To keep pace with the ever-growing requirement to minimize hands-on time while increasing the number of readouts that can be obtained from precious sample material, a wide range of automated immunodetection systems has been developed. Including instruments that combine the entire Western blotting process into a single platform, systems affording more consistent staining of tissue samples, and walk-away solutions for ELISA processing, these sophisticated devices are complemented by automated liquid handling to streamline immunodetection while improving data quality.
Developed as an alternative to Western blotting, Simple Western™ systems from ProteinSimple, a Bio-Techne brand, provide gel-free, blot-free protein separation and detection. Lysates are prepared similarly to those used for traditional Western blots, then Simple Western automates the protein capture, antibody probing, washes and detection steps. This provides greater reproducibility through elimination of user-bias, as well as a larger linear dynamic range for more accurate quantitation, and faster time to results, according to the company.
“Simple Western assays take place in a capillary, within which proteins are separated by size or by charge,” explains Brian Quast, senior director, product marketing. “During size-based assays, antibodies are used to detect target proteins following immobilization of the entire protein population to the capillary wall. Charge-based separation involves a similar process. However, by co-loading ampholytes, protein separation is based on pI to identify discrete changes such as post-translational modifications.”
The most recent model within the Simple Western product range, Jess, features fluorescence detection and total protein staining and normalization capabilities. The instrument can process up to 25 samples in 3 hours while conserving sample with a 3 µL load requirement. “We’ve successfully validated over 1,200 antibodies from Novus Biologicals and R&D Systems for use in the Simple Western application,” adds Bryan Tinsley, marketing director at Novus Biologicals, a Bio-Techne brand, “and we’re continually expanding this list through in-house and customer testing.”
Automation
Immunohistochemical staining of tissue samples is another notoriously time-consuming process that has been improved using automation. “Our Lab Vision™ Autostainers perform all steps of an IHC staining protocol to generate highly reproducible data,” says Daniel Ehler, global marketing manager, Thermo Fisher Scientific. “This includes the application of blocking reagents, primary antibodies, secondary antibodies, or detection reagents, and chromogens, in addition to carrying out all necessary washing steps. By dispensing precise reagent volumes and incubating for the exact length of time required, Autostainers overcome the unavoidable inconsistency caused by applying reagents manually.”
Currently affording a maximum capacity of 48 slides (the 480S-2D model), and suitable for use with FFPE or frozen tissue samples, Lab Vision Autostainers are easily combined with other Thermo Fisher Scientific products. “For automated antigen retrieval of FFPE tissue we offer the Lab Vision PT module,” notes Ehler. “This uses gentle heat to preserve tissue integrity. By avoiding the damaging effects of temperature variations, microwaves or pressure, the PT module delivers unrivalled reproducibility.”
Also benefiting from automation, ELISA processing can now be considered a walk-away procedure that frees up considerable time to be spent on other tasks. “Our ELISA NIMBUS, designed using the Microlab NIMBUS liquid handling platform, can process up to eight 96-well microplates in parallel,” says Corrie Spoon, product support engineer at Hamilton. “The instrument software is highly intuitive, providing the option to process all samples on all assays within a run or to assign individual samples to specific assays using a worklist. End-to-end sample tracking provides an additional level of confidence in the resulting ELISA data.”
Kristina Klette, market segment leader, biotechnology, adds that both off-the-shelf and in-house ELISAs can be performed on the ELISA Nimbus with equal ease. “This system is extremely flexible,” she says. “Although the maximum capacity will be influenced by the choice of labware and any specific assay requirements such as dilutions, by providing the option to load samples in either tubes or microplates almost any ELISA assay can be adapted to run on the ELISA NIMBUS.”
Flow cytometry
Flow cytometry is yet another popular technique to have experienced the advantages of automation. For example, Miltenyi Biotec’s MACSQuant® X is easily incorporated into automated flow cytometry protocols to provide processing and analysis of up to ten parameters in 96- and 384-well formats in only 15 minutes and just under 60 minutes, respectively. According to Johannes Fleischer, global product manager, this is a truly hands-free instrument that offers speed, high throughput, and consistency.
“We’ve developed a whole ecosystem around the MACSQuant instrument family to fulfil automation and high-throughput requirements,” says Fleischer. “Since the MACSQuant instruments possess features that allow automated start-up, calibration, compensation, staining, washing, and shut-down processes we’ve been able to develop so called “Express Mode” assays that can run completely autonomously.” MACSQuant Express Modes simplify flow cytometric analysis via pre-defined experiment settings alongside acquisition and analysis templates. “We also offer a service for integration of Thermo Fisher Scientific’s Cytomat™ plate hotels with the MACSQuant instruments and have supported customers in the integration of liquid-handling systems such as TECAN and HighRes,” adds Fleischer.
Dispense with ease
Dr. Paul Held, applications laboratory manager at BioTek, notes that the company’s EL406™ Combination Washer Dispenser represents an essential workhorse for any laboratory that requires automated dispensing and washing for 96- and 384-well plates. “Multiple dispense and aspiration of wash buffer to each well of a microplate is incredibly arduous,” he says. “The EL406 is easily integrated with BioTek’s BioStack™ Microplate Stacker to automatically process up to 50 plates at a time.”
While the EL406 can be incorporated into many different assay protocols, immunocytochemical staining is one application that has seen considerable benefits. “Typical fix and stain workflows can be challenging to perform manually since cells may easily be damaged or inadvertently removed,” explains Held. “Automation with the EL406 allows for optimization of dispense and aspirate parameters, including x-y-z positioning during these functions. These adjustments enable dependably good results compared to manual methods.”
Image: Use of the EL406™ to fix and stain NIH 3T3 cells expressing GFP. Green: GFP, red: F-actin, blue: DAPI. Image courtesy of BioTek.
As well as its utility for cellular staining, the EL406 can be employed for cell seeding and media exchange. “Using two different target primary antibodies of different species and their corresponding labeled secondary antibodies is quite common,” says Held. “The EL406 uses only slightly more antibody than manual staining methods, and although addition is via a peri-pump dispenser that requires 4 mL to prime the tubing, the antibodies are easily recovered at the end of the dispense by purging.”
The systems described here are just some of the innovative automated immunodetection solutions that have been designed to streamline workflows, increase throughput, and improve data reliability. As the desire for ever more readouts within shorter timescales and requiring less user input continues to grow, instrumentation will certainly evolve further still.