2 Vaccination
This week the laboratory investigation is to measure the development of antibodies in two subjects who have been enrolled in a phase-1 study to assess the effectiveness of a COVID-19 vaccine. You will be using the ELISA again, but with a different set of samples to the previous weeks.
The first 10 minutes of the video describes the samples and data available. It is followed by a section which reminds you how to use the ELISA laboratory. Also note that the video refers to samples from 10 subjects, but in the version of the laboratory available to you, we have only included samples from the two subjects that you are investigating. In this demonstration a different chromogen, OPD was used. You can do this if you like (remember to use the 645nm filter on the plate reader), or you can use TMB as you have done in previous assays.
As you watch the video, be sure to note the vaccination schedule and when the blood samples were taken (Figure 3).
Download this video clip.Video player: Video 1 Detecting antibodies against SARS-CoV-2
Transcript: Video 1 Detecting antibodies against SARS-CoV-2
DAVID MALE
Hello. I'm David Male from the Open University. In this video, I will show you how you can detect antibodies against the coronavirus SARS-CoV-2 using a technique called ELISA, an enzyme-linked immunosorbent assay. This technique is often used to quantitate antibodies in diagnostic laboratories or for research purposes.
We have recreated this technique in a virtual laboratory where you can test a set of serum samples from 10 individuals who have been immunized against SARS-CoV-2 and who have then been monitored for 200 days to test their levels of antibodies against the virus. This will show you how the immune response against the virus develops following vaccination.
This diagram shows the time course of the study. A serum sample was taken on day zero immediately before the subjects were given their first vaccination. Samples were then taken at days 7, 14, and 21 to monitor the appearance of antibodies. Another sample was taken at day 56 before the second dose of vaccine, and the effect of the second vaccination was monitored at day 70.
A final sample was taken about six months later to establish how quickly antibodies declined in the serum. Now, let's see how the antibodies can be measured using the ELISA technique.
This diagram illustrates the key steps in an ELISA. The assay is done on 96-well polystyrene plates that have been sensitized with antigen. In this case, the antigen is the spike protein of the SARS-CoV-2 virus corresponding to the antigen used for the vaccination.
In the first stage, diluted serum is applied to the wells on the plate. If there is specific antibody in the serum, it will bind to the antigen on the plate. The bound antibody is called the primary antibody. Any unbound proteins in the serum, including non-specific antibodies, are then removed in a wash step.
In the second stage, a ligand is applied to the plate which binds specifically to the primary antibody. The ligand also has a coupled enzymatic portion, which is crucial for the following step. In some cases, the ligand itself may be an antibody that binds to the primary antibody, in which case, it would be called a secondary antibody. The plate is then washed again to remove any unbound ligand.
Finally, a chromogen is put into the wells. The chromogen is a colorless chemical which generates a colored end product when acted on by the enzyme of the ligand. The colored end product is detected on a plate reader. The more primary antibody is present, the more enzyme is bound to the plate and the more colored end product is produced. Hence, this technique can quantitate how much antibody is present in the initial serum sample.
Now let's look at how the virtual ELISA laboratory recreates the technique. The virtual laboratory allows many options in choosing different serum samples and the dilution series of these samples. You can also choose the time of the incubations with the antibodies and the number and duration of the wash steps. You can select from three different ligands which detect different classes of antibody.
There is also a choice of three chromogens for the development step. Finally, the results are read on a plate reader. And it is important to select the correct wavelength filter. The results correspond with what you would see in a real laboratory as these experimental conditions are varied. Moreover, if you happen to choose conditions that aren't quite right, then your results may not be optimal and perhaps even provide no usable results at all.
So it's important that you note exactly what you do at each of the steps. It's also important to use controls in each assay. Initially, you should follow the recommended experimental conditions. But once you have your experiments working well, I would encourage you to modify some of the conditions and see how this affects the results. This way, you will also learn a lot about technical aspects of the assay.
The exact appearance and layout of the experiments within the on-screen experiment will depend on the web browser you are using and the screen size. In this introduction, I'm using a desktop computer and Microsoft Edge as the browser.
