PyroCell® MAT HS Rapid System
Instructions for Routine UseCat. # 296408 contains:
Cat. # 279770 - PyroCell® MAT HS Kit
Cat. # 296406 - PeliKine Human IL-6 ELISA Rapid Kit

1
RT-MN038 09/23

The PyroCell® MAT Kit and the PyroCell® MAT HS Kit are Lonza Bioscience
products for the Monocyte Activation Test. Components are manufactured by
Sanquin Reagents B.V. for Lonza Bioscience.
The PeliKine Human IL-6 ELISA Rapid Set A and PeliKine Human IL-6 ELISA
Rapid Set B are manufactured by Sanquin Reagents B.V. and distributed by Lonza
Bioscience for Monocyte Activation Test applications.

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Notices© Lonza 2023. All rights reserved.
No part of this publication may be reproduced, transcribed, or transmitted in any form, orby any means electronic or mechanical, including photocopying and recording, for any
purpose other than the purchaser’s use without written permission of Lonza.

Trademarks
PyroCell® is a Lonza registered trademark.
All trademarks belong to Lonza or its affiliates or to their respective third party owners.

Restrictions and liabilities
Information in this document is subject to change and does not represent a commitment by
Lonza. Changes made to the information in this document will be incorporated in new editionsof the publication. No responsibility is assumed by Lonza for the use or the reliability ofreagents, software or equipment that are not supplied by Lonza or its affiliated dealers.
The PyroCell® MAT HS Kit has been designed and validated for reproducible detection of avariety of different pyrogens using IL-6 cytokine release as read-out for pyrogenic activity.
Other cytokines may be suitable as read-out, but their use with the PyroCell® MAT HS Kit
needs to be validated by the user. A reference sample with a known concentration of apyrogen (not included) may be used for quality control purposes.
pMAT Cells are found to be reactive towards the non-endotoxin pyrogens listed in theCertificate of Analysis (CoA). pMAT Cells are responsive to multiple non-endotoxin
pyrogens, the specific reactivity has to be determined by the user.
The applicable warranty for Lonza reagents and products stipulated in this user guide isfound at www.bioscience.lonza.com. This warranty contains the obligations of Lonza, and noother warranties, expressed, implied, or statutory are given. Other than the express warranty
contained herein, Lonza shall not be responsible for any direct or consequential damages ofany kind. This warranty shall not be changed or modified in any way without the express
written permission of Lonza.
Lonza and Sanquin Reagents B.V. are not liable for any claims, including third party claims,
due to or caused by pyrogenic activity in pharmaceutical products. Consult the relevant
chapters in the European Pharmacopoeia (Ph. Eur. 2.6.30) as well as other documents
relevant for your specific purpose for utilization of the test.
Do not use reagents beyond the expiry date. The expiry date is printed on the packaging or
directly on the vial. pMAT Cells can be used for up to 6 months from the date of shipment.

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Quality control
It is considered good laboratory practice to run laboratory samples according to instructions
and specific recommendations included in the instructions for use of the test to be conducted.
Failure to follow these instructions may result in erroneous test data.
Safety Data Sheets (SDS ) are available from the Lonza product website.
Batch-specific Certificates of Analysis are available from the Lonza website,www.lonza.com/coa.

Regulatory information
The Monocyte Activation Test (MAT) is described in the European Pharmacopeia (Ph.
Eur. chapter 2.6.30) as a compendial, in vitro test for the detection of pyrogens inpharmaceutical preparations. The MAT is therefore, suitable as a replacement test for
the Rabbit Pyrogen Test (RPT) after a product-specific validation1.
The MAT is recognized by other world pharmacopeias such as the United States
Pharmacopeia2, as an alternative method to the RPT. Alternative method validation may
be required3. Please contact your local authorities for country-specific information.

