I have been looking to write this for a while as I have to admit that I have a vested interest in both camps. As an immunologist by background, I’ve always been involved with immunoassays, but more recently my focus has been on liquid chromatography. In this blog I will give a balanced view of the pros and cons of each technique and finish up with my opinion. It will be interesting to see which camp you belong to.
Immunoassays have been used extensively in all areas of life science research and in diagnostic applications for many years. The purpose of an immunoassay is to identify and quantitate specific antigens in a sample, for example in a diagnostic test, where the presence of that antigen could indicate the presence of disease. They use a combination of an antibody or antibody-like molecule to capture the specific antigen or molecule of interest and additionally a reporter molecule to measure the amount of antigen so that quantitative data on the concentration can be obtained when compared to a reference standard curve. The most common format is based on the sandwich ELISA approach where an antibody specific to the antigen of interest is attached to a solid surface (a microplate well or magnetic microsphere, for example) before being exposed to the sample. The antigen in the sample then binds to the antibody and unbound components of the sample are washed off before the addition of a second antibody specific to a different epitope on the target antigen is added. This second antibody has a reporter molecule or tag attached, such as horseradish peroxidase, so that the amount of bound antibody and thus the amount of antigen can be calculated from the signal created by the reporter/tag against a reference standard curve.
Other variations of immunoassays are also used that rely on a single antibody with the array of antigens attached to the solid surface or the antigens are labelled directly, but whichever format is used they all apply the same basic principle of an antibody binding the specific antigen. For more information on immunoassays, download our immunoassay guide.
Advantages of Immunoassays
As would be expected with an established and extensively used technique there are a number of benefits of immunoassays:
- Due to its wide use, the technique is well characterised, well understood, trusted and relatively straight forward to troubleshoot.
- The equipment required is relatively inexpensive, flexible and scalable so you can choose from a relatively manual process with low capital investment to more automated and costly set-ups.
- Typically the training requirements are relatively low for the basic day-to-day operation of immunoassays.
- The technique generally offers good sensitivity and selectivity, has a broad dynamic range and is able to deal with complex samples containing multiple antigens.
Disadvantages of Immunoassay
As expected in science, as in life, there are always compromises to be made in, for example, performance and immunoassays are no exception, Here are some of their drawbacks:
- The availability of a specific antibody is critical, in some cases an antibody or antibody pair may not exist or their specificity may be inadequate. Poor specificity can lead to low sensitivity and false results.
- Coupled to the above, the range of analytes and antigens that can be detected with immunoassay is somewhat limited. Also it is very difficult to identify any post-translational modifications with immunoassay.
- Immunoassays are a multi-step process, using a complex biological molecule (antibody), in a biological reaction and as such reproducibility of the process intra- and inter-lab can be an issue. Lot-to-lot variation in the antibody product itself also has to be carefully controlled.
- While the cost of the capital equipment to perform the assay can be relatively inexpensive, the day-to-day running costs can be quite high due to the reagent usage and especially the antibody cost.
- Sample volumes, especially in ELISA, can be quite high, although the ability to multiplex and application of new technologies are decreasing the sample volumes required.
- Due to the need to bind antigen and antibody and the wash steps, the immunoassay process is quite long (1-3 hours), although again new technologies are decreasing this time.
LC / LC-MS
Liquid Chromatography (LC) and Liquid Chromatography-Mass Spectrometry (LC-MS) both offer the potential to be an alternative to immunoassays are at least complementary. The LC / LC-MS process is similar to that of an immunoassay where the LC portion is performing part of the selectivity by separating your compounds/antigens from one another, but the identification and quantitation is done by the HPLC detector in LC (usually a simple UV based detection mechanism) and the mass spectrometer in LC-MS. So while they look and feel very different from immunoassays, the basic processs is essentially the same. How does LC and LC-MS stack up to immunoassay?
Advantages of LC / LC-MS
Liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS) technology has developed at a rapid pace over the previous decade and what might once have been seen as disadvantages are now strengths:
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- Modern ultra-high-performance LC (UHPLC) and MS instruments, coupled with powerful informatics means that samples can be run and analysed in minutes, giving high sample throughput.
- Mass spectrometry offers the highest sensitivity and precision for the identification and detection of analytes. Liquid chromatography with high precision instruments such as the Thermo Scientific™ Vanquish™ UHPLC system, coupled with sensitive diode array detectors, also offers high performance.
- Required sample volumes are very small, meaning less of a precious sample is required.
- The process is almost completely automated, with few manual steps. When this is coupled to high precision instruments the results are highly reproducible with low coefficient of variance.
- Day-to-day reagent costs are low.
- A high degree of multiplexed analysis of analytes is possible.
- The option is there for further characterisation of your antigen, for example sequence identification, analysis of isoforms and post translational modifications.
- A wider range of compounds and analytes can be analysed.
Disadvantages of LC / LC-MS
There are still barriers to the widespread adoption of LC / LC-MS in immunoassay laboratories, these include:
- High cost of the initial capital investment in instrumentation which can run in to several hundreds of thousands of dollars depending on the type of instrumentation.
- The technology is perceived as being complex to operate with a heavy training commitment; however modern instruments and software have reduced this with a trend to simplification of operation in recent years.
- Selectivity in LC is not as good and as easy to optimise compared with immunoassays. Selectivity is obtained in LC by the column chemistry and does require some optimisation. This especially presents a problem with highly complex samples. In addition, when working with unknown or new samples and liquid chromatography, there is no direct way of identifying your specific antigen from all the others unless coupled to mass spectrometry. When coupled to MS, selectivity can be extremely high.
Can LC / LC-MS Ever Replace Immunoassays?
As indicated, I’m torn on this one, but believe that technological advances mean that soon LC and LC-MS will begin to replace immunoassays. However, I see an intermediate step in the transition that combines the best of both techniques.
I feel the major disadvantage with LC is that potential lack of selectivity and ability to identify accurately that particular antigen in a sea of others. If you could pull out that particular antigen, or small subset of antigens, first, then with a precise UHPLC instrument where you are confident that your antigen peak will elute at exactly the same time each run, then you can get all the LC benefits, while side-stepping immunoassay issues. The MS then adds an absolute layer of confidence and certainty to the identification although with additional cost.
One technology that could help in this respect is the Mass Spectrometric Immunoassay (MSIA™), and, as the name suggests, marries the best of both. It enables you to affinity capture your antigen of interest in a pipette tip format (offering low sample volumes and a degree of automation – tick, tick) and then elute the selected antigen for identification with LC or LC-MS.
In conclusion, I can see LC and LC-MS replacing immunoassays in the not too distant future. Indeed in some areas they have already started to do so. For assay development I can see a MSIA approach or UHPLC coupled to High Resolution Accurate Mass (HRAM) mass spectrometry being used, but when the assay is established and routine and reference standards available it can be run on standalone LC or LC-MS and gain the advantages these techniques offer.
View the LC / LC-MS options currently available from Thermo Fisher Scientific for diagnostics.
Learn more about MSIA technology and publications at the resource library.
Our webpages contain numerous resources and guides on immunoassays.