Before we start, a quick word about timing. In the virtual laboratory, we have telescoped time, so an incubation that would normally take 60 minutes can be done in one minute. The incubations are started and stopped on a timer like this. It means that an assay which would normally take two to three hours can be done in a few minutes.
As mentioned, ELISA is normally done using 96-well polystyrene plates that have been sensitized with a specific antigen. They are relatively cheap, used once, and then discarded. If your ELISA experiment goes wrong, you can come back to the beginning and start again by pressing the reset button. Now, let's take a look at the laboratory.
The first step is to choose serum samples. These are samples taken from individuals that may or may not contain the antibodies you are going to test for. You can see eight tubes containing separated blood samples with serum at the top of the tube and red blood cells at the bottom. Up to eight different serum samples can be selected, one for each row of the ELISA plate.
Select a sample by clicking the box beneath one of the sample tubes. You will see a menu with all the samples available. Each sample you choose will be assigned to one of the rows on the ELISA plate. In this case, I'm going to use a negative control as the first sample which will go on row A. I'm then going to choose a series of seven different samples from one subject, subject one or S1.
The first sample is from day zero, and it goes on to row B. It is listed as S1 d0, meaning that it is from subject one on day zero. The second sample from subject one on day seven, called S1 d7, will go on row C. The third from day 14 will go on row D, and so on.
I keep adding samples until I have one sample assigned to every row of the plate. I have now chosen a set of seven samples from subject one. But if you scroll through the list, you will find the samples from all the other subjects.
Step two shows a 96-well plate. You will see the identities of the samples chosen in step one are now listed beside each row of the plate. The task in this step is to perform a serial dilution of the serum samples. Put simply, a serial dilution is a successive dilution of a starting sample. You will see why this is important as you progress through the experiment.
The serial dilution is performed by adding a known volume of the serum sample to the left-most well on the plate and mixing it with a known volume of diluent. A diluent is simply a liquid medium that is compatible with the sample which we can use to dilute the sample. Every well on the plate contains 100 microliters of diluent. You need to take note of this volume so that you can work out what your serial dilution will be.
To make a serial dilution, a volume of each of the serum samples chosen in step 1 is added to the leftmost well on the plate, well 1. It is mixed with a diluent, and then a defined volume is transferred to well 2 and mixed. The same amount is transferred from well 2 to well 3 then from well 3 to well 4, and so on down the plate to well 12.
This procedure is carried out with the multichannel pipette illustrated. By transferring a volume of the sample from well to well and mixing it with fresh diluent each time, each sample is successively diluted along the plate. You need to decide what volume you want to transfer from well to well by changing the setting on the pipette.
In this case, I'm going to transfer 100 microliter volumes down the plate. So I set 100 microliters on the pipette and press transfer. Since there is 100 microliters of the diluent in each of the wells, transferring 100 microliters will produce a serial dilution where the concentration of the sample decreases by a half each time.
In this case, well 1 is a 1 in 2 dilution of the serum sample. Well 2 would be a 1 in 4 dilution, and so on. You can now see the dilutions are given along the top of the plate. The pipette volume can be set at anything between 20 microliters and 100 microliters. By choosing other volumes on the multichannel pipette, you can make other serial dilutions such as one where the concentration of the sample decreases by a third each time, giving 1 in 3, 1 in 9, 1 in 27, and so on.
The doubling dilution series shown here is a good starting point. But if serum samples have very high titers of the antibody under investigation, then a smaller transfer volume giving a higher dilution series will be required.
In an ELISA, the antibody that binds to the antigen on the plate is called the primary antibody. In step 3, I first have to choose the antigen-sensitized 96-well ELISA plate that I will use for the assay. The dropdown menu lists all the available options. Here I have only one option as I want to detect antibodies to spike protein of the virus.
When I choose the spike protein ELISA plate, I'm using a plate where an equal amount of the antigen is bound to every well on the plate. If there are relevant antibodies in the serum samples, then they will attach to the antigen on the base of the wells. At this stage, we have two 96-well plates. One is our antigen-sensitized ELISA plate, and the other contains the serial dilutions of our serum samples.
We now transfer the contents of the serial dilution plate to the antigen sensitized plate. This allows any spike-protein-specific antibodies to bind to the antigen on the plate. By pressing the Transfer button, diluted samples are transferred across from the serial dilution 96-well plate prepared in step 2.
