Techniques in Molecular Biology: Monoclonal Antibody Production

Antibodies are perhaps one of the most important tools in the arsenal of molecular biologists. They have a wide variety of applications, from targeting macromolecules with fluorescent dyes or other indicators to aid in visualization, or as a component of other molecular techniques, such as immunopercipitation of proteins. Antibodies that specifically target a researcher's molecule of interest, though, have not always been in the biologist's toolbox.

Before researchers had this ability, antibodies were produced using a simple technique: inject your antigen of choice into a mouse (or goat, or rabbit), and the mouse will produce antibodies targeted against the antigen. After taking a blood sample from the mouse and collecting the serum, the antibodies could be purified. Antibodies produced in this manner were said to be polyclonal: that is, they were derived from multiple antibody producing B-cells in the spleen. Each antibody producing cell produces a different antibody¹, and antibodies produced in this way will be a mixture of different antibodies from different cells.

Monoclonal antibodies, in contrast to the polyclonal variety, are derived from a single antibody producing cell. Antibodies from a single cell will all be specific for the same antigen epitope. This confers several advantages over polyclonal antibodies. Perhaps the biggest advantage is that they allow researchers to target specific epitopes on an antigen. Let's say you wanted to mark a specific residue on a protein using antibodies that are labelled with a fluorescent dye. Using polyclonal antibodies, your protein would end up labelled all over, since the polyclonal antibodies would bind of a variety of epitopes on the protein's surface.  Monoclonal antibodies specific to the residue of interest would get rid of all the problematic non-specific binding.

The production of monoclonal antibodies, however, is somewhat different, and pretty cool. The technique starts off just as it would if you were making polyclonal antibodies: inoculate a mouse using your antigen of interest. The antibody producing B-cells in the mouse spleen will begin to make antibodies that target your antigen. Where Köhler, and Milstein's technique differs is in what is done with those B-cells. Normally, the antibody-producing cells only start making antibodies near the end of their life. Isolating individual cells (thus isolating only one type of antibody) and growing them in culture would work to produce monoclonal antibodies, but only for a short amount of time. Milstein and co. got the idea of fusing the B-cells with immortalized mylenoma cells. Cancer cells can, for a variety of reasons, become immortalized², and continue replicating - indeed, this is what makes cancer a problem! By creating hybrids between B-Cells and mylenoma cells - called hybridomas - they were able to created antibody-producing cells that live forever and keep on producing antibodies. Culturing these cells and purifying the antibodies now became a more viable option. Separate cell lines were isolated and cultured, so that each culture contained only cells from an individual lineage, and consequentially, produced only one kind of antibody. These cells are first grown on plates to establish a lineage, but are eventually transferred to large tissue culture flasks. This allows for tons of monoclonal antibodies to be produced, isolated, and used by researchers the world over.

This technique pioneered by  Milstein and Köhler revolutionized the way research is done in molecular biology.  It was important enough that it won Georges Köhler, and César Milstein, the Nobel Prize in Medicine in 1984³. Since then, antibodies that target any antigen imaginable have been developed, and can easily be ordered from companies that specialize in production of monoclonal antibodies. It is unlikely, I think, that monoclonal antibodies will be ever displaced as a staple tool for molecular biology research.

FURTHER READING:

I you're interested in reading more about the history of antibodies, their discovery and the story of Milstein and Köhler's work, I suggest reading through "A Brief History of the Antibody" posted at the Proteintech Group blog [Part I Part II Part III]. Milstein's Nobel lecture also contains some interesting insights into antibodies, antibody research and the development of Hybridoma technology, and can be found here.


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1. The different antibodies produced are specific for the same antigen, but are directed towards different epitopes on the antigen.

2. This is to say that they are not subject to the Hayflick Limit. The Hayflick Limit, named after American researcher Leonard Hayflick, is the number of times a given cell line can divide before stopping. Originally, cells were thought to replicate indefinitely, and failure to keep cell lines alive was thought to be due to ignorance of optimal techniques. Hayflick and Paul Moorhead, working at the Wistar Institute in Philadelphia in 1961, showed experimentally that cell lines impose a limit on the number of times they can divide. The limit differs between cell types, but for humans it is around 52 divisions.

3. The prize was shared with Neils Jerne, who was awarded the prize for his work on the development and control of the immune system.

Next-Generation DNA sequencing: The Future is Now! Part 1: Pyrosequencing

Are you tired of sequencing your gene of interest 800bp at a time? Sick of straining your eyes staring at a fuzzy chromatogram? Fed up with waiting hours for your PCR to finish only to realize you forgot to add ddNTPs to your reaction mix? Well, nex-gen DNA sequencing is for YOU!

Cheesy sales-schtick aside, next-generation DNA sequencing technologies are on the rise (and are poised to soon become current-gen technologies - some already are!). The days of loading your PCR'd sequencing mix onto an automated capillary sequencer may soon be numbered. So, to make the change of power of our mighty sequencing overlords a little easier, this series of posts will be dedicated to how the upcoming technologies work, their advantages over conventional sequencing technologies, and their problems.

