ELISA and advanced detection technologies

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Good morning everyone, and welcome to this webinar, ELISA and Beyond.

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My name is Teresa, and I'm here today with my colleague, Rebecca.

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Together, we will provide you some information about ELISA and other immunoassays.

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First of all, we will introduce ourselves.

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Hi again, my name is Teresa, and I'm working as a bioresearch field application

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scientist since January of 2021. Previously to joining the company, I did my

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PhD about nutrition and type 2 diabetes.

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What do I normally do on my free time?

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Well, I love to try new food, so consider myself as a foodie, and I also love

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reading books. In addition to that, I enjoy traveling, doing yoga, and I have

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started dancing. Now, I will pass on my colleague, Rebecca.

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Thank you, Teresa. Good morning everyone.

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My name is Rebecca, and I'm a field application scientist for Molecular Devices.

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My background is in neuroscience, where I studied in University College, Dublin.

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I joined Molecular Devices about a year ago and currently cover Ireland and the

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Nordics. Some of my hobbies include eating, as you can see here on the left,

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also running and tennis in my spare time, and I'm an avid animal lover.

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Here's a picture of my little dog, Willow, on the right.

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So to start off, here is a brief look at what we will cover today.

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We'll look at what is an ELISA, its three base detection modes, and then finally

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moving on to some more advanced technology such as HTRF and Alpha

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technology.

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So first off, what is an ELISA?

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ELISAs are a type of immunoassay that are commonly used to quantify levels of a

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specific target within a sample.

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Samples include serum, plasma, saliva, which you'll be familiar with the COVID

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tests, and also with pregnancy tests, as the example here is urine.

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There are four main types of ELISAs. The first one here is a direct ELISA.

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An antigen or sample is immobilized directly on the plate, and a conjugated

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detection antibody binds to the target protein.

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The substrate is then added in suspension, producing a signal that is directly

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proportional to the amount of analyte in the sample.

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These are used when assessing antibody affinity and specificity, and also when you

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want to investigate blocking or inhibitory interactions.

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An indirect ELISA is similar to a direct ELISA in that an antigen is immobilized

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again on a plate. But it includes a conjugated secondary antibody, shown here in

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blue, directed against the host species of the primary antibody, shown here in

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green.

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The suspended substrate then again produces a signal directly proportional to the

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amount of analyte

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or antigen bound in the well.

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So these are used when measuring endogenous antibodies.

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Moving on to a sandwich ELISA, which is the most common type of ELISA.

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Two specific antibodies are used to sandwich the antigen, commonly referred to

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as matched antibody pairs. A substrate then in suspension again is added

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and produces a signal that is directly proportional to the amount of antigen

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present in the sample.

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These are usually used when determining analyte concentration in a biological

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sample.

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And lastly, we have the competitive ELISAs, which are commonly used for small

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molecules when the protein of interest is too small to efficiently sandwich

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with two antibodies. So here the antigen competes with the reference antigen for

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binding of a specific amount of labeled antibody.

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The reference is pre-coded on a multi-well plate, and the sample is

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pre-incubated with the labeled antibody and added then to the wells.

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So depending on the amount of antigen in the sample, more or less free antibodies

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will be available to bind to the

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reference

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antigen.

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This means that the more antigen there is in the sample, the less reference

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antigen will be detected and the weaker the

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signal. These are usually used when determining

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concentrations of small molecules or hormones.

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So here is a table showing which ELISA to choose from and their advantages and

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disadvantages.

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For direct ELISAs, they're fast and simple.

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However, they can be less specific as it only uses one antibody and

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has potential for high background if all proteins from the sample are immobilized

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in the well. For indirect, amplification arises

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using the secondary antibody.

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There is, however, potential for cross-reactivity caused by the secondary antibody.

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For a sandwich ELISA, has the highest specificity and compatible with

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small matrices. However, the protocols can be long and challenging to develop.

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For a competitive ELISA,

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they have the ability to quantitate small molecules.

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However, again, they can be less specific since you're only using one antibody and

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requires the conjugate antigen. The illustration above shows

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a workflow for a typical sandwich ELISA.

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You can see here that there is eight steps.

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You have your 96 to 3L microplates where your antibody binds to the well,

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then the addition of your sample. And then here shown in red, you have your two

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washing steps, which can be very time consuming as it can take over an hour for

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these washing procedures. You usually wash the microplate three times in

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each wash steps, which can be a total of six washes.

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Then here with seven and eight,

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the microplate reader is used to detect the color change produced when the target

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antigen is present. And then lastly, you have your software, which is used to plot

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your standard curves and calculate results.

