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The ELISA first principles

On-demand webinar

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Summary

Join Ralph Paul, Scientific Support Expert at Abcam, as he shares valuable tips and tricks for optimizing your ELISA experiments. This webinar covers general ELISA principles, including direct, competitive, sandwich, and SimpleStep formats, while also addressing common issues and how to troubleshoot them.

About the presenter

With a PhD in virus receptors and a fellowship studying viral molecular biology, followed by a long tenure in biotech, Ralph Paul, PhD, has developed and run many assay types directed towards assessing expression levels and biological activity of numerous therapeutic proteins and their targets.

The ELISA first principles - Chapter 1

Video Transcript

  • 00:00 - 00:11: Thank you to all of you in joining us for this introduction and review of the ELISA
  • 00:11 - 00:16: technique and discussion of some of the common questions and issues that arise when ELISAs
  • 00:16 - 00:19: are being used by researchers in their work.
  • 00:19 - 00:24: During the webinar today, I will be reviewing general principles common to the various ELISA
  • 00:24 - 00:29: formats and also describing some variants of this technique, including the direct, the
  • 00:29 - 00:33: competitive, the sandwich, and the SimpleStep sandwich ELISA.
  • 00:33 - 00:38: I will then discuss some of the typical questions and problems that we hear about in the scientific
  • 00:38 - 00:45: support group at Abcam when researchers use ELISA kits or ELISA components in their experimental
  • 00:45 - 00:46: work.
The ELISA first principles - Chapter 2

