On this page
An antibody that recognises exactly one target is one of the most useful tools in biology, but for most of the twentieth century nobody could make one on demand. The body makes a mixture, and the cells that make any single antibody die quickly outside the body. In 1975 two scientists in Cambridge solved both problems at once. This post tells their story from Nobel Prize records and Milstein's own Nobel lecture, explains the hybridoma method step by step, and includes a worked dilution example.
Who were Milstein and Köhler?
César Milstein was born on 8 October 1927 in Bahía Blanca, Argentina, and died on 24 March 2002 in Cambridge, according to his Nobel facts page. His Nobel autobiography describes a PhD in Buenos Aires done with no funding, a fellowship and second PhD at Cambridge, and a return to Argentina that ended when political persecution of scientists forced his resignation. Back in Cambridge from 1963 at the Medical Research Council's Laboratory of Molecular Biology, he moved from enzymes to immunology at Fred Sanger's suggestion.
Georges Köhler was born on 17 April 1946 in Munich and died on 1 March 1995 in Freiburg, as his Nobel facts page records. His Nobel biography shows he finished his PhD in 1974, from work done at the Basel Institute for Immunology, and then spent April 1974 to March 1976 as a postdoctoral researcher in Milstein's laboratory.
What problem were they trying to solve?
The 1984 Nobel press release explains it. Each antibody-producing lymphocyte makes antibody of only one specificity, and a clone of such cells makes identical, monoclonal antibody. But these specialised cells do not survive long when taken out of the body. Myeloma cells, a type of tumour that arises from an antibody-producing clone, can grow in culture indefinitely, but nobody knows what their antibody recognises.
Milstein was not even aiming for a new technology. In his Nobel lecture he explains that his group was studying mutations in antibody-producing cells and needed a cultured cell line that made an antibody with a known target. None they tried was suitable. "And yet in a funny way our lack of success led to our breakthrough," he said, "because, since we could not get a cell line off the shelf doing what we wanted, we were forced to construct it."
How does the hybridoma method work?
Milstein's colleague Dick Cotton had already fused two myeloma cells and found that the hybrid made the antibody chains of both parents. After Milstein described this work in a seminar in Basel, Köhler came to Cambridge. Their 1975 paper in Nature describes cell lines made "by fusion of a mouse myeloma and mouse spleen cells from an immunised donor", which secreted antibodies against sheep red blood cells.
The steps, as the press release and lecture describe them:
Immunise a mouse with the chosen antigen (in the first experiment, sheep red blood cells).
Take spleen cells, which include the antibody-producing cells.
Fuse them with myeloma cells growing in culture. The myeloma line had been made resistant to a drug, azaguanine, and the hybrids were then picked out by growing them in a selective medium called HAT.
In the hybrid, the myeloma partner supplies the ability to keep dividing, and the spleen partner supplies the antibody's specificity.
Dilute the hybrids so that colonies grow from single cells, then test each colony and keep those making the wanted antibody.
A good hybridoma can then produce that one antibody without limit. The press release calls the technique one of the most important methodological advances in biomedicine of the 1970s and lists uses such as purifying substances like interferon and identifying structures on cells for diagnosis.
Worked example: diluting to one cell per well
Step 5 depends on simple arithmetic. These are sample numbers for a classroom exercise, not from the original paper.
Suppose a suspension has 2,000 hybrid cells per mL, and you will put 0.1 mL into each well of a plate. You want about 1 cell per well on average.
Cells needed per mL: 1 cell ÷ 0.1 mL = 10 cells per mL.
Dilution factor: 2,000 ÷ 10 = 200, so dilute 1 part suspension with 199 parts medium.
Check: at 10 cells per mL, 0.1 mL holds 10 × 0.1 = 1 cell on average.
"On average" matters. Some wells will get none and some two, which is why each colony is tested and often re-cloned.
What mistakes do students make with this topic?
Calling any antibody monoclonal. Monoclonal means all molecules come from one clone and are identical. Serum antibodies are a mixture.
Mixing up the partners. The myeloma cell gives immortality; the spleen cell gives specificity.
Getting dilution factors backwards. Going from 2,000 to 10 cells per mL is a 200-fold dilution, not a 1/200-fold one.
If the cell biology here feels like a lot of moving parts, the Fermi Concept Library covers every concept in physics, chemistry, biology, mathematics and computing, so you can revisit lymphocytes and cell culture one idea at a time. For how the antibody molecule itself was worked out, read our post on Rodney Porter, Gerald Edelman and the Y-shaped antibody.
What can a student take from how they worked?
Fix the obstacle in front of you. They needed a cell line that did not exist, so they built one.
Value the side project. Cotton's "little experiment being done in the background", as Milstein called it, became the key method.
Credit the team. Milstein's lecture names the colleagues and the tissue culture assistant, Shirley Howe, who worked on the experiments. He also describes the "unspoken message" he took from his laboratory, which he put as "Do good experiments, and don't worry about the rest".
Next step: redo the dilution example for 5,000 cells per mL and 0.2 mL per well, and check your answer by working out the cells in one well.
