A chance at absolution

The Case Of Kramer

This project started thanks to the American virologist Florian Krammer from mount Sinai hospital in new York. He and his group engaged in respiratory viruses, but the influenza virus. From mid-December — beginning of January, when it became clear that in China the epidemic began, Florian Cramer and his colleagues said, “We need to do a test-system.”

When the infection hit the US, the testing took over the CDC, the organization for the control of infectious diseases (then equivalent of the CPS). And the rest banned. A test she was going very slowly. At some point my test-system for PCR was done at the University of Washington have begun testing, and it is in this region then began a sharp increase in the number of cases — simply because there were to test without waiting for CDC.

Kramer was one of those who said that in fact the infection is much more that the number of infected is ten times higher than in medical statistics. For example, he pointed out that Canada began to detect cases coming from those American States where the CDC cases of the disease have not noticed.

Here I must say that there are PCR tests — they are needed in order to determine whether a person is sick coronavirus infection or not — and there are tests for antibodies to the virus. These tests are needed for slightly different tasks. The first allows you to identify the sick and to isolate (so they infected less healthy). And the second allows us to identify the coronavirus infection have either been at a fairly late period or to determine that this person has acquired immunity to it. And so he will not have to sit in quarantine, he may come in contact with patients and so on is important, for example, for doctors. PCR test-system can’t do.

Therefore, we need a simple, cheap and accurate test for antibodies, i.e. proteins that the immune system produces in response to invasion of the virus. According to some estimates, the antibodies can be detected for at least a year after the disease, and even ten years. And antibody test is the only reliable way to figure out whether the ill person get an infection and formed whether immunity against it.

Plasmid in the envelope

The virus in the laboratory of Kramer was not, like most microbiologists in the world. But he was at the disposal of scientists from Wuhan, Singapore, many others — and in January the first genome of the pathogen COVID-19 was decrypted and made public.

The Americans took the fragment of the viral genome that encodes the viral S-protein spike, “spike.” The virus uses it to stick to the cell (and then to get inside), and it recognizes the same virus the immune system.

The gene’s protein is quite large, nearly 4 thousand nucleotides. Special devices-synths allow you to get only short fragments with a length of about 100 nucleotides. Glue 40 such pieces — already not a trivial task, even in the mode of Abraham it takes a month and a half.

For this you need to take the synthesized pieces, pour the enzyme that glues them to insert into the vector, and run it all through the bacteria that they reproduce, test, and repeat this cycle many times — like in the kitchen. Requires a lot of patience, attention.

Ultimately they received a plasmid — a circular DNA fragment with the gene’s protein. Using this plasmid, they launched mass production of viral S-protein in bacterial cells. And the resulting protein has been used to test system: test who in the body there are antibodies able to contact him.

But Kramer not just made this plasmid. One of the merits of his team that they have optimized the gene’s protein to make it easier to accumulate in cell culture. For example, they artificially altered the gene so that no protein remained in the cell, and immediately exported from the cells into the medium. In addition, they sewed him a special mark, which allows you to track protein (and related antibodies) in the chromatogram. Another modification was needed to make the resulting protein molecule to connect three — in trimers that the test was effective. Finally, they added to the gene marker of bacterial resistance to the antibiotic that the plasmid was easier to accumulate in certain bacteria. In General, a huge design work.

And then they stood up and said we made such plasmids, using them it is possible to produce proteins. They can be placed in the basis of this analysis system, it is possible to catch antibodies. We have them, we are ready to give their all. And sent them to dozens of labs all over the world completely free, because they believe that testing should be widespread and available.

This feels nice! Thank you @crabbyabz ! https://t.co/ue47mZkVY4

— Florian Krammer (@florian_krammer) April 11, 2020

Kramer revisit one of those scientist expelled their plasmids

 

The start cell of the plant

A week after I wrote Krammer, we got the plasmid. Roughly speaking, this is the manual for the production of viral protein. This is not a test system, it is still far. Now, this plasmid should be integrated in the machine that manufactures it.

First, we need to ensure that the plasmid gets inside a bacteria.

Typically, this use of E. coli. If it is to survive in a solution of calcium chloride and some other ions in a certain concentration, if then the temperature begins to fluctuate, it begins to absorb DNA.

So plasmid gets into bacteria and begins replicating using the internal machinery of bacteria. Within the same bacteria can zarabotatj thus about a thousand plasmids. Then these “banks plasmid” reveal with the help of a set of reagents, filtered, and somewhere for an hour or two you get a clean solution with the plasmids.

Navarro therefore plasmids which are derived from Kramer, we are engaged in the production of the viral protein.

Usually use the same protein of Escherichia coli: they are very well accumulated proteins in large numbers — tens, hundreds of milligrams. But we took mammalian cells because many proteins in eukaryotic cells modifierade: squirrels are hung the remnants of phosphoric acid, and sugar. And if you’re doing the test-system, its specificity, the ability to “learn” the antibodies may not only depend on protein, but such post-translational modifications. Bacteria such posttranslational modifications of proteins are not able to do, so we need mammalian cells. For us, for my lab it was the first such experience.

We took two types of cells is a cell line of hamster, often used in this role, factory, and human cell line HEK is human embryonic kidney cells. This line is also there in almost every laboratory.

With the help of another reactant we Packed plasmid DNA in liposomes — lipid capsules. In them the DNA molecule, as in a soap envelope, slip through the membrane into the cells. And further, this cell turned into virus factory S-protein. Due to the design of Crammer this protein accumulates inside the cells, and immediately exported in the culture medium, that is, outside of cells, and we can only build this.

In four days the number of cells equivalent to about six Petri dishes (we did not use themselves Cup, we have multiplied the cells in a special roller bottles) have gained approximately 0.1 milligram of viral protein.

Meet antibodies

Then, almost invisible amount of protein — 2 micrograms — it was distributed in 96 cells in experimental tablet. It only remains to check whether this protein is to find antibodies to the virus.

To do this, we needed the blood of people who have had coronavirus infection, i.e. those who had received a positive result of PCR test for coronavirus, but is now fully recovered — that is, the same PCR test showed a negative result.

Find survivors, we helped a group of “Hematology center” led by Grigory Efimov, because they, like medical organizations, had the right to work with ill.

Drops of blood plasma of people who have had COVID-19, and a control sample dripped in the cell this tablet. The mechanism here is simple: if the blood contains antibodies, they should contact the viral protein. However to see it’s not easy, it requires several stages of processing.

Here’s your viral protein at the bottom of the cell contacted with the antibody to the protein from blood plasma. Then the cell are washed from plasma, and put antibodies in rabbits or mice to human immunoglobulins, i.e., human antibodies. To these second antibodies is attached to the light — fluorescent label or an enzyme for color reactions.

Antibodies of animals join human and makes a triple sandwich, a viral protein, human antibodies, and these secondary labeled antibodies. Then you just have to paint, to make visible proteins, where the presence of antibodies.

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