Common Formats of Lateral Flow Tests Home>>Common Formats of Lateral Flow Tests
Common Formats of Lateral Flow Tests
Different formats are adopted in LFA. Strips used for LFA contain four main components. Detailed description of each is given in technical note 1. There are several formats of Lateral Flow Tests chosen depending on the analyte.
2.1 Sandwich Format
In a typical format, label (Enzymes or nanoparticles or fluorescence dyes) coated antibody or aptamer is immobilized at conjugate pad. This is a temporary adsorption which can be flushed away by flow of any buffer solution. A primary antibody or aptamer against target analyte is immobilized over test line. A secondary antibody or probe against labeled conjugate antibody/aptamer is immobilized at control zone.
Figure 2-1 Schematic of sandwich format of LFA
Sample containing the analyte is applied to the sample application pad and it subsequently migrates to the other parts of strip. At conjugate pad, target analyte is captured by the immobilized labeled antibody or aptamer conjugate and results in the formation of labeled antibody conjugate/analyte complex. This complex now reaches at nitrocellulose membrane and moves under capillary action. At test line, label antibody conjugate/analyte complex is captured by another antibody which is primary to the analyte. Analyte becomes sandwiched between labeled and primary antibodies forming labeled antibody conjugate/analyte/primary antibody complex. Excess labeled antibody conjugate will be captured at control zone by secondary antibody. Buffer or excess solution goes to absorption pad. Intensity of color at test line corresponds to the amount of target analyte and is measured with an optical strip reader or visually inspected. Appearance of color at control line ensures that a strip is functioning properly. Figure 2-1 shows schematic of general sandwich format of LFA.
2.2. Competitive Format
Such format suits best for low molecular weight compounds which cannot bind two antibodies simultaneously. Absence of color at test line is an indication for the presence of analyte while appearance of color both at test and control lines indicates a negative result. Competitive format has two layouts. In the first layout, solution containing target analyte is applied onto the sample application pad and prefixed labeled biomolecule (antibody/aptamer) conjugate gets hydrated and starts flowing with moving liquid. Test line contains pre-immobilized antigen (same analyte to be detected) which binds specifically to label conjugate. Control line contains pre-immobilized secondary antibody which has the ability to bind with labeled antibody conjugate. When liquid sample reaches at the test line, pre-immobilized antigen will bind to the labeled conjugate in case target analyte in sample solution is absent or present in such a low quantity that some sites of labeled antibody conjugate were vacant. Antigen in the sample solution and the one which is immobilized at test line of strip compete to bind with labeled conjugate. In another layout, labeled analyte conjugate is dispensed at conjugate pad while a primary antibody to analyte is dispensed at test line. After application of analyte solution a competition takes place between analyte and labeled analyte to bind with primary antibody at test line (See Figure 2-2).
Recently, a unique change was introduced in conventional design of LFA by introducing a new line (antigen line) in between test and control lines for detection of C-reactive protein (CRP) in serum samples. This format involves somehow a competition between analyte in solution and analyte pre-dispensed on a new line. New line was formed by dispensing CRP antibody solution followed by CRP solution. In case of very low concentration of CRP in sample, most of the labeled conjugate molecules will remain unreacted and migrate to antigen line and CRP present at antigen line will capture these labeled conjugates and it will result in an intense color at antigen line and rest of labeled conjugate will move to control line and will produce relatively a light color. In case of very high concentrations, most of CRP molecules will be captured at test line and will be sandwiched in between labeled conjugate and prefixed antibody at test zone, this complex will move and be captured by control line antibody. In this case very few labeled conjugate molecules will be retained at antigen line. The lesser the color at antigen line, the higher the concentration of analyte. This format can be tried for other clinical and non-clinical analytes.
Figure 2-2 Schematic of competitive format of LFA
2.3 Nucleic acid lateral flow (immuno) assay
The NALFIA set-ups are usually designed for testing the presence or absence of pathogens in food, feed or the environment. In the NALFIA set-ups, the analyte is an amplified double-stranded nucleic acid sequence (ds-amplicon) specific of the organism using primers with two different tags; recognition of the analyte is done by binding to a tag-specific antibody. In a typical layout developed for the detection of pathogenic bacteria the nucleic acid was amplified using PCR with two tagged primers. A ds-amplicon was obtained with one strand labelled with biotin and the other strand labelled with, e.g., fluorescein isothiocyanate or digoxigenine. A solution of antibodies raised against the tag was sprayed at the test line. The biotin will bind to the avidin-labelled nanoparticles and the other tag will bind to the antitag antibody, resulting in the coloured signal. The response is directly proportional to the amount of analyte.
Nucleic acid testing has important applications in food safety analysis, environmental monitoring and increasingly in medical diagnostics. Meeting the emerging paradigm of medicine, in which pharmacogenomics and individualised theranostics are of increasing importance for patient stratification and avoidance of adverse drug effects, there is a clear need for rapid, inexpensive, highly sensitive and simple-to-use companion diagnostic tests for the qualitative/quantitative detection of nucleic acids.
Figure 2-3 Schematic of Nucleic Acid Lateral Flow (Immuno) Assay