- SimpleStep ELISA キットの原理
- SimpleStep ELISA キットの使用例
演者の Russel Neuner は University of Massachusetts で Molecular and Cellular Biology の PhD を取得し、現在はアブカムで Senior Scientist に就いています。
Hello and welcome to this webinar on Introduction to SimpleStep ELISA. Today's principal speaker is Russell Neuner, Senior Scientist at Abcam. Russell received his PhD in Molecular and Cellular Biology from the University of Massachusetts.
Joining Russell today will be Dan Schwartz, Commercial Manager at Abcam. Before we start I'd like to just quickly remind you that questions for the Q&A section at the end of the webinar can be submitted at any time by the Q&A panel on the bottom right hand side of your screen. I will now handover to Russell who will start this webinar.
RN: Thanks, Lucy. Today I would like to introduce a brand new technology from Abcam called the SimpleStep ELISA. This technology provides improved and differentiated performance characteristics, while still retaining the familiar process and standard data outputs of a traditional ELISA. A brief outline of today's webinar will focus on the following topics: an introduction to the SimpleStep ELISA; key product features and assay performance characteristics; advantages over the traditional ELISA; various applications for the SimpleStep ELISA in scientific research; an example product that outlines all of the data and analysis.
The term 'ELISA' stands for Enzyme-Linked Immunosorbant Assay. This laboratory technique is used to detect in quantity a specific analyte from a complex mixture or solution. Compared to other immunoassay techniques, such as western blotting, an ELISA provides better specificity and dynamic range.
The most common type of ELISA is the sandwich ELISA where two antibodies are used to measure a specific target protein in a biological sample, such as human serum or cell culture extract. In a sandwich ELISA, one antibody is immobilized to the bottom of every well in a microtiter plate. This antibody is called the capture antibody and upon sample incubation, binds the target protein. An additional step incorporates a second antibody that can be labelled with a reporter enzyme, such as horseradish peroxidase or HRP. This antibody is called the detector antibody and completes the sandwich ELISA.
The SimpleStep ELISA follows the exact same methodology and it's based on the sandwich ELISA format. A SimpleStep ELISA utilizes a capture antibody that is conjugated to an affinity tag, and a detector antibody that is conjugated to HRP. Together, these two antibodies complete the sandwich ELISA. In order for the sandwich complex to remain bound to the microtiter plate, every well in a SimpleStep ELISA is precoded with a highly-specific anti-tag monoclonal antibody. This antibody binds to the affinity tag conjugated to the capture antibody, and is essential for this technology.
At this time, I would like to show a brief movie describing the SimpleStep ELISA protocol. The SimpleStep ELISA begins with removing the number of microtiter strips needed to setup your assay. As I previously mentioned, every well in a SimpleStep ELISA is precoded with a highly-specific anti-tag monoclonal antibody. To begin, add 50 µl of the protein standard or unknown sample to each well. Next, add 50 µl of the antibody cocktail containing the capture antibody, and detector antibody to each well.
Incubate the SimpleStep ELISA for 1 hr at room temperature with shaking to ensure thorough mixing of antibodies and analyte. During this incubation step, the antibodies bind to the target protein and simultaneously the sandwich complex is bound to the SimpleStep microtiter plate.
Following the 1 hr incubation, a single wash step removes the unbound analyte sample and antibody cocktail, leaving behind only the immobilized sandwich complex.
Next, a detection reagent, also known as TMB is added to every well and incubated for 10 min. Following the incubation, a stop solution is added to every well where color develops in proportion to the bound sandwich complex.
To measure each signal, a SimpleStep ELISA is read on a standard microplate spectrophotometer instrument, at a 450 nm wavelength.
Here is a summary of what I've described thus far, showing a direct side-by-side comparison of the SimpleStep ELISA on the left, to the traditional ELISA on the right. Each technique begins with assay setup or sample and reagent preparations. This typically takes about 1 hr, depending on the number of samples in an assay.
Next, is a 1 hr incubation; in the traditional ELISA this step is for the samples only, and may take up to 2 hr, depending on the manufacturer's instructions. In the SimpleStep ELISA this step is for the samples and antibody cocktail, containing both capture and detector antibodies.
