Endotoxin Test Protocol
For use of the Charles River Endosafe
Endotoxin Portable Test System (PTS)
I.
Materials Needed:
Charles River Endosafe PTS reader
Endotoxin test Cartridges: Catalog # PTS2005F
Sterile, Endotoxin Free Pipette tips
0.25µL Pipette for sample dispensing
Pipettes for dilutions
Sterile, Endotoxin free water for dilutions
Sterile Endotoxin free sample and dilution containers
Gloves
NOTE: Do not use polypropylene containers for storage or dilutions of
samples as Endotoxin in the sample will stick to the container and lower
recovery for the sample.
II.
Purpose and Background:
The Charles River Endosafe System consists of a handheld spectrophotometer and
cartridge system with Limulus Amebocyte Lysate (LAL), Control Standard Endotoxin
(CSE), and Synthetic Substrate for bench top chromogenic analysis all dried onto a single
use cartridge. The purpose of the test is to provide rapid (less than 15minutes)
quantitative Endotoxin results for in-process and point of use samples.
Each lot of cartridges has a pre-calculated CSE reaction time curves already programmed
into the system. Once the lot number of the cartridge being used is entered into the PTS
reader the system will calculate the result based on the pre-programmed standard curve
reaction times.
There are two assay sensitivities available for use: 1-0.01 EU/ml and 5-0.05 EU/ml. The
standard curve reaction times will be based on the sensitivity of the assay.
For 1–0.01 EU/ml Assay sensitivity:
1 EU/ml: Reaction Time: X seconds
0.1 EU/ml: Reaction Time: Y seconds
0.01 EU/ml: Reaction Time: Z seconds
For 5-0.05 EU/ml Assay sensitivity:
5 EU/ml: Reaction Time: A seconds
0.5 EU/ml: Reaction Time: B seconds
0.05EU/ml: Reaction Time: C seconds
Based on the reaction times in the standard curve assay the system will calculate the
Endotoxin value in the sample being tested based on where the sample reaction time falls
on the known standard curve. If no Endotoxin is detected in the sample the result will be
reported as <0.01 EU/ml or <0.05 EU/ml based on the assay’s lowest detection level (or
sensitivity).
III.
Test Procedure and Reader Set-Up:
1. Allow the desired number of cartridges to warm to room temperature prior to use.
All materials for the assay are optimized to be run at 37oC so it is important to
allow all materials, including sterile water for dilutions to warm to room
temperature before using. At the same time turn on the PTS reader to allow it to
perform a self test and warm up to 37oC (this will take 3-5 minutes).
2. While materials are warming up turn the UV light on in the hood if dilutions are
to be performed. All dilutions need to be preformed in the hood to prevent
contamination of the samples prior to testing. Dispensing of the samples into the
cartridge can be done on the bench top.
3.
Dilute all samples except Milli-Q system and some buffer samples to 1:1000
using sterile water for Irrigation. This is especially important for colored samples
(i.e. containing phenol red) as this is a chromogenic assay and any color in the
sample itself will interfere with the spectrophotometer’s ability to read reaction
related color changes.
4. Once the reader has completed its self test the display screen will read “Insert
Cartridge”. Wearing gloves remove the cartridge from the wrapper and insert it
into the reader with the sample wells facing up. Be certain not to touch the
cartridge anywhere but the handle as touching the cartridge itself, especially the
optical cells portion can interfere with the reader’s ability to read absorption
changes properly.
5. Once the cartridge is inserted it will prompt you to enter an OID – operator
identification. Enter the name of the person performing the assay at this time.
6. The prompt will now ask for a lot number. Enter the lot number of the cartridge
at this time. It is critical that you enter the correct cartridge lot number as this is
how the system identifies standard curve reaction times and assay sensitivity. If
you are using a lot number that has not been used with the reader before it will
prompt you for a calibration code. This code can be found on the certificate of
analysis that came with that particular lot of cartridges. Enter this number
correctly as it is tied to the standard curve and archived spike concentration for
that particular lot of cartridges. If the lot number and corresponding calibration
code is already programmed into the system the reader will indicate “Endotoxin
checking” and will go directly to the next prompt skipping the calibration code
prompt.
7. The system will ask you to confirm cartridge lot number. If it is correct hit enter
to continue.
8. You will now be asked to enter the sample lot number. For Media lab samples
enter the batch number of the media being tested followed by a hyphen and the
bottle number the sample was removed from.
9. Now you will be prompted to enter the sample ID. For media lab samples this
will be the product number of the media being tested.
10. Enter the dilution factor. The reader automatically defaults to a dilution factor of
1 (i.e. undiluted). If the sample is undiluted press enter to continue. If the sample
is diluted enter the dilution factor now. If the sample is a 1/10 dilution enter 10, if
it is a 1/1000 enter 1000 etc.
