Antibody to assay: The key to consistent and reproducible immunoassay results

Video transcript

• 00:00 - 00:16: Hello, everyone. Thank you for joining this webinar today. My name is Andrew Ball. I’m
• 00:16 - 00:21: VP of assay development platforms at Abcam, and I’m based at Abcam’s Waltham, Massachusetts
• 00:21 - 00:26: site. And today I’m going to share with you on the topic of antibody to assay, the key
• 00:26 - 00:31: to consistent and reproducible immunoassay results. My aim is to take you on a journey
• 00:31 - 00:36: through the principles and processes that underpin development of robust and reliable
• 00:36 - 00:42: antibody and assay reagents that can be counted upon to support and accelerate your research.
• 00:42 - 00:49: First, I’d like to briefly introduce you to Abcam. We are a global life science company
• 00:49 - 00:54: supporting customers at the forefront of life science research. Our innovative products
• 00:54 - 01:00: are used by hundreds of thousands of scientists worldwide, and we seek to empower life scientists
• 01:00 - 01:05: globally with high quality biological reagents and tools in support of research, drug discovery
• 01:05 - 01:13: and diagnostics applications. Abcam’s core purpose is to serve life scientists to achieve
• 01:13 - 01:19: their mission faster. To do this, we strive to develop best in class reagents and tools
• 01:19 - 01:26: to enable high quality, highly reproducible research data. To achieve our mission of serving
• 01:26 - 01:32: scientists, Abcam has grown into a globally connected organization with centers of excellence
• 01:32 - 01:38: on multiple continents, all interacting closely to deliver high quality tools for our customers.
• 01:38 - 01:43: The work I’ll show you today is the product of collaboration between teams in Hangzhou,
• 01:43 - 01:49: Australia, Eugene, Oregon, Adelaide, Australia, Waltham, Massachusetts, and Cambridge, UK.
• 01:49 - 01:53: And I’d like to acknowledge here that the fantastic work that each of those teams is
• 01:53 - 01:59: doing. Today, I’ll be speaking to you about, first of all, our recombinant monoclonal antibody
• 01:59 - 02:04: development process, which underpins a large part of our overall technical strategy. Then
• 02:04 - 02:09: I’ll talk about the development of matched antibody pairs for use by customers and also
• 02:09 - 02:14: in our own immunoassay platforms. And finally, I’ll show you how we use these antibody pairs
• 02:14 - 02:20: in the development of singleplex and multiplex immunoassays.
• 02:20 - 02:26: This slide serves to highlight why Abcam does what it does. Every one of you watching will
• 02:26 - 02:33: know that many experiments fail and the associated waste of expense is huge. Billions of dollars
• 02:33 - 02:39: are being wasted annually on non-reproducible research. And beyond the economic loss, it’s
• 02:39 - 02:43: well known now that there’s a reproducibility crisis in life science that risks undermining
• 02:43 - 02:49: the credibility of much published research. Several studies have been undertaken to identify
• 02:49 - 02:55: the root causes of irreproducibility, several of which are shown here. But interestingly,
• 02:55 - 03:01: the biggest cause is the reagent used in experiments themselves being inappropriate or not working
• 03:01 - 03:07: as intended. Addressing this issue has been absolutely central to Abcam’s technical strategy
• 03:07 - 03:12: for several years. And we’re investing heavily in and totally committed to providing products
• 03:12 - 03:19: designed to perform correctly and consistently first time and every time.
• 03:19 - 03:24: So although historically associated primarily with antibodies, Abcam has grown significantly
• 03:24 - 03:29: in recent years and our product portfolio has evolved far beyond just being an antibody
• 03:29 - 03:34: provider. Over the past several years, we’ve expanded our offering to include product lines
• 03:34 - 03:40: such as highly validated bioactive proteins, off the shelf CRISPR modified cell lines,
• 03:40 - 03:45: a large portfolio of cellular and biochemical assays. And of course, the topic of today’s
• 03:45 - 03:52: discussion, which is high quality single and multiplex immunoassays.