Importantly, the contents of each well in the serial dilution plate is moved into the corresponding position on the antigen-sensitized ELISA plate. You will notice that when you press Transfer, the wells all turn blue to indicate that liquid has been transferred. Now we incubate the samples on the antigen-sensitized plate by starting the clock. An incubation time of 45 minutes to 1 hour is recommended.
[BEEPING]
I will stop the incubation now. If you incubate for a short time, the antibody has less time to bind to the antigen, and the signal at the end of the assay will be lower. If you incubate for a bit longer than one hour, it will not make much difference. The incubation allows the antigen and any antibodies in the samples to interact. This is such a strong interaction that we can wash the plate quite vigorously to remove any unbound antibodies and other serum proteins.
In essence, washing the plates simply means removing all the liquid from each well and adding a volume of fresh washing buffer. You need to choose how many wash steps to do and for how long you leave the wash liquid to incubate during each wash. Click on the Wash Start button to begin a wash, and press Wash Stop to end a wash. Three washes of five minutes each are the minimum recommended.
[BEEPING]
If you do not wash the plates sufficiently, residual unbound antibodies in the serum samples will neutralize the detection antibody which will be added later.
Step 4 involves detection of any antibody that bound to the antigen in the preceding step. This is done by using an enzyme-conjugated ligand, usually another antibody that is added to all of the wells in the 96-well plate. This detection antibody is called the secondary antibody because it binds to the primary antibody that is bound to the antigen.
In step 4, you are presented with three vials that contain secondary antibodies. There are three major classes of antibody present in serum-- immunoglobulin M, immunoglobulin G, and immunoglobulin A, which are referred to as IgM, IgG, and IgA. Each of the secondary antibodies specifically detects one of the classes of serum antibodies.
In this demonstration, I want to detect IgG antibodies, so I select the vial anti-human IgG, which is conjugated to the enzyme horseradish peroxidase or HPO. You should also notice that the concentration of the secondary antibody is shown on the label. The stock of the anti-human IgG HPO conjugate has a concentration of 1.5mg per mil. This is too concentrated to use neat, so I need to prepare a dilution.
The recommended concentration for the anti-IgG antibody in this assay is 0.6 micrograms per mil. And I need 10 mils of the solution in total to add to the 96-well plate. I therefore need to add 4 microliters of the anti-IgG stock solution to the 10 mils of diluent.
The way to calculate the required volume of stock is shown in the panel. The volume of stock is the final concentration divided by the stock concentration multiplied by the final volume. That is 0.6 micrograms per mil divided by 1,500 micrograms per mil multiplied by 10,000 microliters. If you did not follow that, just take it on trust that you need 4 microliters of the stock anti-human IgG.
The optimum concentration of secondary antibody depends on the reagent and batch. And in a real lab, each new batch of antibody would be tested to find its activity. Typical optimum concentrations will be in the range 0.5 micrograms per mil to 5 micrograms per mil. Recommended final concentrations of these three secondary antibodies are shown in the table.
The pipette volume can be set at anything between 2 microliters and 100 microliters. If you do not use enough of the secondary detection antibody, the signal at the end of the assay will be low. If you use too much, the background values will be high. And in any case, that would be wasteful of an expensive reagent.
In step 5, the secondary detection antibody is added to every well on the plate. As before, this antibody is incubated on the plate for 45 to 60 minutes. This incubation allows the secondary antibody to bind to the primary antibody.
[BEEPING]
If the incubation is too short, the signal at the end of the assay will be reduced. After the incubation, the unbound secondary antibody must be removed from the plate with washes. As before, a minimum of three five-minute washes is recommended. If you add an extra wash or make the washes slightly longer, it will make little difference. But you will find that three washes of five minutes are more efficient than, say, one wash of 15 minutes.
In step 6, the enzyme activity of the horseradish peroxidase linked to the secondary antibody is used to produce a colored product in each of the wells. To do this, I will add a chromogen solution to every well on the plate. In this case, there are three options. I will choose OPD which is o-Phenylenediamine. It produces a red-colored end product when acted on by horseradish peroxidase.
