, the membrane is a very important component of any LFA. The pores of the membrane (ranging from 0.05-12 µm) provide capillary forces that allow the liquid sample to travel across the LFA, as well as allow for the immobilization of the molecules that are necessary for analyte detection on the test line and performance confirmation on the control line [2]. The flow of the liquid through the membrane can be improved by washing the membrane in bovine serum albumin (BSA).  

 Test line 

• • The test line is the portion of the LFA that indicates whether the test is positive or negative (i.e. whether, or to what degree, the target analyte is present in the sample). The test line is composed of immobilized proteins that bind to nanoparticles to generate a signal based on the presence of analyte in a sample. In a qualitative LFA, the test result is simply determined by the presence, or lack thereof, of the test line [2]. In a quantitative LFA, the resulting test line can be imaged, and its strength can be correlated to the concentration of the target analyte in the sample.   

Control line 

• • The control line is the portion of the LFA that indicates to the user whether or not the test is functioning properly. The control line is composed of affinity ligands that bind to nanoparticles regardless of if the target analyte is present in the sample [2]. This function serves to reduce the number of false readings that are recorded.  

Absorbent (Wicking) Pad 

• • The absorbent pad, located at the very end of the LFA, is meant to collect any excess liquid after the sample has completed its journey across the membrane. It is important that the excess liquid be held in the absorbent pad, as this prevents any backflow of the sample.  

Backing Card 

• • The backing card is a stiff piece of thin material that holds all the components of the LFA. The LFA components are held in place on the backing card by an adhesive tape or glue for easier usage and handling of the device by the user.  

Lateral Flow Formats

Sandwich Format: 

In a sandwich LFA, two different antibodies are used (Primary and Secondary). The primary antibody is immobilized at two different positions in the LFA. Primary antibodies are located on the conjugate pad (labelled with nanoparticles) as well as on the test line (not labelled with antibodies) [3]. The secondary antibodies are only immobilized at one position; the control line (not labelled with nanoparticles) [3]. When the liquid sample passes through the conjugate pad, if the target analyte is present, it will form a complex with the labelled primary antibody. When this complex arrives at the control line, it will form a “sandwich” with the unlabelled primary antibody. Whether or not the target analyte is present in the sample, some conjugated molecules (labelled primary antibody) will travel from the conjugate pad to the control line, where they will bind to the unlabeled secondary antibody [3]. Thus, for this type of LFA, two visible lines represents a positive test, while one visible line (control line) represents a negative test. This sandwich format is preferred for larger target analytes.

Competitive Format: 

Competitive format LFAs come in two different arrangements (seen below). In the first arrangement, the target analyte is labelled and immobilized on the conjugate pad, unlabeled primary antibodies are immobilized on the test line, and unlabeled secondary antibodies are immobilized on the control line [3]. If any of the target analyte is present in the sample, it will compete against the labelled analyte to bind to the primary antibody on the control line, while the labelled analyte will exclusively bind to the secondary antibody on the control line [3]. Thus, for a positive test, only the control line will be visible. In the second arrangement, the labelled primary antibody is immobilized on the conjugate pad, target analyte and carrier molecule conjugates are immobilized on the test line, and the unlabeled secondary antibody is immobilized on the control line. If the target analyte is present in the sample it will compete with the analyte-carrier molecule conjugate to bind to the labelled primary antibody. 

Nanoparticles in Lateral Flow Assays: 

LFAs generate an optical signal through the colored nanoparticles on the test lines. Larger nanoparticles will provide a stronger signal and be easier to see however particles that are too large will have trouble flowing through the nitrocellulose membrane which will limit binding on the test line [2]. As a result, nanoparticles between 20 nm and 500 nm are selected for LFAs. The most common nanoparticle utilized as a reporter are 40 nm diameter gold nanoparticles. This sized nanoparticle has a peak absorbance at ~520 nm which produces a strong red colored test line. Another size and shape of nanoparticles that have been used is a gold nanoshell with a 150 nm diameter. This type of nanoparticle provides a 3-20 fold increase in sensitivity compared to the 40 nm gold nanoparticle [2]. Gold nanoshells have a silica core making it less dense than solid gold nanoparticles, which allows it to flow more easily through the nitrocellulose membrane. 

Lateral Flow Assay Design: 

The design of an LFA is flexible and can be tailored to a specific application. The sections below outline typical steps and considerations taken when developing an LFA design.

Step 1: Nanoparticle Selection 

The choice of reporter nanoparticle is important when designing an LFA as it determines the sensitivity and specificity, stability, cost, development time, and final readout signal of the assay [2].

While selecting a nanoparticle, a question you may ask yourself is whether or not the assay requires high sensitivity. Depending on this, the type of conjugation, and the protein used, a nanoparticle size and shape can be recommended. 

Step 2: Antibody Selection 

The sensitivity and selectivity of antibodies are useful in the detection of low concentrations of analyte in a sample [2]. Antibodies are Y-shaped proteins used in recognizing antigens (viruses, chemicals, etc.). The binding properties of antibodies have different optimization parameters, which makes LFA development difficult. 

When selecting an antibody, consider whether to use a polyclonal or monoclonal antibody. Polyclonal antibodies are produced from immunized animals and monoclonal antibodies are antibodies prepared in the lab [2]. Polyclonal antibodies consist of mixtures of different antibodies made from different B cell clones in an animal. Whereas, monoclonal antibodies are made from a single B cell clone. 

Consider the type of antibody selected as the performance of the LFA depends on the antibodies ability to bind an analyte in a sample. 

Step 3: Nitrocellulose Membrane Selection

The choice of nitrocellulose membrane is crucial for the development of a high performance LFA. Different nitrocellulose membranes have unique flow dynamics, sensitivity, specificity, and consistency [2]. 