• 03:52 - 03:57: So let’s start by looking at the key ingredient underpinning our immunoassay portfolio, recombinant
• 03:57 - 04:04: monoclonal antibodies. Here we can see some of the key steps involved in our recombinant
• 04:04 - 04:10: antibody development process. We’re utilizing best in class antibody development platforms
• 04:10 - 04:15: to develop the highest performing antibodies in the market. And we’re focusing our efforts
• 04:15 - 04:19: on generating recombinant rabbit monoclonals. Since the rabbit immune system has a large
• 04:19 - 04:25: B cell repertoire that can generate a very diverse range of antibodies. This recombinant
• 04:25 - 04:30: monoclonal approach results in antibodies that are specific and very sensitive to the
• 04:30 - 04:35: targets of interest. And by taking this approach along with extensive characterization, we’re
• 04:35 - 04:40: able to overcome challenges associated with more traditional polyclonal and non-recombinant
• 04:40 - 04:45: monoclonal antibodies, things like batch to batch consistency issues, gene loss, cell
• 04:45 - 04:50: line drift. And the end result is a portfolio of antibodies that are guaranteed to give
• 04:50 - 04:57: reproducible results, whether for our customers or for our internal assay developers.
• 04:57 - 05:01: By working with recombinant technology, we’re able to improve antibody sensitivity through
• 05:01 - 05:07: antibody engineering and to select the most favorable antibody qualities for our products.
• 05:07 - 05:13: In terms of antibody selection, a recombinant antibody is the most robust format available.
• 05:13 - 05:17: Expression can be carried out at almost any scale and the long-term supply of antibody
• 05:17 - 05:22: is guaranteed. So this makes recombinant antibodies a great solution for long-term studies or
• 05:22 - 05:29: for using the same antibody across multiple applications.
• 05:29 - 05:34: Rabbit antibodies have several advantages over other species, such as rodents. They’re
• 05:34 - 05:40: better at distinguishing subtle epitope variations, post-translational modifications, conformational
• 05:40 - 05:46: changes, or small molecules, so are highly versatile for multiple applications. Rabbits
• 05:46 - 05:51: are also able to generate high affinity antibodies against antigens that are highly conserved
• 05:51 - 05:56: between human and mouse and therefore not immunogenic in mice and have a higher affinity
• 05:56 - 06:01: for target antigen on average than most mouse monoclonal antibodies.
• 06:01 - 06:06: So consequently, recombinant rabbit monoclonal antibodies are highly specific and sensitive
• 06:06 - 06:10: for their targets. And being recombinant, they’re also fully renewable with high lot-to-lot
• 06:10 - 06:16: consistency. As an example of this consistency, I just want to quickly draw your attention
• 06:17 - 06:23: to the figure here, which is a recombinant anti-PD-L1 clone 28.8, which is now an approved
• 06:23 - 06:29: diagnostic. And as shown in the slide here, we see excellent consistency with reproducible
• 06:29 - 06:37: specific staining between six different production batches of the antibody.
• 06:37 - 06:41: In order to have confidence in an antibody, it’s critical to verify antibody specificity
• 06:41 - 06:45: and performance in order to ensure that they only bind to the target of interest and are
• 06:45 - 06:51: appropriate for the context in which they’re being used. The requirements and recommendations
• 06:51 - 06:56: for antibody validation can be different for each context of use. And so we test each antibody
• 06:56 - 07:00: in as many applications as possible that are scientifically relevant to the protein target
• 07:00 - 07:06: of interest. We select the most appropriate applications for a particular protein target
• 07:06 - 07:12: based on detailed assessments of the scientific relevance and of likely end uses. And we verify
• 07:12 - 07:18: antibody specificity and performance in-house via techniques like Western blot, immunocytochemistry,
• 07:18 - 07:25: immunohistochemistry, flow cytometry, ChIP, immunoprecipitation, and many more. And because
• 07:25 - 07:31: the requirements and recommendations for validation are different for each type of assay, all
• 07:31 - 07:36: of our antibody reagents are evaluated for the specific assay and context in which they
• 07:36 - 07:42: will be used. We strive to select the screening materials to match as closely as possible
• 07:42 - 07:46: to the samples that you would use in your own application. And this ensures that when
• 07:46 - 07:54: we say an antibody is qualified for a specific application, that you can trust the performance.
• 07:54 - 07:59: In addition to the application testing methods I just described, one thing I wanted to specifically
• 07:59 - 08:04: mention today is the importance of knockout validation, which has become absolutely integral
• 08:04 - 08:10: to our antibody development process and for which we’ve received several industry awards.
