We are trying to validate anti-C3 testing in gel. I have David Saikin's post from 3/13/09 but when I printed it I lost some of the information. I am trying to find out how long you incubate the cards and at what temperature.

Thanks in advance for your help.

We are trying to validate anti-C3 testing in gel. I have David Saikin's post from 3/13/09 but when I printed it I lost some of the information. I am trying to find out how long you incubate the cards and at what temperature.

Thanks in advance for your help.

Sorry Karen, but when you say you are trying to validate anti-C3 testing in gel, are you asking about a positive control for the DAT (i.e. C3b, C3c, C3d coated red cells), or are you validating the test for when you have an antibody that is capable of initiating the complement cascade in serum?

I take it that, in this day and age, when we are using EDTA anti-coagulated samples almost exclusively (or, at least we are in the UK) you are asking about the former (as EDTA "poisons" the complement cascade by chelating Ca++, Mn++ and Mg++).

Hi Karen,

For now, I'll assume you want to prepare complement coated red cells as a positive control for the DAT.

This method will coat the cells with C3/C4.

(i) To 20 mL of a 10% (w/v) aqueous solution of sucrose, add:

(a) either 4 drops of fresh whole blood, or

( 2 drops of washed, packed red cells and two drops of fresh serum.

(ii) Incubate at 37oC for 15 minutes.

(iii) Wash the cells 4 times and resuspend as a 2-4% suspension in saline. Store at 4oC. Use, at the correct dilution (0.8%?, depending on your gel technique) and discard after 48 hours.

I (a) hope this works and ( hope this will be of help.

I incubate for 5 minutes wth the anti-C3b,-C3d at room temp before centrifugation.

Ditto.

David

Thanks for your quick response. I'm glad you understood what I was asking because apparently I didn't make myself very clear to anybody else.

I did my best - honest!

I think the confusion comes because in Europe there are gel cards with monospecific anti-C3d and anti-C3c (really C3b) already present in the gel. In the States you don't have these (I think....)