Immunoassay
⚠️ A third exam format — read this first
Slide 3 of this deck — from Yu Chunyan, who also gives the Type II and Immunotherapy lectures — sets out the following exam structure:
I. 6 Definitions (3 points each) = 18
II. 56 Blanks (0.5 points each) = 28
III. 20 Single choices (1 point each) = 20
IV. 10 MULTIPLE choices (1 point each) = 10
V. 4 Questions to be answered briefly (6 points each) = 24
Compare that with the 2019 and 2020 papers on the Past Papers page: those have 20 blanks, no multiple-choice section, a 12-mark true/false section, and FIVE brief answers. Three differences matter: the blanks are worth more and are half-mark each; there is a multi-answer MCQ section that does not exist in either past paper; and true/false may be gone.
Do not assume the paper you sit will match the two we hold. Prepare the content, not the format — and expect that a multiple-answer section may appear, which is scored quite differently from single-best-answer.
Slide 2 is blunt and worth obeying: “Please make sure every word is written with clear, neat and standard handwriting when you answer the questions during the exam. Any careless and illegible answers will get deduction of the points. If your handwriting is too difficult to make out, the answer will be directly regarded as ZERO.”
In a paper that is 18 marks of definitions and 24–30 marks of written answers, that is not a throwaway remark.
One further piece of course arithmetic, from Unit 1: the written examination is 70% of your mark and the experiments are 30%. The three practicals covered in this unit — ELISA, neutralization, and observation of immune organs — are therefore worth nearly a third of the subject on their own.
- How many past-paper formats are now known? → Three — 2019, 2020, and the structure on this lecturer's slide
- What section appears here but in neither past paper? → A 10-mark MULTIPLE-choice (multi-answer) section
- What is the experiments component worth? → 30% of the course mark
What immunoassay is ★★
Every technique in this unit rests on one fact you learnt in Unit 2: antigen and antibody bind specifically. That is all. The entire discipline of immunoassay is the business of making that invisible binding event visible, and then countable.
| Dimension | Options |
|---|---|
| Purpose | Qualitative — detect the existence of antigen or antibody Quantitative — detect the concentration |
| Visibility | Visible methods and invisible methods (needing a label) |
| Target | Detection of antigen or antibody Detection of lymphocytes |
When you meet an unfamiliar immunological test, ask which of two things it is doing.
If you have known antibody, you are looking for antigen. Blood typing uses known anti-A and anti-B serum to find out which antigen is on the patient's red cells.
If you have known antigen, you are looking for antibody. A rheumatoid factor test uses known IgG to find out whether the patient has antibody against it.
That is why the same technique appears in a 'direct' and an 'indirect' form: the chemistry is identical, and only the reagent you supply and the thing you are hunting are swapped. The final goal is always the same — clinical diagnosis, therapy assessment, prognosis and research.
- What principle underlies all immunoassay? → The specific binding of antigen and antibody
- What is the difference between qualitative and quantitative methods? → Qualitative detects existence; quantitative detects concentration
- What are the four goals of immunoassay? → Clinical diagnosis, therapy assessment, prognosis, scientific research
Agglutination tests ★★
The interaction of insoluble particulate antigens — intact bacteria or cells — with specific antibodies, producing visible agglutinates after a certain time and at a certain electrolyte concentration.
| Type | Detects | Examples |
|---|---|---|
| Direct agglutination | Antigen | ABO blood typing — anti-A, anti-B and anti-A,B sera identify the antigen on the patient's red cells |
| Indirect agglutination | Antibody | Rheumatoid factor test; pregnancy test (indirect agglutination inhibition, detecting hCG, with a colloidal-gold readout) |
The distinction is worth fixing because it is examinable and easily inverted. In direct agglutination the antigen is already on a particle — a red cell, a bacterium — so adding antibody clumps it directly. In indirect agglutination the antigen is soluble, so it must first be coated onto an artificial particle such as latex; antibody in the patient's serum then clumps those coated particles.
- Define agglutination. → Interaction of insoluble particulate antigen with specific antibody producing visible agglutinates
- Which type detects antigen? → Direct agglutination — e.g. ABO blood typing
- Which detects antibody? → Indirect agglutination — e.g. rheumatoid factor
- How does a pregnancy test work? → Indirect agglutination inhibition, detecting hCG
Precipitation tests ★★
Precipitation is the same reaction with a soluble antigen instead of a particulate one. Because soluble antigen and antibody form a lattice only near equivalence, precipitation tests are naturally quantitative — the position of the precipitin line reports concentration.
| Method | Type |
|---|---|
| Single immunodiffusion | Quantitative |
| Rocket electrophoresis | Quantitative |
| Double immunodiffusion | Qualitative — identity of antigens |
- How does precipitation differ from agglutination? → The antigen is soluble rather than particulate
- Name two quantitative precipitation methods. → Single immunodiffusion and rocket electrophoresis
Labelled immunoassays — and the practicals ★★★
Agglutination and precipitation are visible methods — you can see the clump or the line. But most antigen–antibody binding is invisible, and detecting tiny amounts requires attaching a label that generates a signal: an enzyme, a fluorochrome, a radioisotope or colloidal gold.