• 08:10 - 08:15: Over the last few years, Abcam has invested heavily in a large-scale knockout validation
• 08:15 - 08:21: process to ensure target specificity. In brief, we developed an extensive library of human
• 08:21 - 08:28: knockout haploid cell lines that are generated via CRISPR-Cas9 and certified via Sanger sequencing.
• 08:28 - 08:32: These knockout cell lines provide a complete loss-of-function phenotype that can be used
• 08:32 - 08:38: to confidently determine antibody cell activity. We did this to provide an extra layer of validation
• 08:38 - 08:43: and of confidence for researchers shaken by the reproducibility crisis. And so we’re
• 08:43 - 08:49: now able to provide solid evidence of antibody cell activity for the target of interest.
• 08:49 - 08:55: This initiative has allowed Abcam to review its antibody portfolio and remove underperforming
• 08:55 - 09:00: antibodies from the catalog, even if they were widely used and highly cited. And so
• 09:00 - 09:05: we can instead focus on providing best-in-class clones and creating gold standards for commercial
• 09:05 - 09:10: antibodies. And again, allowing you to focus on research and not having to worry about
• 09:10 - 09:15: the quality of your reagents. At present, we have over 4,000 knockout validated antibodies
• 09:15 - 09:22: available and that number is increasing rapidly.
• 09:22 - 09:26: Our recombinant rabbit monoclonal antibody portfolio is well established now and I’m
• 09:26 - 09:31: sure that many of you in the audience are very familiar with it. But one newer antibody
• 09:31 - 09:36: product line that I’d like to introduce you here today is our new recombinant multiclonal
• 09:36 - 09:43: antibodies. These antibodies recently won the 2022 Citeab Innovation Award, which was
• 09:43 - 09:49: a great honor. And this category of antibodies are a defined mixture of carefully selected
• 09:49 - 09:54: individual recombinant monoclonal antibodies designed to recognize different epitopes on
• 09:54 - 09:59: the same antigen. They have all the benefits of the recombinant monoclonal antibodies,
• 09:59 - 10:05: but are able to recognize multiple epitopes, so are an ideal solution for applications
• 10:05 - 10:10: where a polyclonal antibody would traditionally be used, and yet are able to provide the specificity
• 10:10 - 10:15: and reproducibility only available from a recombinant antibody. We have over 100 of
• 10:15 - 10:19: these multiclonals available already and there’ll be many more to come. And I’m personally
• 10:19 - 10:24: very excited to see some of the ways in which both our customers and also our internal scientists
• 10:24 - 10:27: will utilize these.
• 10:27 - 10:32: So having introduced our recombinant monoclonal antibodies, let’s now move on to a key way
• 10:32 - 10:38: in which we leverage our recombinant antibody portfolio, namely for the development of matched
• 10:38 - 10:46: antibody pairs for use in immunoassays.
• 10:46 - 10:50: When we think about developing an immunoassay, which I’m sure many of you have done before,
• 10:50 - 10:56: there are many steps to consider and to optimize, from antibody selection, making antibody enzyme
• 10:56 - 11:03: conjugates, choosing an appropriate protein standard, buffer selection, mitigating cross-reactivity
• 11:03 - 11:08: and on and on. But at the heart of everything is choosing a high-performing antibody pair,
• 11:08 - 11:13: and if it’s for commercial development or regulated use, or even if it’s just for a
• 11:13 - 11:19: research assay that you’d like to use repeatedly or pass on to others in the lab, specificity
• 11:19 - 11:24: and batch-to-batch consistency of the antibody pair will be absolutely key to your success
• 11:24 - 11:26: or failure.
• 11:26 - 11:31: So to elaborate further on the importance of antibody pair selection, here’s some of
• 11:31 - 11:37: our internal data. The figure here on the left represents a comparison between a rabbit
• 11:37 - 11:43: polyclonal and a rabbit monoclonal antibody pair, targeting human IL-1RA, and you can
• 11:43 - 11:48: see that the monoclonal pair in this case provided a much greater level of sensitivity,
• 11:48 - 11:52: detecting lower concentrations of analyte, which is important for low abundance biomarkers
• 11:52 - 11:56: and also for minimizing sample holding requirements.
