Last time I meet articles where for conjugation enzyme (HRP) - antibody is used polimer-HRP. What for usual-HRP is substituted polimer-HRP?
It's a kind of marketing. The antibody has a MW of 160 kD, HRP 40 kD. From many tests we know, that a molar ratio of 1 : 4 is in most cases sufficient. Higher molar ratios leed to sterical hindrances, higher background reactions. There a only a very few cases where a HRP polymer is helpful.
Thank you very much for your answer. I used HRP 40 kD (high-quality) many years ago and my conjugates according to B. Wilson and P. Nakane (a weight ratio of 2 : 1) were enough good.
We use streptavidin polymerised HRP (about 20). Since we have a biotin-strepatvidin step we see no steric hindrance but we do achieve a 10 fold increase in sensitivity of the ELISA (PeliKine cytokine ELISA). Note that you have to use caseine in your dilution buffer to reduce background.
Dear Dr. John Voorn,
Many thanks for your interesting answer.
I am agree caseine reduce background. Qualitative BSA ( V fraction) is good too for it.
Wish you great successes.
L.Vergun
Hi John, the sensitivity in an optimized ELISA depends only on the affinity of the primary and secondary antibody, not on the labeling. You can increase the slope of the curve and also the background reaction.
In immunohistochemical reactions it's important to have as much as possible enzyme at the point where the antigens was detected ...
Dear Stephan, indeed the affinity of the antibodies is crucial for the sensitivity but the detection system is crucial to show all bound antibodies. The trick is to increase signal without increasing the background and polymerised-HRP diluted in casein can achieve this. To even more increase the sensitivity of the ELISA we perform all incubations on an orbital shaker, this increases the slope and shortens the linear range but we are working with sensitivities < 1 pg/ml for our cytokines.
We also tested poylimerised-HRP in ELISPOT and histochemical stainings and this also worked perfect (comparable to the ABC method).
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