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.
In-cell Western Blot (ICW)
This technique goes by a few different names, including in-cell ELISA, cytoblot, and near-infrared immunocytoblot (see Stockwell, Haggarty, & Schreiber). An in-cell western can quantify the amount of a target protein (or post-translational modification of a target) in a cultured cell. The in-cell western requires comparatively less processing and may, therefore, be more quantitative. Adherent or suspension cells are cultured in a microplate, fixed and permeabilized, and then treated with up to two primary antibodies. This is followed by incubation with secondary antibodies — ideally conjugated to a near-infrared fluorophore (see Chen, et al.).
Start to finish, it’s estimated to take ≤ 5 hrs for 96+ samples.
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:
Imaging Mass Cytometry
Imaging Mass Cytometry was first described by Giesen et al. (2014) - it uses a combination of laser ablation techniques and CyTOF mass spectrometry where the antibodies are actually conjugated to heavy-metals instead of enzymes or fluorophores.
This technology allows for highly multiplexed immunohistochemistry (IHC) or immunocytochemistry (ICC), of up to 37 different protein markers at once. Fluidigm offers an exclusive catalog of heavy-metal conjugated antibodies as well as the Hyperion Imaging System for Imaging Mass Cytometry.
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.
Immuno-PCR combines ELISA and real-time PCR (RT-PCR) by conjugating the detection antibody to an oligo. The antibody detects the analyte, and the amount of associated oligo can be quantified with RT-PCR. Immuno-PCR is extremely sensitive and can detect analytes in the pg-fg range, as well as being highly amenable to multiplexing with the use of multiple unique oligos.
Although immuno-PCR was developed in 1992 by Sano et al., it was not widely adopted due to the difficulty of conjugating antibodies to oligos. Recent developments in antibody conjugation technology are making immuno-PCR much more accessible and customizable.
Multiple vendors now offer conjugation services (Creative Biolabs, Bio-Synthesis) and kits (Novus Biologicals, Abcam). Alternatively, avoid the learning curve of RT-PCR and send your samples to RayBiotech for full immuno-PCR testing.
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!
Antibodies are the keystone reagents for all immunoassays. Thanks to advancements in antibody engineering, multiple new types of antibodies have been developed to address immunoassay challenges such as tissues that are difficult to penetrate, high background signal from pesky endogenous antibodies, and needing to coordinate the hosts when using multiple primary antibodies.
Antigen binding fragments (F(ab) fragments) contain just a single antigen binding domain of the antibody, or one arm of the Y shape, and weigh ~50 kDa.
Since this is much smaller than a whole antibody (~150 kDa), F(ab) fragments can be used to penetrate difficult tissues in assays like IHC, and less accessible regions on the antigen.
Due to the monovalent nature of F(ab) fragments, they can also be used as secondary antibodies in an experiment that applies multiple primary antibodies from the same host species. Similarly, they can allow the same polyclonal antibody to be used for both capture and detection in a sandwich ELISA.
F(ab) fragments can also be used to block pesky endogenous antibodies and reduce the amount of background noise when using a primary antibody from the same species as the sample.
Finally, since these fragments lack the Fc region, they can be used in in vivo assays to reduce the harmful immune response that can occur from recognition of the Fc region of a full-length antibody.
Single-chain variable fragments (scFvs) are actually a linker-fusion of the two variable domains (heavy and light chains). This produces a ~25 kDa fragment that’s even smaller than a F(ab) fragment, while maintaining full antigen binding capacity.
scFvs exhibit a lot of the same benefits as F(ab) fragments and can be even more effective at penetrating difficult tissues, and binding to difficult epitopes such as narrow cavities on the target antigen.
Nanobodies, also called single domain antibodies (sdAbs), are fragments derived from the antibodies produced by camels, llamas, alpacas, or sharks. The fragments of these antibodies only contain a single domain, and are therefore only 12-15 kDa–about half the size of an scFv.
Similar to other antibody fragments, nanobodies are ideal for in vivo assays, and for binding to epitopes that are even more challenging to access such as the active sites of enzymes or structural canyons.
ChromoTek specializes in nanobodies and has an extensive catalog.
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.