1.2 ELISA – experimental conditions
As mentioned previously, you will be using one set of standard conditions in the ELISA laboratory to measure IgG antibodies against SARS-CoV2. To do this, you will watch the video guide again, but this time you will note down the conditions used. You will use these variable(s)/condition(s) when you use the laboratory later this week.
Now go on to the next activity in which you will identify the assay conditions used in the protocol shown in Video 1 for the ELISA: Epidemiology laboratory.
Activity 2 ELISA: Experimental conditions
Look at the table below For each action(s) in each step, note down the condition or variable in the blank cells in the table below.
| Step | Action | Variable(s) /condition(s) |
|---|---|---|
| 1. Serum sample | Select eight samples, one for each row of the plate | |
| 2. Dilution plate | Select transfer volume for serial dilution | |
| 3. Primary antibody incubation | Select ELISA plate Primary incubation time Number of washes Duration of each wash |
|
| 4. Secondary antibody preparation | Select secondary antibody Choose volume of stock reagent |
|
| 5. Second antibody incubation | Secondary antibody incubation time Number of washes Duration of each wash |
|
| 6. Chromogen | Select the chromogen Chromogen incubation time |
|
| 7. ELISA plate reader | Wavelength of filter on plate reader | |
When you have filled in the table, click on ‘Reveal answer’ to check your conditions are correct. These are the conditions you will use in your own assays. Note down these conditions ready for the next set of activities.
Answer
The correct conditions are:
| Step | Action | Variable(s) /condition(s) |
|---|---|---|
| 1. Serum sample | Select eight samples, one for each row of the plate | |
| 2. Dilution plate | Select transfer volume for serial dilution | 100 µl |
| 3. Primary antibody incubation | Select ELISA plate Primary incubation time Number of washes Duration of each wash |
Spike protein 45 minutes 3 5 minutes |
| 4. Secondary antibody preparation | Select secondary antibody Choose volume of stock reagent |
Anti-human IgG, HPO conjugate 4 µl |
| 5. Second antibody incubation | Secondary antibody incubation time Number of washes Duration of each wash |
45 minutes 3 5 minutes |
| 6. Chromogen | Select the chromogen Chromogen incubation time |
TMB 20 minutes |
| 7. ELISA plate reader | Wavelength of filter on plate reader | 450 nm |
If you had chosen ABTS as the chromogen, then the appropriate filter is 450nm.
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If the samples have very high levels of antibody, what adjustment would you make, so that the results will still be in range of the assay?
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Reduce the volume on the dilution plate transfer – eg 50µl transferred will give a 1:3 dilution series.
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If you use a higher concentration of secondary antibody than the recommended amount (1µg/ml), what effect will that have on the end result?
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The background values will be high.
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If you had used O-phenylene diamine (OPD) as the chromogen, what filter should you use on the plate reader?
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The 645nm filter.