In the context of the COVID-19 epidemic, test strips based on the principle of thin-layer chromatography are becoming increasingly relevant.

The LFT (immunochromatographic strip) consists of the following basic elements (Fig. 1):

• A plastic base onto which all other components of the test are glued.
• Sorbents with a particle size of 10-20 µm are applied to the base, usually made of glass, plastic or aluminium;
• Layer thickness ranges from 100 to 250 µm (for analytical plates) to 2 mm (for preparative plates).
• Filter (sample pad).
• Pad or membranes with a conjugate.
• Chromatographic membrane containing one or more immune complex capture zones and a control capture zone.
• Absorption membrane (absorption pad).

The test strip can be placed in a plastic casing, which has a sample well and test window. [1]

The liquid on the test strip begins to migrate along it due to the capillary effect according
to the principle of the aforementioned thin-layer chromatography. [2]

There are several types of lateral flow tests in the world at the moment. Let us consider some of them:

Direct or “sandwich” LFT format. [1], [2], [3]

The “first” antibodies, which are dried in the form of an active zone, are attached to colloidal gold beads (red in the figure) – all this forms a specific antibodies conjugate (Fig. 2).

The sample is put on one of the ends of the test strip so that it moves into the active zone under the effect of capillary forces (Fig. 3).

The “first” antibodies “capture” the target antigen (analyte) and also move along the strip (At-label) (Fig. 4).

• In the test zone (T-zone) on the test strip, the “second” antibodies are dried to the target antigen. When passing through this zone, the target antigen captured by the “first” antibodies is also captured by the “second” antibodies, resulting in the so-called “sandwich” (At-Ag-Am-label), which forms a stained line.
• In the control zone (C-zone) on the test strip, specific antibodies specific to the “first” antibodies (regardless of the binding of the target antigen) are dried.

Thus, some of the colloidal gold beads will in any case attach to the C-zone forming the second stained line (Fig. 5, 6).

Fig. 6: Interpretation of direct LFT results. Source: https://www.monographies.ru/ru/book/section?id=9929 Eventually, + Positive result: there is a target antigen in the sample and two stained lines (T-zone and C-zone) are formed; – Negative result: If there is no target antigen in the sample, only one line (C-zone) is visible.

The direct LFT method is used to detect high molecular weight compounds – viruses, including HIV; various hormones (e.g., in pregnancy tests), infectious disease agents.

The sandwich LFT is not suitable for the detection of low molecular weight substances, because the antigen must have at least two antigenic determinants (i.e., antigen binding sites) to form an At-Ag-At sandwich. Therefore, a different scheme, indirect competitive LFT, is used for the analysis of small molecules, including narcotic substances. Advantages of the method:

• speed,
• ease of use,
• the possibility of using non-instrumental LFT with the visual assessment of the test result.

Competitive LFT method [1], [2]

This method is based on competition between

• analyte (antigens) and
• immobilized analyte conjugate: carrier protein (test zone)

for a limited number of binding sites in the At-label conjugate.

When the sample is applied, the analyte binds to the At-label conjugate on the membrane, forming an immunocomplex. The latter passes through the test zone (the analyte conjugate – carrier protein – is immobilized on it).

Let us suppose that the sample contains antigens in large quantities. In this case, the immunocomplex cannot bind to the conjugate in the test zone: low molecular weight compounds usually have one antigenic determinant (antibodies have one antigen binding site), and that one is already occupied by the antigen from the conjugate.

Next, the immune complex is bound by anti-species antibodies on the control line.

As a result, the absence of a stained stripe in the test zone and the presence of a stain in the control zone indicates that the concentration of the analyte in the test sample exceeds its threshold value for this test.

Thus,

•  + Positive result is indicated by a single stained stripe in the test zone (C-zone),

If there is no analyte in the sample, the At-label conjugate binds to the analyte conjugate: the carrier protein immobilized in the test line zone. The unbound At-label conjugate gets into the control line region and binds to the antispecies antibodies there. Thus, the presence of two stained lines (test and control) is a negative test result. I.e.

• – A negative result will be indicated by the two stained stripes (T- and C-zone).

Various particles with the following properties are used as labels in the LFT:

1. Coloring agents (nanoparticles of colloidal gold or carbon, or particles of stained latex). The use of different staining labels attached to the latex particles allows multi-analysis where lines of a different color correspond to different analytes. The most commonly used label is colloidal gold nanoparticles, which form dark brown (or dark pink) lines.
2. Fluorescent, phosphorescent and bioluminescent labels covalently bound to latex particles.
3. Paramagnetic labels (also attached to latex particles).
4. Enzyme labels are used in the same way as in ELISA (enzyme-linked immunosorbent assay). The test results are recorded by staining the substrates, and the test result is visual, or read using a reader.

When choosing a material for a chromatographic membrane, the following properties of the membrane should be considered:

1. Hydrophilicity is necessary to allow flow (which is aqueous media) through the membrane.
2. Ability to bind proteins: in order to form a test and control line on the membrane, protein conjugates must be fixed in such a way that they are not washed away by the lateral flow. Proteins are “attached” to the membrane through electrostatic interaction: the dipoles contained in nitrocellulose bind the protein dipoles.

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References

1. Научная электронная библиотека [Электронный ресурс]. URL: https://www.monographies.ru/ru/book/section?id=9929

2. Капиллярность [Электронный ресурс]. URL: https://ru.wikipedia.org/wiki/%D0%9A%D0%B0%D0%BF%D0%B8%D0%BB%D0%BB%D1%8F%D1%80%D0%BD%D0%BE%D1%81%D1%82%D1%8C

3. Иммунохроматография [Электронный ресурс]. URL: https://www.youtube.com/watch?v=xWPOnh_xdts