• 11:56 - 12:02: And at Abcam, we now use recombinant rabbit monoclonals exclusively for our immunoassay
• 12:02 - 12:08: development in-house. I’ve spoken already of the challenges with batch-to-batch consistency
• 12:08 - 12:13: in polyclonal antibodies, and the figure in the middle illustrates the significant differences
• 12:13 - 12:18: in assay performance and background observed when changing lots of a polyclonal detector
• 12:18 - 12:24: antibody. In contrast, using the recombinant rabbit monoclonal pair, as shown on the right,
• 12:24 - 12:29: the standard curves line up beautifully, even after changing the lots of the detector. And
• 12:29 - 12:34: this consistency is really important, and it’s often overlooked actually, but if developing
• 12:34 - 12:38: an assay that you plan to use repeatedly, maintaining consistency in the antibody pair
• 12:38 - 12:42: from lot to lot is absolutely critical.
• 12:43 - 12:50: Abcam has developed a large portfolio of matched recombinant rabbit monoclonal antibody pairs
• 12:50 - 12:56: that we make available commercially for use in immunoassay development. It’s important
• 12:56 - 13:02: to note that our pairs are designed and developed specifically to work together. We’re not mixing
• 13:02 - 13:07: and matching individual antibodies here. Our antibody pairs have their own development
• 13:07 - 13:12: pipeline, and they’re specifically designed to work together. Some of the key steps in
• 13:12 - 13:19: that pipeline are shown here. So we produce and evaluate multiple clones per target. We
• 13:19 - 13:24: screen them for optimal performance, including specificity, immunoassay sensitivity, and
• 13:24 - 13:31: the optimal orientation for sandwich ELISA. Once we’ve selected the two best pairs, they’re
• 13:31 - 13:35: screened against a full standard curve and with appropriate biological samples to ensure
• 13:35 - 13:41: specificity in complex matrices. And then we evaluate assay linearity. We screen for
• 13:41 - 13:47: cross reactivity before we approve the pair with the most optimal combination of performance
• 13:47 - 13:56: attributes. We then make these pairs commercially available as quickly as possible. Increasingly,
• 13:56 - 14:01: based on customer feedback and expectations, we make these pairs available in a carrier-free
• 14:01 - 14:08: format for maximal flexibility and for ease of conjugation. We currently offer over 1,500
• 14:08 - 14:13: such matched antibody pairs and, again, are adding more on a regular basis. These offer
• 14:13 - 14:18: an economical alternative to buying and screening multiple, several, and separate antibodies on
• 14:18 - 14:22: your own, and come with all the benefits of recombinant rabbit monoclonals that I’ve discussed
• 14:22 - 14:28: previously. This has become a popular product line for both academic researchers, but also
• 14:28 - 14:32: for industry partners who want to use these pairs in their own immunoassay platforms.
• 14:36 - 14:40: Of course, as we discussed at the beginning of this section, there are many steps in developing
• 14:40 - 14:46: robust and reliable immunoassays, and depending on your context of use, the validation requirements
• 14:46 - 14:52: may be extremely rigorous. Examples of the type of data you might need to generate to consider
• 14:52 - 14:58: an assay well validated are shown on this slide, such as precision, dilution linearity,
• 14:58 - 15:04: interference, and cross-reactivity testing. Antibody pair selection is certainly the most
• 15:04 - 15:09: critical step in a successful immunoassay development project and often the most time
• 15:09 - 15:13: consuming and expensive, but there are other factors to consider and optimize around, too,
• 15:13 - 15:18: so I thought I’d spend just a couple of minutes here sharing some tips from our immunoassay
• 15:18 - 15:22: development team for those of you who do use our matched antibody pairs.
• 15:25 - 15:30: Titrating the capture and detector antibodies by testing multiple different concentrations
• 15:30 - 15:36: is an important early step. Using more antibody isn’t necessarily a good thing.
• 15:36 - 15:41: Often, sensitivity and assay background can be improved upon by finding the optimal working
• 15:41 - 15:47: concentrations, so it’s good to test these early on. Likewise, the concentration of the reporter
• 15:47 - 15:53: and of the antibody enzyme conjugate can significantly influence assay signal-to-noise,
• 15:53 - 16:00: and again, more isn’t always better here, so some experimentation and titration here is recommended.
• 16:03 - 16:07: Another really important thing to note is that the selection of the protein standard
• 16:07 - 16:12: used to calibrate the assay also is a significant determinant of assay performance.
