2. CONTENTS
• ANTIGEN
• ANTIBODY
• Ag – Ab REACTION
• PROPERTIES OF Ag-Ab reaction
• STAGES
• FACTORS INFLUENCING Ag-Ab REACTION
• References.
3. • Any molecule which binds to an antibody and
generates an immune response.
• Proteins / Polysachharides.
• TYPES – Complete ( MW>10000D) – Both antigenic
and immunogenic.
– Hapten (MW<10000D) – Only antigenic.
ANTIGEN
5. FACTORS INFLUENCING IMMUNOGENICITY
• ANTIGEN SIZE
• CHEMICAL NATURE
• STUCTURAL COMPLEXITY
• DOSAGE
• ZYGOSITY
• DEGREE OF FOREIGNESS
• ROUTE OF ANTIGEN ADMINISTRATION
6. ANTIBODY
• Specialized glycoprotein known as IMMUNOGLOBULINS
produced from Plasma cells in response to an Antigen.
• FIVE CLASSES – IgG, IgM, IgA, IgD, IgE
7. IgG
• GAMMA heavy chain
• Monomeric
• Bivalent
• Serum half life – 23 days (IgG3 – 8
days)
• Warm antibody
• 70 – 75% of serum concentration
• Produces Extravascular Hemolysis
IgM
• Mu Heavy chain
• Pentameric
• Valency is 10
• Serum half life – 5 days
• 10% of serum concentration
• Cold antibody
• Intravascular hemolysis
8. Immune antibodies
• Produced on exposure to foreign
red cells
• IgG
• React best at 370C
• Antibodies formed for Rh, Kell,
Kidd, Duffy, Ss etc.
Naturally occurring
antibodies
• Produced in response to
substances in environment that
resemble RBC antigens.
• IgM
• React best at room temperature
or lower
• Antibodies formed for ABH, Hh,
Ii, Lewis, MN, P group systems.
9. ANTIGEN-ANTIBODY REACTIONS
• A biomolecular association where antigen and antibody combine
specifically, in a observable manner in varying proportions.
• TYPES:
• AGGLUTINATION
• PRECIPITATION
• AGGLUTINATION INHIBITION
• HAEMOLYSIS
10. PROPERTIES OF Ag-Ab REACTIONS
• SPECIFIC
• NON COVALENT INTERACTIONS
• HYDROGEN BONDS
• ELECTROSTATIC INTERACTIONS
• HYDROPHOBIC INTERACTIONS
• VAN DER WAALS FORCES
• STRENGTH
• AFFINITY
• AVIDITY
11. ANTIGEN AND ANTIBODY REACTION
• The Ag-Ab reaction is Reversible.
• in accordance with law of mass action, it can be represented as
Ab + Ag AbAg
• At Equilibrium,
[AbAg]
[Ab] * [Ag]
k1
k2
=
K[Ag]
k1
k2
= K
[AbAg]
[Ab]
=
Higher the Equilibrium constant, the greater the antibody combining with antigen.
12. AFFINITY
• Strength of a single Ag-Ab bond
• Produced by summation of
attractive and repulsive forces.
• It is the sum total of non
covalent interactions between
single epitope with its
corresponding paratope.
• IgG has higher affinity than IgM
AVIDITY
• Binding strength of a multivalent
antigen with antisera in an
immunized individual.
• FUNCTIONAL AFFINITY
• Better indicator of strength of
Ag-Ab reaction.
• IgM has higher Avidity than IgG
(IgM has 10 valencies)
• Avidity increases with Time
13. STAGES OF Ag-Ab REACTION
• Primary / Stage of Sensitization
• Secondary / Stage of Lattice formation
14. FACTORS AFFECTING Ag-Ab REACTIONS
STAGE OF SENSITIZATION
• TEMPERATURE
• INCUBATION TIME
• pH
• IONIC STRENGTH
STAGE OF LATTICE FORMATION
• Ag-Ab CONCENTRATION
• CENTRIFUGATION
• DISTANCE BETWEEN RED CELLS
• POTENTIATORS
• ENHANCEMENT MEDIA
15. STAGE OF SENSITIZATION
1. TEMPERATURE
• IgM antibodies react at room temperatures or below 22oC at
Immediate spin phase.
• IgG antibodies react at 37oC and react optimally at AHG phase
16. STAGE OF SENSITIZATION
2. INCUBATION TIME
• For optimal Ag-Ab reaction, a suitable time is required to reach
equilibrium
• Depends on the antibody being detected and the reaction
medium.
• IgG antibodies can be detected after 30-45 min incubation.
• IgM antibodies can be detected with an Immediate spin
without any incubation.
