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🧬 IMMUNOGLOBULINS & ANTIGEN-ANTIBODY REACTIONS

2nd MBBS Microbiology | MUHS Exam Notes

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PART 1 - ANTIBODIES / IMMUNOGLOBULINS

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📌 DEFINITION OF ANTIBODY

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📌 CLASSIFICATION OF IMMUNOGLOBULINS

Subclasses:

• IgG: IgG1, IgG2, IgG3, IgG4
• IgA: IgA1, IgA2
• IgM, IgD, IgE: No subclasses

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📌 BASIC STRUCTURE OF IMMUNOGLOBULIN (LABELLED DIAGRAM)

Basic Four-Chain Unit:

        Antigen-binding sites
           ↓           ↓
    [VL][VH]        [VH][VL]
    [CL][CH1]  ←Fab→[CH1][CL]
          \   Hinge  /
           \  region/
           [CH2][CH2]  ← Fc region
           [CH3][CH3]

• 2 Heavy chains (H) - ~55,000 Da each (IgG/IgA/IgD) or ~70,000 Da (IgM/IgE)
• 2 Light chains (L) - ~24,000 Da each
• Held together by interchain disulfide bonds
• Light chains: Kappa (κ) or Lambda (λ)
• Variable regions (V): N-terminal, antigen binding - contains 3 CDRs (Complementarity Determining Regions / hypervariable regions)
• Constant regions (C): C-terminal, effector functions

Enzymatic Fragments:

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📌 IgG - STRUCTURE & FUNCTION ⭐(MUHS Frequent)

Structure:

• Monomer - basic 4-chain unit (2H + 2L)
• Heavy chain: γ (gamma), MW 55,000 Da
• Domains: VH - CH1 - Hinge - CH2 - CH3
• Hinge region present between CH1 and CH2 (flexible, protease-sensitive)
• 4 subclasses: IgG1 > IgG2 > IgG3 > IgG4 (differ mainly in hinge region)
• Contains N-linked carbohydrate on CH2 domain
• MW: ~150,000 Da
• 2 antigen-binding sites (divalent)

Functions of IgG:

1. Major serum antibody - 75-80% of total serum immunoglobulins
2. Only Ig that crosses the placenta - provides passive immunity to newborn (via FcRn receptor)
3. Opsonization - Fc region binds Fc receptors (FcγR) on phagocytes, enhancing phagocytosis
4. Complement activation - IgG1, IgG2, IgG3 activate classical pathway (IgG4 cannot)
5. Neutralization of toxins and viruses
6. ADCC (Antibody-Dependent Cell-mediated Cytotoxicity)
7. Secondary immune response - main antibody produced
8. Half-life: ~23 days (longest of all Ig classes)
9. Involved in Type II and Type III hypersensitivity

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📌 IgM - STRUCTURE, PROPERTIES & FUNCTIONS ⭐⭐⭐ (MUHS Very Frequent)

Structure:

• Pentamer - 5 monomeric units joined by J chain and disulfide bonds
• Heavy chain: μ (mu), MW 70,000 Da
• No hinge region - has extra domain CH2 instead (domains: VH-CH1-CH2-CH3-CH4)
• Has tailpieces at C-terminus of heavy chains (help polymerization)
• MW: ~900,000 Da
• 10 antigen-binding sites (decavalent) - but functionally 5 are usually accessible
• J chain (joining chain, MW ~15,000 Da) links the pentamer

Properties of IgM (Enumerate): ⭐⭐

1. Pentameric structure - largest immunoglobulin
2. Confined mainly to intravascular compartment (large size prevents tissue entry)
3. First antibody produced in primary immune response
4. Indicator of acute/recent infection
5. Most efficient complement activator (classical pathway) - one IgM molecule sufficient
6. Best agglutinating antibody - 10 combining sites
7. Does NOT cross placenta
8. IgM in cord blood = intrauterine infection (congenital infection marker)
9. Found on surface of B lymphocytes as BCR (monomeric form)
10. Half-life: ~10 days
11. Isohemagglutinins (ABO blood group antibodies) are IgM
12. Cold agglutinins are IgM
13. Rheumatoid factor (RF) is IgM anti-IgG

Functions of IgM:

1. Primary humoral immune response
2. Agglutination of particulate antigens
3. Complement activation (classical pathway)
4. Bactericidal/bacteriolytic reactions
5. Acts as BCR on naive B cells

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📌 IgA - STRUCTURE & DIAGRAM ⭐

Forms:

Secretory IgA Diagram:

    [Fab][Fab]       [Fab][Fab]
        \               /
    [Fc]—J chain—[Fc]
         |       |
    [Secretory Component (SC)]
    (wraps around the dimer - protects from proteases)

Functions of IgA:

1. Mucosal immunity - first line of defense at mucosal surfaces
2. Prevents microbial attachment to mucosa ("immune exclusion")
3. Present in colostrum - passive immunity to newborn via gut
4. Secretory component protects sIgA from proteolytic digestion
5. IgA1 - more in serum; IgA2 - shorter hinge, more resistant to bacterial proteases

