Agglutination test in micro
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Agglutination Tests in Microbiology
Basic Principle
Agglutination is the visible clumping of particulate antigens (bacteria, RBCs, latex beads) when cross-linked by specific antibodies. Antibody molecules act as bridges between antigen-carrying particles, forming a lattice that precipitates as macroscopically visible aggregates.
- IgM antibodies are far more efficient at producing complete agglutination than IgG due to their larger size and higher valence (10 binding sites).
- When only IgG is involved, enhancement is needed (e.g., lowering ionic strength, adding polymerized albumin, dextran, or PEG).
- The zeta potential (net negative charge on RBCs/bacteria) creates mutual repulsion that must be overcome for agglutination to occur.
- Antigen-to-antibody ratio is critical: extreme excess of either inhibits aggregation (the prozone/hook effect).
Principles of passive particle agglutination: (A) reverse agglutination - antibody-coated particle detects antigen; (B) passive agglutination - antigen-coated particle detects antibody - Henry's Clinical Diagnosis and Management by Laboratory Methods
Types of Agglutination Tests
1. Direct (Active) Agglutination
The natural surface antigens of a particulate agent (whole bacteria or RBCs) react directly with antibodies in the patient's serum. No prior sensitization of the particle is needed.
Examples:
- Widal test - patient serum is serially diluted and tested against Salmonella O (somatic) and H (flagellar) antigens. A single O antigen titer >1:320 and H antigen titer >1:640 is considered positive. Requires two specimens 7-10 days apart to show a rising titer. - Jawetz Medical Microbiology
- Weil-Felix test - cross-reacting antibodies from Rickettsia infections agglutinate Proteus OX-19 strains.
- Blood grouping (ABO/Rh typing) - direct testing of erythrocytes with known antisera (anti-A, anti-B, anti-D).
2. Indirect (Passive) Agglutination
Soluble antigens are artificially adsorbed or covalently coupled onto a particle carrier (RBCs, latex, gelatin, bentonite). Patient antibody then cross-links these sensitized particles.
Examples:
- TPHA (Treponema pallidum hemagglutination assay) - T. pallidum antigens coated onto fixed sheep RBCs; detects syphilis antibodies.
- HIV antibody detection - sensitized human or avian erythrocytes.
- Passive latex agglutination - soluble antigens coated on latex beads; antibody in specimen agglutinates them.
3. Reverse Passive Agglutination
Here the antibody is fixed to the particle, and the test detects soluble antigen in the specimen.
Examples:
- Latex agglutination for cryptococcal antigen - antibody-coated latex beads agglutinate in the presence of Cryptococcus neoformans polysaccharide antigen in CSF or serum (sensitivity 60-95%).
- Detection of HBsAg - reverse passive hemagglutination.
- Lancefield typing of beta-hemolytic streptococci - latex beads coated with type-specific antibodies agglutinate when exposed to streptococcal group antigens.
- Group A Strep antigen detection (pharyngitis rapid testing).
4. Agglutination Inhibition
A competitive technique for detecting small antigens (haptens) that cannot cross-link particles themselves.
- Principle: A fixed amount of antibody is mixed with the test specimen. If antigen (hapten) is present in the specimen, it occupies the antibody. When the antibody-laden specimen is then mixed with hapten-coated particles, no agglutination occurs - inhibition = positive test.
- Classic example: Pregnancy test (latex agglutination inhibition for hCG) - if hCG is present in urine, it blocks anti-hCG antibody from agglutinating hCG-coated latex beads.
- Also used to identify specific viral hemagglutinins (hemagglutination inhibition test for influenza).
5. Coagglutination
Similar to latex agglutination, but uses Staphylococcus aureus (Cowan I strain) - rich in Protein A - as the carrier particle instead of latex. Protein A binds the Fc region of IgG, leaving the Fab arms free to react with antigen.