After the 1 hr incubation, the SimpleStep ELISA is ready to be washed, developed and read on a spectrophotometer. The traditional ELISA is washed and the detector antibody is added for an additional 1 hr incubation.
Most commercially-available ELISA products on the market utilize a biotinylated detector antibody. This type of detector antibody requires an additional reporter incubation step. A common reporter for a biotinylated detector antibody is a streptavidin molecule conjugated to HRP, therefore, an additional 1 hr incubation step is required for the traditional ELISA.
After the final 1 hr incubation, the traditional ELISA is ready to be washed, developed and read on a spectrophotometer.
In summary, a traditional ELISA can take up to 4.5 hr of continuous bench time. While, in comparison, the SimpleStep ELISA only takes about half the amount of bench time, or 2.5 hr.
The SimpleStep ELISA technology has many great benefits for use in the lab: first, the SimpleStep ELISA protocol is, as the name infers, simple! Additionally, the SimpleStep ELISA has a shortened protocol time, having reduced complexity and a shorter assay duration allows for easier and faster data generation. The SimpleStep ELISA retains the familiar process and standard data outputs of a traditional ELISA. This requires no special training or costly instrumentation. The liquid phase incubation kinetics of both the capture and detector antibody, drives the assay to superior sensitivity. In addition, allowing both the capture and detector antibody to interact with the target analyte in solution, imparts a more efficient binding process. This is compared to having an immobilized capture antibody in a microtiter plate common to the traditional ELISA. The SimpleStep ELISA has a single incubation step and a single wash step, having reduced the number of sample handling steps can lead to a greater reproducibility and accuracy in an assay. Finally, the SimpleStep ELISA technology allows for concurrent, parallel target analysis in a single microplate. The next section of this webinar will discuss Abcam's multi-target SimpleStep ELISA kits.
Every SimpleStep ELISA kit from Abcam will contain the following reagents for use in your research. A SimpleStep ELISA microplate capable of performing 96 tests; this arrives pre-coated with a highly-specific monoclonal antibody. A 5X cell extraction buffer; this is specially-formulated for cell culture and tissues extraction samples. A concentrated 10X capture antibody, and concentrated 10X detector antibody, 10X wash buffer for use in the single wash step, TMB detection reagent and stop solution.
Additionally, every SimpleStep ELISA will include a protein standard, this is a highly-purified protein used to generate a standard curve similar to the one shown on the right. Each standard curve is plotted using a 4 parameter logistic, non-linear regression model. A protein standard is also very useful in an assay to measure or interpolate the amount of target analyte in an unknown sample. Every SimpleStep ELISA will include a sample diluent, and this is alternatively referred to as a standard diluent, or calibrator diluent. A sample diluent is a specially formulated buffer that is optimized to correct for non-specific interactions, such as a matrix effect and an immunoassay. SimpleStep sample diluents generate maximum recovery of target analyte in a variety of sample types. As seen in the figure on the right, when compared to two other ELISA kits, SimpleStep ELISA assays have near 100% recovery of target analyte across a 5-point serial dilution.
Lastly, every SimpleStep ELISA will include an antibody diluent, this is alternatively referred to as an assay diluent or blocking buffer. The antibody diluent is a specially formulated buffer that is optimized to reduce non-specific antibody conjugate interactions. It eliminates any interference due to heterophilic antibodies, such as HAMA or Human Anti-Mouse Antibody. Heterophilic antibodies are present in complex serum or plasma sample types, and are capable of generating strong false/positive results in immunoassay techniques. The figure on the right shows how our SimpleStep antibody diluent eliminates heterophilic antibody interference versus common immunoassay blocking buffers.
Next, I would like to discuss a few applications in research for the SimpleStep ELISA. First, is the validation of a secreted protein biomarker. The quantification of a biomarker in healthy versus diseased patient samples could shed light into biomarker discovery, and lead to advances in research. The example shown is for the protein clusterin, or apolipoprotein J. The figures show a serial titration of normal human serum, and interpolated clusterin levels across a panel of ten individual, normal, healthy serum donors.