Today's post: Pyrosequencing

Pyrosequencing is perhaps the one Next-Gen sequencing technology that is the most like current generation automated sequencing (if you need a reminder on how that works, I've written about it in the past). It still requires primer design, and rounds of PCR, but the method of detection of incorporated nucleotides differs.

Pyrosequencing starts by adding your PCR'd sequence to a reaction mix that contains DNA polymerase, and three important enzymes: DNA sulfurylase, apyrase and luciferase. DNA sylfurylase is an enzyme that converts a pyrophosphate molecule into ATP. Luciferase is an enzyme which uses ATP to convert luciferin into oxyluciferin, resulting in the emission of light. Apyrase's job is to degrade unincorporated nucleotides. Given that the addition of a nucleotide into a growing DNA strand results in the release of a pyrophosphate molecule, keen readers might already see how pyrosequencing works.

With conventional automated chain-termination sequencing techniques, we add all of our dNTPs to the reaction mix at once; they can easily be distinguished because each base has a different fluorescently labelled dye bound to it. With pyrosequencing, however, one cannot toss in all the nucleotides at the same time. Rather, we have to add each one sequentially for each nucleotide in the sequence. That is, we first do a reaction with A, then with T, then with C and lastly with G. We then repeat this cycle over and over until the sequencing reaction is complete (if this sounds confusing, hopefully the diagram and video below will clarify it). Why we do this will be apparent in a moment.

When a nucleotide is incorporated into the DNA strand, a pyrophosphate molecule is liberated¹. This pyrophosphate molecule can then be converted by the DNA sulfurylase into a molecule of ATP. Luciferase picks up this ATP² molecule and uses it to catalyze the conversion of luciferin to oxyluciferin. This chemical conversion results in the emission of a photon. This chain of events, then, means when a nucleotide is added by DNA polymerase to the growing DNA chain, we get the emission of light. A computer with a suitable detector could detect this light, and we would have an indication of when a nucleotide was added in our sequencing reaction.

But if we add all of our dNTPs at once, then how do we know which ones are being ? This is why we only add one nucleotide to the mix at a time. First the reaction is run using dATP. We then add apyrase to remove any remaining nucleotides and repeat with dCTP, and so on. If we add the nucleotides one at a time, then they will be incorporated (or not incorporated) into the sequence one at a time, and consequently we get one light signal at a time.

The light signals are recorded on a chart called a pyrogram. This chart records not only which nucleotides resulted in the emission of light but also the intensity of that signal. If three dTTP molecules were incorporated at once, then there would be three photons emitted and three times as much light; this would result in a triple peak on the pyrogram. From the pyrogram, then, one could easily read off the exact DNA sequence. The figure to the left shows one such pyrogram. The sequence in this example would be GCAGGCCT.




The following video puts the whole process together nicely.



So why would one choose pyrosequencing over automated chain-termination methods? Well, for one, it's cheaper (though, not as cheap as some of the other upcoming next-generation sequencing methods). Practically, it's easier to do, since it doesn't require running through gels or capillaries. Analyzing the resulting pyrogram is also easier than analyzing a chromatogram. Chromatograms can often be spotted with "N"s when the computer cannot tell if the wavelength of light from a dye is one color or the other; however, with pyrosequencing, detection is binary - either a photon is emitted or not - so results are more accurate and clearer.

Though, pyrosequencing does have it's drawbacks. It requires a greater number of PCR cycles than traditional sequencing does, so it may take longer to complete, especially for longer sequences. Currently, a typical read of sequence data from pyrosequencing is about 300 to 500bp - shorter than the typical 800 to 1200bp you get from chain-termination methods. This, however, is likely to improve as the technology advances and becomes more refined. The shorter reads, though, make it tough to sequence genomic regions containing high amounts of repetitive DNA.

So that is pyrosequencing in a nutshell. Next time: Helicos sequencing!

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1. NTPs are triphosphates (that's what the TP stands for!), meaning each nucleoside (base+sugar) is attached to a phosphate molecule. To add a nucleotide to a growing DNA strand, the reaction requires an input of energy. This energy comes from the breaking of the triphosphate chain in each nucleotide; two phosphates are broken off and released as a pyrophosphate (PP_i) molecule, and the remaining portion of the nucleotide is attached to the hydroxyl group on the 3' carbon of the previous nucleotide in the sequence.

2. Observant readers might be confused here. Since luciferase requires ATP to convert luciferin to oxyluciferin, won't this cause a problem when we add dATP to the sequencing reaction? Won't there be competition between DNA polymerase and luciferase? Well, if you thought that, then you'd be right! For this reason, we use dATPαS instead of dATP for pyrosequencing. This molecule has a sulfur atom attached to the α phosphate of the nucleotide, and cannot be used as a substrate by luciferase. Problem solved!