Complement coated sensitized cells

[steve w]

Can anyone help me, I've been asked to prepare C3 and C4 sensitized cells, which I last prepared in 1982! Has anybody got an up to date method for this preparation?

Thanks Steve

[Rh-fan]

We use fress serum and sucrose to sensitice cells with C4 (for detection of anti Ch/Rg antibodies), I know that these cells also have C3 on the surface but I have never tested it.

What do you want to do with those cells after sensitization?

Peter

[steve w]

It's to validate polyspecific cassettes for C3b and C3d

Steve

[Rh-fan]

http://www.hemobioscience.com/PDFs/C3_Press_Release_Final.pdf

Have you seen this ad?

It are commercial C3 coated cells.

[steve w]

Yes I have thanks, but we'll need in house reagents.

Steve

[Rh-fan]

I have made some copies from our Methodes book (from 1993) from the American Red Cross about C3b, C3d and C4 coating.

I hope this will be of use.

Peter

[steve w]

Thanks!

[David Saikin]

I have found that the "home made" complement cells have a very short life span.

[PDS]

Hi,

 

After prepping cells using the Fruitstone method (American Red Cross #139) the resulting cells are reacting with our Anti-C3 reagent AND our Monospecific Anti-IgG. I was expecting a positive response from the C3 reagent only. Does anyone have any idea of why IgG is non-specifically binding to the cells along with C3b?

 

Thx

[galvania]

Most methods for coating cells with complement will also coat them with a certain amount of IgG.  Whether you then detect the IgG depends a bit on the method and the reagent you are using.  Basically  most complement control cells are only suitable for checking that you have a positive reaction with anti-Complement reagents, but NOT for controlling that other reagents do not detect anti-complement

[PDS]

Thanks for the response. 

 

If this is the case, when we are controlling AHG tests ,which contain anti-IgG and anti-complement components, how can we be sure that any positive reaction is due to only complement coating of the cells and not IgG (or a combination of both)?

[tupton]

Question - why are you making these cells in-house when they are readily available from a manufacturer? Cost?

[PDS]

I am not based in a clinical lab, only require cells for research purposes. Commercial cells require at least a 12 month standing order, something i don't need. In any case i think i may have solved the problems i was having with non-specific IgG binding by altering my incubation procedure at 0-1C. Results now show i have pure C3b coating.