Unit 2: Serological techniques and immune response
Immunology and Mycology notes · PTU syllabus (BMLS407-18)
On this page
Unit summary
Antigen–antibody reactions are the basis of serological diagnosis. This unit covers antigen–antibody reactions, complement fixation, immunofluorescence, ELISA, CCIEP, RIA, SDS-PAGE and western blotting, the Widal, VDRL, ASO, CRP, Brucella and Rose–Waaler tests, complement, humoral and cellular immunity, hypersensitivity, vaccines and India's immunisation programme.
After this unit you can
- Explain antigen–antibody reactions and serological techniques
- Perform and interpret common serological tests
- Explain complement and humoral and cell-mediated immunity
- Classify hypersensitivity and describe vaccines and the immunisation schedule
PTU syllabus topics
- Antigen-antibody reactions and their applications
- principle
- procedure and applications of complement fixation test
- immunofluorescence
- ELISA
- CCIEP
- RIA
- SDS-PAGE and western blotting
- serological tests — Widal
- VDRL
- ASO
- CRP
- Brucella tube agglutination
- Rose-Waaler
- complement system and activation pathways
- humoral and cellular immune response
- hypersensitivity reactions
- vaccines and India's Extended Programme of Immunization (EPI)
Widal
Antibodies to Salmonella O and H antigens
Typhoid fever
VDRL
Non-treponemal antibodies
Syphilis (screening)
ASO
Anti-streptolysin O
Rheumatic fever after strep infection
CRP
C-reactive protein
Inflammation, infection
RF / Rose-Waaler
Rheumatoid factor
Rheumatoid arthritis
Topic 1
Antigen–antibody reactions
Precipitation
Soluble antigen + antibody form a visible precipitate
Ouchterlony double diffusion, VDRL (flocculation)
Agglutination
Particulate antigen clumped by antibody
Widal, blood grouping, latex tests
Complement fixation
Antigen–antibody complex consumes complement; indicator system (sheep RBC + haemolysin) does not lyse
Classic for syphilis (Wassermann)
Neutralisation
Antibody blocks toxin or virus
ASO
Labelled assays
Enzyme, fluorescent, radioactive or chemiluminescent labels
ELISA, IF, RIA, CLIA
- Prozone phenomenon: antibody excess can give false negatives — dilute the serum.
Topic 2
Serological techniques
- Immunofluorescence
- Direct (labelled antibody on tissue) and indirect (patient antibody then labelled anti-human Ig) — ANA, FTA-ABS
- ELISA
- Enzyme-labelled detection — HIV, hepatitis
- CCIEP (counter-current immunoelectrophoresis)
- Antigen and antibody driven towards each other in agarose — rapid precipitation lines
- RIA
- Radiolabelled competitive assay
- SDS-PAGE
- Proteins separated by molecular weight
- Western blot
- SDS-PAGE proteins transferred to nitrocellulose and probed with patient serum — confirming antibodies to specific antigens
Topic 3
Common serological tests
Widal (typhoid)
Agglutination with Salmonella O and H antigens
Titre of 1:160 (O) or more, or fourfold rise, suggests typhoid
VDRL (syphilis)
Flocculation with cardiolipin antigen
Reactive; titre; false positives in pregnancy, malaria, SLE — confirm with TPHA
ASO (streptococcal)
Neutralisation or latex agglutination for anti-streptolysin O
Above 200 IU/mL supports recent infection — rheumatic fever
CRP
Latex agglutination or turbidimetry
Raised in acute inflammation and bacterial infection
Brucella tube agglutination
Standard agglutination test
Titre 1:160 or more significant
Rose–Waaler (rheumatoid factor)
Sensitised sheep red cells agglutinated by IgM anti-IgG
Positive in rheumatoid arthritis (latex RF test is simpler)
Topic 4
The complement system
Classical
Antigen–antibody complexes (IgM, IgG) bind C1q
C1, C4, C2, C3
Alternative
Microbial surfaces activate C3 directly
Factors B, D, properdin
Lectin
Mannose-binding lectin on microbes
MASPs cleave C4 and C2
- All converge on C3 convertase → C3b (opsonisation), C3a and C5a (inflammation, chemotaxis) and the membrane attack complex (C5b–C9) that lyses cells.