• 16:13 - 16:16: You’ll need to find a protein that delivers a high signal-to-noise ratio
• 16:16 - 16:22: across all the concentrations in your standard curve and that closely mimics the native protein
• 16:22 - 16:27: found in your biological specimens. Abcam does have a large number of high-quality proteins
• 16:27 - 16:32: available and is investing heavily in this area, but wherever you source your proteins from,
• 16:33 - 16:37: it’s important to use a reputable vendor who’s capable of providing high-purity,
• 16:37 - 16:43: correctly-folded proteins and with a robust process for ensuring good batch-to-batch consistency,
• 16:44 - 16:48: as both native and recombinant proteins can be highly variable between batches.
• 16:51 - 16:55: It’s also important to establish a standard curve and a sample dilution that puts your specimens
• 16:55 - 17:00: within the measurement range of the assay. If there’s existing literature in your target
• 17:00 - 17:05: of interest, check to see what concentrations others have observed and try to establish a
• 17:05 - 17:11: curve that will accurately quantify control and experimental samples. If no published data exists,
• 17:11 - 17:16: then try a wide range of standard curve conditions and sample dilutions until your
• 17:16 - 17:22: samples come within the measurable range. As shown here, a saturated signal from a sample
• 17:22 - 17:28: will produce erroneous extrapolated results, and in general, larger sample dilutions help to
• 17:28 - 17:33: minimize against matrix interference and also to save on sample volumes. So, if your assay
• 17:33 - 17:40: is sensitive enough, a higher sample dilution may be beneficial. Another point of note to consider
• 17:41 - 17:44: is the use of blocking reagents to eliminate assay interference.
• 17:45 - 17:49: Depending on your sample matrix, there may be many other components in there that
• 17:49 - 17:54: could interfere with your immunoassay performance, things like heterophilic antibodies,
• 17:54 - 18:01: rheumatoid factor, human anti-mouse antibodies, and many more. And often just using BSA as a
• 18:01 - 18:06: blocker is insufficient, and there’s frequently a risk of false positive or negative signals from
• 18:06 - 18:12: interference in the matrix. The good news is that there are many commercial sources of
• 18:12 - 18:18: good blocking reagents available. So, if you suspect an artificially elevated signal or if
• 18:18 - 18:23: you’re seeing unexpected interference, try using some additional blocking agents within your buffers
• 18:23 - 18:30: and diluents. So, having told you a little about using matched antibody pairs for your own use in
• 18:30 - 18:36: assay development, I’d like to spend the rest of my talk today focusing on how we use them internally
• 18:36 - 18:41: to produce our own commercially available assay kits. And I’ll start with our popular
• 18:41 - 18:48: SimpleStep ELISAs. These ready-to-use assay kits are a great solution for people who
• 18:48 - 18:52: don’t want to spend time performing the types of assay optimization I just described.
• 18:54 - 18:59: As we’ve discussed, measuring proteins in biological samples is a complex business,
• 18:59 - 19:04: but the interest in biomarkers and in proteomic studies continues to grow rapidly, and
• 19:04 - 19:11: getting it right is key. As we’ve seen already, extensive assay optimization and validation is
• 19:11 - 19:15: required in order to have confidence that the results you’re getting are reliable and
• 19:15 - 19:21: reproducible. We talked at the beginning about the reproducibility crisis and the amount of money
• 19:21 - 19:26: that’s wasted in research, and there are other challenges and frustrations too, such as just
• 19:26 - 19:33: wasting valuable lab time, needing to repeat your experiments, wasting samples, and delayed projects.
• 19:34 - 19:40: Abcam wants to help researchers achieve their goals faster. And so for single-plex
• 19:40 - 19:47: immunoassays, our solution is a product line we call the SimpleStep ELISA. In conventional
• 19:47 - 19:53: sandwich ELISA, as shown on top here, the capture antibody is immobilized to the bottom of each well
• 19:53 - 19:59: of a microtiter plate. After adding the sample and incubating, the unbound materials are then
• 19:59 - 20:05: removed with a wash step, and then a detector antibody is added. This will also have to incubate
• 20:05 - 20:10: for another hour or so to allow the sandwich complex to form. Finally, another wash, and then
• 20:10 - 20:15: enzyme substrate is added. Color development will proceed until stopped by an addition of a stop
• 20:15 - 20:21: solution. This type of assay typically takes between about three to five hours and involves
• 20:21 - 20:28: multiple steps. It’s quite labor-intensive and certainly time-consuming. Our SimpleStep ELISA
• 20:28 - 20:34: is also based on the sandwich ELISA format, but in contrast involves fewer assay steps and can
• 20:34 - 20:41: be completed in under 90 minutes with a simple mix, wash, read workflow. So how do we do this?
