Endotoxin Test ProtocolFor use of the Charles River Endosafe Endotoxin Portable Test System (PTS)

I. Materials Needed:

Charles River Endosafe PTS readerEndotoxin test Cartridges: Catalog # PTS2005F Sterile, Endotoxin Free Pipette tips0.25µL Pipette for sample dispensingPipettes for dilutionsSterile, Endotoxin free water for dilutionsSterile Endotoxin free sample and dilution containersGloves

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: For 5-0.05 EU/ml Assay sensitivity:1 EU/ml: Reaction Time: X seconds 5 EU/ml: Reaction Time: A seconds0.1 EU/ml: Reaction Time: Y seconds 0.5 EU/ml: Reaction Time: B seconds0.01 EU/ml: Reaction Time: Z 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 Termsa. 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/Mi. K = the threshold pyrogenic dose in endotoxin units/kg

Experimentally determined in rabbits, confirmed in humansK = 5 EU/kg for drugs with a route of administration other than intrathecalK = 0.2 EU/kg for drugs administered intrathecally

ii. M = maximum human dose of product/kg of body weight in a single hour periodIf 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 limitii. MVD = (Endotoxin Limit)(Concentration of sample solution)

λWhere: Endotoxin Limit = K/MConcentration of sample solution = concentration in units/mLλ = 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 Parametersa. 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 interpretationa. 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.