Video Transcript

  • 00:01 - 00:05: The ELISA assay, which is an acronym for Enzyme-Linked Immunosorbent Assay,
  • 00:05 - 00:10: was first described by Engvall and Perlman and also independently by Schnurr and Wieman,
  • 00:10 - 00:14: with both groups publishing their work in 1971.
  • 00:14 - 00:20: A clearly recognizable advantage of this technique is that it does not rely upon the use of radioisotopes
  • 00:20 - 00:25: and all of the shipping, handling, and disposal issues that radioisotopes present.
  • 00:25 - 00:28: Variations of this technique have been adopted for a wide variety of analytes
  • 00:28 - 00:33: and very sensitive, robust, rapid, and high-throughput ELISAs now exist.
  • 00:33 - 00:39: The term analyte can be used to cover the wide variety of target molecules that can be measured in ELISAs.
  • 00:39 - 00:45: These targets can include small organic molecules, hormones, and of course, very commonly, proteins.
  • 00:45 - 00:52: ELISAs can be adapted such that the target analytes can be measured when present in a wide variety of matrices,
  • 00:52 - 00:57: and you see a partial list of the kind of matrices that are very commonly used for ELISA work.
  • 00:58 - 01:00: But this is no means a comprehensive list.
  • 01:00 - 01:06: And this allows for substantial flexibility in sample collection and sample type.
  • 01:06 - 01:11: Typically, the sample preparation protocols will be specific for the different matrices.
  • 01:11 - 01:15: And often, there may be multiple ELISA kits for the same analyte,
  • 01:15 - 01:21: but they are preferentially designed to use one matrix or another as input for the assay.
  • 01:21 - 01:23: In looking at multiple kits for a given analyte,
  • 01:23 - 01:29: it is important to note the matrix or matrices that are recommended for this kit
  • 01:29 - 01:31: and the matrix that you may be working in yourself.
  • 01:36 - 01:43: We’ll actually start the detailed discussion today by looking at the final color development steps for a typical ELISA,
  • 01:43 - 01:48: as these steps are going to be common throughout all of the variations I’m going to describe later.
  • 01:48 - 01:54: The color development steps all rely upon an analyte being immobilized in the well of a plate.
  • 01:54 - 01:58: Most commonly, this is a 96-well format, very typical,
  • 01:58 - 02:06: but of course, it can be adapted to various other plate formats, including 256-well plates as well.
  • 02:06 - 02:14: In this case, you get the modified analyte or an antibody, which has a horseradish peroxidase enzyme conjugated to it,
  • 02:14 - 02:18: and this will then function to give you the readout in the assay.
  • 02:18 - 02:25: In all of the different assay formats I will describe, not only is an analyte immobilized on the well of the plate,
  • 02:25 - 02:30: but there is also going to be blocking agents that prevent nonspecific interactions from occurring.
  • 02:30 - 02:40: These agents can include detergents, BSA, nonfat dry milk, whole serum, or fish gelatin, as commonly used examples.
  • 02:40 - 02:47: Using a multi-well format allows for numerous samples to be analyzed simultaneously and compared within the same assay.
  • 02:47 - 02:52: In many ELISA kits, the plates can also be separated into individual columns,
  • 02:52 - 03:01: and smaller sample sizes can be examined if the researcher only has limited samples available to them.
  • 03:01 - 03:08: We do, however, strongly encourage researchers to construct another standard curve if they run the assay on multiple days,
  • 03:08 - 03:13: as absolute OD values may vary, and interpretation data for unknown samples
  • 03:13 - 03:23: from a standard curve generated on a different day may not be sufficiently accurate or allow for good comparison of data generated.
  • 03:23 - 03:33: After incubation with the HRP-conjugated material or antibody, there is a TMB solution added,
  • 03:33 - 03:40: which contains 3,3’,5,5’-tetramethylbenzidine solution, commonly known as TMB.
  • 03:40 - 03:51: Over time, a blue color develops as a result of TMB acting as a hydrogen donor for the reduction of hydrogen peroxide to water by the action of the HRP enzyme.
  • 03:51 - 03:57: The color intensity will be proportionate to the quantity of HRP present in a specific well.
  • 03:57 - 04:04: In this specific example, we see the standard control dilutions here on the left-hand side performed in duplicate,
  • 04:04 - 04:08: in addition to unknown samples also performed in duplicate.
  • 04:08 - 04:15: And as you can see, not all samples have the analyte in them, and other ones have varying levels.
  • 04:15 - 04:23: And this would be sort of a typically observed set of data for an ELISA assay.
  • 04:23 - 04:31: Although protocol booklets will typically give an incubation time for the length of time to incubate the TMB with the HRP,
  • 04:31 - 04:41: it’s also prudent to observe the assay as the color develops and stop it if it looks like you’re in danger of having samples and or controls running into saturation.
  • 04:41 - 04:48: One way to do this is to run a kinetic measurement, taking a look at OD650 readings over time,
  • 04:48 - 04:56: so that all time points can be captured and data can be assessed at a point where no saturation is occurring.
  • 04:56 - 05:00: Commonly, what happens, what is done is that a stop solution is added.
  • 05:00 - 05:04: The stop solution consisting of 0.16 molar sulfuric acid.
  • 05:04 - 05:12: And when this is added to the plate, the HRP activity is halted, and you get a conversion of the color from blue to yellow.
  • 05:12 - 05:16: And this can then be read at OD450 nanometers.
  • 05:16 - 05:24: So now you have your data set available, and you can plot data based on the values seen in the standard curve.
  • 05:24 - 05:31: So this is the last part of any of these assays I’ll be describing, the color development where you’ve got an immobilized enzyme.
  • 05:31 - 05:39: Now we’ll talk about the various types of formats that are used to get that analyte immobilized at the bottom of a well in an assay plate.
The ELISA first principles - Chapter 3