Nitrocellulose membranes are commercially available and when choosing a membrane the following characteristics should be considered: 

1. 1. Pore Size (µm)
   2. Porosity (%) 
   3. Thickness (µm) 
   4. Chemical Treatment

These characteristics should be considered as the pore size and porosity affect the rate of speed at which a sample travels along the membrane [2]. Manufacturers also treat the nitrocellulose membrane beforehand with certain chemicals and surfactants which could affect the performance. As a result, looking into these factors before purchasing a membrane is important. 

Vendors for nitrocellulose membranes: 

• • Whatman/GE
  • Millipore
  • Sartorius 
  • MDI 

Step 4: Conjugate Pad Selection 

The choice of conjugate pad is important for long-term stability of the conjugate, binding of conjugate or sample, and to ensure that the conjugate is released after wetting [2]. 

Conjugate pads are commercially available and when choosing a conjugate pad the following factors should be considered. 

1. 1. Density (g/m²)

   2. Thickness (µm)

These two factors are important as they control the conjugate uptake and release. 

Vendors for conjugate pads: 

• • Millipore 
  • Ahlstrom 

Step 5: Sample Pad Selection 

The sample pad is important in an LFA as it controls the release of the sample onto the membrane or conjugate pad as well as acts as a filter for unwanted components while allowing the analyte to pass through [2]. 

Sample pads are commercially available and when selecting a sample pad the following should be considered: 

1. 1. Thickness/Weight 
   2. Tensile Strength 
   3. Material

These factors should be considered as a thicker and heavier membrane will allow a higher volume of sample to be added however there may be issues with compression. A sample pad with low tensile strength can affect the lamination process and the type of fiber used in sample pads can affect both the retain volume and tensile strength [2]. 

Ultimately, the sample (blood, saliva, urine, etc.) has the greatest affect on the type of sample pad being used for the LFA. 

Step 6: Absorbent Pad (Wick) Selection 

The absorbent pad is crucial to the LFA as it increases the amount of sample that can enter the strip. The bed volume (density and thickness) of a membrane is limited and an absorbent pad at the end of the strip will act as a sponge for excess sample. The absorbent pad can help in preventing non-specific binding and sensitivity [2]. 

When selecting an absorbent pad, one should be chosen so that backflow is minimized so that the stability window for the strip's signal is maximized. Absorbent pads are commercially available and when selecting an absorbent pads the same factors considered for sample pads can also be considered for absorbent pads. However, a difference is that the absorption capacity for an absorbent pad should be much greater than that of a sample pad [2]. 

Vendors for absorbent pads: 

• • Millipore 
  • Whatman/GE
  • Ahlstrom

Step 7: Test Strip Assembly 

To assemble an LFA test strip the following three things must be done: 

1. 1. Lamination 
   2. Cutting 
   3. Cassette 

Lamination 

• • This is the umbrella term for the assembly of all LFA components into one strip. The assembly starts with a backing card which has an adhesive side covered with a liner. The liner is removed and a nitrocellulose membrane is placed in the middle of the strip. The laminated side of the nitrocellulose membrane goes on the adhesive side of the backing card. Next, the absorbent pad and conjugate pad is placed on the adhesive side of the backing card where there is 1-2mm overlap with the nitrocellulose and these two components. Finally, the sample pad is applied where there is overlap with the conjugate pad. 

Cutting 

• • Once the strip has been laminated, it can be cut into strips. A guillotine can be used to cut strips of the same size. Ensure to look out for strip width inconsistencies and frayed edges. Quantitative assays are cut to around 5-6mm in width.

Cassette 

• • Cassettes are important for reproducibility and reliability of an LFA [2]. The cassette applies pressure at points on the strip for fluid to pass through. 

Step 8: Running the Assay 

There are numerous methods in which an LFA can be run. Two in particular are dipstick assays and full assays. 

Dipstick Assay 

• • A dipstick assay is where the conjugate is in liquid form. As a result, the LFA is prepared without a sample or conjugate pad. The sample and conjugate is mixed together in a test tube and the strip is dipped in the solution. 

Full Assay 

• • A full assay is a fully assembled strip in which the conjugate is dried on the conjugate pad. An appropriate volume of sample is applied to the sample pad and the test runs through. 

Step 9: Analyzing the Strip 

Analyzing an LFA depends on whether the assay is quantitative or qualitative. Assays can be analyzed through a variety of methods, one of which being by eye. For qualitative tests this is acceptable as it delivers a yes/no answer however for quantitative tests it is not. For semi-quantitative tests an assay may be determined by eye however a gradient score to go along with the test must be provided. 

A camera set up with controlled lighting should be used to take an image of the strip for a quantitative test. Under this set up, the line strength (colour) can be analyzed utilizing image analysis software's such as ImageJ and produce a value based on the color. 

Step 10: Assay Optimization 

Optimizing an LFA is related to developing a high sensitivity assay. When optimizing an assay the items outlined in the image below should be considered: 

Optimization helps overcome issues encountered during test runs such as instability, non-specific binding, and more.

References

[1] Cytodiagnostics Inc. "Lateral Flow Assay," [Online]. Available: https://www.cytodiagnostics.com/pages/lateral-flow-assay [Accessed: 29-Jan-2021]

[2] nanoComposix. "Introduction to Lateral Flow Rapid Test Diagnostics," [Online]. Available: https://nanocomposix.com/pages/introduction-to-lateral-flow-rapid-test-diagnostics#target [Accessed: 29-Jan-2021]

[3] Elif BurcuBahadır, Mustafa KemalSezgintürk, "Lateral flow assays: Principles, designs and labels" Elsevier, vol. 82, pp. 286-306, 2019, doi: 10.1016/j.trac.2016.06.006