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Back to Basics: Why blood group antibodies have temperature preference?

A Blog from Eric Ching:
In blood group serology, there is one basic rule: for every given rule, there are exceptions!
We, at least, I, tend to use words such as: mostly, almost always, might, not usually, rare, highly unlikely, exceedingly rare….. to cover these exceptions.
In our training days, our teachers taught us that most IgM antibodies are cold-reacting while almost all IgG antibodies prefer an incubation period at 37oC for optimal antigen binding before IAT.
I think most of us have experienced  IgG cold reactive auto and alloantibodies such as anti-M, -N, -P, -IH or even –Pr while rare warm reactive IgM Rh antibodies detectable only after 37oC but not by IAT, caused debates in the 1980’s as to whether “spin and read” after 37oC incubation could be omitted.
What governs the temperature preference of an antibody?
It is NOT the antibody class! It is the antigen’s chemical structure that dictates the temperature preference of its corresponding antibody.
If you recall from your training days, that there are four types of non-covalent molecular interactions between the epitope of the antigen and the paratope (complementarity determining or the hypervariable region) of the antibody.
These are:
  • Electrostatic or Ionic bonds
  • Hydrogen bonds
  • Van der Waal’s interactions and London Repulsive Forces
  • Hydrophobic forces
The first two types of bonds are exothermic and are involved with carbohydrate and glycoprotein antigens: ABH, Ii, P1, Lewis M,N etc.
The last two types of bonds are endothermic and are involved with protein and lipoprotein antigens: Rh. Kell, Kidd, Duffy, etc.
Although temperature does not affect the Equilibrium Constant (Ko) of a given antibody antigen interaction, it does affect the rate of antibody uptake and dissociation of antibody.
For those who are interested in this topic, read on for more details!
Covalent Bond  vs  Non Covalent Forces
             Relationship between                                       Stabilization
Non-covalent bonds  force and distance                     energy  (Kcal/M)             
     Electrostatic                   1/d2                                                 5-10
     Hydrogen                       1/d2                                                   2-5
     Hydrophobic                   1/d7                                                  1-5
     Van der Waal's               1/d7                                                   0.5
      stabilization energy= energy required to break the bond.
For covalent bonds, the stabilization energy is between 40-140 Kcal/M; in other words, it takes
much more energy to break a covalent bond than the weaker non-covalent forces.
The above also shows how both hydrophobic forces and Van der Walls interactions are only
operative in extremely short distance. It decreases 10 million folds when the distance between   
the reacts only doubles!
Electrostatic Bonds
These are attractive forces between opposite charge sites. eg. COO- and NH3+. The number of charge groups depends on the pH of the suspending medium. The Coulombic  Law states that the force between two charged particles e1 and e2 (antigen and antibody) is indirectly proportional to the dielectric constant(ability to dissipate charge) and the square of the distance between these charged particles.            
          f= e1e2/Dd    where f= attractive force(+)                                D= dielectirc constant
                                                    repulsive  force(-)                                 d=distance
Hydrogen Bond
When a hydrogen atom which is covalently linked to a electronegative atom (e.g.,  an oxygen atom) gets close to another electronegative group (e.g., C=O), then the hydrogen atom is shared by the two negatively charged groups forming a hydrogen bond. The formation of hydrogen bonds is exothermic (gives off heat) and is stabilized by an aqueous environment. Therefore, a decrease of temperature will increase antigen-antibody interactions. Hydrogen bonds are more significant in carbohydrate antigenic determinants than those of protein or lipoprotein in nature.
Van der Waal’s and London
These are interactions of electron clouds surrounding the polar groups. The strength of Van der Waal's is indirectly proportional to the seventh power of the distance between the two atoms.
In contrast, London repulsive forces are due to the penetration of electron fields. Electrons of the like spin tend to stay away from each other (Pauli Exclusion principle- only two electrons can occupy the same atomic orbit and they must be of the opposite spin). London repulsive force is indirectly proportional to the 12th power of the distance between the two interacting groups.

Hydrophobic Effect
Hydrophobic effects are results of Van der Waal's and London's interactions. Hydrophobic (non polar) groups have a strong tendency for self association. Water molecules are squeezed out in the process. Hydrophobic effects may contribute up to 50% of the total bond strength in certain antigens defined by warm reacting antibodies. The squeezed-out water molecules lead to a decrease in the free energy of the system, because they assume a more random orientation when released. In other words, they gained entropy(randomness). Hydrophobic effects are entropy driven and are endothermic (absorb heat). Therefore, an increase in temperature will encourage antigen-antibody interactions.
Effects of temperature on the rate of antibody uptake or sensitization
Generally, temperature variation may or may not affect ab-ag reaction. In exothermic reactions (e.g., anti-I and the I antigen), an increase in temperature decreases the association constant. In contrast, in exothermic reactions involving immune antibodies, a temperature increase has a little effect on their Ko. However, the rate of antibody uptake exceeds the rate of dissociation at higher temperatures
Have you tried to incubate a known anti-D with a panel for an hour at RT before IAT to see if it works? :)
The release of energy takes two forms:
1. As Heat in the exothermic reactions: Hydrogen bonds are predominantly exothermic and associated with carbohydrate antigens, hence antibody directed against these antigens will proceed further to completion at lower temperature.
2. As a Change in Entropy(randomness): Hydrophobic effects are endothermic and entropy driven, therefore antibodies to protein antigens favor reaction at higher temperature.
If you would like learn more on this subject, there is an old but easy to read AABB publication entitled “Antibody-Antigen Interactions Revisited”  published in the early 80”s. Since I am away from my home office, I’ll send the exact reference upon your request.
Best Regards,


Very informative and interesting post thanks
9/28/2022 12:22:55 PM

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