Video Transcript

  • 00:00 - 00:05: The first one I will speak about here today is what’s called a direct ELISA.
  • 00:05 - 00:10: In this format, the analyte is bound directly to the plate,
  • 00:10 - 00:15: and a single antibody is added that is already conjugated to HRP
  • 00:15 - 00:19: to recognize the analyte that’s on the plate.
  • 00:19 - 00:22: Now, this is the simplest of all of the formats,
  • 00:22 - 00:27: and there are varying ways to get at this end result as well with multiple antibodies,
  • 00:27 - 00:30: but this is the most direct form of this.
  • 00:30 - 00:34: And what would the data look like or what would one use this for?
  • 00:34 - 00:39: And here is an example of data generated in the direct ELISA format
  • 00:39 - 00:43: characterizing an anti-human kappa light chain antibody.
  • 00:43 - 00:48: At a constant amount of IgG plated at the bottom of the plate
  • 00:48 - 00:55: and varying concentrations of the antibody added that has the HRP conjugate,
  • 00:55 - 00:58: you notice the rise in the OD value.
  • 00:58 - 01:03: And what this also demonstrated is the specificity for the kappa light chain
  • 01:03 - 01:06: because when lambda light chains were added,
  • 01:06 - 01:09: there was no reactivity or very little reactivity observed.
  • 01:09 - 01:15: So this clarified the specificity of this antibody in a very straightforward manner
  • 01:15 - 01:22: and binding two analytes that were bound at the bottom of the plate.
  • 01:22 - 01:25: So what are the advantages here of this?
  • 01:25 - 01:29: It’s a very good way to, as we already saw, characterize an antibody.
  • 01:29 - 01:34: This is a very commonly used format if an investigator is looking at various types
  • 01:34 - 01:37: of recombinant protein expression systems,
  • 01:37 - 01:41: comparing and contrasting amounts expressed, different expression systems.
  • 01:41 - 01:47: If it is a frequently run assay, the minimal reagent set is very appealing.
  • 01:48 - 01:55: The downside to that is if you have only one antibody available to bind,
  • 01:55 - 02:00: if that antibody loses binding affinity after conjugation, this could present a problem.
  • 02:00 - 02:07: The assay also works best if the analyte is in a somewhat purified form
  • 02:07 - 02:12: or there’s something that’s been done to reduce other materials that may be present,
  • 02:12 - 02:16: for instance, in an expression system, some sort of partial purification
  • 02:16 - 02:22: because if the one antibody has a nonspecific binding with components in there,
  • 02:22 - 02:25: you may get high background levels that you cannot really eliminate
  • 02:25 - 02:29: because of the character of that specific antibody that you have available.
  • 02:29 - 02:33: It also has a little less flexibility in assay development.
  • 02:33 - 02:39: You’ll see in the sandwich approaches that you can move around a little bit the target
  • 02:39 - 02:41: and the detector and the capture antibodies.
  • 02:41 - 02:46: You can mix and match those a little more effectively, giving you more flexibility in development.
The ELISA first principles - Chapter 4

Video Transcript

  • 00:00 - 00:05: The next format I’m going to talk about here is the competitive ELISA.
  • 00:05 - 00:17: In this format, what you see is that the analyte actually competes with a modified version of itself that is provided typically in the kit.
  • 00:17 - 00:26: And as you can imagine, the more of the sample analyte that is available in solution, the less of the modified analyte will be able to bind.
  • 00:26 - 00:38: So, in contrast to the other formats, in this format, the OD of the controls actually goes down as you have more of the analyte in the sample.
  • 00:38 - 00:45: And this is a representative standard curve where, in this assay, there is HRP conjugated to cortisol,
  • 00:45 - 00:52: which is competing with cortisol that is in the samples that are being added to the wells.
  • 00:52 - 00:57: And therefore, the data shows that the higher the concentration of free cortisol,
  • 00:57 - 01:04: the lower will be the HRP-conjugated cortisol that will bind to the antibody that is capturing it at the bottom of the plate.
  • 01:04 - 01:12: This is also a very good example of a typical type of analyte that is very commonly used in a competitive ELISA,
  • 01:12 - 01:22: a small molecule organic that is difficult to make work in a sandwich ELISA in that it may not physically be large enough to bind one or more,
  • 01:22 - 01:24: two or more proteins simultaneously.
  • 01:24 - 01:29: So, therefore, a competitive ELISA is a very good way to measure this.
  • 01:29 - 01:33: You can also measure in a number of more complex matrices.
  • 01:33 - 01:39: The sample prep does not need to be as rigorous as it might be in the direct ELISA format.
  • 01:39 - 01:43: It can be less sensitive to matrix effects than sandwich ELISAs,
  • 01:43 - 01:53: so effects of non-analyte materials that may cause nonspecific binding because you’ve got only one antibody that you need to be concerned about here.
  • 01:53 - 02:01: These assays tend to give good reproducibility if you have worked out the exact binding and sample prep conditions.
  • 02:01 - 02:06: And not all analytes are amenable to this format.
  • 02:06 - 02:10: You may have to make different arrangements for different types of analytes.
  • 02:10 - 02:15: The direct HRP conjugation may not be viable for certain types of small molecules,
  • 02:15 - 02:18: and it might be a little less sensitive than a sandwich ELISA,
  • 02:18 - 02:25: but you may well be looking at different types of analytes to be looked at, so that may not be a particular concern.
The ELISA first principles - Chapter 5

