The ELISA Test Explained
The stages of the ELISA test, from antigen binding to enzyme-linked antibody, washing and the substrate colour change, and what the result shows. Includes the 5-mark exam question, a worked model answer and the examiner trap on the colour change and the enzyme-linked antibody.
Chapters (9)
Exam question [5 marks]
The ELISA test can be used to find out whether a patient's blood sample contains a particular antigen. Describe how the test is carried out and explain how it shows that the antigen is present.
Try it on paper first, then check the mark scheme below. The question is original and was written for this lesson.
Mark scheme
| Mark | Mark point | Also accept | Do not accept |
|---|---|---|---|
| 1 | Sample added to the well and any antigen sticks / attaches to the surface | "antigen is immobilised / fixed to the well" | "antibodies stick to the well" |
| 2 | Primary antibody, complementary to (specific to) the antigen, is added and binds to it | "antibody with a complementary binding site", "specific antibody" | "antibody is the same shape as the antigen"; antibody binds to "any" antigen |
| 3 | Well is washed after each stage / to remove unbound antibody (so only bound antibody remains) | "washed to remove unbound / excess antibody or enzyme", "prevents false positives" | "to clean the well"; washing mentioned with no reason |
| 4 | Enzyme-linked (secondary) antibody added, which binds to the primary antibody | "second antibody with an enzyme attached"; "enzyme-linked antibody binds to the primary antibody" | "enzyme binds to the antigen"; "enzyme-linked antibody binds directly to the antigen" (not the format taught; no mark) |
| 5 | Substrate added, enzyme converts it to a coloured product, so a colour change shows the antigen is present | "enzyme only present if antigen present", "no colour change = no antigen" | "it changes colour" with no mention of enzyme and substrate |
The stages must be in order (sample, primary antibody, enzyme-linked antibody, substrate). Mark 3 needs a reason for washing or a statement that it follows each stage. Mark 5 needs both parts: the enzyme converting substrate to a coloured product AND the link from colour back to the antigen; only one half scores no mark. Candidates who describe the enzyme as binding to the antigen directly lose mark 4.
Model answer (5/5)
The patient's sample is added to a well, and any antigen sticks to the surface. The primary antibody, which is complementary to the antigen, is added and binds to it. The well is washed after each stage to remove any unbound antibody. An enzyme-linked antibody is then added, which binds to the primary antibody. After a final wash, the substrate is added. The enzyme converts the colourless substrate into a coloured product. The enzyme is only held in the well if the antigen was present, so a colour change shows that the antigen is present.
Examiner trap
- Forgetting wash steps between each stage
- Not explaining why a colour change shows the antigen is present
- Not explaining the role of the enzyme-linked antibody
Key terms
- ELISA
- enzyme-linked antibody
- substrate
- colour change
- washing
- specific
- antigen
- primary antibody
- secondary antibody
Lesson notes
A written version of the video, chapter by chapter, for revision. Times link to the video.
0:30The well and the sample
ELISA uses antibodies to detect an antigen. Remember that an antibody has a binding site that is complementary to one specific antigen. So an antibody will only bind to its own antigen, and that specificity is what makes the test reliable. The test is done in a well, in a plastic plate with lots of wells, so many samples can be tested at once.
0:56Antigen sticks to the well
Stage one. The patient's sample is added to the well. If the antigen is in the sample, it sticks to the inside surface of the well. The well is then washed out, to remove anything that has not stuck.
1:10Primary antibody binds
Stage two. A solution of the primary antibody is added. This antibody has a binding site complementary to the antigen we are looking for, so it binds to it, and only to it. Now wash again. Why? Any primary antibody that did not bind is rinsed away. If it stayed, it could cause a false result later.
1:33Enzyme-linked secondary antibody
Stage three. A second antibody is added. This one has an enzyme attached to it, so we call it the enzyme-linked antibody. It binds to the primary antibody. So if the antigen was there, we now have a chain: antigen, then primary antibody, then enzyme-linked antibody. And yes, wash once more, to remove any enzyme-linked antibody that has not bound.
Otherwise, loose enzyme would give a colour even when there is no antigen.
2:04Substrate and colour change
Stage four. Now the substrate for the enzyme is added. The enzyme is specific to this substrate, and it converts the colourless substrate into a coloured product. So the well changes colour.
2:17Positive versus negative result
Here is the logic examiners want. The enzyme is only held in the well if the antigen is there to start the chain. If there is no antigen, the antibodies have nothing to bind to, they all get washed away, and no enzyme is left. So a colour change means the enzyme is present, which means the antibodies bound, which means the antigen was in the sample.
No colour change means no antigen.
3:23Examiner trap and recap
The trap. Saying it changes colour, so the antigen is present, is not enough. You must explain that the enzyme turns the substrate into a coloured product, and that the enzyme only stays in the well if the antigen is there.
And never forget the washing between each stage. Recap. Antigen sticks, primary antibody binds, enzyme-linked antibody binds, substrate turns coloured.
Next time, we ask the bigger questions about vaccines and monoclonal antibodies: ethics, and how to judge the evidence.
Watch first
This lesson builds on:
- B Cells, Clonal Selection and Antibodies · AQA 7402 · 3.2.4
- Monoclonal Antibodies in Medicine · AQA 7402 · 3.2.4
Keep going
More from 3.2 Cells
- Antigens and Cell Recognition · AQA 7402 · 3.2.4
- Phagocytosis: How Phagocytes Work · AQA 7402 · 3.2.4
- T Lymphocytes: The Cellular Immune Response · AQA 7402 · 3.2.4
- B Cells, Clonal Selection and Antibodies · AQA 7402 · 3.2.4
Independent revision resource, not affiliated with or endorsed by AQA. The exam question is original and was written for this lesson; it is not an AQA question. Spotted an error? Email info@medservatory.com with the lesson code AB-049.