How do you produce monoclonal antibodies in the lab. using B-cells? What's the stepbystep process?

Question

More important, where do i get information regarding a protocol of cloning antibodies using B-cells in the lab? I want to know exactly how to do this (materials, methods, etc).

Answer 1

this is generally accomplished by immunizing mice then fusing their spleen cells to make a hybridoma to produce a cell line secreting the antibody.

Monoclonal Antibody Production (1999)
Institute for Laboratory Animal Research (ILAR)

http://books.nap.edu/openbook.php?record_id=9450&page=12

if you can find this book it might be helpful. Best of luck, it's a difficult process!

Answer 2

Well,
First I think that if you want a detailed answer like that you should do some searching on pub med or google. That is half of understanding a technique. But I can give you a short version to give some basic background.

First you screen antibody producing cells for one or two that produce antibodies to your antigen. Second, you have to immortalize and propagate the cell buy fusing it to a cancer cell thus making a hybridoma cell. Then you can either use tissue culture and harvest the supernatant or, more commonly, people inject the fused cell line into mouse and collect the ascites fluid. After that it is a matter of purifying the ascites fluid, I would recommend using affi-gel blue from bio-rad first and then if it still needs to be cleaner, immobilize the antigen on a solid matrix (you can use nitrocellulose or a cyanogen-bromide activated resin) get the antibody to bind, wash, and elute using glycine pH2 or 3.


Google monoclonal antibody production, and look around

Answer 3

To produce monoclonal antibodies, one removes B-cells from the spleen or lymph nodes of an animal that has been challenged several times with the antigen of interest. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer) and that have lost the ability to produce antibodies. This fusion is done by making the cell membranes more permeable by the use of polyethylene glycol, electroporation or, of historical importance, infection with some virus. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely. Large amounts of antibodies can therefore be produced. The hybridomas are sufficiently diluted to ensure clonality and grown. The antibodies from the different clones are then tested for their ability to bind to the antigen (for example with a test such as ELISA) or immuno-dot blot, and the most sensitive one is picked out.

In the above process, one uses myeloma cell lines that have lost their ability to produce their own antibodies or antibody chain, so as to not contaminate the target antibody. Furthermore, one employs only myeloma cells that have lost a specific enzyme (hypoxanthine-guanine phosphoribosyltransferase, HGPRT) and therefore cannot grow under certain conditions (namely in the presence of HAT medium). These cells are preselected by the use of 8-azaguanine media prior to the fusion. Cells that possess the HGPRT enzyme will be killed by the 8-azaguanine. During the fusion process many cells can fuse. Myeloma with myeloma, spleen cell with spleen cell, 3 cells of different types etc... The desired fusions are between healthy B-cells producing antibodies against the antigen of interest and myeloma cells. These are relatively rare, but when one succeeds, then the healthy partner supplies the needed enzyme and the fused cell can survive in HAT medium. This is the trick to detect the successfully fused cells. The medium must be enriched during selection to favour hybridoma growth. This can be achieved by the use of a layer of feeder cells or supplement media such as briclone.

Monoclonal antibodies can be produced in cell culture or in live animals. When the hybridoma cells are injected in mice (in the peritoneal cavity, the gut), they produce tumors containing an antibody-rich fluid called ascites fluid. Production in cell culture is usually preferred as the ascites technique may be very painful to the animal and if replacement techniques exist, may be considered unethical. Fermentation chambers have been used to produce antibodies on a larger scale. Nowadays, bioengineering allow production of antibodies in plants.

And also look in this web page for information ragarding cloning antibodies
http://www.mpiib-berlin.mpg.de/research/MolecularImmu.htm

hope it will help