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Andrew: Hi, yes. Thank you for the question. That's certainly possible. We do test a lot of different sample types for each assay. And we try to determine that based on what we believe are the most likely use cases and samples of interest for the analysis, but it's very possible that, you know, new conditions may emerge, and it's possible you might want to test a different sample type. If that is something you want to try, I'd suggest you refer to some of the things I said earlier, actually, in the section on pairs of immunoassay validation, perform some characterization experiments to determine that the assay works for your matrix of interest, and that the sample diluent is appropriate.
For instance, I'd recommend doing spike recovery and dilution linearity experiments in your sample matrix to ensure that the matrix and the analysis perform appropriately with the kit, and check, you know, control and positive samples to ensure they're falling within the standard curve range of the assay. So there's no guarantee of success, but it's certainly possible. And if you'd like additional help, we do have a great scientific support team that you can always reach out to with questions about a specific sample or sample type.
Moderator: Okay, great. Fabulous. Thanks for that one, Andrew. The next question I've got here for you is, "Are the SimpleStep ELISA® kits single-use only?"
Andrew: Okay. No, thank you for that question too. No, not necessarily. Generally, the SimpleStep ELISA kits are designed to support at least two separate experiments. The kits contain sufficient reagents for 96 separate tests, and those are run on 12 8-well strips that come with the assay. So the strip well format allows for some flexibility, and the strips that are not being used can be stored for a second use. And we also provide two vials of the protein standard with each kit. So one vial can be reconstituted on the first day when you want to do experiments, you know, with the assay and then, you know, hold on to the other vial, and it can be reconstituted and the assay can be run on a second date with some of your remaining wells. So at least two uses are certainly possible.
Moderator: Great, thanks a lot for that one. The next question I've got here for you is, "Can an extracellular matrix like Matrigel interfere with antibody diffusion through the sample, that is the 3D organoids, and what properties of the antibody should we take into account to overcome this?"
Andrew: Okay. That's a really interesting question. I assume that's referring to some of the slides I was showing earlier where we used immunohistochemistry staining. I am certainly not the most qualified person that I've come to talk about IHC, but what I would refer the question to is go to our website. We have a immunohistochemistry guide there, which contains a lot of protocols about tissue processing on how to handle different sample types with the assays. I know that it's sort of an interesting area of research at the moment.
Actually, there was a paper I saw quite recently on bioRxiv.org where people were looking at techniques to do just this actually. So if I can... I think it was a group of authors. In fact, if you wanna email me at firstname.lastname@example.org, I'd be happy to send you the reference, but yeah, I know this is an area of interest. I would suggest you go check our immunohistochemistry standing guide on our website. And again, our scientific support team can certainly help there too.
Moderator: Great. Thanks a lot. Thanks for directing our audience to that resource. The next question I've got from an attendee here is, "If I want to scale up my matched pair assay, how can Abcam help?"
Andrew: Okay. That's another interesting question. Interesting in that prior to me joining Abcam, actually, I was a match pair customer myself at a different organization. So I can speak to this first hand. Those match pairs are, I think I mentioned in the talk, but there's something we're really taking quite seriously, and we want to help people who are using those. We've had a lot of resources dedicated to supporting people with this scale up. We have some dedicated business development and technical support for people who are doing this. So you can certainly reach out to Abcam directly.
But specific things that we can do in terms of scaling up the matched pair is we can provide larger sizes, sort of larger vial sizes so you get more antibody. We can reserve large quantities of single lots as well, if that's something you're interested in. We have a lot of different conjugation kits as well that we can provide to help you conjugate the antibodies. And we can also do, you know, customized diluents and buffers as well. So there's quite a bit of flexibility here, and this is something we do spend a lot of time on and want to help people succeed. So please, you know, contact us directly, and we should be able to help them with any scale up questions you have.
Moderator: Great. Thanks a lot. The next question that's come through here is, "Is SimpleStep ELISA available in a 384-well format?"
