There are several possibilities to obtain the recovery over 100% in the
Bacterial Endotoxins Test (BET).
Assuming you used a kinetic LAL method and the spiked endotoxin was
prepared from an endotoxin dilution used for the standard curve, there
are three possibilities.
(1) Bias from the shape of the standard curve
A standard curve with a kinetic BET assay is usually bent. Most of
KTA and KCA with microplate readers usually give concave standard
curves. Since the regular kinetic BET techniques use the linear
regression, there comes a bias from the shape of the standard curves.
Assuming the standard curve used was concave, a calculated endotoxin in
the middle of the standard range is larger than the actual endotoxin
concentration. Spiked endotoxin concentrations are usually in the
middle of the standard range. Therefore, the recovery of the spiked
endotoxin is usually higher than 100% with a concave standard curve, if
there is no interference. You can see this bias if you calculate
endotoxin concentrations from the onset times of the standard dilution
used. I published some of our data relating this issue on the BioProcess
International in 2010. (See the website
(2) Endotoxin activity change by the sample
Endotoxin activity can be changed by the sample. We see more
inhibition but some of them are enhancement. For example, detergents and
proteins can change the micelle size of endotoxin in the solutions to
change the activity.
(3) Enhancement of the LAL reaction by the sample
If the pH of the reaction mixture is changed, the sensitivity of
the LAL reagent will be changed. The magnesium concentration in the
reaction mixture is also critical for the sensitivity of LAL reagent.
Denatured protein may increase the degree of turbidity change, resulting
in enhancement by providing shorter onset times in KTA.
I recommend you checking the bias from the shape of the standard curve
first because this bias is often observed and the degree of the bias is
usually beyond expectation. If the bias is not so large, then the
interference is enhancement. It is hard to know which caused the
enhancement, endotoxin or the LAL reaction. However, more dilution
should provide a recovery close to 100%. It is a good idea to test a
positive water control prepared by the same manner for the positive
The acceptance criteria of the endotoxin recovery in the sample is
between 50% and 200%, according to the BET. I guess this was decided
because of the agreement of the acceptance range for the gel-clot
method. I personally like to see the recovery at least 75%-150% (maybe
80%-130%) for my samples with a kinetic method, although there is no
I hope this information is helpful for you.
Thank you and best regards,
Masakazu Tsuchiya, Ph. D.
Senior Research Scientist
Endotoxin and Microbial Detection
E-mail: [log in to unmask]
Accelerating Drug Development. Exactly.
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From: The Pharmaceutical Microbiology Forum Email List
[mailto:[log in to unmask]] On Behalf Of ADET (Anders Thorn)
Sent: Monday, May 16, 2011 5:10 AM
To: [log in to unmask]
Subject: [PMFLIST] Interference in the LAL test
When you make a screening of different dilutions of a sample for LAL
test that cause inhibition of the assay the recovery of the positive
product (%PPC recovery) increase with the dilution. Often you will see
that the curve (% PPC as function of the dilution) level off when the
%PPC recovery is above 100 % (for instance at around 130 % which
corresponds to the %PPC recovery of LAL reagent water or WFI tested in
your assay) and not when the %PPC recovery is exactly 100 %.
When is a solution considered free for interference in the LAL test? Is
it when you dilute your sample so you get a %PPC recovery at around 100
% or is it when you dilute your sample to a degree where the curve (%
PPC as function of the dilution) level off (for instance at around 130
Thanks and best regards,
Novo Nordisk A/S
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