Intended use statement
The PyroCell® MAT HS Kit, consisting of cryopreserved human peripheral blood mononuclear
cells (PBMC) pooled from four healthy donors (pMAT Cells), and MAT Human Serum (HS)Supplement, is used as an in vitro assay for the detection of pyrogenic components in raw
materials, intermediates, parenteral preparations or medical devices.
This PyroCell® MAT HS Kit is for laboratory use only and not intended for diagnostic purpose,
detection of pyrogens in man or animal, treatment of patients or patient management.

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Product support and technical service
If your Lonza product fails to function properly, if you have questions about how to use or
maintain our products, or if you need to send an instrument to Lonza for repair or otherservice, please contact our Scientific Support.
North America
Customer Service: 800 638 8174 (toll free)
Scientific Support: 800 521 0390 (toll free)scientific.support@lonza.com
Online ordering: bioscience.lonza.com
EuropeCustomer Service: + 32 87 321 611
Scientific Support: + 32 87 321 611
scientific.support.eu@lonza.com
Online ordering: bioscience.lonza.com
For technical support for countries outside of the USA or Europe, please contact your local
Lonza sales office or your regional distributor. See contact information on back cover.

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Revision History
Rev Date

Changes

2023

First issue

Document Conventions
This icon identifies information that protects the safety of the operator and theintegrity of data.
Warning!

A Warning indicates the potential for bodily harm and tells you how to avoid the
problem.

CautionA Caution indicates the potential for damage and tells you how to avoid the
problem.

Note:

Bold text is primarily used for emphasis.
This icon calls attention to important information.

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Warnings and precautions
NOTE: All reagents described in this manual are intended for research use only.
Warning!

Not suitable for in vitro diagnostics or medical use.

Warning!

Not intended to detect pyrogens in man or animal or for diagnosis of human or animal
diseases.

Warning!

Not intended for patient management.

Warning!

pMAT Cells contain traces of fetal bovine serum (FBS) sourced in North America, as astabilizer. FBS components shall be handled in laboratory environments only.

Warning!

pMAT Cells and MAT Human Serum Supplement are derived from human blood donations.
Human blood used for preparations was tested negative for human infectious disease
markers. Please refer to the CoA for the pMAT Cell lot. However, primary human cells cannot
be assumed to be free from infectious agents and must be handled with appropriate care.

CautionHandle all reagents according to Good Laboratory Practice (GLP) using appropriate
precautions.

CautionApply aseptic and non-pyrogenic handling of the PyroCell® MAT HS Kit. The MAT shall be
carried out by authorized and well-trained laboratory personnel only.

CautionPyroCell® MAT HS Kit components must be stored at ≤ -80°C under controlled conditions.
Temperature excursions impair the functionality of components. Thawed reagents must be
used immediately and cannot be refrozen.

Caution

Do not use vials that show signs of temperature excursion such as ice crystals inside the vial or
uneven surfaces. Please contact Lonza scientific support.

Caution

Do not use leaking or damaged vials.

Caution

Do not use vials of pMAT Cells where the medium color changed from pink to yellow.

Caution

Do not use reagents that show turbidity or other signs of microbial contamination.

CautionExpiry. Do not use reagents beyond the expiry date. The expiry date is printed on the
packaging or directly on the vial.
pMAT Cells can be used for up to 6 month from the date of shipment.

Caution

Disposal. Dispose of reagent containers according to local regulations.
Please refer to the Safety Data Sheet (SDS) for product safety information.

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Abbreviations
Abbreviation
BET
BRP
CLC
CO2
CoA
Complete IMDM
ELISA
EU/mL
EEU/mL
HPE
HRP
HS
IMDM
IL-6
IPA
LAL
LOD
MAT
MVD
mL, µL
N/A
NEP
NM
OD
PBMC
Pcs
Ph. Eur.
pMAT Cells
RPT
RSE
SDS
4-PLM
5-PLM

Description
Bacterial Endotoxin Test
Biological Reference Preparation
Contaminant Limit Concentration
Carbon dioxide
Certificate of Analysis
Iscove’s Modified Dulbecco’s Medium with added MAT Human
Serum Supplement (included in the PyroCell® MAT HS Kit)
Enzyme-linked Immunosorbent Assay
Endotoxin Units per Milliliter
Endotoxin Equivalent Units per Milliliter
High performance buffer
Horseradish peroxidase
Human serum (if used in combination with PyroCell® Kits)
Iscove’s Modified Dulbecco’s Medium
Interleukin-6
Isopropyl Alcohol
Limulus Amebocyte Lysate
Limit of Detection
Monocyte Activation Test
Maximum Valid Dilution
Milliliter, Microliter
Not applicable
Non-Endotoxin Pyrogens
Nanometer
Optical Density
Peripheral Blood Mononuclear Cells
PiecesEuropean Pharmacopeia
Pooled PBMC qualified for the MAT
Rabbit Pyrogen Test
Reference Standard Endotoxin
Safety Data Sheet
Four parameter logistic model
Five parameter logistic model

8

Table of Contents

Notices ......................................................................................................................... 3
Trademarks..................................................................................................................... 3
Restrictions and liabilities................................................................................................ 3
Quality control ................................................................................................................. 4
Regulatory information .................................................................................................... 4
Intended use statement .................................................................................................. 4
Product and technical service ......................................................................................... 5
Revision history .............................................................................................................. 6
Document conventions ................................................................................................... 6
Warnings and precautions .............................................................................................. 7
Abbreviations .................................................................................................................. 8

1.

Background – Monocyte Activation Test (MAT)....................................................... 111

2.

PyroCell® MAT HS Rapid System – Assay principle ................................................. 12

3.

Material information and storage conditions .............................................................. 13
3.1 PyroCell® MAT HS Kit ............................................................................................. 13
3.1.1 Package content............................................................................................. 13
3.1.2 Storage and stability ....................................................................................... 13
3.2 PeliKine human IL-6 ELISA Rapid Kit ..................................................................... 14
3.2.1 PeliKine human IL-6 ELISA Rapid Set A ……………………...………............. 14
3.2.1.1 Package content ................................................................................ 15
3.2.1.2 Storage and stability .......................................................................... 14
3.2.1.3 Sensitivity ........................................................................................... 15
3.2.1.4 Reference standard ........................................................................... 15
3.2.2 PeliKine human IL-6 ELISA Rapid Set B ........................................................ 15
3.2.2.1 Package content ................................................................................ 16
3.2.2.2 Storage and stability .......................................................................... 16
3.3 Further reagents and consumables ........................................................................ 16
3.4 Laboratory equipment needed ................................................................................ 17
3.5 Recommended literature......................................................................................... 18

4.

Definitions and calculations ....................................................................................... 19
4.1 Calculating the Maximum Valid Dilution (MVD) and the Contaminant Limit
Concentration (CLC) ..................................................................................................... 19
4.2 Calculating the Limit of Detection (LOD) ................................................................. 20
95.

Test procedure .......................................................................................................... 20
5.1 Use of qualified cells ............................................................................................... 20
5.2 Preparatory testing – Choosing the right method.................................................... 21
5.3 Routine testing – Monocyte activation .................................................................... 23
5.3.1 Preparation of complete medium.................................................................... 24
5.3.2 Preparation of endotoxin dilutions ................................................................. 25
5.3.2.1 Method A............................................................................................ 25
5.3.2.2 Method B ........................................................................................... 26
5.3.3 Preparation of product sample dilutions ......................................................... 28
5.3.4 Incubation of test samples with pMAT Cells ................................................... 29
5.4 Routine testing – human IL-6 ELISA ....................................................................... 30
5.4.1 Preparation of working strength buffers .......................................................... 31
5.4.2 Harvest the cell culture supernatant and prepare dilutions…………………… . 32

5.4.3 (Optional) Preparation of an IL-6 standard curve ........................................... 33
5.4.4 ELISA incubation and wash steps ................................................................. 34
5.5 Microtiter plate reading ........................................................................................... 36
6.

