We are focused on three main immunoassays: ELISpot, FluoroSpot, and ELISA. They are sensitive methods, useful for answering questions on specific immune responses.
We like them all, for the various reasons described below.
To learn what product format suits you best,
go to Product guide.
Mabtech ASTOR 2 is ELISpot's best friend
T cell ELISpot: Human IFN-γ
B cell ELISpot: Human IgG and IgA
If one cell responds, you will find it
The enzyme-linked immunospot (ELISpot) assay is an extremely sensitive immunoassay performed in a 96-well plate format to quantify protein secreting cells.
The assay principle is straight forward: cells are cultured inside a plate coated with capture antibody. Cytokines, immunoglobulins, or other target proteins secreted by the cells are captured immediately after secretion and throughout the stimulation process. After cell removal, secreted proteins are identified using a detection antibody. Visible spots form after adding a precipitating substrate. Each spot corresponds to an individual analyte-secreting cell.
Identify rare cell populations
With minimal manipulation of the cells, ELISpot lies closer to reality than most other cell-based immunoassays down to the single-cell level. Due to capacity to find one cell in a million, ELISpot is particularly valuable for studies of rare cell populations causing immune responses.
Capture transient analytes
As ELISpot target analytes immediately after secretion and throughout the stimulation process, the method can detect cytokines that otherwise disappear from samples. For example, ELISpot can detect analytes that are rapidly degraded by proteases (IL-2), are quickly taken up by bystander cells (IL-4), or bind to soluble receptors (TNF-α).
Easy to scale up
ELISpot is robust and easy to perform, making it suitable for analyzing many samples in parallel or at different timepoints during a clinical trial. Standardization of the ELISpot assay has been achieved in specific settings and the method is the basis of an FDA-approved diagnostic test for tuberculosis. The assay is carried out on a 96-well plate and analyzed with an automated ELISpot reader, enabling rapid analysis of several plates in a row.
For assessment of T cell immunity
ELISpot is commonly used to investigate antigen-specific immune responses and to discriminate between subsets of activated T cells. This is applied in studies of infectious diseases, cancer, allergies, and autoimmune diseases. In vaccine research, ELISpot is the gold standard to define vaccine efficacy by measuring the capacity to elicit T cell responses, for example by assessing IFN-γ secretion. Diagnostic assays based on ELISpot are available, including tests to detect patients with tuberculosis or SARS-CoV-2 infection by measuring IFN-γ secretion from T cells responding to defined peptides.
Also perfect for the study of antibody-secreting cells
The B cell ELISpot assay is one of few assays measuring immunoglobulins directly
upon secretion. There are two strategies: Firstly, the B cell ELISpot can be used to assess antibody-secreting cells (ASCs). Due to its sensitivity, the method enables identification of rare ASCs a specific antigen. Secondly, you can evaluate circulating antigen-specific memory B cells after polyclonal activation. The B cell ELISpot is regularly used to detect B cell responses elicited by infection or vaccination.
To get the most out of your assay, download our Step-by-step guide to ELISpot.
Tell the story of every cell
FluoroSpot is the multiplex version of ELISpot. Just like in the ELISpot assay, target proteins secreted by cells are captured by specific antibodies immediately after secretion and throughout the stimulation process, making it an extremely sensitive sandwich assay. The difference to ELISpot lies in the mode of detection: where ELISpot relies on an enzymatic reaction, FluoroSpot utilizes fluorescence. This opens up for multiplex analyses of several analytes simultaneously, enabling studies of cell populations with different functional profiles.
Study analytes with different kinetics
Like intracellular cytokine staining (ICS) in flow cytometry, FluoroSpot is ideal for delineating the functional pattern of each cell. However, where ICS detects produced analyte clogged up inside the cell, FluoroSpot opens up for a more physiologically relevant study of analyte secretion. Analytes released directly after activation can be combined with others that have slower kinetics without manipulating intracellular processes. By capturing all secreted analyte during the entire stimulation, FluoroSpot has been shown to be up to 500 times more sensitive. Thus, for many research questions, flow cytometry panels may be better complemented with FluoroSpot than with intracellular staining.
