Re: Antibody titrations (2004)

Hmmm.  Most of the time I agree with you, Mario.  But this time I partially 
disagree.  For those labs that do titrate each new lot of conjugated 
antibody they receive, cell number does become a factor.  Especially if the 
goal is to use the antibody concentration just at the top of the saturation 
curve.	We routinely recommend this type of titration to avoid the 
increased backgrounds and waste of reagent that you indicate.  We routinely 
see poor staining results based on staining intensity when cells are not 
counted prior to staining and antibody concentrations are not accounted for 
with regard to cell number.

I would agree that if most labs use the manufacturer recommended 
concentration for staining, that a wide range of cell numbers can be 
accommodated.

Another twist is immune assays that involve competition of antibodies.	In 
this case, antibody/cell ratios are critical as the labeled antibody must 
be a sub-saturating levels to see the competition.

All the best,
Julie

At 04:35 PM 4/22/2004 -0400, Mario Roederer wrote:
>It is time for my annual reminder that antibody amounts for staining are 
>based on the concentration of the antibody, not the number of cells being 
>stained*.
>
>I.e., the antibody:antigen ratio is such that, until you stain more than 
>50 million cells, in general, you are staining in vast antibody excess and 
>the cell number is irrelevant.
>
>So if you stain 2 million or 5 million (or 100,000) cells, do NOT change 
>the amount of antibody you are using, you simply waste reagent and might 
>increase background!
>
>mr
>
>(* PS, as a side note:  very high affinity antibodies are actually "bad" 
>in this regard.  The optimal titre for these reagents is so low that in 
>fact they can become cell-number (antigen-amount) dependent at typical 
>cell numbers, about 1 million per stain.  On the other hand, low affinity 
>antibodies are nice in that you could stain 1 billion cells with the same 
>amount that you stain 1 million cells and have no decrease in fluorescence!
>
>For a full explanation of these issues, and a brief description of how to 
>titrate antibodies properly, see Kantor, A. and Roederer, M. (1997). FACS 
>analysis of lymphocytes. In: Handbook of Experimental Immunology (Fifth 
>Edition), Herzenberg, L. A., Weir, D. M., Herzenberg, L. A. and Blackwell, 
>C. (ed.), Blackwell Science, Cambridge, pp 49.1-.13.
>
>2.4MByte PDF available upon request)
>
>
>At 12:59 PM +0800 4/22/04, Michael Wong wrote:
>>Dear Noosheen,
>>
>>Our lab has just purchased the eBioscience ZAP-70 FITC antibody. I wonder 
>>how much antibody is needed to incubate with 1x10^6 cells. According to 
>>the NEJM article, they are using 1.5ug antibody per 500,00 cells. But 
>>they are using Upstate unconjugated antibody. Is this the same for this 
>>eBioscience antibody?
>>
>>Regards,
>>Michael