Analysis of results...................................................................................................... 36
6.1 Method A – Quantitative test................................................................................... 38
6.1.1 Acceptance criteria for the endotoxin standard curve..................................... 39
6.1.2 Calculation of pyrogenicity ............................................................................. 39
6.2 Method B – Semi-quantitative test .......................................................................... 40
6.2.1 Acceptance criteria ......................................................................................... 41
6.2.2 Calculation of pyrogenicity ............................................................................. 41
6.3 Method C – Lot comparison test ............................................................................. 41
6.3.1 Acceptance criteria ......................................................................................... 41
6.3.2 Calculation of pyrogenicity ............................................................................. 42

Contact Us ....................................................................................................................... 43

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1. Background – Monocyte Activation Test (MAT)
Pyrogenic substances in parenterally administered pharmaceutical products can lead tofever, and life-threatening reactions in patients. In order to ensure patient safety, it isrequired to demonstrate that parenteral preparations do not contain harmful levels of
pyrogens. Pyrogenic substances can originate from microorganisms such as bacteria or
fungi, from viruses, or from non-organic sources such as chemicals and primary packaging
materials. The most potent pyrogen is bacterial endotoxin derived from gram negative
bacteria. All other pyrogens are summarized under the term non-endotoxin pyrogens (NEP).
The purpose of a pyrogen test is to demonstrate that the amount of pyrogenic contaminants
contained in a preparation is below the contaminant limit concentration (CLC) in order torelease the product as being safe. Different tests acknowledged by regulatory agencies areavailable to determine whether a product or test preparation is free from pyrogenic
contaminants (Fig. 1).

Endotoxin

Pyrogen

Test type
Monocyte Activation Test (MAT)
Ph. Eur. 2.6.30
Rabbit Pyrogen Test (RPT)Ph. Eur. 2.6.8, USP <151>

Mechanism
In vitro: measures inflammatory
cytokines released by human blood
monocytes
In vivo: measures raise in bodytemperature after injection of a drug

Recombinant Factor C Assay (rFC) In vitro: recombinant alternative to
LAL. Based on factor C, the first
Ph. Eur. 2.6.32, USP <86>component of the LAL clotting cascade

Identification
Bacterial endotoxins and nonendotoxin pyrogens (e.g.
bacteria, viruses, yeasts andmolds)

Specific for bacterial endotoxin(gram-negative bacteria)

Limulus Amebocyte Lysate (LAL) In vitro: measures the initiation of the
Test (= BET Assay)clotting cascade in horseshoe crab
blood amebocytes by endotoxin
Ph. Eur. 2.6.14, USP <85>

Fig. 1: Compendial endotoxin and pyrogen detection methods

The Monocyte Activation Test (MAT) is an in vitro assay based on the detection of proinflammatory cytokines released by human blood monocytes upon stimulation with
pyrogenic substances. The MAT is capable of detecting endotoxins as well as nonendotoxin pyrogens in raw materials, intermediates, process samples and manufactured
products. The MAT has been qualified and validated by the European Center for the
Validation of Alternative Methods (ECVAM) in 20052 and by the Interagency Coordinating
Committee on the Validation of Alternative Methods (ICCVAM)3 in 2008. It has been
adopted by some main pharmacopeias, for example, the European Pharmacopeia (Ph. Eur.
Chapter 2.6.30)1 as a compendial method and is considered to be suitable, after a productspecific validation, to replace the rabbit pyrogen test (RPT)1. Other pharmacopeias describe
the MAT as an alternative method to the RPT. For example, the United States
Pharmacopeia (USP) requests the MAT to be validated according to USP <1225>4,5.
Please contact your local authorities for more information.
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2. PyroCell® MAT HS Rapid System – Assay Principle
The PyroCell® MAT HS Rapid System measures a response of the human innate immune
system. Monocytes, the key cells of innate immunity, respond to the presence of pyrogens(endotoxin and non-endotoxin pyrogens) by secreting pro-inflammatory cytokines, which
can be measured by an Enzyme-Linked Immunosorbent Assay (ELISA). On the day of theexperiment, the pMAT Cells are thawed and incubated with the product or test samples inan overnight cell culture at 37°C in a humidified cell incubator. On the next day, the cell
culture supernatants are harvested and the cytokine interleukin-6 (IL-6) released by the
monocytes is detected with the PeliKine Human IL-6 ELISA Rapid Kit. Presence of IL-6 isthen measured in a microplate reader as optical density (OD). Using a standard curve
generated with reference standard endotoxin (RSE), the OD values are finally converted
into Endotoxin Equivalent Units (EEU/mL).