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This workflow can also be fully automated.

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We all know too well that manual microplate washing with pipettes can be very

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painful and laborious, quite like this picture here on the left.

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I can remember when I was doing my ELISAs in the lab, carrying out the washing

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steps can be very laborious. However, with the use of a microplate

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washer, you can reduce these time-consuming steps by washing the wells without the

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need for pipettes, similar here to the picture on the right.

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It would have made my life a lot easier in the lab.

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By using a microplate washer for the washing steps three and five, you will achieve

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efficient cell washing with minimum cell loss and optimal assay performance.

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It allows for precise dispense and aspiration control, allowing

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processing without cell disruption.

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The washer does not require any external pumps or computer to operate,

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minimizing bench space.

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The washer is easy to integrate with the latest automation tools, and it is

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compatible with the Molecular Devices StackMax and many robotic plate handlers.

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Now we will move on to absorbance analyzer.

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To start off, we will ask the question, what is absorbance?

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Well, it is when a molecule absorbs light at specific wavelengths and efficiencies.

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Optical density donates to the amount of light absorbed by the solution.

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You can see on the left picture below where absorbance follows the Beer-Lambert

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law, where OD is directly proportional to the concentration of the sample being

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tested.

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On the right

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is a picture giving a present representation of the optics within a microplate

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reader and how the OD read is achieved.

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You have your light source here above, which is usually LED or xenon flash lamp,

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then your monochromator or filter, your sample, then below, and your

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photodiode for detection.

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Here are some absorbance applications below,

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and in shown in red are your ELISA and amino assays.

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These can be read in single endpoint, in kinetic,

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well scan, and so on.

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Below also you have the ability for micro volume applications, which you can use

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the SpectraDrop that can quantify as little as two to four microliters

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of a sample.

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So the next slide here is very fitting with the Irish culture.

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If you can't tell from my accent, this is where I'm from.

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So a lot of people in my country will be very happy with this research.

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This is an example of how gluten was quantified in beer using an ELISA method.

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I will just briefly summarize the study.

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There has been recent rise in the prevalence of celiac disease,

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and monitoring gluten levels in food and beverages has become increasingly

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important as more people strive to avoid gluten.

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The RIDA Screen Gliadin competitive ELISA can detect small peptide fragments using

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a standard material, hydrolyzate of wheat, rye, and barley.

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The R5 antibody used in this kit recognizes potential toxic peptide

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sequences of gliadins from wheat and prolaminins from rye and barley.

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The competitive ELISA provides a quantitative solution for gluten detection in

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beer. The hands-on time required to run this assay is reduced by using a

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multi-wash washer to automate steps.

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Detection of absorbance with the SpectraMax ABS Plus reader and the calculations of

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results using our SoftMax Pro software further simplify the ELISA

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workflow and flag any results that warrant further attention.

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So now we all can enjoy a beer at the pub.

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This is the family of single-mode readers from Electro Devices which are compatible

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with absorbance. The SpectraMax QuickDrop, the EMax Plus, the

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VersaMax, and the SpectraMax ABS.

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Now we will move on to fluorescence and ELISA.

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So again, we'll look at the principle of fluorescent intensity.

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It is when a molecule absorbs energy, and it is transformed from a ground to an

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excited state, shown here below.

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It then releases the energy in the form of light of a lower intensity and longer

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emission wavelength.

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Due to the energy dissipation during the excited state lifetime, the emission

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energy of a photon is lower. It therefore has a longer wavelength than the

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excitation, as you are losing some of the energy in the form of heat.

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The difference in energy or wavelength is called the Stokes shift.

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The Stokes shift is fundamental to the sensitivity of fluorescent techniques

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because it allows emission photons to be detected against a low background,

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isolated from the excitation photons.

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Depending on your fluorophore, the Stokes shift can flux in nanometers.

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A microplate reader with fluorescence intensity detection uses a light source,

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which is a xenon flash lamp or LED, to excite the

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fluorophore. The wavelength required to excite the sample can be selected using

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either a filter

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of a specific wavelength or a monochromator tuned to the required

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wavelength. The fluorophore then emits light of a different wavelength

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selected by a second filter or monochromator.

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This emitted fluorescence is detected by a photomultiplier tube, and the

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fluorescence intensity of a sample is expressed as relative fluorescent units.

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With our readers, there is little optimization necessary due to the unique

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automatic PMT.

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When selected, the software will automatically normalize the data within each well,

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so it is independent of the PMT gain selected.

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Therefore, the data from each well and plate can be comparable.

