Lateral Flow Assay

Lateral flow assays (LFAs) is 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 [5]. 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) [5].  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, thus making 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.

 Figure 2: Lateral Flow Assay Components [2]

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.

Figure 3: Sandwich Lateral Flow Assay Format [3]

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, unlabelled primary antibodies are immobilized on the test line, and unlabelled 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 unlabelled 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. 

Figure 4: Competitive Lateral Flow Assay Formats [3]

Nanoparticles in Lateral Flow Assays: 

Lateral Flow Assay Design: 

• Nanoparticle Selection 
• Antibody Selection 
• Nitrocellulose Membrane Selection
• Conjugate Pad Selection 
• Sample Pad Selection 
• Absorbent Pad (Wick) Selection 
• Test Strip Assembly 
• Running the Assay 
• Analyzing the Strip 
• Assay Optimization 

References

Contributors: