Endotoxin Test Protocol
For use of the Charles River Laboratories
Endoscan-V Kinetic Turbidimetric Software and Reader
I.
Materials Needed:
Charles River Laboratories Endoscan-V Software and PC
BioTek ELx808 reader
KTA Lysate 0.06EU/mL Sensitivity (Catalog #R15006)
Control Standard Endotoxin -CSE (Catalog # E110)
Lysate Reagent Water – LRW (Catalog #W110)
Pyrogen-free 96 well test plates (Catalog #M905)
Sterile, Pyrogen Free Pipette tips
Pipettes for sample dispensing
Pipettes for dilutions
Pyrogen-free sample and dilution containers (Fisher Catalog#149592A)
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 Laboratories Endoscan-V Kinetic Turbidimetric system consists of
spectrophotometer, PC and software system using Kinetic Turbidimetric (KTA) Limulus
Amebocyte Lysate (LAL) and Control Standard Endotoxin (CSE) for the creation of a
known value standard curve. The purpose of the test is to provide quantitative Endotoxin
results for in-process and end product samples.
The assay sensitivity available for use is 0.06EU/mL using a standard curve of 5-0.05
EU/mL
III.
Making Standard Curve Solutions
1. Allow all reagents to warm to room temperature before being testing. Also
remember to turn the reader on prior to testing to allow it to run through its start
up process and warm the unit to 37°C.
2. Once the CSE has warmed to room temperature it can be used to make dilutions
for your known standard curve. If the CSE has not been rehydrated, rehydrate it
now per the instructions on the Certificate of Analysis that came with that lot of
CSE (see CRL CofA folder located next to the Endoscan-V computer).
a. NOTE: When it is first rehydrated the CSE vial must be vortexed for a
minimum of 5minutes prior to initial use and vortexed day of use for 12minutes at minimum.
3. Depending on the concentration of the CSE once rehydrated as described in the
CofA you will make dilutions to obtain at minimum 1mL of standard solutions of
the following values: 5.0 EU/mL, 0.5EU/mL and 0.05 EU/mL
a. EX: CSE labeled as – 1000EU/mL
i. Perform a 1/20 dilution → 0.5mL/9.5mL → 50 EU/mL
↓
ii. Perform a 1/10 dilution → 0.5mL/4.5mL → 5.0 EU/mL
↓
iii. Perform a 1/10 dilution → 0.5mL/4.5mL → 0.5 EU/mL
↓
iv. Perform a 1/10 dilution → 0.5mL/4.5mL → 0.05 EU/mL
IV.
Setting up Reader Plate Template
1.
Open the Endoscan-V software on the computer in NB1-10 by clicking on
the Endoscan-V icon on the desktop and log on to the system. The screen
will prompt you to choose to open an existing file or create a new file –
choose new plate.
2.
Next the Properties screen will pop up. On this screen you can see the
default setting for the system (i.e. read times, wavelength setting, read
intervals). You may also enter plate properties at this time (i.e. CSE,
lysate and LRW lots and expirations) and well group properties. After
closing this screen you will be able to begin setting up your plate template.
Each 96 well plate will run one set of standards, one negative control and
20 samples.
3.
First begin by clicking and dragging the 5.0; 0.5; 0.05 standard curves to
the top left hand corner of the plate. Next type CTRL1 into the next set of
wells below (this is your LRW negative control and the system will not
provide an automatic spike for this well group; you only need to type it in
one well – the other will populate automatically).
4.
At this point you may start setting up the plates depending on the samples
you have to run. If you are running standard stock product samples, use
the product database (you will see this below your template) to “drag and
drop” the samples you would like to test. You will need to add/enter the
lot number of the product you are testing each time as well as check and
confirm the dilution is correct.
5.
If you are testing samples that are not in the database (i.e. customs or other
samples) simply type the name of the sample in the appropriate well and
hit enter – the system will automatically create one sets of replicates (this
it’s default setting) and one set of automatic spike wells (positive product
control). Continue adding samples until the plate is full
a. NOTE: It is advisable to run a full plate of samples if at all possible
to ensure cost efficiency
b. NOTE: One bottle of lysate will allow you to run 10 samples +
standards and negative control; you will need 2 full bottles of lysate to
run a full plate of 20 samples, standards and control.
c. NOTE: If you are running dilution series of one sample it is
advisable for you to add these samples in succession on the plate as
this will make reading and interpreting results easier.
V.
Pipetting the 96 well plate
1.
2.
3.
4.
Once the template is complete on the computer you can begin to
aliquot the samples onto the 96 well plate per your template.
i. NOTE: Be cautious when pipetting so as not to splash into
adjacent wells and to ensure the samples are dispensed into the
proper wells as laid out in the template you created.
Aliquot 100µL of all samples, standards and negative controls. Begin
by aliquoting the LRW, then the standards from lowest to highest
concentration (you may use the same pipette tip for this task). Next
aliquot your samples as laid out on the computer template.
i. NOTE: A minimum of 400µL of sample is required to run this
test (100µL for each well, 2 sample replicates and 2 spike
replicates).
Next aliquot the spike into the designated spike wells. Use 10µL per
spiked well of the 5.0 EU/mL standard to do this. Ensure you do not
splash into adjacent wells when you do this.
i. NOTE: The spike concentration should be the equivalent of the
middle of your standard curve (i.e. 0.5 EU/mL). In order to
achieve this concentration on the plate you must use the 5.0
EU/mL standard as 10µL of spike into 100µL of sample is
effectively a 1/10 dilution providing you with your desired 0.5
EU/mL spike concentration.
Lastly aliquot 100µL of rehydrated lysate (rehydrate lysate per
instructions on the vials – do not vortex, simply rotate up and down
until solution turns clear before use) to each well. Once the lysate is
dispensed into the wells the test has effectively begun.
i. Look over the plate quickly and check for bubbles and that you
have dispensed lysate into all the wells prior to placing the plate in
the reader.
NOTE: Once lysate is rehydrated discard the rubber stopper
and replace with parafilm.
5.
VI.
Immediately upon dispensing lysate place the plate in the reader with
the top off and close the lid and hit the green arrow on the top just
below the tool bar. Or click Tools and Start Collection. The unit will
shake for a short period of time (this is normal and acts to remove
bubbles from the wells and mix the samples) and then data collection
will begin and the system will run the test automatically and shut off
automatically.
Reading Reports, Interpreting Results and Troubleshooting
1.
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 standard and control CV of <10% and a
sample CV of <20%. Anything higher than this indicates a significant
variation in the reaction times of the two replicates indicating an
invalid assay.
2.
Endotoxin Calculations:
a. 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
b. 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*****
VI.
Media Lab Sample Acceptance Criteria Table
Description
Grace’s Insect Cell Media
Cell Culture Media
MilliQ
Buffers
L-Glutamine
Penicillin/Streptomycin
Cell Culture Media Concentrated
Acceptance Criteria
≤20.0EU/mL
≤1.0EU/mL
≤0.05EU/mL
≤1.0EU/mL
≤0.5EU/mL
≤0.5EU/mL @ 5mL/500mL in DMEM
≤1.0EU/mL of a 1X Solution