11. The reader will now prompt you to enter your sample. Carefully pipette 0.25µL
of sample into each of the sample wells. Ensure good pipette technique as any
bubbles will cause the test to abort because the bubble will interfere with the
optical reader. After you press enter the sample will be automatically drawn up
into the cartridge. The reader will indicate that it is prepping sample (this will
take approx. 4mins.). Once the PTS finishing prepping the sample it will indicate
test in progress.
12. Once the test is complete the reader will display the results of the test on the
screen and prompt you to remove the cartridge. Once you remove the cartridge
place it back into its wrapper and discard it into the biohazard container.
IV.
Retrieving and Transferring Results from PTS system
1. Connect the reader to the computer in the appropriate COM port. Open the PTS
logger folder on the desktop or the R drive. Click on the PTS logger icon to open.
Choose connect and indicate which COM port you want to connect to – the
computer in NB1-15 has only one COM port so enter 1 in the box and click
connect. The reader will confirm connection. If a connection cannot be made
check your connection to the reader itself and the computer and ensure it is tight
and retry. If this does not work close the logger and restart before attempting to
connect again.
2. Next select log and open/close log. This will complete connection to reader.
Enter the print menu on the reader by pressing 4 from the main menu. Print menu
will appear on the screen, continue pressing menu till you get to the option you
want. You can print all tests, print by date, print last test etc.
3. If you print last test the last test performed will automatically scroll onto the log
screen. If you want to print all tests on a certain date, enter the date desired and
press enter and all tests performed on that date will scroll on the screen. Close or
minimize the log window and open the notepad file in the PTS logger folder (this
is where the information scrolled onto the logger screen is temporarily stored).
Save this file to the R-Drive/Cell Core/My Documents/Endotoxin Test Results.
V.
Reading and Interpreting Results
1.
Definition of Terms
a. Spike – positive product control. A sample “spiked” with a known
level of Endotoxin. The archived spike concentration for a particular
lot of cartridges can be found on the Certificate of Analysis for that lot.
This and the standard curve reaction times are entered in the PTS
system in the form of the “Calibration Code”.
b. Endotoxin Limits: = K/M
i. K = the threshold pyrogenic dose in endotoxin units/kg
Experimentally determined in rabbits, confirmed in humans
K = 5 EU/kg for drugs with a route of administration other
than intrathecal
K = 0.2 EU/kg for drugs administered intrathecally
ii.
M = maximum human dose of product/kg of body weight
in a single hour period
If the pediatric dose/kg is higher than the adult dose, then
the pediatric dose/kg = M
c. Maximum Valid Dilution:
i. MVD provides an upper bound for dilution that still provides
for endotoxin detection at the endotoxin limit
ii. MVD = (Endotoxin Limit)(Concentration of sample solution)
λ
Where:
Endotoxin Limit = K/M
Concentration of sample solution = concentration in units/mL
λ = confirmed label claim sensitivity of the GC lysate or lowest
point on the referenced standard curve
*If the limit is in EU/mL, the concentration of the sample
solution is not needed
*****MVD is unitless…it is a dilution factor*****
2.
Valid Test Parameters
a. Spike Recovery: A valid test has a spike recovery between 50-200%
i. ([Spike Well – Neg Product well)/Spike Value]*100 = Spike
Percent recovery. If this value is out of range dilute the sample
until it is brought within range – allowing for maximum valid
dilution.
b. CV: Coefficient of Variation = (Standard deviation/mean) of the test
replicates. A valid test has a spike and test CV of <25%. Anything
higher than this indicates a significant variation in the reaction times of
the two replicates indicating an invalid assay.
3.
Results interpretation
a. Wells 1 and 3 are the sample test replicates and wells 2 and 4 are the
spiked control replicates.
b. The reaction onset times for the spiked wells should correspond with
the middle of the standard curve for that assays sensitivity level (i.e. an
assay with a sensitivity of 0.05 EU/mL has a standard curve with
reaction times taken at 5 EU/mL, 0.5 EU/mL and 0.05 EU/mL the
spike concentration should be close to 0.5 EU/mL or the middle of the
standard curve. The reaction times of the spiked wells should
correspond with this point on the standard curve and these times can
be found on the Certificate of Analysis for the lot of cartridges being
used.
c. The reaction onset times of the sample wells will correspond to the
level of Endotoxin detected in the sample based on where the reaction
time lies on the standard curve for that lot of cartridges.
d. If a sample is diluted the result given is adjusted for the dilution factor
and reflects the Endotoxin Level in the undiluted sample.