30 September 2011

Monoclonal?

I am making antibodies in the lab. These are recombinant IgG1 derived from human plasmablasts. Variable heavy and light chain domains, originating from a single cell, are obtained via PCR. The variable regions are cloned into a plasmid vector which contains the IgG1 constant region. The IgG plasmid construct is transfected into a HEK cell line (human embryonic kidney) and the antibodies are purified from the culture media.

  • Do these antibodies better represent a monoclonal or a polyclonal?
  • Is this another method of producing a human monoclonal antibody?
  • At what point(s) in the process could variability increase?
  • How may these rAb's (recombinant antibodies) differ from the antibody being made by the patients plasmablasts?

Bennett JL, Lam C, Kalluri SR, Saikali P, Bautista K, Dupree C, Glogowska M, Case D, Antel JP, Owens GP, Gilden D, Nessler S, Stadelmann C, Hemmer B.

Ann Neurol. 2009 Nov;66(5):617-29.

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2 comments:

  1. AlexE7630October 4, 2011 at 4:40 PM

    1. I would say these rAb's fall into the "monoclonal category", saying that the single cell RT-PCR makes sure that the IgG1 VH and Vk DNA of only that single Plasmablast found through sorting is cloned into the pCEP4 vector, not the VH and Vk of EVERY Plasmablast found during the sort. This would produce IgG rAb's only produced by that one single Plasmablast colony.
    2. I definitely think this is monoclonal method separate from the hybridoma model, since there is no fusion of cells needed and the transfected cells are not immortalized. This means that although a monoclonal rAb is being produced, new cells must be transfected every 2-3 weeks, which is quite different to the hybridoma model.
    3.There are TONS of places where variability could increase. Any place where restriction enzymes are used to cut the plasmid DNA adds variability. Also, there must be some variability added into the process during transfection. Maybe one of the transfected cells starts to chew away at the pCEP4 vector after taking up the Lipofectamine-DNA complex. More variability is added every time a transfected cell proliferates, as this increases the chances for incorrect transcription or translation of the pCEP4 vector, leading to a mutated version of IgG to be produced. Also, any point ligation is needed could lead to variability and a non wild type IgG. The answer, EVERYWHERE!
    4.Other than posttranslational modification, if any, I would think these rAb's work identically to the antibody being made by the patient's plasmablasts. The binding assays confirm that the rAb IgG binds the same target (AQP4) with a similar affinity to IgG found in patient CSF. Unless the two IgG variants are incredibly similar, I would not expect to see these results.

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  2. pSIVANovember 17, 2011 at 12:22 AM

    Hello,

    This is the perfect blog for anyone who wants to know about this topic. Monoclonal antibodies are antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent...

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