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You can also have the option of reading from the bottom, which is advantageous for

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coated plates or cell placed assays where the signal is coming from the bottom of

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the well.

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A lot of molecules may absorb light, however, a lot less will emit light.

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Therefore, fluorescence can have less background compared to absorbance.

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So fluorescence applications are similar to absorbance, but they will have a lower

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background and therefore improved sensitivity.

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Here are some of the fluorescence applications and where fluorescence ELISAs are

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highlighted in red.

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This is a schematic workflow depicting sequential molecular binding events of a

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sandwich fluorescent ELISA.

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Within each well, the capture antibody is absorbed on the reactive surface,

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followed by surface passivation by a blocker, blocking buffer to reduce

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any non-specific antigen binding.

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Upon target binding to capture the antibody, alkaline phosphatase

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type detection antibody specific to the antigen is added.

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Additional fluorescent substrate activated by AP generates a detectable

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fluorescent signal, indicating successful binding events.

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Here is an example of the CatchPoint simple step ELISA kits, which have been

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developed using a fluorescent substrate to provide improved linearity over an

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extended dynamic range, providing better quantification of both the lower

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and upper ends of the curve. Both an absorbance and fluorescent ELISA kit were

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plotted on the same graph for comparison, shown on the left here.

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On the y-axis are logged values. On the left-hand side shows a log

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OD standard curve for a basic colorimetric ELISA kit.

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You can see that the lower part of the curve to the upper part are only

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approximately a 1.5 fold range in OD.

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In comparison to the right-hand side, which shows an approximate three log fold in

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RFU values for the CatchPoint ELISA kit, depicting a greater increase in

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dynamic range for the assay.

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You can also see in the colorimetric ELISA kit that the linearity starts to drop

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off at around 1,000 picomol per mil on the x-axis, whereas the CatchPoint

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ELISA is linear up to 10,000 picomol, greatly increasing the detection

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limit of the human ICAM-1.

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Some of the other additional benefits include the extended dynamic range, which I

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mentioned, stable signal for readout flexibility, no compromise in

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sensitivity. It's only a one-wash with only 90 minutes,

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and high-quality antibodies from batch to batch provide consistency.

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The single-mode reader from Electro Devices, which is compatible with fluorescence,

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is the Gemini.

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Now we will have a look into luminescence

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ELISAs. Firstly, again, we will look at the

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principle of luminescence.

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It is the emission of light by a substance as a result of a chemical or

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enzymatic reaction.

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No excitation component is needed.

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However, it needs a dedicated detector, which is a photomultiplier tube in photo

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counting to ensure assay sensitivity.

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Below is an example of a chemiluminescent reaction.

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On the left-hand side of the reaction, you have your reactants, which are catalyzed

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by firefly luciferase,

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and in turn producing products

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and light.

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There are two principal chemiluminescent reactions, which are shown here in the

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figure below.

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The graph on the left shows a flash reaction. It causes a flash of light.

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As you can see here in the graph in red, the reaction is extremely rapid and it

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generally needs injectors as the time period is within seconds, shown below here on

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the x-axis.

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The graph on the right, shown here in green, is a glow reaction.

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As you can see, the RLU values increase slowly over a period of time,

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and they remain stable for usually minutes or hours.

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One of the advantages of luminescence is that most molecules and atoms

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either absorb or fluoresce, not many luminesce.

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Therefore,

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the sensitivity of luminescence is the greatest.

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Here are some of the luminescent applications where the luminescence ELISAs is

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highlighted here in red.

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With COVID, I had a lot of spare time and binge-watched a lot of crime series, and

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I always wondered why they would turn off the lights during crime scene

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investigations and see a blue glow spot in certain places.

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So they actually use an organic compound called luminol,

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which exhibits a chemiluminescence with a blue glow when mixed with an appropriate

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oxidizing agent.

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Therefore, forensic investigators use luminol to detect trace amounts of blood at

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crime scenes as it reacts with the iron in the hemoglobin.

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So this is just showing the compatible luminescence reader, the spectrolometer from

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Molecular Devices.

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This table then shows a brief overview of the detection strategies for

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ELISAs. All modes use the same enzyme probes, which

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are horseradish peroxidase

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and alkaline phosphatases. However, from absorbance to fluorescence,

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there is an increase in sensitivity,

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and also from fluorescence to luminescence, there's a thousandfold in sensitivity.

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Below here are some of the advantages to take into consideration when picking which

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read mode to use.

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For absorbance, there is direct visualization of the OD, and there's high

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reproducibility between plates.