Topic 5
Humoral and cellular immune responses
Cells
B lymphocytes → plasma cells
T lymphocytes — CD4 helper, CD8 cytotoxic
Effector
Antibodies
Cytotoxic killing, cytokines activating macrophages
Targets
Extracellular bacteria, toxins
Intracellular pathogens (viruses, TB), tumours, grafts
Antigen presentation
B cells recognise native antigen
T cells recognise peptides with MHC
- Primary response: lag of 7–10 days, mainly IgM. Secondary response: faster, larger, mainly IgG, due to memory cells.
Topic 6
Hypersensitivity reactions
Type I (immediate)
IgE on mast cells releases histamine
Anaphylaxis, asthma, hay fever
Type II (cytotoxic)
IgG or IgM against cell surfaces
Transfusion reactions, haemolytic disease of the newborn
Type III (immune complex)
Antigen–antibody complexes deposit and activate complement
Post-streptococcal glomerulonephritis, SLE, serum sickness
Type IV (delayed)
Sensitised T cells
Mantoux test, contact dermatitis, graft rejection
Topic 7
Vaccines and the immunisation programme
- Live attenuated
- BCG, oral polio, measles–rubella, rotavirus
- Killed (inactivated)
- Injectable polio (IPV), rabies
- Toxoids
- Tetanus, diphtheria
- Subunit and recombinant
- Hepatitis B, HPV
- Conjugate
- Pneumococcal (PCV), Hib
- mRNA and vector
- COVID-19 vaccines
- India's Expanded Programme on Immunization (1978) became the Universal Immunization Programme (1985); Mission Indradhanush improves coverage.
Birth
—
BCG, OPV-0, hepatitis B birth dose
6, 10 and 14 weeks
—
OPV, pentavalent (DPT-HepB-Hib), rotavirus, fractional IPV, PCV (as per schedule)
9–12 months
—
Measles–rubella 1, JE (endemic areas), vitamin A
16–24 months
—
MR 2, DPT booster, OPV booster
5–6 years and later
—
DPT booster; Td at 10 and 16 years; Td in pregnancy
Key terms
- Agglutination
- Clumping of particulate antigen by antibody
- Prozone
- False negative due to antibody excess
- Complement
- Plasma proteins aiding lysis and inflammation
- Hypersensitivity
- Exaggerated immune response causing damage
- Toxoid
- Inactivated toxin used as a vaccine
Quick revision
- Precipitation, agglutination, CFT, neutralisation, labelled assays; prozone.
- IF, ELISA, CCIEP, RIA, SDS-PAGE, western blot.
- Widal, VDRL, ASO, CRP, Brucella SAT, Rose–Waaler.
- Classical, alternative, lectin pathways; MAC.
- Humoral vs cellular; primary and secondary response; types I–IV hypersensitivity; vaccine types; UIP.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.What is the prozone phenomenon?
- Q2.What is the principle of the Widal test?
- Q3.Why is VDRL called non-specific?
- Q4.What does the membrane attack complex do?
- Q5.Give an example of type III hypersensitivity.
- Q6.Name two live attenuated vaccines.
Long-answer questions
- Q1.Describe antigen–antibody reactions and their applications.
- Q2.Describe the Widal, VDRL, ASO and CRP tests.
- Q3.Explain the complement system and immune responses.
- Q4.Classify hypersensitivity reactions and describe the immunisation schedule.
Stuck on this unit?
Message SBS on WhatsApp for help with Immunology and Mycology, or to ask about studying B.Sc MLS at Synetic.