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Dispositivi Lateral Flow

LATERAL FLOW DEVICES

Lateral flow devices (LFD) are field tests based on a kind of immunochromatography. All the reagents are generally inside the device and this makes the test very easy to perform, by anyone and anywhere. LFDs are less precise than other screening immunoassay-based methods, like ELISAs, but can be quantitative, sensitive, and accurate enough. LFDs are often called "strips”.

Typically LFDs have a sample pad where the sample is dropped, a conjugate pad where the tracer is adsorbed, a membrane where the binding assay occurs (having two active sites, one for the test, one for control), and an absorbent pad that is responsible for the sample and reagent flow through the membrane. When the sample is loaded on the sample pad, it moves to the conjugate pad, where it dissolves the tracer (generally a conjugate of anti-analyte antibodies to colloidal gold particles). Because of capillary forces, the flow of liquid moves through the membrane, where the reactive parts (lines of dry immuno-reagents) are located.


Generic example of a competitive lateral flow device.

A binding partner is adsorbed on the first line. In case of a sandwich immunoassay the reagent would be a capture antibody, while in case of a competitive immunoassay it would be an analyte - protein conjugate (competitor). When the sample is analyte-free, no conjugate binds to the test line in a sandwich assay (i.e. a LFD for GMO or for pathogen microorganisms); in case the test is a competitive one (i.e. a LFD for mycotoxins or drug residues), then the conjugate binds to the test line, because nothing inhibits the antigen-antibody reaction. In case the sample contains the analyte, the test line will become coloured when the LFD is a non-competitive one, and will be blank or colourless in case of competitive LFD, since the contaminant binds the gold-labelled antibodies, making them unreactive to the analyte immobilized on the test line. Thanks to the absorbent pad, even when the whole membrane is wet, the flow of liquid continues moving more sample through the membrane to react thoroughly with both reactive lines. The second line is made by antibodies that will bind the tracer whatever is the analyte concentration in the sample. In case the control line would not be coloured, at the end of the assay time, the test is to be considered invalid.


In a competitive lateral flow device, both lines will be developed for
uncontaminated samples.
For a contaminated sample, the test line of a competitive lateral flow
will be absent or slightly coloured.

One relevant feature of the LFDs is that the test kits are more stable and the shelf life is often longer than ELISAs ones, because no enzyme or other liquid bio-reagent is employed. Another feature is that the test results are much less temperature-sensitive, because the colour is not developed by an enzyme and because the assay happens in a few minutes.

Test and control lines are clearly visible by eyes. Nevertheless, in order to avoid differences in result evaluation, as well as to allow quantitative measurements, refractometer readers have been developed.


ELISA IMMUNOASSAY

ELISA (Enzyme Linked ImmunoSorbent Assay) is an assay based on the highly specific binding of antibodies to a molecular structure of an analyte or a group of molecules that share it. The binding of antigen to antibodies take place generally in a microwell and the extent of binding is revealed by the amount of colour measured in the well at the end of a series of reaction steps. The colour is due to the enzyme activity, being the enzyme (mainly horseradish peroxidase) linked either to antibodies or antigens: it is the so-called "conjugate" or tracer.

In food diagnostics both competitive and non-competitive immunoassays are employed. Non-competitive (sandwich) immunoassays can be used to detect high molecular weight compounds (macromolecules) or micro organisms (bacteria or viruses). Competitive immunoassay are more complex assays developed in order to measure small molecular weight compounds, whose structure cannot allow the simultaneous binding of two antibodies. While in case of the non-competitive ELISAs the design of the assay is basically all the time the same (capture antibodies on the reaction vessel, developing antibodies added after the first incubation and washing step) in case of competitive ELISAs different designs are possible.

The most common design of competitive ELISAs is the direct one. The antibodies are adsorbed on the reaction wells and the sample is added to the wells together with a tracer or conjugate. The conjugate is the signal generating partner, being a complex between an analyte analogue and the enzyme. In a reference well ("B zero" or "zero binding" well) no analyte is added. The whole amount of tracer can bind the antibody and, after the washing step that removes any unbound molecule, the enzymatic reaction of a colourless substrate will develop an amount of colour. In all the other wells, both where standard analyte or contaminated samples are added, the binding of the conjugate is inhibited by some extent, since part of the antibodies binding sites are saturated by the free analyte. The higher is the analyte concentration, the lower is the conjugate binding and the developed colour.


A direct competitive immunoassay.

Another possible design of competitive ELISAs is the indirect enzyme immunoassay (originally described in literature as IEMA). In this kind of assay the binding partner coated on the microwells is the antigen instead of the antibody. The reference or "B zero" well is the well where the highest signal is induced by the binding of the conjugate (antibody-enzyme complex) to the antigen without any inhibition. In all the other wells the free analyte in standards or contaminated samples will bind part of the antibodies, lowering the amount of conjugate binding to solid phase. After the washing step the different extent of binding is hence converted in different amount of colour by the enzyme reaction.

Both direct and indirect ELISAs can be developed using just the specific anti-analyte antibodies or employing anti-antibodies too. In case of the direct ELISA, anti-antibodies are coated on the microwells, while the specific anti-analyte antibody has to be added to the well. As a result, just one more liquid reagent has to be added, and the test maintains just one washing step.



A direct competitive immunoassay with double antibody.

n case of indirect, or antigen-coated ELISAs, the double antibody version has a longer assay implementation. In a first incubation just the core binding step is performed (competition of free antigen and immobilized antigen for the antibodies binding sites in the liquid phase). After a washing step the bound antibodies are detected by means of anti-antibodies labelled by an enzyme. A second washing step is followed by the addition of the colourless substrate: the assay is hence made by three incubations separated by two washing steps.

Of course both the precision and the accuracy of the liquid handling, as well as the quality of the washing step are very important in order to get consistent test results.

At the end of the process the amount of colour (absorbance) in each well is measured by a photometer. The photometer is generally a microplate reader whose computer can calculate the analyte concentration of samples by interpolating the absorbance values into the calibration curve (either a batch-reference curve or the curve performed in the test itself).



IMMUNO-AFFINITY COLUMNS

Immuno-affinity columns (IAC) are based on a specific antibody-analyte binding. These tools are developed in order to enable sample purification and concentration, thanks to their capability of collecting the contaminant of interest and removing the interfering compounds that can generate background noise both in screening or instrumental (chromatographic methods) analysis. The columns contain a gel bed coupled with specific antibody for the analyte of interest. When the sample flows through the column, the antibodies retain only the molecules of interest, draining interfering compounds into the flow through. By means of an apposite elution solution, the analyte can be collected for further analysis.