Associates of Cape Cod – LAL Assay

Product Reconstitution

• CSE is reconstituted with LAL Reagent Water (LRW) (or equivalent). Please refer to the Certificate of Analysis when using CSE. A Certificate of Analysis exists for each CSE-lysate lot combination.
• Chromo-LAL lysate is reconstituted with LAL Reagent Water (LRW). It can also be reconstituted with Glucashield®(1→3)-ß-D-glucan inhibiting buffer, to render the assay endotoxin specific.
• Pyrochrome lysate is reconstituted with an optimized Pyrochome reconstitution buffer (C1500). Pyrochrome can also be reconstituted with Glucashield buffer, a (1→3)-ß-D-Glucan inhibiting buffer, to render the assay endotoxin specific (CG1500).
• Pyrosate is reconstituted directly with the sample by adding 0.5 mL to the sample tube (SPL). After approximately 60 seconds of gentle mixing, 0.25 mL is transferred to the endotoxin tube (PPC). The lot-specific incubation time at 37±1°C is given on the Certificate of Compliance.
• Pyrotell multi-test vials may be reconstituted with LAL Reagent Water (LRW), Pyrosol^® buffer or Glucashield^® buffer. Glucashield (1→3)-ß-D-glucan inhibiting buffer is used to render the assay endotoxin specific.

Product Packaging

• The most commonly used size is 0.5 µg/vial. The 0.5 µg/vial is also used for testing depyrogenation ovens. The 125 µg/vial is typically used for special purposes, e.g., making endotoxin challenge articles for validation of depyrogenation processes. The 10 ng/vial CSE is available in packs of five vials.
• Chromo-LAL lysate is sold by the vial for greater ordering and testing flexibility. This allows you to order the amount of lysate that is right for you.
• Pyrochrome is offered with a choice of reconstitution buffer and is recommended for use with the 10 ng/vial Control Standard Endotoxin (CSE, EC010). Certificates of Analysis, specific to the Pyrochrome and CSE lot, can be obtained from ACC.
• The Pyrosate kit is available in a 10 test kit and a 30 test bulk package for each sensitivity. Each kit contains sample test tubes (SPL) and endotoxin test tubes (Positive Product Control-PPC). A Certificate of Compliance, specific to the Pyrosate and CSE lot, can be obtained from ACC or online.
• Pyrotell lysate is available in single test vial (STV), 2 mL or 5 mL fill sizes. The 2 mL and 5 mL vials are sold individually. STV’s are sold in packs of ten vials. Control Standard Endotoxin (CSE) is provided separately with a Certificate of Analysis, specific to the Pyrotell lysate lot with which it will be used.
• Pyrotell^®-T lysate is available in individual multi-test vials. Each vial contains reagent for approximately 96 tests.

Product Stability

• CSE is reconstituted with LRW or equivalent, and is stored at 2–8°C before and after reconstitution. Once reconstituted, the maximum storage time for the different CSE preparations is 10 ng/vial: 7 days, 0.5 µg/vial: 4 weeks, 125 µg/vial: 3 months. *CSE should not be frozen.
• The reconstituted lysate is stable for 24 hours at 2–8°C or for two weeks at -20°C or colder if frozen immediately after reconstitution. Reconstituted Chromo-LAL lysate may be frozen and thawed once.
• Once reconstituted, Pyrochrome is stable for 8 hours when stored at 2–8ºC.
• Pyrosate is stable at room temperature and does not require refrigeration for shipping or storage.
• The reconstituted lysate is stable for 24 hours at 2–8°C or for up to 3 months at or below -20°C. May be frozen onceand will retain activity for as long as 3 months if stored at or below -20°C.
• Once reconstituted, Pyrotell^®-T lysate is stable for 24 hours, if stored at 2–8°C. Pyrotell^®-T lysate may be frozen onceand will retain activity for as long as 3 months if stored at or below -20°C.

