New technologies are flipping traditional antibody-based assays on their heads. These assays are quicker, more sensitive, can be conducted high-throughput, and require minimal sample. It’s an exciting time to be a bench scientist!
I've organized these new technologies according to the corresponding traditional immunoassay that they’re improving upon. Surely there are more that I'm not yet aware of, so please let me know about them in the comments below.
Western blots can take 2 days of manual labour to run, require a large amount of sample (20-30 µg), and typically detect a single protein at a time. Not anymore if you’re lucky enough to get your hands on some of this new tech!
Western Blotting Using Capillary Electrophoresis (WesCE)
A hybrid between capillary electrophoresis (CE) and conventional western blotting was developed where each capillary contains stacking and separation matrices, and immunostaining can be done right in the capillary.
This technology is available in the Simple Western platform by Protein Simple, which automates your westerns from protein separation all the way through detection. It requires only 3uL of sample per capillary and can run up to 96 samples in a mere 3 hours.
Single-cell Western Blotting (scWestern)
Since many cell populations are actually heterogeneous, Hughes et al., at UC Berkeley developed an approach to measure protein expression within each individual cell.
The method uses a thin polyacrylamide gel prepared with micro-wells (20 µm diameter) that fit approximately a single cell. Lysis of each cell is performed right in the gel, gel electrophresis is carried out, proteins are immobilized to the gel-matrix with UV-light, and immunoprobing is conducted.
A scWestern can measure the protein abundance in thousands of individual cells on a single micro-gel in just 4-6 hours.
Traditionally the sample preparation for IHC is manual and therefore variable, and the results are interpreted by eye. The following technologies automate the IHC protocol to reduce variability in the results and use artificial intelligence to interpret the resulting images, which can discover trends that even a human scientist might miss.
If you’re in a high-volume pathology lab, you’ll want to check out these advancements.
Automation of IHC
Systems are now available to automate various stages of the IHC protocol, which helps to standardize the process and reduce variability in the results. These systems will improve your workflow, as well as the quality and reproducibility of your results:
- Dako Omnis (Agilent)
- BOND series (Leica)
- Ventana BenchMark XT and ULTRA ISH/IHC Systems (Roche)
- SNAP i.d. 2.0 IHC system (MilliporeSigma)
IHC Image Analysis Using Artificial Intelligence
Many of the above systems create IHC images at an increased rate, resulting in a bottleneck when a human scientist is needed to interpret the results. Now, multiple companies are applying image recognition artificial intelligence (AI) to analyze these results, and can even identify trends across images that a human eye may not pick up on.
Check out the image recognition AI technology provided by the following companies:
Flow cytometers can accurately characterize individual cells within a population and physically separate the resulting sub-populations, yet this characterization is limited by the number of cell parameters that can be measured.
These new technologies allow for the detection of 50-100 parameters at once!
Multiparameter Flow Cytometry
The award for the highest number of parameters that can be detected through fluorescence-based flow cytometry goes to the FACSymphony by BD Biosciences.
Their instrument can measure up to 50 parameters across 10 laser wavelengths, allowing the analysis of rare or complex cell types and events.
Mass Cytometry (CyTOF)
Instead of the traditional fluorescent probes used in flow cytometry, mass cytometry employs heavy-metal labeled probes to significantly reduce signal overlap and increase the number of detectable parameters to over 100.
The Helios mass cytometer by Fluidigm is currently the only platform on which mass cytometry can be performed, and helps you get the most detailed information from every sample.
Enzyme-linked Immunosorbent Assay (ELISA)
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay that uses antibodies to detect and quantify their ligands (often proteins) within a liquid sample. Traditional ELISAs can only detect a single analyte and have limited sensitivity, which makes the detection of low abundance analytes difficult.
The following two new ELISA platforms are addressing these shortcomings.
Single Molecule Array (SIMOA)
Single molecule array (SIMOA) technology can count the presence or absence of a signal for each molecule within a sample, resulting in a 1,000x increase in sensitivity compared with traditional ELISAs. This allows for the detection of proteins that were previously difficult or impossible to measure.
SIMOA technology was developed by Quanterix and can be run on various platforms that they provide. Don’t have access to the platform? Multiple companies are also offering ultra-sensitive SIMOA biomarker testing services.
Multi-Array Assay Technology (Meso Scale Discovery)
Meso Scale Discovery’s electrochemiluminescence (ECL) technology uses SULFO-TAGTM labels that emit light upon electrochemical stimulation initiated at the electrode surfaces of the microplates. By combining this with patterned arrays, their technology allows for the detection of multiple analytes in a single well with improved sensitivity and no washes are needed.
In immunofluorescence (IF), fluorescently labeled antibodies are used to detect analytes in a sample that can be visualized with a fluorescence microscope.
Traditionally, IF is conducted on a single layer of cells or a thin slice of tissue to determine the distribution of the biomolecule, and only a few analytes can be measured in each sample.
Tissue Clearing Methods
Several types of tissue clearing methods have been developed to visualize protein localization within 3D tissue samples.
These methods can render whole organs or even whole organisms transparent, allowing for the visualization of internal proteins with antibodies that have been fluorescently labelled. The see-through tissue allows the passage of light required to excite the fluorescent tags.
Check out the following tissue clearing methods:
- 3DISCO (3D imaging of solvent-cleared organs)
- uDISCO (ultimate DISCO)
- vDISCO (nanobody(VHH)-boosted DISCO)
- SeeDB (see deep brain)
- PACT (passive CLARITY technique)
Iterative Indirect Immunofluorescence Imaging (4i)
4i was developed by Gut et al. (2018) as a high-throughput immunofluorescence method that uses iterative hybridization and antibody removal to detect more than 40 different proteins at once in a biological sample.
The best thing about 4i is that you can use conventional fluorescence microscopes and off-the-shelf antibodies!
That's All for Now (What Did I Miss?)
I wish I had access to some of these technologies when I was at the bench!
Which of these would be the most useful to you and your lab?
Is your favorite tech missing from this list?
Please let me know in the comments.