Robotic method to isolate antibody secreting Single B cell and RT-PCR

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Philipp
Philipp's picture
Robotic method to isolate antibody secreting Single B cell and RT-PCR

Hi there!

I want to know where's a scientific group who isolate B cells from human blood.
My concern is that if I want to get mAb gene for cloning, I should find a way to isolate single B cell and conduct RT-PCR.
Cell based On-chip microarray format will be the best if there is.
But there was actually a group in japan that published a paper in NATURE.
Except that group, hope to know if there's anyone else.

1. those who can screen B cells high-thoughput.
2. And they should pick up just a single Antibody Secreting B cells to do RT-PCR
3. and then They should perform Cloing to have a antigen-specific antibody as a mount of human mAb.

FACS sorting or anyother multi-well plate format will be also fine.

Can such there be????

 Thanks!

xm833
xm833's picture
I've done this quite a bit

I've done this quite a bit and its not too difficult once you work out RT-PCR conditions-especially if you have access to automation. There are several papers describing primers and conditions for single-cell PCR of variables from B cells-both mouse and human. You should try several of the protocols and see which works best for you. I believe Novagen sells sets of primers for this purpose. Because Ig RNAs are expressed so strongly in B cells there is a lot of flexibility in getting RT-PCR to work, compared with more rare genes.

You can take freshly isolated B cells from human PBMC or splenocytes from mice, stain for B220+ and IgM and/or IgG (I used pan-IgG but be sure to validate your reagents by FACS first) then sort for B220+ cells with the desired Ig (I wanted cells that underwent affinity maturation so I sorted B220+ IgG+)

Deposit the cells in 96 well plates with the RT-PCR buffer already in them. If you want to be strict, you can do 1 cell/well but expect wells that don't work. You can do 3/well and that will fill plate but you will have wells with multiple cells-depends upon how tolerant you are of mixes that you have to deconvolute later. If using HT approaches it might not matter.

Carry out RT-PCR for heavy and light chain variables-you can do these as separate reactions or do both together in same reaction. If you don't want to lose anything you can do separately, run on a gel and isolate bands of the appropriate size for subcloning. If you are doing HT you can simply do both reactions, clone into a vector such as topo-TA, and just isolate enough bacterial clones to ensure you get heavy and light chain.

I've seen differences in how well heavy vs light variable PCR works-I typically could get about 80% of my wells to yield heavy AND light chain variables if I did single cell deposition by FACS. This took some optimization, however, and I started at about 40-50%.

If you want to spend extra time with the primer design, you can set them up so that whatever you clone is ready for immediate use in an expression vector for full antibodies and clone in directly from your RT-PCR, then transfect minipreps into CHO or some other mammalian line and just screen for function. This is a lot of extra optimization so I would start with basic RT-PCR first before moving on to this. However I can say that it does work