Running into problems validating Anti-C3 in buffered Gel

[gene20354]

Some time ago I asked for advice on validating Immucor's Anti-C3d with Ortho's bufferd gel card. I got the help that I needed but I have run into a problem. I have tested 4 samples that produced weakly positive to 1+ reactions with anti-C3d in the tube but all were negative when I tested them in gel. I have been converting Immucor’s complement control cells to 0.8% and using them as my positive control. The control always works but I can't figure out why my samples are coming up negative. Has anyone experienced this?

[SbbPerson ☆]

On 7/15/2010 at 5:03 PM, gene20354 said:

Some time ago I asked for advice on validating Immucor's Anti-C3d with Ortho's bufferd gel card. I got the help that I needed but I have run into a problem. I have tested 4 samples that produced weakly positive to 1+ reactions with anti-C3d in the tube but all were negative when I tested them in gel. I have been converting Immucor’s complement control cells to 0.8% and using them as my positive control. The control always works but I can't figure out why my samples are coming up negative. Has anyone experienced this?

 

I know your post is 13 years old, but it got me thinking. Usually we QC out Anti-C3 reagent with the tube method, but I was curious to see what would happen if I used buffered gel card instead.  This is what happened: 

 

1) I converted my 3% C3 control cells to 0.8%.

2) I pipetted 50 uL this 0.8 cells into 2 wells of the buffered gel card, one labelled "pos" and the other "neg". 

3) I pipetted 25 uL of Anti-C3 into the "pos" well.

4) I pipetted 25 uL of saline into the "neg" well. 

5) I centrifuge the card for 10 minutes. 

 

The results are as expected. The "pos" was 4+ and the "neg" was 0.  

This was a stronger positive result compared to the tube method, in which we usually get is a 1+ to 3+ reaction.

 

My conclusion is that maybe there was something wrong with your gel cards. Did you use MTS diluent or saline when you converted your cells to 0.8? I am not sure if that would matter, but considering your samples were weakly positive to begin with, maybe the conversion "diluted" the samples.