Available Models

Chromo-LAL

Kinetic Chromogenic Formulation

Chromo-LAL lysate is lyophilized with substrate reagent and buffers. It is optimized for the kinetic chromogenic LAL test method in microplate readers. Chromo-LAL is a buffered, stable and robust lysate, suitable for quantitative testing of a wide range of samples.

Product Sensitivity

The sensitivity for chromogenic assays is determined by the lowest standard concentration on the standard curve used for the assay. The maximum sensitivity of Chromo-LAL is 0.005 EU/mL.

Sample to Lysate Ratio

In the Chromo-LAL test, reconstituted Chromo-LAL reagent is used at a ratio of 1:1 and a volume of 100 µL.

Test Performance

The Chromo-LAL/sample mixture is incubated at 37 ± 1°C in a microplate reader equipped with a 405 nm filter. Software is used to construct the standard curve and calculate the endotoxin concentrations.

Control Standard Endotoxin (CSE)

Control Standard Endotoxin (CSE) is a widely used standard for endotoxin testing. It is a purified extract of E. coliO113:H10, the same strain used for the United States Pharmacopeia and the European Pharmacopeia reference standard endotoxin (RSE).

CSE is used as an economic alternative to the RSE. CSEs are standardized against the RSE as indicated on the Certificate of Analysis, so that results can be reported in Endotoxin Units (EU) and International Units (IU). CSE can be used for all LAL testing. A 10 ng/vial CSE is made specifically for use with our Pyrochrome chromogenic reagent.

Test Performance

CSE is used to make standard curves and controls when performing the LAL assay. The concentrations used are dependent on the type of assay (e.g., chromogenic, gel-clot, or turbidimetric) and for photometric methods, the detection range required.

Key Features

• CSE is a stable, preparation of endotoxin that can be used in all LAL testing
• CSE is especially useful for depyrogenation studies
• Certificates of Analysis for each CSE-lysate lot pairing gives a potency that is specific to the unique lot combination
• CSE can be reconstituted to achieve specific endotoxin concentrations

Pyrochrome

Chromogenic Endotoxin Testing Reagents

Pyrochrome is a versatile quantitative reagent that is used to perform kinetic or endpoint assays. It is a sensitive and flexible reagent that can be used for testing in compliance with the USP, EP and JP bacterial endotoxins test chapter. Pyrochrome is offered with either Pyrochrome Buffer or for endotoxin-specific testing, with Glucashield^® Buffer. Pyrochrome can be used with the new Pyros Kinetix^® Flex tube reader at a 1:1 and economical 4:1 ratio.

Pyrochrome can be used for a wide variety of endotoxin tests, ranging from standard water testing to samples requiring high sensitivity, such as intrathecal products and those requiring high dilutions to overcome interference.

It is also offered in a diazo kit for endpoint tests. (The diazo reagents shift the absorption spectrum making it especially useful for testing samples with color interference.)

Product Sensitivity

The sensitivity for chromogenic assays is determined by the lowest standard concentration on the standard curve used for the assay. The maximum sensitivity of Pyrochrome is 0.001 EU/mL when run in Pyros Kinetix Flex tube reader or incubating microplate reader with Glucashield Buffer.

Sample to Lysate Ratio

In the Pyros Kinetix Flex tube reader, Pyrochrome can be used at an economical ratio of 4:1 using 50 µL of reagent per well or at 1:1 using 100 µL/well. In a microplate reader, the reagent is used at a ratio of 1:1 and a volume of 50 µL/well (60 tests/vial) or 100 µL/well (30 tests/vial).

Test Performance

The Pyrochrome/sample mixture is incubated at 37±1°C and read in a microplate reader equipped with a 405–410 nm filter or 540–550 nm filter for diazo method. The time of incubation is dependent on the lowest standard concentration in the standard curve. Software is used to construct the standard curve and calculate the endotoxin concentrations.

