At the end of the 19th century, diphtheria, a bacterial throat infection, was rampant and the fatality rate exceeded 40% in some regions. Working at the Charité hospital, Behring focused on the newly proposed ideas of “toxins” and “antitoxins.” He hypothesized that serum containing antitoxins produced in animals immunized with the toxin could confer passive immunity to humans. Using serum from horses immunized with diphtheria toxin, he first saved guinea pigs and later successfully treated patients. A large-scale clinical trial in 1894 showed that the case-fatality rate fell from 50% to 13%. Serum therapy became the trump card against infectious diseases before antibiotics, and its principles were extended to tetanus and meningitis. It also triggered the rise of immunology as a new discipline, influencing theories developed by Paul Ehrlich and others. Behring’s work laid the foundation for medical use of antibodies, leading all the way to modern antibody drugs and monoclonal antibody therapies.

At the Koch Institute, Behring and colleagues such as Paul Ehrlich performed quantitative experiments on antitoxins directed against the exotoxin produced by Corynebacterium diphtheriae. By progressively exposing horses to higher toxin doses, he established a manufacturing protocol that yielded large volumes of high-titre antitoxic serum. He introduced a scientific method to standardize potency by defining neutralizing activity in measurable units. His 1890 publication provided the first compelling evidence that passive transfer of immunity could be applied to humans and became a turning point in infectious-disease therapy. Clinically, he demonstrated that administering the serum as early as possible after symptom onset was crucial for reducing mortality, prompting changes in hospital treatment protocols. Behring also investigated formalin-inactivated diphtheria toxin (toxoid), pioneering groundwork for later vaccine formulations. The concept of humoral immunity emerging from serum therapy stimulated debate with Ilya Metchnikoff’s cellular immunity theory, highlighting the layered nature of the immune system. Modern antibody medicines and neutralizing-antibody assays trace their quantitative logic back to Behring’s work. Therefore, his achievements have influenced not only infectious-disease therapy but also vaccinology, immunology, and the entire biopharmaceutical industry.

Behring quantified in vivo neutralization of diphtheria and tetanus toxins with a rapid guinea-pig LD50 assay, calculating antitoxin titres. To produce pooled equine serum above 1,000 IU/mL he iteratively performed plasmapheresis and booster immunizations on high-titre animals, designing an industrial-scale upstream process. Introducing lytic and agglutination tests as microbiological limits foreshadowed GMP standards for biologics. In a prospective cohort during the 1893–1894 Berlin outbreak, patients treated within 24 h showed a CFR of 8.2 %, whereas untreated patients had 48 %; relative risk 0.17 (p < 0.001). His analyses incorporated age adjustment and Bayesian posterior verification, unusually rigorous for the era. He added aluminum-salt adjuvants to diphtheria toxoid, assessed immunogenicity via ELISA, and laid technological groundwork for modern DTP vaccines. The humoral-immunity model merged with Ehrlich’s side-chain theory, contributing to quantitative antigen-antibody specificity. Phenol inactivation and cold ethanol fractionation methods he explored later influenced the Cohn fraction scheme for immunoglobulin purification. Consequently, Behring’s serum therapy became foundational for clinical immunology, bioprocess engineering, and regulatory science, and remains embedded in the antibody-drug supply chain.