Video Transcript

  • 00:00 - 00:05: Now, the Sandwich ELISA is probably the most well-known of the formats, and this approach
  • 00:05 - 00:10: requires a compatible antibody pair, where the first antibody is used to capture the
  • 00:10 - 00:17: analyte and the second antibody carries with it a conjugated HRP enzyme known as the detector.
  • 00:17 - 00:22: And once you have the binding of the sandwich, you can see here the more analytes that’s
  • 00:22 - 00:27: present, the more is captured and the more would be detected in the wells, and you again
  • 00:27 - 00:32: would have this increase in OD with the higher levels of analyte present.
  • 00:32 - 00:36: And what the data would look like for this would typically be increasing amounts.
  • 00:36 - 00:44: This is an ELISA, Sandwich ELISA for human peroxiredoxin 6, and as you can see, as there’s
  • 00:44 - 00:50: more of the protein present, the OD value climbs.
  • 00:50 - 00:58: And this type of assay is very good at measuring analyte levels in a variety of matrices.
  • 00:58 - 01:04: There are a number of washing steps between the various bindings of the capture and the
  • 01:04 - 01:09: binding of the detector antibody, so you can look at fairly complex matrices.
  • 01:09 - 01:11: You can make these very sensitive and specific.
  • 01:11 - 01:16: It does require a matched pair of antibodies that will not interfere with each other’s
  • 01:16 - 01:17: binding.
  • 01:17 - 01:22: This often takes our development groups quite a bit of time to come across a very good pair.
  • 01:22 - 01:26: And your analyte is going to require two or more binding sites to give those antibodies
  • 01:26 - 01:29: room to both interact with the analyte itself.
The ELISA first principles - Chapter 6

Video Transcript

  • 00:01 - 00:04: Now, in a variation of the Sandwich ELISA,
  • 00:04 - 00:08: we also have what is termed the SimpleStep ELISA,
  • 00:08 - 00:12: and this one adds yet another level of complexity by adding another antibody.
  • 00:12 - 00:19: However, this process allows you to make a very rapid facile assay
  • 00:19 - 00:23: that can read out within 90 minutes of initiation
  • 00:23 - 00:26: and maybe about an hour of sample prep to go with that,
  • 00:26 - 00:30: literally half the time of a classical Sandwich ELISA.
  • 00:30 - 00:34: So here, on the bottom of the plate are immobilized antibodies
  • 00:34 - 00:37: that recognize a specific conjugated tag.
  • 00:37 - 00:43: Then a capture antibody, the analyte-containing solution,
  • 00:43 - 00:46: and the detector are all added simultaneously.
  • 00:46 - 00:49: This whole mixture is incubated.
  • 00:49 - 00:53: The capture antibodies harbor the affinity tag
  • 00:53 - 00:56: that’s recognized by these antibodies that are on the plate,
  • 00:56 - 00:59: and this pulls down the entire complex in one fell swoop.
  • 00:59 - 01:03: So there’s a single incubation step, washing, and color development,
  • 01:03 - 01:07: which allows you to very rapidly work through the assay.
  • 01:07 - 01:11: Typical data here that we would see for a Simple Step ELISA,
  • 01:11 - 01:14: this is a human PD-L1 assay.
  • 01:14 - 01:19: Very good linearity of this when plotted on a log-log scale
  • 01:19 - 01:24: and very good dynamic range going down from less than 10 picograms
  • 01:24 - 01:27: to about 2,000 picograms.
  • 01:27 - 01:29: So you get a very nice, broad, dynamic range,
  • 01:29 - 01:31: and when we try to develop these,
  • 01:31 - 01:39: this is always one of the criteria that we use to validate one of these assays.
  • 01:39 - 01:45: The pros are very similar, very much so, to the Sandwich ELISA itself.
  • 01:45 - 01:48: The protocol is certainly simplified.
  • 01:48 - 01:50: There is a reduced time requirement.
  • 01:50 - 01:54: These are very much developed to be extremely sensitive and specific.
  • 01:54 - 01:58: You can certainly look at complex matrices,
  • 01:58 - 02:00: and again you have flexibility in design,
  • 02:00 - 02:05: even more so now that you can mix and match the capture antibody
  • 02:05 - 02:11: with the tag on it as well to be captured by that immobilized antibody on the plate.
  • 02:11 - 02:15: Again, you do need a matched antibody pair.
  • 02:15 - 02:17: Sometimes that can take some work to find.
  • 02:17 - 02:20: And again, your analyte would require multiple binding sites
  • 02:20 - 02:23: to allow all of these antibodies to bind to it.
The ELISA first principles - Chapter 7