• 20:41 - 20:47: Well, SimpleStep ELISA can streamline the workflow by using a format wherein the antibody
• 20:47 - 20:53: analyte sandwich complex is formed in solution in a single step. In SimpleStep ELISA, each capture
• 20:53 - 20:59: antibody is conjugated to an affinity tag, and every well is precoated with a highly specific
• 20:59 - 21:05: monoclonal antibody against that affinity tag. So to begin the assay, you simply add your sample
• 21:05 - 21:10: and the antibody cocktail containing the capture and detector antibodies, and then you incubate.
• 21:10 - 21:16: And so in just one step, the complete sandwich complex forms in the well and then is anchored
• 21:16 - 21:23: to the plate by the immunoaffinity tag. Following a one-hour incubation, a simple wash step removes
• 21:23 - 21:28: the unbound analyte and antibody, leaving behind only the immobilized sandwich complex.
• 21:29 - 21:33: Next, the detection reagent is added and incubated for 10 to 30 minutes, and following that,
• 21:33 - 21:39: add stop solution, and the signal can be read on a standard microplate spectrophotometer
• 21:39 - 21:44: at 450 nanometer wavelength. So no specialized equipment is required here.
• 21:45 - 21:50: This rapid protocol is very consistent across all of the many hundreds of SimpleStep ELISAs.
• 21:50 - 21:55: So another benefit here is that it’s very easy to run assays to multiple targets using the same
• 21:55 - 22:00: simple and familiar workflow. In addition to a simplified workflow,
• 22:01 - 22:06: SimpleStep kits offer greater sensitivity and consistency than many other commercial ELISA kits.
• 22:07 - 22:12: The figure on the right here shows an example of the assay sensitivity of several SimpleStep
• 22:12 - 22:17: kits versus products from another vendor. And you’ll see there for most analytes, SimpleStep
• 22:17 - 22:24: offers higher assay sensitivity, often by several orders of magnitude. This performance is in large
• 22:24 - 22:29: part because of the high affinity of the recombinant rabbit monoclonal antibodies used, and also because
• 22:29 - 22:34: of the liquid phase incubation kinetics, which allow the capture and detector antibodies to
• 22:34 - 22:39: interact with the target analyte in solution. In the previous section on antibody pairs,
• 22:39 - 22:44: I mentioned that assay validation is a critical and often technically challenging aspect of
• 22:44 - 22:49: immunoassay development. In our SimpleStep ELISA, we perform extensive optimization and
• 22:49 - 22:54: verification testing to ensure that the product will meet rigorous technical specifications.
• 22:55 - 22:59: I’d like to spend the next few minutes talking about this work in a little more detail.
• 23:01 - 23:05: We optimize our SimpleStep assays to ensure that they have the best possible sensitivity,
• 23:05 - 23:11: a broad range of detection, and to be highly reproducible. We begin the development of each
• 23:11 - 23:16: kit by evaluating multiple recombinant rabbit monoclonal antibody pairs, created here at Abcam
• 23:16 - 23:22: within the antibody pairs pipeline I discussed earlier, in order to identify the most sensitive
• 23:22 - 23:29: pair. The limit of detection, or minimal detectable dose, shown here in red on the left. In this
• 23:29 - 23:35: example, the human IL-1RA assay reports a limit of detection of four picograms per milliliter.
• 23:36 - 23:41: One of the benefits of using an antibody with good sensitivity is the ability to detect proteins that
• 23:41 - 23:48: are normally in low abundance in healthy donors, like IL-1RA. It also saves on sample volume.
• 23:49 - 23:54: The dynamic range of the assay is impacted by both the background of the assay and the instrument
• 23:54 - 24:01: specifications, shown here as the plate reader saturation. We optimize our assays to give the
• 24:01 - 24:04: broadest possible dynamic range with the standard colorimetric plate reader.
