The unit ‘milliliter’ (mL) has little utility outside of laboratory. Perhaps it would be tremendously hard to quench thirst with one thousandth of a liter of water. White blood cells, the subject of my research, hover around 4,000 – 11,000 of individual cells per mL of blood¹.

With a vial of enriched peripheral blood cells², the cell density hovered around 10,000,000 cells/mL. Perhaps one might think this would be enough to interrogate the diversity of immune cell populations, and one is not wrong. But that is not the case with certain cell populations and certain questions one would have in mind.

And this was the case with the work I carried for my doctoral dissertation.

Rarity Class S

In my public defense seminar, I made it clear that the odds were stacked against me. The primary subject of my research was antigen-specific memory B cells in human, a subset of white blood cells, which present in peripheral blood at a minute frequency. A manual counting with hemocytometer at 200 cells per minute allows for a person to count 50,000 cells in a little over 4 hours. If this person was lucky, s/he could have found 1 antigen-specific memory B cells between his/her breakfast and lunch. To obtain ~100 antigen-specific memory B cells would have taken more than 2 weeks with no breaks.

Without sophisticated technology, it was impossible to interrogate such rare population of immune cells. Indeed, it is very astounding that despite their extreme rarity, we are all not dead from infections.

Technology allows for rapid progressions of discoveries in the field, and allowed me to interrogate probably close to 500 vials of enriched white blood cells. For a total of 5 billion cells, I probably had queried 100,000 – 500,000 of individual antigen-specific memory B cells within 36 months³ pursuing my dissertation projects.

In the game of chance, that is an awful bet. But immunology is much more complicated than that and this complexity allows us to… simply be alive.

Rarity and Utility

Combined rarity and utility fetch for a high price. Gold satisfies both attributes; its use extends from cosmetics to electronics. Sand, satisfies only the latter; but when carefully molded and manipulated, silicon also comes with an exorbitant price tag, made possible with technologies borderline wizardry.

B cells and its product, antibodies, exist between the two spectra of being gold and also being sand at the same time. Polyclonal preparation of antibodies is not a mythical art, and procuring them only takes several weeks. Inject several laboratory mice today with antigens and inject it again come two to three weeks afterwards, followed by another waiting period of several weeks, the mice would yield bountiful and presumably high quality antibodies⁴. Acquiring the polyclonal antibody mixture from mice requires minimal efforts nowadays.

However, this mixture is a finite resource. Use them frequently, you will run out of it. But worry not, because since 1975⁵, the field has figured out approaches for immortalizing B cells that produce these antibodies. The first iteration of this approach is probably a little hacky, it involves coaxing antibody-producing B cells to physically fuse with myeloma cell partner⁶. The efficiency for generating these immortalized cells was not great, yet this technology revolutionized the field of biomedical research.

A single hybridoma (hybrid of antibody-producing B cell and myeloma fusion partner) produces antibody with a single specificity. Therefore, the antibody its produced is called ‘monoclonal antibody’, that is, of one clone. Hybridoma comes with two major advantages: (a) it is immortal, if you properly keep it in culture or freeze it away for later exploits, and (b) it continuously produce antibodies, so one could harvest the monoclonals over and over again.

It cannot be overstated how important this notion of ‘monoclonal antibody’ is. With polyclonal antibodies, you cannot be sure of the composition that makes up their constituents. It could be a mix of 70% high quality antibodies and the rest are of questionable characters. With monoclonal antibody, all doubts are removed. It does one job, recognizes its target in one way, and that’s it. Knowing precisely what they do enables for high fidelity dissections into many biological phenomena.

Close to half a century since the method was described, the field has many tricks to acquire for monoclonal antibodies. Searching for B cells with defined specificities became less of a gold mining but more of a refining sand to generate silicon. The efficiency soared higher from mere sub-10% with the 1975 method to close to 80% with contemporary strategies. Single B cell antibody cloning, which took off in the second half 2000s decade, is how the field prefers to acquire monoclonals.

Hybridoma allowed for mining monoclonals. Single B cell antibody cloning gives us ways to find the elusive ones.

To Many Billions More

A mouse spleen can yield 50,000,000 – 90,000,000 total cells. A spleen is similar to a big regional airport hub: everything routes through there. Every single blood cells transit through it, and some take residence within the spleen for a while. Spleens from roughly 20 mice already equals to 1 billion of cells.

I completed my doctoral dissertation without taking advantage of the advances in the single B cell antibody cloning methodologies. Though, antibody cloning requires a marriage between immunology, genetic engineering, and bioinformatics to be able to make sense of it all⁷. But for my postdoctoral work, I am eager to be at the bleeding edge of this technology. I am eager to hunt for the elusive monoclonals, and there are many of them out there awaiting characterization.