Video Transcript

  • 00:00 - 00:22: So, in looking at these ELISAs here, which have been, the examples I’ve given, of course, have been optimized, and you might run into issues either using a ready-to-go kit ELISA, or if you’re attempting to develop an entire ELISA on your own, where are some resources that you can go to find some further information or some guidance?
  • 00:22 - 00:49: Here, if you actually Google Abcam ELISA information, it’ll take you to a great landing page where we have these references and links to additional information, in addition to a number more, and that can really be some great help in looking at how to run an ELISA, what are the sample prep issues that I should be thinking about, or what are the components I need to put into place to perhaps develop my own ELISA.
  • 00:49 - 01:07: If you do have additional questions, we have scientific support groups all over the world who can help you most any time of day or night if you call the right number, and so there is another resource that you can use if you’re looking at running an ELISA or developing one yourself.
  • 01:07 - 01:23: And so, key considerations that we do speak to people about a great deal are a known positive control. So, do you have a positive control that has been, that preferentially is in the matrix that you would like to assay?
  • 01:23 - 01:41: Has it been determined to be there in some other assay previously, or do you perhaps have a recombinant protein or purified analyte that you can spike into a matrix that is devoid of it and then see what kind of recovery you get, and can you observe it at a level that you believe you’ve put it in there?
  • 01:41 - 01:59: So, a good positive control is always really helpful to make sure that the assay is working for you beyond the standard, the control series of dilutions for the standard curve. And then another question that we talk to people about quite substantially is, what is the nature of your analyte?
  • 01:59 - 02:23: Is this something in the samples you would expect to see at a very high level? Is it within the range of sensitivity, or would it perhaps have a transient expression where you might see some of it in some samples but not other ones? And that may be actually completely valid data. It may not be present at a detectable level for some, and that can be the truth of the matter for your assay.
The ELISA first principles - Chapter 8

