Are antibodies already HRP-conjugated more sensitive than the two steps "antibody + washing + anti species HRP-conjugated"?
If yes, kind you suggest any kit to do it?
I would say that increases your signal (the already-conjugated) and you can expect better signal/noise ratio. If you are looking to be more sensitive, you may want to biotinylate your first antibody and use streptavidin-polyHRP for detection. Very easy-to-use kits out there.
I'm currently using an anti-HIs-HRP antibody in a sandwich ELISA to detect secreted proteins. I don't think it is as sensitive as the biotin-extravadin methods, but it definitely eliminates the need for a secondary antibody, and thus saves quite a bit of time.
the two steps method of "antibody + washing + anti species HRP-conjugate" is better as it increases sensitivy and specificity.
Hi.If you want to give increase sensivity until pgr/ml. you will use biotin and avadin antibody kit .
Hello, the two steps procedure is absolutely more sensitive. The secondary anti-species antibody i can use has to be tested in ELISA. For me, the avidin/streptavidine coupled to biotin strategies have show a limited field of application dependent of the samples you have to test. Be carefull of the natural biotin in your sample for example that induce high background.
It's a kind of marketing arguments.
You can compare the direct antigen detection with an direct HRP labeled antibody vs. the indirect method (antigen-specific antibody, washing, anti-species-antibody-HRP). You should be sure to use the same antigen-specific antibody for an experiment like this. The lower detection limit will be the same. You can find some increased background, an in general elevated signal.
Why is it so: The highest influence on the detection limit (sometimes it's mixed with the sensitivity: see also ICH guidelines) has the affinity of your solid phase antibody to your antigen, the second highest has the secondary antibody. You can estimate this from the basic chemical low of mass action.
Thats why the quality of the antigen-specific antibodies is so important.
PS: the specificity could never be changed due to the same chemical basics.
Hi,
keep in mind that you need to find the right assay for your application. In some cases you might find better performance (ratio of sensitivity/specificity) using the one step procedure, while for other applications you will have to use a two step procedure to gain good sensitivity and specificity.
In my experience the two step procedure promotes better specificity and using the Biotin/Strep system usually gives you better sensitivity while at the same time preserving good specificity.
Hi Ralf,
make sure that you are using the right terms. The sensitivity of an assay is the proportion of right positive results.
The Specificity is the proportion of negatives in a binary classification test which are correctly identified.
This terminology can be used for instance in the detection of disease specific antigens/antibodies/or commonly analyts.
Here in a two site binding assay the specificity is defined as the specificity of the used antibodies. This could never be changed within a given assay procedure until the antibodies are not changed. The sensitivity is much better named by "the lower detection limit". The smallest difference in concentrations is named "analytical detection limit"
see also ICH guideline ICHQ2_R1: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf
Hi Stephan,
of course you are right. Excuse me for being imprecise on the terms. Please refer to my answer in the case of medical diagnostics (e.g. detection of disease marker in a western blot or dot blot system).
Hi Ralf,
the best summary ever about the biometrical terms was given by Abel, U.(1993)
Die Bewertung diagnostischer Test. Hippokrates-Verlag, Stuttgart. He has done this for all kinds of diagnostic tests used commonly in tumor diagnostics (including radiological screening etc.) The basics behind are always the same. The only problem, this book not available on the market only in second hand bookshops.
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