Immunoassays have been around for many decades, with the development of the first ever immunoassays being in the 1950s. These tests measure the concentration of a molecule using antibodies or antigens. A label is detected with a plate reader to measure the signal received by the binding of the antibody with the analyte (fluorescence, radiation, color change, etc.).
The LDTD technology, on the other hand, relies on a laser beam that heats the back of a specially-designed well-plate to desorb the sample and carry it into a mass spectrometer, where the molecules are analyzed.
So, why should you choose the LDTD technology over a well-known and safe technology like the immunoassay for high-throughput screening?
Simplicity and speed.
Let’s break it down into a few categories:
One of the limitations of immunoassay is the selectivity of antibodies. Selectivity in immunoassay can be influenced by matrix effects, cross-reactivities and also by autoantibodies and human anti-reagent antibodies. Analytes that belong to a same family of molecules are not distinguishable because of their similar reaction to the antibody used. Furthermore, families of molecules are usually analyzed with a single assay. This causes false positives, and sometimes false negatives to occur. Of course, this can be bypassed by working on producing highly-selective and specific antibodies, but this process can take up to 2-3 months and is very costly.
To compare with the LDTD technology coupled to mass spectrometry, the detection of the same analyte, with sample extraction steps done before hand, would generate a result with a parent mass and a specific daughter mass attached to it. This way, very similar molecules can be distinguished by optimizing the MS/MS conditions or the sample extraction steps. Multiple sample extraction procedures have already been developed for the usual fluids used (urine, blood, plasma, hair, saliva, …). As such, when a new designer drug is created for example, its transition can just be added to the method and analyzed with every other molecule in the sample.
An immunoassay, such as ELISA, requires between 100 and 200 µL of sample. This can cause problems with samples that are scarce. Multiplexed immunoassays were developed to lower the sample volume used, but the volumes are still higher than other techniques use. The volume deposited on the LazWell™ plate to be used with LDTD can vary between 0.1 µL and 10 µL. In certain applications, the actual sample volume collected from an individual can be as low as 10 µL.
An ELISA test for a single analyte (or a molecule group) can range from 4 to 5$. This cost is heavily influenced by the cost of the antibody used. Of course, the cost per analyte can be reduced by multiplexing and depends on the platform used. As for the LDTD technology, the cost per sample can be as low as 0.1 to 0.5$. This cost can also be lowered by multiplexing samples and depends on the LazWell™ model used (96-wells, 384-wells or 1536-wells).
If we consider the antibody-antigen binding and the wash steps, an assay of a single plate can take up to 2-4 hours to be accomplished. So, for a 96-well plate, this is approximately 150 seconds per sample. Depending on the instrument, multiplexing and the protocol used, the assay time can be lowered or increased.
The analysis time sample-to-sample with the Luxon Ion Source® can be below 1 second without considering the sample preparation. Sample preparation can differ a lot depending on the matrix and the molecule analyzed. For example, a simple liquid-liquid extraction done manually on 96 samples can take about 45 minutes, so 30 seconds per sample. A single sample can contain dozens of different molecules to be analyzed and its preparation can also be done with an automated liquid handler.
All-in-all, the LDTD technology coupled to a mass spectrometer (LDTD-MS/MS) can easily replace immunoassays in high-throughput screening.
Check out our toxicology section for more information on our applications.