Second, is the investigation of membrane protein cleavage. The detection of the soluble extracellular domain, such as a cell receptor or ligand and cell culture supernatant, could give clues to a cell-shedding or cleavage event. The example shown is for the protein fas ligand, or CD95L. The soluble fas ligand can be detected in cell culture supernatant upon proteolysis by MMP7, or upon simulation of peripheral blood mononuclear cells with the drug PHA.
Third, is the quantitation of an intracellular protein analyte. The ability to measure the presence of an intracellular protein, or a specific post-translational modification site, could give invaluable information related to a cell signaling event. The example shown is for the mitochondrial protein, frataxin; frataxin is measured in various cancer cell lines to display differing levels in each cancer type. Shown on the right is the analysis of frataxin and Friedereich's ataxia, the most common inherited human ataxia. B lymphocyte cell extracts from control, carrier and FA patients show varying levels of frataxin based on their genetic inheritance.
Fourth, is the analysis of a protein level upon drug treatment. The importance of confirming an unknown drug treatment to a cancer cell line, could yield faster and more specific drug discovery efforts. The example shown is for the hypoxic-induced protein HIF-1 alpha. The figure shows the serial titration of deferoxamine or DFO-treated hela cells, and the up-regulation of HIF-1 alpha in response to drug treatment.
Next, I would like to present the type of data generated in the analysis for all SimpleStep ELISA products. The validation data is included in every Abcam product booklet, with recommendations for sample preparation and typical dynamic ranges by sample type. Today, I'll review the human stem cell factor, SimpleStep ELISA, or ab176109. Stem cell factors are widely expressed ligand for the kit cell receptor. It is membrane-bound and proteolytically cleaved into a soluble ligand. The soluble form is heavily glycosylated and exists as a homodimer outside of a cell.
Kit receptor activation by autophosphorylation occurs upon ligand binding, and results in the subsequent activation of downstream signaling pathways. Stem cell factor is critical in the early stages of haematopoiesis to stimulate masked cell proliferation. Additionally, stem cell factor can mediate cell-to-cell adhesion events.
Defects found in the gene that encode for a stem cell factor cause familial progressive hyperpigmentation and, lastly, the abnormal function of this growth factor can lead to specific types of leukaemia.
The reproducibility or precision of each SimpleStep ELISA assay reports both intra-assay CV and inter-assay CV. Intra-assay CV is based on values interpolated from the protein standard curve in a single experiment, while inter-assay CV is based on values interpolated across multiple experiments over a specific time period. All intra-assay CV and inter-assay CV are less than 10%, and conform to industry standards.
These values are very important, because precision reflects the performance of an assay in the hands of the user, and ensures consistency over time. The sensitivity is calculated as the minimal detectable dose. The human stem cell factor SimpleStep ELISA reports a sensitivity of 0.51 pg per ml. Benefits of using an assay with a low sensitivity can save on limited and costly sample types. Example protein standard curve data is shown in two forms, so the user has references for data validation. Representative raw optical density values from a duplicate experiment are reported in the table shown on the right. Normalized or background subtracted protein standard curve data, is shown in the figure on the left.
The recovery data is performed by setting up a spike recovery assay. Recombinant protein is spiked at three different concentrations into various sample matrices. The recovery of the spiked protein indicates if a component of the sample interferes with the assay. Common sample matrices used in our recovery analysis include cell culture supernatant, human serum and human plasma collected with three different anticoagulants to ensure maximum recoverability of all plasma-based proteins.
Spiked or unspiked sample matrices are serial diluted to test for the linearity of dilution. The linearity of an assay for a given sample indicates whether the sample interferes with the accurate detection of a target analyte at a specific dilution. All calculated recovery and linearity of dilution data is plus or minus 20%, and conform to industry standards. If the recovery or linearity of dilution needs to be corrected for a given sample type, then a specially formulated sample diluent is included in each SimpleStep ELISA kit.
Every SimpleStep ELISA is validated using multiple biological samples for assay specificity. Human stem cell factor is measured in a panel of ten individual, normal, healthy serum donors. Values are in pg per ml and interpolated from the protein standard curve. All secreted serum or plasma-based targets are tested and fall within World Health Organization blood reference ranges. When available, the SimpleStep ELISA is calibrated against a known NIBSC international standard, and includes a conversion factor for data comparison.