ELISA — practical 1
The enzyme-linked immunosorbent assay is the workhorse and one of your three assessed practicals. An enzyme is conjugated to an antibody; when its substrate is added, a colour change reports the amount of antigen or antibody present. It is quantitative, sensitive, and can be run on many samples at once — which is why it is used for everything from hormone assays to infectious serology.
It also connects directly to Unit 13: the lecture's own question — a patient is allergic to pollen, dust mites, seafood and peanuts; which antibody and which cytokine are involved, and how would you detect them? — is answered by IgE and IL-4, measured by ELISA.
Neutralization — practical 2
The neutralization practical uses tetanus exotoxin and tetanus antitoxin: the antitoxin blocks the toxin's activity, and the readout is whether the toxic effect occurs. This is Unit 3's neutralisation — the one antibody function that needs neither complement nor cells — demonstrated directly.
Observation of immune organs — practical 3
The third practical is anatomical and histological: the thymus behind the manubrium sterni, and the nude mouse, which is congenitally athymic. That animal is the living proof of Unit 1's claim about what the thymus does — no thymus, no mature T cells, no graft rejection.
- What does ELISA use to generate its signal? → An enzyme conjugated to antibody, producing a colour change with substrate
- Which antibody and cytokine underlie allergy, and how are they measured? → IgE and IL-4, by ELISA
- What does the neutralization practical demonstrate? → Tetanus antitoxin blocking tetanus exotoxin — antibody neutralisation
- What is a nude mouse, and what does it show? → A congenitally athymic mouse — no thymus means no mature T cells
Detecting and evaluating lymphocytes ★★
The second branch of immunodetection assesses the cells rather than the molecules, and it does so on two axes: number (normal range, higher, lower) and function (normal, stronger, deficient). A patient can have a normal lymphocyte count and profoundly defective function, which is why both must be measured.
| Test | What it measures |
|---|---|
| Lymphocyte transformation test | T-cell function — PHA stimulation converts small lymphocytes into lymphoblasts over 48–72 hours (Unit 2's mitogens) |
| E-rosette test | T cells — CD2 is the sheep-erythrocyte receptor (Unit 7) |
| Phagocytosis assay | Phagocyte function |
Notice that both cell tests are things you already know from other units. The E-rosette test works because CD2 (LFA-2) is the receptor for sheep erythrocytes — stated in the T-cell lecture. The transformation test works because PHA, Con A and PWM are polyclonal mitogens — from the antigen lecture, and the subject of True/False Q1. The assays are applications of facts you have already learnt.
- On what two axes are immune cells evaluated? → Number and function
- What does the lymphocyte transformation test use, and over what period? → PHA stimulation, over 48–72 hours
- Why does the E-rosette test work? → CD2 on T cells is the sheep-erythrocyte receptor
Revision layer
Why this unit matters even though no past-paper item comes from it
Two reasons. First, the experiments are 30% of the course mark, and all three practicals live here. Second, this deck contains the exam-format warning in §1, which affects how you prepare for everything else.
| Question | Answer |
|---|---|
| Principle of all immunoassay? | Specific antigen–antibody binding |
| Qualitative vs quantitative? | Existence vs concentration |
| Direct agglutination detects? | Antigen — e.g. ABO blood typing |
| Indirect agglutination detects? | Antibody — e.g. rheumatoid factor |
| Pregnancy test method? | Indirect agglutination inhibition, detecting hCG |
| Quantitative precipitation methods? | Single immunodiffusion, rocket electrophoresis |
| ELISA signal? | Enzyme + substrate → colour change |
| Allergy — antibody and cytokine? | IgE and IL-4 |
| Neutralization practical? | Tetanus antitoxin blocking tetanus exotoxin |
| Lymphocyte transformation test? | PHA stimulation, 48–72 h |
| E-rosette test works because? | CD2 is the sheep-erythrocyte receptor |
| Nude mouse? | Congenitally athymic — no mature T cells |
- What single principle underlies every immunoassay? → Specific antigen–antibody binding
- Which agglutination type detects antigen, and which antibody? → Direct detects antigen; indirect detects antibody
- How would you detect a patient's allergen-specific IgE? → By ELISA
- What are the three practicals? → ELISA, neutralization, observation of immune organs
- What is the experiments component worth? → 30% of the course mark
- What exam-format difference does this deck reveal? → A 10-mark multiple-ANSWER section, 56 half-mark blanks, and possibly no true/false