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📌 TABLE: PROPERTIES OF ALL IMMUNOGLOBULINS

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PART 2 - ANTIGEN-ANTIBODY (Ag-Ab) REACTIONS

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📌 ANTIGEN-ANTIBODY REACTIONS - DEFINITION & GENERAL FEATURES ⭐

General Features (Enumerate): ⭐

1. Specificity - Ag reacts only with its specific antibody (lock and key)
2. Non-covalent bonds - reversible
3. Both Ag and Ab must be multivalent for visible reactions (precipitation/agglutination)
4. Equivalence zone - optimal ratio of Ag:Ab for maximum precipitation/agglutination
5. Temperature dependent - most reactions optimal at 37°C (some at 4°C, e.g. cold agglutinins)
6. Electrolyte (NaCl) required for lattice formation in precipitation
7. pH - optimal 6.5-8.0
8. Prozone and postzone phenomena occur at extremes of ratio

TYPES of Ag-Ab Reactions:

1. Precipitation - soluble Ag + Ab → precipitate
2. Agglutination - particulate Ag + Ab → clumping
3. Complement fixation
4. Neutralization (toxin/virus)
5. Opsonization
6. ADCC
7. Immunofluorescence
8. RIA (Radioimmunoassay)
9. ELISA
10. Western Blot

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📌 PROZONE PHENOMENON ⭐

Explanation:

• In prozone: Antibody excess → each antigen molecule is saturated by antibodies → no lattice formation → no visible clumping or precipitation
• When dilutions are made, optimal ratio is reached → reaction becomes visible
• Also called "hook effect" in immunoassays

Postzone:

• Antigen excess → no lattice formation

Equivalence Zone:

• Optimal Ag:Ab ratio → maximum lattice → visible precipitate/agglutinate

        PROZONE      EQUIVALENCE    POSTZONE
        (Ab excess)     ZONE        (Ag excess)
   Rxn:   ❌ ❌ ❌       ✅✅✅         ❌ ❌

Clinical Significance:

• Widal test - if undiluted serum shows no agglutination but diluted serum does → prozone
• RPR/VDRL for syphilis - prozone common in secondary syphilis
• Always test serial dilutions to avoid missing the reaction

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📌 PRECIPITATION REACTIONS ⭐⭐

• Requires multivalent antigen and antibody
• Precipitate = lattice of cross-linked Ag-Ab complexes
• Visible only at equivalence zone (not in prozone or postzone)

Types of Precipitation Reactions:

A. In Liquid Medium:

B. In Gel (Immunodiffusion):

Agglutination vs Precipitation - Difference: ⭐

Two examples of Precipitation tests:

1. VDRL / RPR - for syphilis (flocculation/precipitation)
2. Ouchterlony double diffusion - for fungal antigens (Histoplasma, Aspergillus)

Two examples of Agglutination tests:

1. Widal test - for typhoid fever (tube agglutination)
2. ABO blood grouping - slide agglutination

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📌 AGGLUTINATION REACTIONS ⭐⭐ (LAQ - MUHS Very Frequent)

• Antibody acts as a bridge between particulate antigens
• IgM is the best agglutinating antibody (10 binding sites)
• Requires electrolytes and optimal pH/temperature
• Lattice formation → visible clump

Types / Enumerate:

1. Direct (Active) Agglutination:

2. Passive (Indirect) Agglutination:

3. Reverse Passive Agglutination:

• Example: Latex coated with anti-HBsAg antibody → detects HBsAg

4. Agglutination Inhibition:

• Example: Latex pregnancy test - detects hCG
• HI test (Hemagglutination Inhibition) - detects antibodies to viruses

5. Coagglutination:

• Used for: Rapid identification of bacterial antigens (Streptococcus, N. meningitidis)

6. Antiglobulin / Coombs Test:

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📌 WIDAL TEST ⭐ - DESCRIBE IN DETAIL

Principle:

• Patient serum (containing antibodies) + killed Salmonella suspensions (O and H antigens) → agglutination
• The titer (highest dilution showing agglutination) is determined
• A rising titer in paired sera (≥4-fold rise) is diagnostic

Antigens Used:

• O antigen (somatic, lipopolysaccharide) - appears in 2nd week
• H antigen (flagellar) - rises later, persists longer
• Vi antigen (capsular) - detects carriers

Procedure (Tube Agglutination):

1. Take a row of 8 tubes
2. Add 0.9 mL normal saline to tube 1; 0.5 mL to tubes 2-8
3. Add 0.1 mL patient serum to tube 1 → mix → transfer 0.5 mL to tube 2 → serial dilution up to tube 7; tube 8 = antigen control (no serum)
4. Dilutions: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640
5. Add 0.5 mL of specific antigen suspension to all tubes
6. Incubate at 37°C for O antigen and 50°C for H antigen for 4-16 hours
7. Read results: H agglutination - large fluffy clumps; O agglutination - compact granular deposit

Interpretation:

Significance / Applications:

1. Diagnosis of enteric fever (typhoid) - especially where blood culture is unavailable
2. Retrospective diagnosis
3. Useful in resource-limited settings

Limitations:

1. False positives: other Salmonella infections, malaria, liver disease, immunization
2. False negatives: early disease, antibiotic treatment, immunocompromised
3. Single serum specimen - unreliable; need paired sera
4. Cannot differentiate typhoid from paratyphoid with anti-O only
5. Prozone phenomenon possible

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📌 PASSIVE AGGLUTINATION TESTS ⭐

Why "Passive"?