This concludes my section of today's webinar. Please feel free to send in any questions you may have regarding the SimpleStep ELISA technology. At this point, I would like to introduce my colleague, Dan, who will be discussing SimpleStep ELISA marketing resources that are available, as well as upcoming promotions and events. I will return later at the end to answer any of your questions.
DS: Thank you, Russ. I'd like to briefly just recap the primary features we're hoping users will discover, really differentiate these kits from others that are available on the market. Foremost, the simplified protocol making these kits very easy to use. The kits require no methods, techniques or instrumentation different from that which is already being used for existing standard ELISA platforms. Additionally, new users should find this method particularly easy to adopt. The accelerated assay duration will save time, reducing total assay run time to about 2 hr, and this is essentially connected to the liquid phased binding kinetics which drives superior sensitivity and specificity. Additionally, the SimpleStep ELISA kit method is developed in-house by Abcam. This ensures a number of product line benefits for the user, consistent batch-to-batch reproducibility is guaranteed, batch-specific stocking, large bulk batch sizes, or specific lot requests can be provided. The SimpleStep kits are in stock and available for next day delivery, and the knowledge base for kit development and utilization is in-house. This enables Abcam to provide the fastest, most reliable and accurate customer support possible.
The SimpleStep catalogue currently contains kits of a variety of targets, primarily extracellular and serum biomarkers, such as cytokines and chemokines, cancer biomarkers, self-signaling or inflammation proteins. Other important research areas which are addressed include metabolism, apoptosis, epigenetics, protein-phospho modifications and many other important targets. The product pipeline is constantly active with 10 to 15 new kits added to the catalogue every month. So if your target of interest is not yet covered, please check back again in the future. Currently, there are over 100 individual kits in the catalogue, this includes over 75 unique single target kits and about 25 dual target kits. Note also that custom combinations of all existing published targets are available in bespoke two or four target kits, through customer support enquiry.
The SimpleStep homepages on the Abcam website provide a great deal of information on the platform if you're interested to learn more. This URL link will deliver you to the homepage. Here you can find the most current list of published kits along with their species' reactivities and sensitivities, as well as technical information about the method of the platform, including the animation of how the method works, which Russ just shared with us. Also, there are examples of validation data, which accompany each kit on its individual product page, links to each product's individual technical datasheet, as well as an overview of the general protocol. Alternately, if you would like to find SimpleStep ELISA kits by utilizing a search for your specific target of interest, this can be accomplished on the Abcam homepage by first entering your target of interest into the keyword window, select 'Kits' on the product pull down menu and then click 'Go'. After the search results are loaded, simply filter the results by the brand keyword for ‘SimpleStep’ and this will give you your results.
Due to the nature of the platform's universal anti-tag coated well plates and buffer systems, SimpleStep kits make it very easy to evaluate multiple targets in parallel. As you can see in our catalogue, we have pre-selected target combinations which might be useful for researchers to measure total and modified protein, or related signaling pathways. Here's some examples; however, custom target combinations are also available. To enquire about generating a bespoke multi-target kit from any published SimpleStep single targets, please visit the homepage for multi-target kits located at this link. These bespoke kits can be ordered by contacting customer support to discuss your needs.
Having been launched in July of this year, SimpleStep is a relatively new product line for Abcam and throughout the last quarter we have been enquiring with early adopters their impressions of how the SimpleStep platform has worked for them, and what they like about it. Thus far, the feedback has been quite good; in our customers' hands the kits are performing as good, or better than the comparable standard ELISA product and most users really appreciate the ease of use and time-savings offered by the platform.
As a special thank you for attending the webinar, we are offering an opportunity for all attendees to try out the SimpleStep kits at a reduced cost. All webinar attendees can use this coupon code for a $100 discount off the purchase price of a kit, and the ELISA team will contact you to learn your impressions of how the kit worked for you. Also, we're hoping to gather feedback from webinar attendees; anyone who is willing to take a brief moment to complete the post-event survey will receive a fun SimpleStep Molly temporary tattoo, and a SimpleStep wobbler stress ball.