• 24:06 - 24:13: The reproducibility or precision of each SimpleStep ELISA reports both intra-assay CV
• 24:13 - 24:19: and inter-assay CV, shown on the right there. Intra-assay CV is based on values interpolated
• 24:19 - 24:25: from within a single experiment, while inter-assay CV is based on values from at least three separate
• 24:25 - 24:32: experiments. By virtue of reducing the number of assay steps in the workflow, less pipetting is
• 24:32 - 24:38: involved with SimpleStep ELISAs, and so a major source of potential error in high CVs is mitigated,
• 24:39 - 24:45: so SimpleStep ELISAs have excellent precision. The human IL-1RA SimpleStep ELISA shown here has
• 24:45 - 24:51: CVs of under 5%, which exceeds industry standards. These values are very important because precision
• 24:51 - 24:56: reflects the likely performance of an assay in the hands of the user and ensures consistency over
• 24:56 - 25:03: time. SimpleStep ELISAs are qualified for multiple sample types, depending on the context of use.
• 25:04 - 25:11: Examples may include serum, plasma, cell culture supernatant, tissue extracts, saliva, CSF, etc.,
• 25:12 - 25:17: and if an important new context of use emerges for an assay, for instance for ACE2, which is
• 25:17 - 25:23: shown here, is for additional blood sample data to support COVID-19 research, we will go back and
• 25:23 - 25:30: add additional validation data to the product data sheet. Other tests that we do include linearity of
• 25:30 - 25:36: dilution and spike recovery. Dilution linearity determines the range of biological that can be
• 25:36 - 25:40: tested and whether samples with high levels of analyte above the upper limit of detection can
• 25:40 - 25:47: still provide reliable quantification after dilution. Samples displaying a little deviation in
• 25:47 - 25:52: concentration after dilution are said to display good linearity, and this confirms a high degree
• 25:52 - 25:58: of accuracy and shows flexibility of the assay at varying sample dilutions. For a passing result,
• 25:58 - 26:07: we require dilution recoveries of 80-120%. Spike recovery determines the differences in percent
• 26:07 - 26:14: recovery of antigen signal between sample matrix and the diluent. The many proteins contained in
• 26:14 - 26:19: a sample matrix, such as serum, may result in underestimation or overestimation of the target
• 26:19 - 26:26: concentration. To assess this, known concentrations of protein are spiked in and quantified, and
• 26:26 - 26:32: results from the matrix and diluent are compared. If the spike recoveries are significantly different,
• 26:32 - 26:37: then components in the sample matrix may be interfering with the analyte detection,
• 26:37 - 26:40: and so the assay diluent may need to be reformulated by us to better
• 26:41 - 26:47: represent the test matrix before we launch the assay. As you can see in the example here for
• 26:47 - 26:52: our IL-1RA SimpleStep ELISA, we show excellent recovery and linearity in multiple matrices.
• 26:52 - 26:57: If our target of interest for the assay has high homology with other proteins, we test for the
• 26:57 - 27:03: possibility of cross-reactivity. For example, D-dimer is a breakdown product of fibrinogen
• 27:03 - 27:08: and is found in the blood during the clotting response. On the left here, you can see we tested
• 27:08 - 27:13: fibrinogen, plasminogen, and others with our D-dimer kit and found that it has low cross-reactivity.
• 27:15 - 27:19: Binding partners like ligands, receptors, cofactors can also alter the measurable
• 27:19 - 27:24: concentration of the analyte or alter the antibody binding, which could potentially
• 27:24 - 27:29: result in immunoassay interference. We test for interference during assay development by spiking
• 27:29 - 27:35: potentially interfering molecules and quantitating the analyte with and without the spiked material.
• 27:35 - 27:40: For example, our D-dimer SimpleStep ELISA here was tested for interference with TPA
• 27:40 - 27:47: and additional binding partners and showed no interference. In addition to interspecies homologs,
• 27:47 - 27:53: some targets have high homology with other animal species. We test for cross-reactivity with mouse,
• 27:53 - 28:00: rat, and bovine serum and other animals when necessary. As an example shown here is the
• 28:00 - 28:05: data supporting that the human D-dimer SimpleStep ELISA kit is only suitable for use with human
• 28:05 - 28:13: samples. This slide shows some of the additional parameters we consider when qualifying an assay.