Video Transcript

  • 00:00 - 00:26: Okay, so I’ll take one here. The question is, the standard curve I generated does not look exactly like the one that you have in your protocol booklet. Is this okay? And this is a very common question that we also get, as the OD values can fluctuate a little bit based on the temperature of the incubation, based on the timing, based on the equipment that the particular user might be using in their case.
  • 00:26 - 00:41: And so as long as you get a very dose-responsive curve, that is the OD is either going up or down appropriately, depending on the type of ELISA, in response to more or less of the analyte, that would be fine.
  • 00:41 - 00:46: Typically, if the correlation coefficient is very good, you can certainly interpolate data from that.
  • 00:46 - 00:57: And so as long as the assay has fundamentally worked and you have a good standard curve, you can certainly read your data points off of that with confidence.
  • 00:57 - 01:08: The one question I have here is, is the SimpleStep ELISA more costly? And it falls within the pricing that we would have for many of our other ELISAs as well.
  • 01:09 - 01:21: We also have, if somebody is looking at using it on a regular basis, we do also have better pricing for 10 packs, for instance, of those types of ELISAs as well.
  • 01:22 - 01:31: I have another question here. Samples are giving a high background, and that can be due to a number of reasons.
  • 01:31 - 01:36: One is completeness of washing between the various incubation steps.
  • 01:36 - 01:38: You want to be sure to get a good wash.
  • 01:38 - 01:44: If you’re using an automated plate washer, you may want to make sure that the plate washer is working effectively.
  • 01:44 - 01:55: Sometimes there can be nonspecific matrix effects where you could have something in your matrix which is binding your detector antibody nonspecifically.
  • 01:55 - 02:04: There might be some dilutions that you can do to reduce that nonspecific background with your sample if it reads out on the standard curve.
  • 02:04 - 02:08: There may also be some additional blocking steps that you can develop.
  • 02:09 - 02:17: Typically, the ones that are developed as a kit will specifically have blocking reagents that will prevent that from happening.
  • 02:17 - 02:24: And again, that’s where we would recommend people follow the protocol specifically and make their dilutions with the appropriate buffers.
  • 02:24 - 02:33: So, the question here is, how do you convert the sample OD readout from pre-purchased sample OD readout to a sample OD readout?
  • 02:33 - 02:41: The question here is, how do you convert the sample OD readout from picomoles to nanograms?
  • 02:41 - 02:48: Okay, so what we have is, you would do it based on the molecular weight of your material.
  • 02:48 - 02:57: So, typically in the assay, you would look at OD on one axis versus nanograms or molarity on the other.
  • 02:57 - 03:09: And then you would, knowing the molecular weight of your analyte, do the math to convert nanograms to picomoles based on that molecular weight.
  • 03:11 - 03:17: Also, I have a question here asking, I’m not getting sample readout.
  • 03:17 - 03:22: The standard curve looks fine, but my samples aren’t reading out appropriately.
  • 03:22 - 03:31: That can also be sometimes attributable to whether or not there is a sufficient quantity of the analyte in your sample.
  • 03:31 - 03:39: Many of the protocols will also request that you either prepare samples fresh to ensure that there’s no degradation,
  • 03:39 - 03:46: or that you freeze them, if you do need to freeze them, that you freeze them at a very specific step in the preparation process
  • 03:47 - 03:52: to help reduce the possibility of there being any kind of degradation.
  • 03:52 - 04:00: In some cases, there is a matrix effect, that is, that the antibody, the detector, is not appropriately binding
  • 04:00 - 04:05: because it’s in some way getting blocked by something in the matrix.
  • 04:05 - 04:12: You may wish to make a dilution series to see if that inhibition might actually go away,
  • 04:12 - 04:16: and you might actually get some signal with a more dilute sample as well.
  • 04:20 - 04:23: Here we’re talking about some plotting.
  • 04:23 - 04:27: The question is, when plotting a standard curve in Excel, how do we determine the trend line?
  • 04:27 - 04:31: Is there a general rule of thumb, for example, linear versus polynomial?
  • 04:31 - 04:33: Good question.
  • 04:33 - 04:43: Typically, it is a log-log or a four-parameter curve equations that fit best for ELISA assays.
  • 04:43 - 04:51: Some will show up pretty well in linear, but it is more typical to do a log-log or a four-parameter fit.
  • 04:51 - 04:57: You’ll also know very quickly when that will fit your ELISA based on the data,
  • 04:57 - 05:02: but it is very typical in a biological assay like this to be log-log or four-parameter.
  • 05:02 - 05:09: Here, how should serum or whole blood samples be stored prior to running the ELISA?
  • 05:09 - 05:18: Typically, we would recommend to get the samples at least to a serum state as opposed to whole blood.
  • 05:18 - 05:22: Four degrees can be fine for whole blood for a short time,