Key Features

• Pyrochrome^® sets the standard for quality and redefines the way chromogenic reagents should perform
• Can achieve a maximum sensitivity of 0.001 EU/mL
• Option to use a sample to lysate volume ratio of 4:1 or 1:1
• Highly cost effective chromogenic reagent
• Use of polynomial regression for calculations

Pyrosate Kit

Rapid Endotoxin Detection*

The Pyrosate kit is developed as an easy-to-use LAL gel-clot test. The assay does not require special training or laboratory supplies. The step-by-step illustrated instructions allow the user to perform assays within minutes. The Pyrosate kit provides rapid results and is especially convenient for research, testing water and dialysate.

Product Sensitivity

The Pyrosate kit is available in sensitivities of 0.03 EU/mL, 0.125 EU/mL and 0.25 EU/mL. The test may be as short as 30 minutes, depending on the sensitivity.

Test Performance

The Pyrosate kit is a rapid gel-clot test that contains a 2λ* endotoxin tube (PPC) matched to the sample (SPL) tube for each sensitivity. This feature is unique to the Pyrosate assay and ensures the validity of the test. The endotoxin tube (PPC) assures that the sample does not interfere with the test, ruling out false negatives. Pyrosate is formulated to eliminate false positives due to (1→3)-ß-D-glucans. This endotoxin specific reagent does not require additional blocking buffers.

Applications

• Hemodialysis
• Water and Water Systems
• Filter Industry
• Research
• Final Product Release

Key Features

• Shorter Assay Time
• Endotoxin Specific
• No Dilutions Required
• No Refrigeration Required
• Matched Positive Product Control

*λ (lambda) is the lowest concentration of endotoxin to cause a positive test result under standard conditions.

Pyrotell

Gel-clot Formulation

Pyrotell^® lysate was the first LAL gel-clot reagent licensed by the US FDA. It is easy to use and is available in both economical multi-test vials and convenient Single Test Vials (STV’s). Pyrotell lysate is a robust reagent, producing firm, easily read clots and is resistant to interfering substances. The gel-clot test does not require sophisticated capital equipment and software and is the simplest LAL test to implement.

Product Sensitivity

Pyrotell lysate is available in multiple sensitivities: 0.03 EU/mL; 0.06 EU/mL; 0.125 EU/mL; 0.25 EU/mL.

Sample to Lysate Ratio

For standard use, a 1:1 mixture of Pyrotell lysate to sample is used.

Test Performance

For 2 mL and 5 mL multi-test vials, 100 µL of lysate is mixed with 100 µL of sample in a reaction tube. For single test vials 200 µL of sample is added to the vial, which serves as a reaction tube. Test tubes are incubated at 37°C for 60 minutes ± 2 minutes. A positive test is indicated if the clot remains solid after the inversion of the test tube.

Pyrotell-T

Turbidimetric Formulation

Pyrotell^®-T lysate is used to quantify endotoxin in kinetic turbidimetric tests. Pyrotell^®-T lysate can be used with the Pyros Kinetix^® Flex tube reader and also incubating microplate readers. When used with the Pyros Kinetix^® Flex tube reader, Pyrotell^®-T gives a highly economic, flexible and sensitive LAL assay.

It can be used for a wide variety of endotoxin tests, ranging from standard water testing to samples requiring high sensitivity, such as intrathecal products and those requiring high dilutions to overcome interference.

Product Sensitivity

When used in a Pyros Kinetix^® Flex tube reader, the maximum sensitivity is 0.001 EU/mL, which is 5 times more sensitive than any other LAL assays. The unique formulation of Pyrotell^®-T lysate allows a wide range of standard curves to be used, giving the user flexibility, speed, and ease in performing assays. When used in a microplate reader, the maximum sensitivity is 0.005 EU/mL.

Sample to Lysate Ratio

The ratio of sample: LAL is determined by personal preference and sample chemistry (interference patterns). A sample: LAL ratio for Pyrotell^®-T of 1:1 or 4:1 can be used according to user choice (and validation requirements).

Test Performance

The sample and lysate are added to test tubes or a microplate and incubated at 37°C. The time of incubation is dependent on the lowest standard concentration in the standard curve. Software is used to construct the standard curve and calculate the endotoxin concentrations.

Pyrotell-T Training