Titration of Various Antisera | Principle, Procedure & Interpretation

Описание: TITRATION OF VARIOUS ANTISERA
Definition
Titration of antisera is a laboratory procedure used to determine the highest dilution of an antiserum that still produces a visible antigen–antibody reaction (agglutination or precipitation).
This dilution is called the antibody titre.
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Purpose / Importance of Antisera Titration
• To determine strength (potency) of antisera
• To standardize antisera before use
• To ensure accuracy in serological tests
• To compare different batches of antisera
• To avoid false positive or false negative results
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Principle
When antiserum is serially diluted and reacted with a known antigen, the highest dilution showing a positive reaction represents the titre of that antiserum.
Antigen–antibody reaction is observed as:
• Agglutination (clumping)
• Precipitation
• Hemolysis (in some systems)
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Types of Antisera Commonly Titrated
1. Blood Group Antisera
o Anti-A
o Anti-B
o Anti-AB
o Anti-D (Rh)
2. Bacterial Antisera
o Widal antisera (O & H)
o Brucella antisera
o Weil-Felix antisera
3. Viral Antisera
o Dengue antisera
o Hepatitis antisera
4. Other Antisera
o ASO (Anti-streptolysin O)
o CRP antisera
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Materials Required
• Specific antiserum to be tested
• Known antigen (RBC suspension or bacterial antigen)
• Normal saline
• Test tubes or microtiter plate
• Micropipettes / droppers
• Water bath / incubator (if required)
• Centrifuge (for blood grouping antisera)
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General Procedure for Titration of Antisera
Step 1: Preparation of Serial Dilutions
• Label test tubes (1:2, 1:4, 1:8, 1:16, 1:32, etc.)
• Add equal volume of saline to each tube
• Add antiserum to the first tube
• Perform two-fold serial dilutions
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Step 2: Addition of Antigen
• Add equal volume of specific antigen to each tube
o RBC suspension for blood group antisera
o Bacterial antigen for Widal, Brucella, etc.
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Step 3: Incubation
• Incubate at room temperature or 37°C as required
• Time varies from 15 minutes to 2 hours
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Step 4: Observation
• Observe for agglutination or precipitation
• Record the last tube showing visible reaction
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Result Interpretation
• The highest dilution showing positive reaction is the titre of antiserum
• Example:
o Agglutination seen up to 1:64
o No reaction at 1:128
o Titre = 1:64
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Specific Examples
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1. Titration of Blood Group Antisera (Anti-A / Anti-B)
Antigen: Known A or B red cells (2–5% suspension)
Procedure Highlights
• Serial dilution of antisera
• Add RBC suspension
• Centrifuge gently
• Observe for agglutination
Result
• Highest dilution with agglutination = antibody titre
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2. Titration of Widal Antisera
Antigens Used
• Salmonella Typhi O & H antigens
Procedure
• Serial dilution of antisera
• Add bacterial antigen
• Incubate at 37°C for 16–18 hours
Significance
• Used to standardize antisera for enteric fever diagnosis
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3. Titration of Brucella Antisera
• Similar serial dilution method
• Positive reaction = granular agglutination
• Used for diagnosis of brucellosis
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4. ASO Antisera Titration
• Detects antibodies against streptolysin-O
• Helps in diagnosis of rheumatic fever
• Titre above normal indicates recent streptococcal infection
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Precautions
• Use clean and dry glassware
• Prepare accurate dilutions
• Use fresh antigen
• Avoid contamination
• Read results under proper lighting
• Do not shake tubes vigorously
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Sources of Error
• Improper dilution
• Weak or expired antiserum
• Incorrect antigen concentration
• Wrong incubation conditions
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Clinical & Laboratory Significance
• Ensures quality control of antisera
• Improves diagnostic accuracy
• Essential in blood bank and microbiology labs
• Helps in standardization of serological reagents
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Conclusion
Titration of antisera is a fundamental immunological technique used to determine the potency of antibodies. Proper titration ensures reliability, reproducibility, and accuracy of serological tests in clinical and diagnostic laboratories.