Uses:
- Identification of bacterial cultures: Streptococcus pneumoniae, Neisseria meningitidis, N. gonorrhoeae, beta-hemolytic streptococci.
- Less useful for direct antigen detection in specimens compared to latex agglutination.
- Jawetz Medical Microbiology, 28E
6. Hemagglutination and Hemagglutination Inhibition (HI Test)
- Certain viruses (influenza, measles, mumps, rubella, adenoviruses) directly agglutinate RBCs via viral surface proteins (hemagglutinins).
- Hemagglutination Inhibition (HI): Patient antibodies block viral hemagglutinin, preventing RBC agglutination. Inhibition = positive (antibody present). Widely used for serological diagnosis of viral infections and antibody titering.
7. Heterophile Agglutination
Antibodies from one species react with antigens of an unrelated species.
- Paul-Bunnell test / Monospot test: EBV (infectious mononucleosis) induces heterophile antibodies that agglutinate sheep RBCs (Paul-Bunnell) or horse RBCs (Monospot). The monospot test is ~75% sensitive, ~90% specific.
- Scott-Brown's Otorhinolaryngology; Henry's Clinical Diagnosis and Management
Particles Used as Carriers (Summary Table)
| Particle | Method | Key Applications |
|---|---|---|
| Human erythrocytes | Direct hemagglutination | ABO blood typing, HIV antibody |
| Fixed animal erythrocytes | Passive hemagglutination | T. pallidum antibody (TPHA), HBsAg |
| Latex | Reverse passive agglutination | CRP, cryptococcal Ag, Strep Ag, RF |
| Latex (colored) | Immunochromatography | hCG (pregnancy test) |
| Gelatin particles | Passive agglutination | HIV antibody, T. pallidum |
| Staphylococci (Protein A) | Coagglutination | Strep, Neisseria typing |
| Bentonite | Passive agglutination | Trichinella antibody |
Henry's Clinical Diagnosis, Table 45.2
Important Concepts
Prozone Phenomenon (Hook Effect)
When antibody concentration is very high relative to antigen, each antibody molecule binds antigen but cannot form cross-linking lattices, resulting in a falsely negative agglutination test. The solution is to dilute the serum further. This is classically seen with cryptococcal antigen testing and in syphilis serology.
Incomplete Agglutination
Primary antigen-antibody reaction occurs but no visible aggregation results - commonly with IgG antibodies due to insufficient bridging distance to overcome the zeta potential of RBCs. Detected by the Coombs/Antiglobulin test (add anti-human IgG which bridges the IgG-coated cells).
Grading of Agglutination
- 4+: One large clump, clear background
- 3+: Several large clumps, clear background
- 2+: Many medium clumps
- 1+: Small clumps, turbid background
- m+ (microscopic): Visible only under microscope
- Negative: No clumping, smooth suspension
Key Clinical Applications in Microbiology
| Test | Organism/Disease | Type |
|---|---|---|
| Widal test | Enteric fever (Salmonella Typhi) | Direct tube agglutination |
| Weil-Felix test | Rickettsia spp. | Direct (cross-reacting) |
| TPHA | Syphilis (T. pallidum) | Passive hemagglutination |
| Latex agglutination | Cryptococcal meningitis, Strep, meningitis | Reverse passive |
| Monospot/Paul-Bunnell | Infectious mononucleosis (EBV) | Heterophile |
| Blood grouping | ABO/Rh compatibility | Direct |
| HI test | Influenza, rubella, measles | Hemagglutination inhibition |
| Coagglutination | Strep, Neisseria identification | Coagglutination |
| Pregnancy test (latex) | hCG detection | Agglutination inhibition |
Sources: Jawetz, Melnick & Adelberg's Medical Microbiology 28E | Henry's Clinical Diagnosis and Management by Laboratory Methods | Tietz Textbook of Laboratory Medicine 7E | Sherris & Ryan's Medical Microbiology 8E
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