From basic research to clinical trials
Like ELISpot, FluoroSpot is useful in e.g., vaccine research and evaluation of cancer immunotherapies, but giving a broader picture of the response by enumerating highly functional triple-secreting IFN-γ/IL-2/TNF-α cells, or killer cells secreting IFN-γ and Granzyme B simultaneously.
Multiplex analysis requires an accurate reader
Because the assay principle is similar to ELISpot, if one cell secretes the analyte, it is detected and visualized as one spot. However, whereas spots in ELISpot can be seen with your naked eye, fluorescent spots need to be analyzed in an automated reader equipped with an exciting light source and suitable emission filters. In addition, the reader needs a spot counting algorithm capable of identifying spot centers and creating an overlay analysis. Our reader Mabtech IRIS™ 2 is optimized for FluoroSpot analysis.
To get the most out of your assay, download our Step-by-step guide to FluoroSpot.
FluoroSpot 500x more sensitive than ICS
Increasing numbers of transfected cells were mixed with non-transfected cells.
Mabtech IRIS 2 is optimized for ELISpot and FluoroSpot.
Ready for reality
ELISA is a straightforward research tool for quantifying analytes in solution. The analytes measured are proteins, such as cytokines, chemokines, immunoglobulins, hormones, or other biomarkers. Typical samples are cell supernatants, plasma, serum, and other bodily fluids. The uncomplicated detection procedure and the versatility of sample choice strengthen the broad use of ELISAs across research fields and diagnostics.
Detect native proteins
We put considerable effort into characterizing our antibodies and finding a perfectly matched antibody pair. The use of monoclonal antibodies makes our ELISAs specific, reproducible, and sensitive. We don’t cut corners during assay development and always validate that our ELISAs recognize the native proteins, i.e., those actually secreted from cells. This makes our ELISAs ready for the reality researchers are uncovering.
Tailored for clinical samples
Our ELISAs are sandwich ELISAs, and therefore, the risk for interference by heterophilic antibodies must be considered. These antibodies might crosslink the assay antibodies and lead to false-positive results. For this reason, we developed a sample dilution buffer, called ELISA diluent, that reduces heterophilic antibody interference. This buffer is included in our ELISA Pro kits but can also be purchased separately.
Samples from patients containing rheumatoid factor are known to have an increased risk of generating false-positive results in ELISA. For such clinical samples, we developed specialized kits to withstand heterophilic interference even more efficiently. We call this kit format ELISA PathRF.
To get the most out of your ELISA experiment, please read our Step-by-step guide to ELISA.
Count foci with ease
FociSpot is our new assay platform that includes both kits and a brand-new analysis capability of Mabtech IRIS™ 2 allowing for automated counting of foci in a 96-well plate format. Focus forming assays (FFAs) can have a variety of different setups for different research goals such as focus-reduction neutralization tests (FRNT) or Tissue Culture Infectious Dose (TCID50) assays. FRNT and TCID50 can be used to quantify viral titers or virus-specific antibody neutralization titers and are commonly used in vaccine and infection research. We know that each viral assay is unique, though, using different cell lines, media, and viruses, but counting foci doesn’t have to be. In these assays, foci are detected with immunostaining using virus-specific mAbs and a precipitating substrate reaction similar to ELISpot. The developed foci can now be easily counted using the FociSpot application in IRIS 2
You spoke, and we listened. We’ve created a handful of virus-specific kits that can be used to detect virus-specific foci. Based on the same development principles of ELISpot, we utilize a biotinylated virus-specific detection mAb followed by a precipitating enzymatic reaction to visualize foci. Can’t find your virus of interest? We have the reagents needed to set up your very own virus-specific FociSpot development.
Automated Foci Counting
In the Apex software of IRIS 2, you can now find a new assay format below ELISpot and FluoroSpot. Simply choose FociSpot and the plate format, insert your plate, and press read. Counting foci has never been easier and, most importantly, more accurate.