Old ideas, new technologies for immunochemistry

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.

Immunochemistry for analyzers are becoming more and more popular globally as these are proving to be an effective tool to diagnose cancer, hepatitis, illegal drugs, fertility problems, sodium levels, endocrine function, and the detection of blood clots. Globally, the immunochemistry market represents approximately 40.1 percent of the global in vitro diagnostics (IVD) market. Immunochemistry is by far the largest segment by volume, mostly due to routine testing. It is primarily driven by growing access to healthcare. It is important to know which analyzer to use as different analyzers have different analysis time, and need different reagents, which make these instruments highly sophisticated. With the rapid technological advancement in healthcare industry, the immunochemistry market is expected to grow from USD 1.69 billion in 2018 to USD 2.77 billion by 2025, reflecting a healthy CAGR of 7.27 percent, according to Market Insights Reports.

Under the strong rules and regulations, laboratories find it difficult to have healthy revenues. Thus, it is very important to find ways to sustain in such a cost-crunched environment. Due to cost cutting in clinical lab fees, profitability per test is decreasing, which makes it necessary for the laboratories to focus on volume rather than the value. There is also heavy pressure for quality, error-free results to ensure patient satisfaction. This forces the labs to lean toward more automated systems with effective workflow solutions. Laboratories are encouraged for automation due to heavy influx of patients with insurance coverage. But with a limited workforce in the clinical lab, it is difficult to manage the huge inflow of patients. On the other hand, patients need prompt and error-free results. Thus, this scenario demands laboratories to seek the help of systems that have high accuracy, with managing high-growing volumes and also offer remote data-acquisition capabilities. The automated analyzers are available with barcode readers, rack detection systems, and sample/plate identification modules to avoid plate or sample switch.

Demand for immunochemistry analyzers is slowing in the US and Western and Eastern Europe. Europe’s challenges in this segment include laboratory consolidation in France and economically troubled Greece, Italy, Spain, and Portugal. China’s growing rural hospital market lacks basic diagnostics laboratory infrastructure and represents an untapped opportunity for affordable immunochemistry analyzers.

The market is likely to witness stiff competition among the prominent players that are engaged in the market across the globe. The high development of the medical industry and the increasing investments by the players for research activities are projected to ensure the development of the market in the next few years. Moreover, the high number of collaborations and acquisitions is considered as another key factor that is predicted to ensure the market development in the coming years.

Technology trends
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.

Western blot. Western blots can take two days of manual labor to run, require a large amount of sample (20–30 µg), and typically detect a single protein at a time.

Not anymore, with some of these new techs!
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 3 uL of sample per capillary and can run up to 96 samples in a mere three 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 (20µm diameter) that fit approximately a single cell. Lysis of each cell is performed right in the gel, gel electrophoresis 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.

Immunohistochemistry (IHC). 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.

Automation of IHC-systems is 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 workflow as well as the quality and reproducibility of results.

IHC image analysis using artificial intelligence. Many of the 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.

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 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 detection of 50–100 parameters at once!

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 get the most detailed information from every sample.

Enzyme-linked immunosorbent assay. 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 two new ELISA platforms are addressing these shortcomings.

Single molecule array. Single molecule array (SIMOA) technology can count the presence or absence of a signal for each molecule within a sample, resulting in a 1000× 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. Do not have access to the platform? Multiple companies are also offering ultra-sensitive SIMOA biomarker testing services.

Multi-array assay technology. 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. 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. Multiple vendors now offer conjugation services and kits.

Immunofluorescence. 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.

Antibody engineering. 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.

Outlook
Innovation and affordability are the two key growth factors and manufacturers are moving toward development of compact and affordable solutions. Cartridge-based immunology testing systems are gaining momentum because of the simple operation, compact hardware, and smaller pack sizes. Through all of the technological advances, systems are evolving according to the needs of users in terms of operator convenience, accuracy, specificity, speed, robustness, and sensitivity. Despite these tremendous advances, there is a critical need for novel diagnostic platforms in order to increase the outreach of immunodiagnostics to remote settings.

Second Opinion
Interference in immunoassays

Prof (Dr) Viyatprajna Acharya
Professor, Dept of Biochemistry, Siksha ‘O’ Anusandhan University,
IMS & SUM Hospital, Bhubaneswar, Odisha

Immunoassays are playing a pivotal role in diagnosis and prognosis due to their high specificity, sensitivity, and a wide array of test parameters including hormones, cancer biomarkers, toxicology parameters, and nucleic acid assays. Currently, fourth-generation immunoassays in terms of ECLIA (electrochemiluminescence) have taken the front seat leaving behind radio-labeled isotopes in liquid-phase assays to solid-phase assays, based on monoclonal antibodies.

However, all the immunoassays face some setbacks in terms of interferent, be it endogenous or exogenous. The endogenous factors can be hormone-binding proteins like SHBG, albumin, thyroid-binding globulin (TBG), or cortisol binding globulin (CBG); analyte auto-antibodies, heterophilic antibodies, and human anti-animal antibodies. A new entrant in this list is biotin, which otherwise known as vitamin H or B7 is now being used as a supplement for different indications like type 1 DM for improved glycemic control, neurological diseases, or improving nail, hair, and skin health, or to maintain a healthy pregnancy. It has been seen that due to biotin excess in samples, there is abnormally high or low (as per the assay format) value of the parameters utilizing biotin-streptavidin-linkage principle.

Exogenous factors like hemolysis and icterus do not affect immunoassays as other analytes and lipemia can affect in some immunoassays. There is also high dose-hook effect due to analyte excess that can saturate both capture and detector antibodies. Thus, as per the assay format there may be falsely low or high values for the analyte.

The optical limitations are light leaks, light piping, and high background luminescence from assay reagents and reaction vessels (plastic cuvette). The extreme sensitivity of chemiluminescence warrants stringent controls on purity of reagents and the solvents and an efficient injector, facilitating adequate mixing when the triggering agent is added to the reaction vessel.

Since the diagnosis and treatment modality as well as dose titration depend on the values of assay parameters, it is the concern of laboratory physicians to minimize errors from their end. No methodology is fool-proof and free from interferences. It is the role of laboratory physicians to be aware of the interferences and they should add a note of caution at the bottom of the report as and when required.