Types:

1. Passive Hemagglutination (PHA):

• Soluble Ag adsorbed to tanned (tannic acid-treated) sheep RBCs
• Patient serum added → agglutination = Ag-specific Ab present
• Examples:
  • HA test for ABO typing
  • Detection of antibodies to soluble polysaccharide or protein antigens

2. TPHA (Treponema pallidum Hemagglutination Assay):

• T. pallidum antigens coated on sheep RBCs
• Detects anti-treponemal antibodies (syphilis)
• Confirmatory test for syphilis

3. Latex Agglutination:

• Latex beads coated with antigen
• Rapid, simple, no need for blood cells
• Examples:
  • Rheumatoid factor detection (latex coated with IgG)
  • CRP test
  • Cryptococcal antigen test (meningitis)
  • Bacterial antigen tests in CSF (meningococcal, pneumococcal)
  • Pregnancy test (hCG detection - agglutination inhibition)

4. Passive Agglutination Inhibition:

• Soluble antigen in patient specimen competes with latex/RBC-bound antigen for limited specific antibody
• If patient has antigen → no agglutination (positive test)
• Example: Latex pregnancy test, HI (Hemagglutination inhibition) test

5. Reversed Passive Agglutination:

• Antibody coated on particles
• Detects antigen in patient specimen
• Example: Latex coated with anti-HBsAg → detects HBsAg in blood

Advantages of Passive Agglutination:

• More sensitive than direct agglutination
• Can detect soluble antigens/antibodies
• Rapid (minutes to hours)
• No need for intact organisms

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📌 ELISA - ENZYME-LINKED IMMUNOSORBENT ASSAY ⭐⭐

Principle:

• Antigen or antibody is immobilized on a solid surface (polystyrene microtitre plate)
• Enzyme-labeled antibody or antigen binds to the analyte
• On addition of substrate, the enzyme converts it to a colored product
• Color intensity is proportional to the amount of antigen/antibody
• Read by spectrophotometer (ELISA reader)

Enzymes Used:

• Horseradish peroxidase (HRP) - substrate: TMB (turns blue/yellow)
• Alkaline phosphatase - substrate: p-nitrophenyl phosphate (turns yellow)

Types of ELISA:

1. Indirect ELISA (Most Common for Ab detection):

Solid phase (Ag) → Patient Ab → Enzyme-labeled anti-human Ig (secondary Ab) → Substrate → Color

• Detects: Antibody in patient serum
• Used for: HIV antibody detection, ANA, anti-HCV

2. Sandwich / Double Antibody Sandwich ELISA (Best for Ag detection):

Solid phase (Capture Ab) → Antigen → Enzyme-labeled Detection Ab → Substrate → Color

• Detects: Antigen
• Two antibodies bind different epitopes of antigen
• Used for: HBsAg, HCV antigen, hormone quantitation (TSH, hCG, LH)
• High sensitivity and specificity

3. Competitive ELISA:

• Patient antigen competes with enzyme-labeled antigen for limited antibody
• High antigen = less color (inverse relationship)

4. Direct ELISA:

• Antigen immobilized → directly add enzyme-labeled antibody
• Simple but less sensitive

Applications of ELISA: ⭐

Advantages:

1. Very high sensitivity and specificity
2. Can be automated
3. Suitable for large-scale screening
4. No radioactive material (unlike RIA)
5. Long shelf life of enzyme-conjugated reagents
6. Quantitative

Limitations:

1. False positives (rheumatoid factor interference)
2. Prozone/hook effect at very high antibody concentration
3. Technical expertise needed
4. Cannot distinguish active from past infection (IgG)

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📌 COMPARISON: AGGLUTINATION vs PRECIPITATION

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📌 QUICK REVISION TABLE - MUHS EXAM POINTS

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📌 EXAM TIP - MUHS PATTERN

• SAQ (5 marks): Define + enumerate/list + brief description + diagram if applicable
• LAQ (10 marks): Define + classify + elaborate each type with principle + examples + table/diagram
• Most repeated: IgG structure+function, IgM properties, Widal test, ELISA, Ag-Ab reactions, Prozone
• Always draw the Y-shaped Ig diagram with labels (VH, VL, CH1-3, hinge, Fab, Fc, disulfide bonds, CDRs)

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