12.1 Assays that use antibody #
12.1.1 ELISA #
An ELISA attaches an enzyme, often horseradish peroxidase or alkaline phosphatase, to a reagent, washes away unbound material, and reads a colourless substrate. The format decides what the signal means. In a direct ELISA, antigen is coated and enzyme-labelled antibody is added: simple, poorly amplified, and needing a labelled reagent for every target. In an indirect ELISA, antigen is coated, unlabelled primary antibody (often diluted serum) is added, and a labelled antibody against that primary’s Fc detects it. One labelled secondary serves every primary of that species, and the test measures antibody. A negative result in the first weeks of infection means antibody has not yet been made; it does not mean the person is uninfected. A sandwich ELISA coats a capture antibody, adds the sample, and detects antigen with a labelled antibody against a second epitope. Signal means the antigen bridged two antibodies, so the assay measures antigen in a mixture. A hapten is usually too small to bind both. In a competitive ELISA, labelled and unlabelled antigen compete for limited antibody, so the signal falls as analyte rises.
12.1.2 Western blot and immunofluorescence #
A western blot denatures polypeptides, separates them by size, transfers them, and probes, usually with indirect detection. A band at the expected mass supports specificity; bands at the wrong masses mean the reagent is seeing something else. Denaturation is the limit. Many antibodies see discontinuous epitopes that SDS-PAGE destroys, so a serum can be positive by ELISA or immunofluorescence and negative on a blot of the same antigen. Immunofluorescence asks where the antigen is: membrane, nucleus, glomerulus, or one cell in a mixture. Direct detection labels the primary; indirect detection uses a labelled secondary and is brighter.
12.1.3 Precipitation and agglutination #
Soluble antigen and antibody precipitate only when they form a lattice, which needs antibody of at least two sites, antigen of at least two epitopes, and a mixture near equivalence. In antibody excess (the prozone) or antigen excess (the postzone) the complexes stay small and soluble, so a strong serum can look negative until it is diluted. Agglutination clumps particles. IgM, with high valence, agglutinates well; IgG often coats red cells without clumping them. ABO grouping is the classical test: people have IgM against the A or B structures they lack, and clumping with known sera assigns the group. The Coombs, or antiglobulin, test adds anti-human IgG to bridge those coats. The direct test asks whether antibody or complement is already on the patient’s cells, as in autoimmune haemolysis and haemolytic disease of the newborn. The indirect test asks whether serum contains antibody against test cells, which is the cross-match. A titre is the reciprocal of the last dilution that still reacts, and a rise between paired sera reports a recent response.
12.2 Cells as the readout, and antibody as the drug #
12.2.1 Flow cytometry and CD markers #
Forward scatter, side scatter, fluorescence, and sorting are described with the instrument in the Cell Biology notes. The immunological content is the CD marker: a molecule defined by monoclonal antibodies and numbered so that laboratories share a name. A single marker seldom defines a lineage. Panels do.
| Marker | Read as | Caveat |
|---|---|---|
| CD45 | Leucocytes | Not on mature red cells |
| CD3 | T cells | Required before CD4 or CD8 is called a T cell |
| CD4 | Helper T cells, with CD3 | Also on monocytes, at lower level |
| CD8 | Cytotoxic T cells, with CD3 | Some NK cells express it |
| CD19, CD20 | B cells | Plasma cells lose CD20 |
| CD16 and CD56, CD3-negative | NK cells | Some T cells are CD56-positive |
| CD14 | Monocytes | Use with scatter |
| CD25 and Foxp3, with CD4 | Regulatory T cells | CD25 alone also marks recently activated T cells |
12.2.2 Monoclonal antibodies as drugs #
A therapeutic antibody uses a chosen site to neutralise a cytokine, block a receptor, or mark a cell for complement or ADCC. The traditional name records the source: -omab murine, -ximab chimeric, -zumab humanised, -umab human. A fully mouse antibody, given repeatedly, is itself foreign and is cleared by an anti-antibody response. Biotechnology and Genetic Engineering owns how the protein is built. Infliximab, a chimeric anti-TNF antibody, is the teaching example of neutralisation, used in rheumatoid arthritis and inflammatory bowel disease. The same TNF maintains granulomas, so blockade can let latent tuberculosis reactivate. Rituximab, anti-CD20, marks B cells for killing. Plasma cells have lost CD20 and are spared, so existing antibody persists while new B-cell responses stop. Antibodies against CTLA-4 or PD-1 are a third design: they block an inhibitor so that an existing T-cell response continues.