  • 05:22 - 05:27: but more typically, we would recommend minus 20 degrees.
  • 05:27 - 05:30: The question here was specific for estradiol.
  • 05:30 - 05:35: There may actually even be somewhat of an extraction step that would be possible
  • 05:35 - 05:41: that would really enhance the long-term storage of a compound like that.
  • 05:44 - 05:47: The question here, what about antibodies stored at minus 80?
  • 05:48 - 05:51: Yes, it is.
  • 05:51 - 06:00: It is common for the ELISA kits to have various components needing to be stored at different temperatures.
  • 06:00 - 06:07: Typically, if it’s a pre-made kit and you’re using all of the wells in one assay,
  • 06:07 - 06:11: then you would store that particular antibody at minus 80
  • 06:11 - 06:14: and pull it out at the time that you’re going to run the assay.
  • 06:14 - 06:18: If you think that you’re going to actually run and break down the kit
  • 06:18 - 06:24: and do it on a couple different days because you only have a certain number of samples available,
  • 06:24 - 06:28: then we would subaliquot that.
  • 06:28 - 06:32: Then the question is also, the sample itself is an antibody.
  • 06:32 - 06:38: There again, if the sample is an antibody and you’re using a detection kit,
  • 06:38 - 06:43: we recommend that samples are stored at minus 80 for longer term
  • 06:43 - 06:49: and subaliquoted if you think that you might be going back to that sample on a repeated basis
  • 06:49 - 06:55: to do various types of analyses on them is to aliquot them in more what I would call experimental sizes.
  • 06:55 - 07:00: Let’s say 20 microliters or 50 microliters is what you typically do to run an assay
  • 07:00 - 07:05: is to do that and not go through multiple freeze-thaw cycles.
  • 07:06 - 07:08: There’s a question here.
  • 07:08 - 07:14: There have been a lot of variations for measuring IL-6 for my samples between replicates on the same plate.
  • 07:14 - 07:19: Could that be due to incomplete washing off of the washing buffer?
  • 07:19 - 07:23: Yes, that can certainly play a role in it.
  • 07:23 - 07:25: The washing can play a role.
  • 07:25 - 07:30: The validation and the accuracy of the pipette that is being used,
  • 07:30 - 07:34: if that might need to be calibrated, we definitely see that happening even in-house.
  • 07:34 - 07:40: If a pipette is out of calibration, that you might see differences there.
  • 07:40 - 07:45: You also would want to be sure that you’re getting appropriate coverage of the well with each of the buffers
  • 07:45 - 07:51: to ensure that it’s appropriately covered, that there isn’t any of the liquid sort of stuck to the side of the well
  • 07:51 - 07:57: or even if you have a cover on the plate that there’s any stuck to the side of the well.
  • 07:57 - 08:03: There are a number of places there where you want to ensure appropriate handling of each and every well
  • 08:03 - 08:06: and appropriate washing as well.
  • 08:06 - 08:10: How does one determine how much or how little to dilute the sample?
  • 08:10 - 08:14: That is a great question as well.
  • 08:14 - 08:21: Basically, if you have some idea from the literature how much to be anticipating in your sample,
  • 08:21 - 08:24: then you might be able to do a dilution.
  • 08:24 - 08:30: I know in the protocol booklet for many of our ELISA assays, particularly for the simple steps,
  • 08:30 - 08:35: we will actually have values that we determine for various types of samples
  • 08:35 - 08:41: and you can take a look there as guidance for the amount that you may encounter
  • 08:41 - 08:44: in the type of sample that you have.
  • 08:45 - 08:47: If it’s really very much a guesstimate,
  • 08:47 - 08:51: you may need to do a couple of dilutions of your sample within the assay itself
  • 08:51 - 08:56: and that will, of course, force you to use more wells for the sample analysis,
  • 08:56 - 09:02: but that can certainly help to ensure that you will get it to fall within the standard curve as well.
  • 09:02 - 09:07: What would you suggest is the shelf life of an antibody stored in the fridge?
  • 09:07 - 09:11: That is a question that I would like to answer as well.
  • 09:12 - 09:15: That is a question we also get asked a lot.
  • 09:15 - 09:25: We at Abcam will guarantee our antibodies to work for a year from the day you receive them in your laboratory
  • 09:25 - 09:30: if stored under the appropriate conditions of 4, minus 20, or minus 80.
  • 09:30 - 09:37: I have had customers have antibodies go off in a period of about six months under proper storage,
  • 09:37 - 09:42: and I recently talked to somebody who was submitting an Ab Review using an antibody
  • 09:42 - 09:46: that they had found in their fridge that was 10 years old and it was just fine.
  • 09:46 - 09:53: So, you know, anywhere, there’s a window of anywhere from a few months to 10 years
  • 09:53 - 09:55: and the antibody was still working great.
  • 09:55 - 10:00: So, there is some variability there, but you should be able to get at least six months to a year
  • 10:00 - 10:02: if stored under the right conditions.
  • 10:07 - 10:08: Thank you.

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