Generation of scFv - how to do without a kit (Jun/30/2007 )

Hi everyone,
i am going into the next big step of generating scFv (single chain variable fragment) after generating and spending considerable amount of time in the generation of monoclonal antibodies. can someone please tell me how to construct scFv without the use of any kit as scFv kits are no longer available from amersham
all the suggestions are highly welcomed
thanking you all in advance
Leelaram

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Well, first you have to isolate RNA from your hybridomas. There are a number of kits available for this. I prefer the ones that utilize membranes instead of Trizol and I think they are basically in the same price range.
Then you do your RT reaction using the total isolated RNA as the template. You can use constant region isotype-specific primers for this reaction or you can use oligo-dT primers that will anneal to all cellular mRNAs.
The next step is the toughest. You have to amplify only the variable regions of your monoclonals, which in itself is not that difficult, but you don't want to introduce any mutations along the way due to the degenerate primer you use.. There is a paper on this subject from Essono, 2003, Journal of Immunological Methods. An alternative that a colleague of mine does is 5' RACE, but it's tedious and very time consuming.
Then you separate the PCR products on an agarose gel. I would certainly sequence them as well, especially if there is a cluster of bands in the approximate size, where your products should be.
For assembly you first have to cut your amplicons and then assemble them either first using a linker between the heavy and light chain and then ligating this product into an expression vector or you can use an appropriate vector to clone both chains directionally (which is of course much easier, I just don't know where you can find the vector).
Then you have to transform E. coli cells with your recombinant vector, induce expression and voila - your recombinant scFV being produced by E. coli.

I hope I haven't left anything out.
Regards,
Miha

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hai miha,
your references were very useful
thank you very much
leelaram

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