We are focused on three main immunoassays: ELISpot, FluoroSpot and ELISA.
They are all 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 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, please read 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™ is optimized for FluoroSpot analysis.
To get the most out of your assay, please read 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 is optimized for FluoroSpot
Ready for reality
ELISA is an immunoassay that enables sensitive quantification of analytes in solution. Common samples are thus cell supernatants, plasma, serum, and saliva. In our ELISAs, an antibody is coated onto the plate to capture the protein of interest. A second antibody allows analyte detection. This detection antibody is labeled with biotin, facilitating subsequent binding of a streptavidin-enzyme conjugate. The addition of the substrate results in a color reaction that is directly proportional to the amount of protein bound. The concentration of protein in the sample is determined by comparison with a standard curve of known protein concentrations.
Detect low levels of analyte
Antibody features such as affinity, avidity, and antigen interaction are essential for a successful ELISA. We put considerable effort into characterizing our antibodies, and the use of monoclonal antibodies makes our ELISAs extremely specific and sensitive. Importantly, we always validate the ELISAs for recognition of native proteins as this often differs from how the antibodies bind to the recombinant protein.
A complex world requires creative kits
The accuracy of most of our ELISA kits is established by calibration with an external reference standard from NIBSC or NIAID. When analyzing pico- and microquantities, the risk for interference by heterophilic antibodies must be considered. For this reason, we have 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.
For problematic clinical samples, containing e.g., rheumatoid factor, we have a developed an even more efficient product to eliminate false-positive signal, ELISA PathRF.
To get the most out of your assay, please read our Step-by-step guide to ELISA.