• 28:14 - 28:19: When available, SimpleStep ELISAs are calibrated against a reference international standard
• 28:19 - 28:24: and include a conversion factor for data comparison. Calibration of an international
• 28:24 - 28:31: standard enables cross-comparison between orthogonal datasets. Every SimpleStep ELISA
• 28:31 - 28:37: is evaluated using multiple biological sample types. All secreted serum or plasma-based targets
• 28:37 - 28:44: are tested with multiple individual donor samples. When possible, we also compare clinical
• 28:44 - 28:48: samples between healthy and diseased states as shown in the lower left here for PSA.
• 28:50 - 28:57: Many of the Abcam-matched antibody pairs, SimpleStep ELISAs, and multiplex assays actually share
• 28:57 - 29:03: the same recombinant monoclonal antibody pairs. This can be especially useful if samples will be
• 29:03 - 29:08: detectable across assay formats or if the assay will be scaled up for higher throughput or
• 29:08 - 29:14: multiplexing. Shown here is an example of a pair for human IL-1RA, which was used in simple
• 29:14 - 29:21: step ELISA, also in a sequential ELISA built using our matched antibody pairs, and also in our
• 29:21 - 29:27: multiplex Fireplex platform. You can see here that the interpolated concentration of the signal
• 29:27 - 29:31: was very consistent across multiple matrices for all three assay formats.
• 29:33 - 29:38: When you purchase a SimpleStep ELISA, you’ll receive an extensive data package with information
• 29:38 - 29:43: relating to the assay performance and the validation studies performed and also everything
• 29:43 - 29:49: you need to execute the protocol. This information is also readily available on our website if you
• 29:49 - 29:56: search for your target of interest. So, to summarize this section, SimpleStep ELISA is
• 29:56 - 30:02: a straightforward solution which addresses most single-plex protein quantification needs.
• 30:03 - 30:08: I hope I’ve demonstrated that the workflow is fast, straightforward, and delivers high-quality
• 30:08 - 30:13: outcomes with no need for specialized equipment. I should also mention that it’s scalable.
• 30:14 - 30:19: Although 96-well strips are the standard format, we offer a 384-well plate coated with the same
• 30:19 - 30:26: anti-affinity tag monoclonal antibody, so each assay can be easily ported over to the 384-well
• 30:26 - 30:32: format. We’ve leveraged the high performance of our recombinant rabbit monoclonals to deliver
• 30:32 - 30:37: high-performing and robust assays, and we maintain a very active development pipeline for
• 30:37 - 30:44: SimpleStep ELISAs at our Eugene, Oregon facility. Currently, we have over 1,200 kits available,
• 30:44 - 30:49: and we continue to add more regularly, spanning a broad range of research areas, including
• 30:49 - 30:55: inflammation, cell signaling, neuroscience, immuno-oncology, and beyond. So, whatever your
• 30:55 - 30:59: biomarker of interest is, it’s likely that we have a SimpleStep ELISA for it, and if not,
• 31:00 - 31:06: it’s probably in our assay development pipeline. So, to summarize everything I’ve discussed today,
• 31:07 - 31:12: starting with the development of high-quality recombinant monoclonal antibodies in response to
• 31:12 - 31:18: the reproducibility crisis, we’ve built a large interconnected suite of assay solutions on that
• 31:18 - 31:25: recombinant monoclonal antibody technology, from matched antibody pairs through to single-plex
• 31:25 - 31:31: ELISA with SimpleStep, and we build using high-quality starting materials that are specifically
• 31:31 - 31:38: designed to work together and to perform consistently for the long term. Then, by
• 31:38 - 31:43: innovating to simplify assay workflows and by performing extensive optimization and verification
• 31:43 - 31:49: of assays, we’re able to provide you with robust and reliable solutions, letting you spend more
• 31:49 - 31:55: time on your research and less time on trying to identify and source appropriate reagents.
• 31:55 - 32:01: We’ve done our job well if we’re helping you reach your goals faster, and we’d love to hear
• 32:01 - 32:07: from you and learn more about how we can help accelerate your research. And so, with that,
• 32:07 - 32:11: I’d like to thank you very much for your interest and for your attention,
• 32:11 - 32:14: and we’ll now turn it over to questions.