Finally, as an addendum, I have included a list of upcoming events sponsored by Abcam's scientific support group, which you may be interested in learning more about and perhaps attending.
Contact information for Abcam scientific support teams is included here. These people should be able to help you with any product technical or catalogue enquiries for your reference after the webinar.
So now we can transfer back to Russ who can answer some of your questions that have been submitted.
RN: Thanks, Dan. During the webinar there were a few questions submitted, so in the time remaining I'd like to go over and answer any of the submitted questions. One question is: What is the shelf life of the SimpleStep ELISA kit? All SimpleStep ELISA kits have been validated and guaranteed for one year from the date of delivery.
Another question: How do I store the kit components before and after use? This is a great question. The recommended temperature for all SimpleStep ELISA kit components is in the refrigerator at 4°C, prior to use at the bench we recommend that you remove and equilibriate some of the reagents to room temperature. Storage instructions for any leftover reagent after use is specifically stated in the Abcam booklet and is specific to that reagent.
Another question is: Are the kits species specific? Yes they are. Each of the kits listed on the Abcam website, clearly state whether they are active to human or mouse. Currently, most of the products are human only validated SimpleStep ELISAs, with mouse SimpleStep ELISAs up and coming very soon.
Another question: Do I have to extract the cells with any special extraction buffer before using these kits? So, as I mentioned in my presentation, each SimpleStep ELISA kit comes with a 5X cell extraction buffer. This has been formulated for use in our assays. It’s highly recommended to use this reagent for cell our tissue extraction sample preparations. Alternative extraction buffers have been tested on certain products, so this would come down to a specific SimpleStep product question. If so please contact our scientific support team who would be happy to help you out in this particular area.
So, another question: What would the cost of one kit be? $470.
Another question: Can I use a sample type that is not recommended in the booklet? This is a great question. Each SimpleStep ELISA kit has been validated for a number of biological samples. Samples or sample types not listed in the Abcam booklet for a specific product generally have not been tested. If you would like additional help, or have further questions related to a specific sample or sample type, please consult our scientific support team and you can ask about our AbTrial program.
Do I need to wash the plate before use? No, you do not need to wash the SimpleStep microplate prior to use. They are ready to use upon arrival. We recommend removing the number of microtiter strips needed to run your assay according to your plate layout, and store the remainder in the supplied foil pouch with included desiccant at 4°C.
Ok so a few questions have come in asking if we have specific SimpleStep ELISAs to your protein of interest. As Dan mentioned in his section of the presentation, please visit the SimpleStep ELISA webpage at Abcam to see if your protein of interest is available yet. If it’s not listed, feel free to contact a member of our scientific support team and they can help out in finding if the product is up and coming or specifically when it will become available.
Another question is: Are the kits good for only one use? No, each of the kits is sufficient for 96 tests and, like I had said previously, the microtiter strips that are not being used can be stored at 4°C. An additional use can be used later. There are also multi-target kits, which are two and four target sets, available as well in our catalogue.
What would happen if I changed the SimpleStep ELISA incubation conditions? Altering the SimpleStep ELISA incubation conditions, such as the temperature, the shaking speed, the incubation time, or wash time, these will all lead to undesirable results. Each SimpleStep ELISA has been optimized for maximum performance and it is highly recommended to follow the protocol included in the Abcam booklet.
Another question is: Is this good enough for 96 samples or plates? Each kit is supplied with 96 wells, so that’s 12x8 well strips. This does include a protein standard, which is highly recommended to use if you plan on using unknown samples that you would like to measure or interpolate your protein of interest from.
This concludes the Q&A section. I'd like to thank all of you for attending today's webinar. If your specific question did not get answered, we will follow-up via email with answers to those questions after today's webinar. Again, if you have any other further outstanding questions, please feel free to contact our scientific support team at any of our offices worldwide.
Thank you Russell and Daniel for this webinar. We have had several questions and I hope they have been answered for you. Also, to let you know a PDF copy of the webinar presentation will be emailed to all attendees within the next 48 hours. Also, when you log-off from this webinar you will be directed to a webpage where you can find more information about the promotion. If you have any extra questions please don’t hesitate to submit them to our scientific support team at firstname.lastname@example.org.