Andrew: Okay. Yes, it is. So as I was mentioning earlier, the assay, the standard configuration is essentially in a 96-well format. So we don't sell them directly in a 384-well assay format. But as I mentioned earlier, one of the key and unique features of the SimpleStep assay is that it utilizes this affinity tag system that I talked about where the plates are coated with an antibody which recognizes an affinity tag, which is on the capture antibody. So what that means is essentially we have a universal SimpleStep plate design which works with any of the kit reagents.
And so we do sell at really very little cost a 384-well SimpleStep plate, which is coated with the same anti affinity tag antibody as the 96-well plate. And that can then be matched up with any SimpleStep kit. So we provide a protocol for how to do that. If you search on our website for SimpleStep 384, you'll probably find that, but briefly to provide sufficient reagent volume for 384-well plates, we recommend using two with a SimpleStep 96-well kit with the 384-well plate. And otherwise it's a pretty simple protocol conversion. So yeah, that's absolutely possible.
Moderator: Fabulous. Thanks for that one. And the next question I've got here from an attendee is saying, "I don't have a high content imager. Can I run FirePlex on a microscope?"
Andrew: Okay. That's an interesting question. Let me think about that. The answer is probably, it depends. To run a FirePlex assay on a microscope probably requires too many wells for manual image acquisition, unless you're very patient with that. But if you have a microscope with an automated stage, it certainly could be possible. You know, high content images, of course, in many cases are quite similar in their specifications to automated microscopes. So theoretically, that should be possible. In fact, I'm aware of at least one lab that has done this quite recently, actually, with our FirePlex 384-well reagents on an automated scope.
There's some restrictions around that in terms of the imager specifications that we recommend. And again, those are on our website. But broadly speaking, to be compatible with the FirePlex 384 reagents, you'd need a scientific CMOS camera, 4 or 5X microscope objective that can capture a whole well. And the ability to capture, you know, image files as TIFs independently on green, yellow, and red channels. So, you know, with an automated stage, many fluorescent microscopes will have that capability. So it's an interesting question. And I think if you want to follow up on that, I'm sure our field applications team could probably advise for everyone how to get that running.
Moderator: Okay. Fabulous. Thanks for that. And the next question I've got here, Andrew, is, "Can you use fluorescently labeled secondary antibodies in an ELISA assay?"
Andrew: Yes, absolutely. Fluorescently labeled secondary antibodies... I suppose the question is aimed primarily at fluorescently labeling our matched antibody pairs with fluorophores. But yeah, before I get to that, we actually do have some fluorescent SimpleStep ELISAs available. I didn't cover those in the presentation. Those are called Catchpoint® Simplestep assays and Abcam developed them in partnership with Molecular Devices. And those assays don't use fluorescently labeled antibodies per se. Rather they use a fluorescent HRP substrate instead of a color metric one.
So if you're working with our matched antibody pairs, you could do something similar to that with a fluorescent HRP substrate and a fluorescent plate reader. And that approach is probably more common, more generally than using fluorescently labeled detector antibodies directly. That approach is sometimes used more commonly, I think, for high throughput screening applications, but yeah, you can in theory take an antibody, conjugate it to a fluorescent dye, and then just detect that fluorescence directly by exposing the plate to light at a particular wavelength that your fluorescent plate reader can read.
If this is something you do want to try, I would suggest, we have a lot of great kits Abcam does for antibody fluorophore conjugation. These are Lightning-Link® Conjugation kits, and they're very fast, and simple, and straightforward protocols. They're available for direct conjugation of antibodies to most of the widely used fluorophores. So, yeah, very possible.
Moderator: Fabulous. Thanks for clarifying that one. And I think we have just time for a few more questions. So we've got an attendee here who is saying, "We use Abcam HSA SimpleStep ELISA for our low concentrated samples. We saw kind of metric effects by high delusions with water compared to buffer during pre-validation. Can you explain that phenomenon?"