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With fluorescence, you're getting an increase in dynamic range.

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However, with luminescence, it is the most sensitive detection strategy, and again,

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you have a wide dynamic range.

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When choosing which plates to use, it is recommended that a clear plate is used for

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absorbance, and if you're in the UV range below three nanometer, you use a UV

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plate.

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And with fluorescence, it is recommended to use a black microplate.

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And for luminescence, it is recommended to use a white

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microplate. Our multi-mode readers shown here are compatible

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with all three detection modes explained, absorbance, fluorescence, and

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luminescence.

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Now I will pass the presentation on to Teresa, where she will explain further ELISA

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detection technologies.

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Thanks, Rebecca, for giving us a detailed explanation about how an ELISA is

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working. Now, I would like to move to the ELISA workflow again and pointing out

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some challenges from this technology.

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The first challenge to mention is the washing steps, increasing assay length and

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time.

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Also, having a fixed plate format, which is commonly used a 96-well plate,

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which makes difficult to increase the throughput,

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limited sensitivity and dynamic range, and finally, short signal stability,

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which is normally around one hour except for luminescence ELISAs.

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As you can see in the picture, eight steps are needed in order to complete the

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whole ELISA workflow.

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So now the question to ask is whether do we have other type of immunoassays which

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can be alternative to ELISA assays, improving the sensitivity and dynamic range.

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Also, where no washing steps are required, increasing the signal stability and

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possibility of changing to 384-well plates, increasing the throughput.

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This alternative can be HTRF and Alpha technology.

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Despite their fancy names and that you might not be familiarized with them, they

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are fluorescence and luminescence-based technologies.

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Let us start with HTRF, which stands for Homogeneous Time Resolved

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Fluorescence. As you can see in this workflow, in this case, no

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washing steps are needed. Also, the assay time is shorter,

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only one to two hours in comparison to an ELISA assay, which is between four hours

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and 24 hours.

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Also, the signal stability is from 24 hours up to days, and

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finally, the dynamic range is up to four logs.

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The increased signal stability will allow you go home and read the following

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morning in case you finish an assay late in the afternoon.

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Finally, this workflow is only four steps in comparison to the eight steps in the

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case of the ELISA assay.

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But now the question is: how does HTRF work?

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As I have just mentioned, HTRF stands for Homogeneous Time Resolved Fluorescence

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and is a TR-FRET technology developed by Sisbio.

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But what does TR and FRET means? Well, let us start with FRET.

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From one side, FRET means fluorescence resonance energy transfer.

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Basically, there are two fluorophore, a donor and an acceptor, which can be, for

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example, protein, DNA. These two molecules are conjugated to two

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fluorophore, the donor and the acceptor.

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The peculiarity of these two pair of fluorophore is that in close proximity, the

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emission of the donor can excite the acceptor.

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In the other side, TR means Time Resolved, and it's based in fluorophore with

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long-lived fluorescence lifetime.

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I will explain a bit more in depth in the following slides.

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So the standard fluorophore like fluorescein has a short-lived fluorescence

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emission in the range of nanoseconds, as you can see in the graph in the orange

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line. Excitation and emission light are measured with a really small delay,

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producing a high background signal.

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The background signal is normally coming from the solution.

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Other fluorophores such as europium or terbium emits long-lived fluorescence,

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which lasts four milliseconds. In this case, excitation and emission are measured

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with a delay, so the signal-to-noise ratio is increased, increasing also the

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sensitivity of the assay.

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Because of the high background might interfere with the signal, we need to use

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filters in order to achieve the sensitivity needed for the HTRF assays.

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Therefore, as we can see in this picture, the plate reader is provided with an

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excitation filter for the excitation of the donor and with two emissions filter,

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one for the emission light of the donor and one for the emission light of the

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acceptor.

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Because the donor will excite the acceptor, only one excitation filter is needed,

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in this case, for the donor.

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But how is this technology being measured in our plate reader and software?

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Well, as mentioned at the beginning, some important and differential parameter from

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this technology is a delay time between the excitation of the sample and the

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collection of the signal. This parameter can be changed in our software in order to

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set up your assay. In general, you will need more optimization

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than for the ELISAs. As you can see, more parameters need to be optimized in order

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to set up your assay, and we can offer you some already optimized protocol from all

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multimode readers.

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Regarding our multimode reader, here I'm showing two of them, which are compatible

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with HTRF measurements.

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These instruments are combined with dedicated filters in order to fulfill your HTRF

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specification. In the case of the iD5, a filter slider is

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combined, and in the case of the i3x, a cartridge is being

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added.