12.3 Key terms, summary, and self-check #
Key terms. Indirect ELISA; sandwich ELISA; competitive ELISA; western blot; equivalence; prozone; direct Coombs; indirect Coombs; CD marker; anti-TNF; anti-CD20.
Summary. Indirect ELISA detects antibody, sandwich ELISA detects antigen, and a competitive format makes the signal fall as analyte rises. A blot assigns binding to a polypeptide of known size only if the epitope survives denaturation. Precipitation and agglutination need equivalence and fail in antibody excess, which is why dilution and a Coombs reagent reveal IgG that did not clump cells. Flow cytometry counts CD phenotypes, and CD4 or CD25 means a helper or a regulatory T cell only inside the right panel. Therapeutic antibodies neutralise, deplete, or release a checkpoint.
Self-check.
1. Which ELISA measures antibody in serum, and which measures antigen in a mixture? Why does a competitive signal fall as analyte rises?
2. Why can an antibody that binds in an ELISA fail on a western blot of the same protein?
3. What is the zone of equivalence, and how can excess antibody cause a false-negative agglutination?
4. What does a direct Coombs test ask, and what does an indirect Coombs test ask?
5. Why is CD25 alone not a regulatory T cell, and why is CD4 alone not a helper T cell?
# Closing: how the chapters fit together
Innate recognition, in Chapters 3 and 4, is both a defence and the decision to start adaptive immunity. Barriers and pattern receptors buy time and mature the dendritic cells of Chapter 2. Those cells, in the paracortex, present the peptides of Chapter 7 to T cells released by the thymus in Chapter 8. B cells meet them at the edge of the follicle with the repertoire of Chapter 6 and the Fc functions of Chapter 5. Cytokines are the language in which these cells choose a programme.
The clinical chapters are that machinery mis-aimed or missing. Hypersensitivity is an effector used against the wrong antigen. Autoimmunity is clonal selection without the tolerance Chapter 1 required. A primary immunodeficiency removes one named step, which is why the infections teach the pathway. HIV removes the CD4 cell that ties the steps together. Transplantation is polymorphism seen by a repertoire selected on different MHC. Tumour escape reuses missing self, anergy, and checkpoints. Vaccines are deliberate primary responses, designed around whether antigen reaches class I, receives T-cell help, or does neither. The assays are how any of these claims is checked.
Revision checklist #
1. Contrast innate and adaptive immunity on speed, specificity, memory, and receptor origin, and state clonal selection in three sentences.
2. Place neutrophils, macrophages, dendritic cells, mast cells, NK cells, B cells, and CD4 and CD8 T cells by function, and say where in a node a naive T cell meets antigen.
3. Pair TLR4, TLR3, TLR7/8, and TLR9 with their ligands, and say what the inflammasome and cGAS–STING each add.
4. Draw three complement routes to C3, name the membrane-attack complex, and state the infection pattern if C5–C9 are missing. Name the enzyme missing in chronic granulomatous disease.
5. Give IgM, IgG, IgA, and IgE one job and one site each, and separate affinity from avidity.
6. State the order of heavy- and light-chain rearrangement, the 12/23 rule, and the two reactions AID starts.
7. Contrast MHC class I and II in structure, cells, peptide source, and T-cell partner, and explain cross-presentation in one sentence.
8. Separate positive from negative selection, list the three activation signals, and give one signature cytokine for Th1, Th2, Th17, Tfh, and Treg.
9. Match hypersensitivity types I–IV to a mechanism and an example, and match SCID, X-linked agammaglobulinaemia, chronic granulomatous disease, terminal complement deficiency, and HIV to the missing function.
10. Separate direct from indirect allorecognition, say where PD-1 and CTLA-4 act, and explain why a conjugate succeeds with a polysaccharide that the pure capsule does not.
Selected sources #
These notes are an original teaching summary, not a transcript of any textbook.
- Murphy K, Weaver C, Berg LJ. Janeway’s Immunobiology. Current editions. W. W. Norton.
- Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology. Current editions. Elsevier.
- Parham P. The Immune System. Current editions. W. W. Norton.
- Burnet FM. The Clonal Selection Theory of Acquired Immunity. Cambridge University Press, 1959.
- Zinkernagel RM, Doherty PC. Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 248:701–702 (1974).
- Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497 (1975).
# Part C. Visual Figures & Scientific Diagrams Gallery
Comprehensive high-resolution visual reference repository for Immunology from the B10logy Visual Atlas.
Antigen Presentation
# Part C. Visual Figures & Scientific Diagrams Gallery
Essential high-resolution reference diagrams for Immunology from the B10logy Visual Atlas.