17. STAGE OF SENSITIZATION
3. pH OF THE REACTION MEDIUM
• IDEAL pH – 6.5 to 7.5
• Anti-M & anti-Pr(Sp1)antibodies are detected best in acidic
medium.
18. STAGE OF SENSITIZATION
4. IONIC STRENGTH
• The Na+ and Cl- ions, in an Isotonic Saline medium, gets
attracted to the oppositely charged Ag & Ab molecules,
• An ANIONIC BARRIER IS FORMED, slowing the rate of
sensitization.
• This barrier is reduced by lowering the IONIC STRENGTH.
• Mediums such as LISS are used
19. STAGE OF LATTICE FORMATION
1. Ag – Ab CONCENTRATION (MARRACK’S LATTICE HYPOTHESIS)
• In ideal conditions, an equivalent amount of Ag and Ab bind
and produce reaction.
• Any deviation causes decrease in efficiency.
21. DOSAGE EFFECT
• It is the amount or degree of expression of an antigen on
RBCs.
• Inheritance of genotypes heterozygously results in a weaker
phenotypical expression.
• Ex: M-positive RBCs from an individual with genotype MM
(homozygous) have more antigens than from Heterozygous
genotype.
• In Rh system, the C,c,E,e antigens also show DOSAGE effects.
22. CENTRIFUGATION
• Enhances agglutination by increasing the gravitational forces
and bringing the reactants closer together.
• Sensitized RBCs overcome their zeta potential (repulsive
effect) for each other and agglutinate.
• Enhances the Lattice formation as the RBCs are in close
proximity.
24. POTENTIATORS / ENHANCEMENT MEDIA
• Used to reduce the ZETA POTENTIAL and enhance the agglutination.
• Examples:
• Anti-Human Globulin (AHG),
• LISS
• COLLOIDS
• Albumin
• Polybrene
• Polyethylene Glycol (PEG)
• Polyvinylpyrrolidine (PVP)
• Protamine
• Enzymes - Papain
25. POTENTIATORS / ENHANCEMENT MEDIA
• ANTI HUMAN GLOBULIN (AHG)
• Has Specificity for Fc portion of Heavy chain of human IgG or
complements.
• It acts as a bridge by crosslinking sensitized red cells and makes them
to agglutinate.
• POLYSPECIFIC and MONOSPECIFIC
• Polyspecific AHG - both IgG and C3d.
• Monospecific AHG – IgG or C3d.
• Used in DAT, Antibody screening, cross matching, Autocontrol
26. POTENTIATORS / ENHANCEMENT MEDIA
• LOW IONIC STRENGTH SOLLUTION (LISS)
• Contains 0.2% NaCl, glycine, salt poor albumin
• Ionic strength is 0.03M. (saline – 0.17M)
• Lowers the ionic cloud around the red cell membrane, thereby
reducing the Zeta Potential.
• This enhances stage of sensitization and thereby decreased
Incubation period.
• It may enhance Cold Autoantibodies leading to False Positivity,
when they are present.
27. POTENTIATORS / ENHANCEMENT MEDIA
• ALBUMIN 22%
• Decreases Zetsa potential by increasing the dielecltric effect of
water.
• Enhances the Lattice formation.
28. POTENTIATORS / ENHANCEMENT MEDIA
• POLYETHYLENE GLYCOL & POLYBRENE
• Increases Test sensitivity
• Polybrene can detect ABO incompatibility and IgG
alloantibodies
• PEG produces very specific reactions and reduces the false
positive reactions.
29. POTENTIATORS / ENHANCEMENT MEDIA
• PROTEOLYTIC ENZYMES
• Reduces RBC surface charge by depressing some antigens and
enhancing other RBC antigens
• Enzymes include
• FICIN
• PAPAIN
• TRYPSIN
• BROMELIN
• Enhanced antibody reactivity to Rh, Kidd, P, Lewis, I antigens
• Decreased reactivity to Fya, Fyb, M, N and S antigens.
30. References
• Harmening DM, Modern blood banking & transfusion practices, 6th
edition, Philadelphia, F.A. Davis Company, 2012, chapter 3; 56-70.
• DGHS, Transfusion medicine technical manual, 3rd edition, MoHFW,
GOI, 2023, section 6; 88-94.
• Sastry AS, Bhat S, Essentials of medical microbiology, 3rd edition
revised print, New Delhi, Jaypee Brothers Medical Publishers, 2021,
section 2; 169-191.
• Klein HG, Antsee DJ, Mollison’s blood transfusion in clinical medicine,
11th edition, Victoria 3053, Blackwell publishing Ltd, 2005, chapter 3;
85,86.