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As summary, here are shown some key features from HTRF like

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long-lived fluorophore like europium and terbium, high signal stability,

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homogeneous assays where no washing is needed,

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able to miniaturize, high sensitivity due to low background, and data

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normalization, allowing direct data comparison.

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In this technology, a ratiometric ratio between the fluorescence from the acceptor

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and the donor is being calculated.

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Now, let's move to Alpha technology.

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As we can see in the workflow, no washing steps are required.

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The assay time is shorter, so only between one and two hours.

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The sensitivity is improved, reaching one picogram per milliliter, and the dynamic

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range is five logs.

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An additional advantage of the Alpha technology in comparison to the ELISA is the

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capacity of multiplexing, where up to three different analytes can be detected at

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the same time, in comparison to ELISAs, where only one analyte is detected.

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Also, only five steps are needed in comparison to the eight steps for ELISA.

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So how is Alpha technology working?

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Alpha technology have been developed by PerkinElmer and is a non-radioactive assay.

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Alpha stands for Amplified Luminescence Proximity Homogeneous assay.

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It is a bead-based assay in which the donor and acceptor beads are conjugated to

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biomolecules, for example, receptor-ligands or antibody-antigen.

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When in close proximity, a luminescence fluorescence signal is produced.

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Now, I will move to the next slide where I will shortly explain in more details how

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this technology works.

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First of all, a laser will excite at 680 nanometer a molecule from the donor

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bead. This produces the conversion of the oxygen into a singlet oxygen that reacts

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with different chemiluminescence of the acceptor beads, activating fluorophores

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that emits light.

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Depending on the type of fluorophore that's been activated, there are three main

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type of technologies, AlphaScreen, AlphaLISA, and AlphaPlex.

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Here, I'm showing the molecules in the acceptor beads that are activated and the

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signal cascades that is being generated, producing light at different wavelengths.

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For example, for AlphaScreen, the signal is from 520 to 620

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nanometer.

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For AlphaLISA at 650 nanometer due to europium.

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And finally, for AlphaPlex at 545 and 645 nanometer

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due to terbium and samarium.

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In order to be able to measure Alpha technology, one of our multimode readers, the

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SpectraMax i3x, together with a filter cartridge, is compatible for measuring

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AlphaScreen and AlphaLISA.

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Some benefit from these cartridges is the use of a strong light source, a laser

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diode, and also the use of an excitation and emission filter.

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As you can see having a look at the wavelength of the filters, the excitation

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wavelength is higher than the emission wavelength.

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We can see in this graph the AlphaScreen emission is broad, so the emission filter

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we use is 100 nanometer bandwidth, passing as much light as possible and increasing

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the sensitivity of the assay. This is the reason why we can measure also the

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AlphaLISA technology, so the emission filter is catching the signal at 620

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nanometer.

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However, Alpha technology can also have some possible limitation.

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So it is known that the signal produced is really bright, generating a glowing

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effect and increasing this crosstalk.

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As you can see here in this picture, sometimes the instrument is catching light

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from adjacent well. This effect is called crosstalk and will depend on instrument

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design.

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Some possible solution for that are a light blocking tube available in the

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AlphaScreen cartridge and the interlaced reading available in our software.

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I will shortly explain a bit more in details.

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So as you can see in the picture, the cartridge is provided with a light-blocking

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tube, which will move out during the read and situated on top of the well in

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order to block away any light from the neighboring wells, reducing the crosstalk.

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An additional solution for reducing crosstalk is the interlaced reading in our

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software. The instrument is reading the plate as a chessboard.

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As you can see in the lower picture, the software is reading A1, A3, A5,

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and so on, and then reading again the plate, in this case, the wells A2, A4,

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A6, and so on. This software feature is helping to reduce crosstalk in addition to

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the light-blocking tube. Some benefits from the interlaced reading are a decrease

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of crosstalk by 13%, lower CV values, and in general, better

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curves.

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Now, I will show you some data from an AlphaScreen assay run together with my

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supervisor in the lab. In these two graphs, we can see the effect of the interlaced

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reading on versus off in three different samples.

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So as you can see in the lower part of the graph, the CV values are reduced and in

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general, we can observe better curves when the interlaced reading was on in

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comparison than when the interlaced reading was off.

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As summary, Alpha technology has the following advantages, high sensitivity assay,

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low background, high signal background ratio, able to miniaturize, a

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cost-effective assay, versatility, flexibility, and low affinity to high affinity

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interactions.

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Thanks a lot for your time, and please let us know if you have any questions.