The Discovery of the Complement System: Pfeiffer and Bordet’s Experiments

The story of the complement system is a fascinating chapter in immunology. It began with the ground breaking experiments of Richard Pfeiffer and Jules Bordet, whose work laid the foundation for our understanding of antibody-mediated immunity. Lets dive into this blog post on Discovery of the Complement System: Pfeiffer and Bordet’s Ground breaking Experiments Explained

---

Richard Pfeiffer and the Pfeiffer Phenomenon (1894)

The Experiment
Richard Pfeiffer worked with guinea pigs and the bacterium Vibrio cholerae.

1. Immunized Animal: He injected live Vibrio cholerae into the abdominal cavity of guinea pigs that were already immune to Vibrio cholerae infection.
2. Observation: The bacteria rapidly disappeared or were lysed inside the animal.
3. Transfer Test: Pfeiffer then injected non-immune guinea pigs with both the bacteria and serum from immune animals.
4. Result: The bacteria were still destroyed.

Pfeiffer’s Conclusion
Pfeiffer believed the destruction was due to phagocytic cells (such as macrophages) in the peritoneal cavity. He thought the serum simply “marked” the bacteria for destruction.
While this showed cooperation between body fluids and cells, he overemphasized the role of the cells alone.

In short: Pfeiffer discovered that immune serum could rapidly destroy bacteria — a phenomenon that became known as the Pfeiffer Phenomenon — but he did not identify the soluble factor responsible

---

Jules Bordet and the Discovery of the Complement System

Bordet’s experiments were designed to uncover the identity of the molecule in immune serum responsible for destroying bacteria (bacteriolysis). He successfully proved that this function requires the cooperation of two distinct components.

Step 1: Establishing the Baseline Lytic Activity

Bordet began by mixing Vibrio cholerae (cholera bacteria) with fresh serum from an immune animal — one that had previously recovered from infection.
The result was dramatic: the bacteria were quickly destroyed (lysed).

This confirmed that something in the immune serum was responsible for bacterial killing.

Action	Observation	Immediate Conclusion
Mix: Bacteria (V. cholerae) + Fresh Serum from Immunized Animal	Lysis (Bacteria are destroyed).	The serum possesses a powerful killing mechanism.

Step 2: Testing the Effect of Heat (55°C)

Next, Bordet tested whether this killing factor could survive heating.

He heated the immune serum to 55°C and repeated the experiment.
This time, no bacterial lysis occurred.

This result was puzzling. Antibodies — the specific immune molecules that recognize bacteria — are known to be heat-stable, meaning they survive moderate heating. So why did the serum lose its killing ability?

Bordet reasoned that a second substance, one that was heat-labile (destroyed by heat), must also be required for lysis.

Action	Observation	New Conclusion (The Puzzle)
Mix: Bacteria + Heated Serum from Immunized Animal	NO Lysis (Bacteria remain intact).	The killing factor is Heat-Labile (destroyed by 55 degree Temp) The specific antibody must be heat-stable, meaning the antibody is not the direct killing factor.

Step 3: Testing the Effect of Time (Unstability)

After discovering that heating immune serum destroyed its bacteriolytic power, Bordet wanted to know whether time alone could do the same thing — even without heat.

He took serum from an immunized animal and simply stored it for several days at room temperature. Then he repeated the same experiment by mixing this old serum with the bacteria.

Observation:
No bacterial lysis occurred. The bacteria remained completely intact.

Interpretation:
This meant that the active killing substance in the serum naturally loses its function over time, even without heating. In other words, it is time-labile — an unstable molecule that gradually degrades or becomes inactive during storage.

Action	Observation	New Conclusion (Reinforced)
Mix: Bacteria + Old (Stored) Serum from Immunized Animal	NO Lysis (Bacteria remain intact).	The killing factor is also Time-Labile (unstable and degrades quickly). This confirms the factor is a fragile, unstable molecule, unlike the durable antibody.

Conclusion:
This finding strengthened Bordet’s argument that the bacteriolytic power of the serum does not come from antibodies.
Antibodies are stable proteins — they can survive heat and storage without losing their ability to recognize bacteria.
But this mysterious lytic factor was fragile, unstable, and easily destroyed by either heat or time.

Step 4: The Crucial Restoration Experiment (The Solution)

This final step proves that the antibody and the killing factor are two separate molecules. To test his theory, Bordet performed his most brilliant experiment:

1. He took heated immune serum (which still contained antibodies but lacked the heat-labile factor).
2. He added fresh serum from a non-immune animal (which had no specific antibodies but contained the mysterious heat-labile substance).
3. When the two were mixed with bacteria — lysis occurred again!

This was the turning point. The destruction of bacteria could be restored only when both components were present:

1. the non-specific heat-labile factor (destroyed by heat, found in all fresh serum). 2. the specific antibody (survives heat), and

Action	What Each Serum Contributes	Observation	Final Conclusion
Mix: Heated Immune Serum + Fresh Non-Immune Serum	Heated Immune Serum: Has the specific antibody (“flag”). Fresh Non-Immune Serum: Has the fresh, non-specific killing factor (“grenade”).	Lysis is RESTORED! (Bacteria are destroyed).	Two Factors are Required: The non-specific killing factor is provided by fresh serum and needs the specific antibody to direct its destructive action.

Bordet’s Deduction

Bordet concluded that the two essential components are:

1. The Specific Component (Antibody): The heat-stable molecule that recognizes the pathogen.
2. The Non-Specific Component (Complement): The heat- and time-labile molecule that executes the lysis.

Would you like to continue our discussion by exploring the key biological consequences of complement activation?

---

Bordet’s Conclusion: The Two-Factor System

From these elegant experiments, Bordet concluded that bacteriolysis required two cooperating elements:

1. The Specific Component — the Antibody
   • Heat-stable and unique to each pathogen.
   • Binds to the bacterial surface and marks it as a target.
2. The Non-Specific Component — the Complement
   • Heat-labile and present in all fresh serum.
   • Once activated by the antibody, it destroys the target cell by punching holes in its membrane.

Bordet originally called this second factor “alexine,” meaning “protector.” It was later renamed Complement, because it complements the action of antibodies — antibodies recognize, and complement executes.

---

Summary: Discovery of the complement system

Bordet’s discovery laid the foundation for our understanding of antibody–complement cooperation, a core principle of the immune system. His work showed that the body’s defense is not just about recognizing invaders — it’s also about activating the right molecular tools to eliminate them efficiently.