immunofluorescence: direct vs. indirect

Martin Offterdinger a8803349.nospam at
Wed Jun 24 01:47:46 EST 1998

On 23 Jun 1998 05:15:06 -0700, peter.cherepanov at
("P.C.") wrote:

>What would be the best choice for doing immunofluorescence:
>to obtain labeled monoclonal antibodies or to use 
>labeled secondary antibody ?
>I heard that using secondary antibodies increases sensitivity - 
>is that true ? 
>(As I understand the primary antibody is usually an IgG, and 
>the bulk of the secondary is IgGs too, which are monomeric. 
>Therefore, I would expect only a drop in sensitivity and 
>specificity. Using biotinilated antibodies may have an advantage, 
>since avidine is tetrameric and can bring more FITC residues to 
>the spot (?))
>I need not only sensitive, but also very specific detection. 
>(surface staining of monocytes, I plan to use CD14 antibody).
>Would like to hear any hints.
The use of sec. Antibodies increases sensitivity; because normally
sec. Antidodies are normally polyclonal, that means that more than one
AB molecule will bind to your primary antibody thus leading to more
fluorochromes at your target  site. This is even more true for
biotinylated sec. ABs and avidin-FITC as a tertiary compound. The sec.
AB (polyclonal!!) has usually more than one biotin linked - more than
one avivdin will usually bind to one sec. AB molecule. You can expect
an increase in sensitivity ( but also an increase in background ).
Martin Offterdinger
Internal Med.I,Dept. Oncology
University of Vienna
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