Andrew: That's the HSA SimpleStep ELISA?
Andrew: Okay. I'm not so sure. I think I need to see the data, and we could talk about that some more. I think... Again, my email address is available. I would suggest you just reach out directly and we'd be happy to chat about that. I think without seeing any of the data upfront, it's sort of difficult to assess that.
Moderator: Okay, great. Thanks a lot for that one. That can be followed up via email. The next question I've got here for you is, "Is it possible to design an ELISA in a 384-well plate?"
Andrew: Yes. absolutely. I was just thinking about that previous question again, and if you said they were diluting the samples with water, I'm not quite sure why they were doing that instead of the sample diluent, but again, happy to follow up. For 384-well ELISA development, yeah, that's absolutely possible. You know, all the same principles essentially apply. As I was talking about there with the matched pairs, although that data was 96-well. You know, I think...thinking about the volume adjustments, you know, for a 384-well plate, you know, just reducing everything down. I'd certainly recommend using automated liquid handling for those because otherwise high CVs can be low, you know, for manual pipetting, but yes, absolutely 384-well ELISA development is certainly possible, often just using smaller volumes than we would use for the 96.
Moderator: Okay, great. Excellent. Thanks for that one. The next question here is, "How do I increase the sensitivity of an ELISA?"
Andrew: Okay. That's a good question. I suppose, you know, there's so many variables that go into ELISA performance. You know, choosing the antibody pair is absolutely key, you know, first of all, but I think, you know, in general, there are few sort of tricks and tips you can use there. I would say, be very careful with the detector antibody concentration used in a sort of absolute minimum amount of detector antibody where possible, because sensitivity is usually driven by the amount of background or non-specific signal at the lower concentrations or at the blank. So, you know, reducing detector antibody concentrations could do that.
If you're building the assay yourself, sometimes using a chemiluminescent readout can slightly improve sensitivity over a color metric detection. Minimizing the assay volume as well, use sort of the minimum possible so that the signal isn't diffusing within the well. And I suppose another thing maybe to suggest is you can always sort of increase your incubation times as well, or even heat up the reactions for the incubations. Each of those things might improve sensitivity as well. So there are several different things to look at, but each of those might help.
Moderator: Okay, great. Fabulous. I think we have time to squeeze in one more quick question. And that is, "What are the correct controls to use for an ELISA?"
Andrew: Okay. That's a good question. It probably depends on the context of use a little bit. I think with the SimpleStep ELISA is we absolutely recommend using the sample diluent as a negative control, and at least, you know, duplicate or triplicate. Then you should always have a positive control just to show that the assay is working, you know, in the detection range of the assay. What we would normally, I think, recommend there for SimpleStep is just, again, diluting some of the protein standard, and then running that to make sure the assay is performing as expected within the dilution range.
For different applications, I think, you know, depending on how robust you want to be, if you're measuring precision, it's sometimes better to do that right across the range of the assay. So I'd recommend, as well as a negative control, having one that's just right above the limit of quantitation of the assay. So as close to that LOQ as possible, and then some close to the upper limit of detection so that you can span the whole range of the assay. And again, if it's a more clinical application, I would recommend that those controls are made up in actually the sample matrix that you're going to be running the assay in. So for most applications, just diluting standard in the sample diluent and using a blank is sufficient, but you can be more stringent if you wish, depending on your context of use. So hopefully that's helpful.
Dr. Andrew J. Ball Ph.D
Andrew leads the Assay Development Platforms R&D organization at Abcam, which encompasses single- and multi-plex immunoassays, cellular, and biochemical assays. He is based in Abcam’s Waltham, Massachusetts location. Previously, Andrew held roles as Vice President of Assay Development at Quanterix Corporation, Vice President of R&D at Quad Technologies, a cell therapy technology company now part of Bio-Techne, and served in a variety of innovation- and development-focused R&D roles at EMD Millipore. His research interests have included neurodegenerative disease, cell therapy, and precision medicine.
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