Once the reaction has started, the color develops progressively with time. At an appropriate time of your choosing, the activity of the horseradish peroxidase enzyme should be stopped by adding sulfuric acid. You should allow enough time for the color to develop so that you can see visible differences between successive wells. If you run the reaction for too long, eventually, every well will develop color and background values will increase.
An incubation time of 5 to 30 minutes is typically used for these assays. In this case, I will stop the reaction after 20 minutes by the addition of sulfuric acid.
[BEEPING]
Finally, in step 7, we get to see the results of the assay by using a 96-well plate reader that can detect the concentration of the red end product in each of the wells. Plate readers shine light at the 96-well plate and record how much light passes through each of the wells. Most plate readers can detect light of several different defined colors. The color is selected by placing an optical filter in the light path.
To detect the red-colored end product, a filter of 645 nanometers is appropriate, so I select that here. If I had chosen a different chromogen in the preceding step, then I would need a different filter at this point. For example, ABTS produces a green end product, and the 450 nanometer filter is appropriate.
Now we can read the absorbance values. The 96-well plate is taken into the plate reader and the absorbance read. The plate is then returned on the tray.
In the final step, you can view the results which are presented in an 8 by 12 array corresponding to their position on the ELISA plate. These results can now be taken for analysis. After you have read the plate using the appropriate filter, you should have a data set that looks like this. I've added a bar at the bottom showing the reciprocal of the serum dilution in each of the wells.
The titer of antibody in a sample is usually read as the highest dilution that gives a detectable signal above the background of the assay or the negative control. If I look at the negative control in row A, there is a background value in wells 3 to 12 which is about 0.09. And the highest value in well A1 is 0.108.
We can therefore say that an absorbance value above 0.11 is a good cutoff point for assessing whether there is a significant level of absorbance above the background. I have now highlighted all the wells above this threshold. From this, we can read off the titer in each sample. And this is now entered on the right-hand side.
Titer is reported as the reciprocal of the highest serum dilution that gives a detectable signal in the assay. The result for subject number one shows a progressive increase in antibody titers up to day 21 following vaccination on day zero. The antibodies are then maintained at this level up to day 56 when the second vaccination was given, and this resulted in a further rise in the titer.
The results would be more accurate and reliable if the samples were done in duplicate or triplicate. But it is really important to note that each assay will be slightly different depending on the exact conditions you use. You need to work this out for yourself by looking at your own data, and it will be different on each assay. Also, be aware that duplicate samples will usually give slightly different results reflecting the variation that is seen in this type of assay.
I've now shown you how to determine the titer of IgG antibodies against SARS-CoV-2 spike protein using ELISA. You can do a lot of different analyses using this set of serum samples. Here are some suggestions.
You could test all 10 subjects at day 21 to find the range of antibody titers induced by the first dose of vaccine. This will also show whether any of them failed to produce an antibody response. You could compare the titers of antibodies after the first and second vaccination to see what effect the second vaccination has on antibody titers.
One important question is how long antibodies last for. This can be seen by comparing the titers in all 10 subjects at day 70 and day 200. You could measure IgA antibodies. IgA is important in protecting mucosal surfaces and is therefore very important in immunity to respiratory viruses. The intramuscular immunization with SARS-CoV-2 vaccine is expected to give a strong IgG response, but the IgA response may be less strong.
Given information on the age and sex of the individuals, you could compare antibody titers in women and men or between younger and older individuals. The tables on this page give some extra information on the age and sex of the 10 subjects in the vaccine trial, which may be useful for your investigations. And there is a reminder underneath of when the samples were taken in relation to the vaccinations. As you can see, there are many investigations that could be done with this set of serum samples.
I hope you have found this introduction to the ELISA technique useful and its application to investigation of COVID-19 antibodies interesting.
Video 1 Detecting antibodies against SARS-CoV-2
Interactive feature not available in single page view (see it in standard view).
This protocol has a primary and secondary dose of vaccine separated by 8 weeks. Samples were taken in the first 21 days to detect appearance of antibodies. A sample was taken just before the second dose of vaccine and again 14 days after the second dose when maximum antibody titres were anticipated. The sample taken 6 months later was intended to detect how long antibodies remained in the serum.