AnalysisIL-6 ELISA

Monocyte
Activation

Process•

Prepare endotoxin dilutions•

Prepare test samples on the cell culture plate•

Thaw pMAT Cells, transfer to plate•

Incubate for 18-24h•

Harvest the cell culture supernatant•

Detect IL-6 cytokines with a Human IL-6 ELISA•

Read results in a microplate reader•

Convert the OD values into concentrations (EEU/mL)•

Determine the pyrogen content in the test sample

Fig. 2: Monocyte Activation Test - Assay overview

12•

See section 5.3•

See section 5.4•

See section 6

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3. Material Information and Storage Conditions
The PyroCell® MAT HS Rapid System (# 296408) combines reagents to conduct the MAT ina ready-to-use kit. The following materials are included: the PyroCell® MAT HS Kit (#279770), comprising 3 vials pMAT cells (M2016LC), and 3 vials Human Serum Supplement,(M2017LS) and the PeliKine Human IL-6 ELISA Rapid Kit (# 296406, comprising the
PeliKine Human IL-6 ELISA Rapid Set A (Sanquin, M2018) and the PeliKine Human IL-6
ELISA Rapid Set B (Sanquin, M2019).

3.1 PyroCell® MAT HS Kit
pMAT Cells (M2016LC) are manufactured and qualified according to the compendial
requirements of Ph. Eur. chapter 2.6.30. Each preparation of pMAT Cells is generated bypooling equal amounts of peripheral blood mononuclear cells (PBMC) isolated from four
qualified human blood donations, and subsequent cryopreservation of the pooled PBMC
under controlled conditions. Please refer to the Certificate of Analysis (CoA) for more
information on quality assurance.
To ensure traceability, each vial of pMAT Cells is labelled with two codes; the first code
(68-XXXX) refers to the original production lot number (lot org) which can also be foundon the CoA. The second code (P-XXX) refers to the individual vial. The lot number of the
MAT Human Serum Supplement is printed on the vial label, and on the CoA.

3.1.1 Package content
Amount3 vials

Component
pMAT Cells (M2016LC)

ClosureGreen

Volume3 x 1 mLLot #68-xxxx (Lot org.)

3 vials

MAT Human Serum
Supplement (M2017LS)White

3 x 2 mL

8000-xxxxxx

3.1.2 Storage and stability•

Important! Components of the PyroCell® MAT HS Kit
need to be stored at ≤ -80°C. Temperature excursion will
impair the functionality of the vial content.

•

Important! Components cannot be re-frozen after thawing
and must be used immediately.

•

Important! pMAT Cells expiry is 6 months from the shipment
date.

•

Consult the CoA for additional information.

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3.2 PeliKine Human IL-6 ELISA Rapid Kit
The PeliKine Human IL-6 ELISA Rapid Kit (# 296406) is comprised of two reagent kits, thePeliKine Human IL-6 ELISA Rapid Set A (M2018) and the PeliKine Human IL-6 ELISA
Rapid Set B (M2019).
•

Only use reagents and microtiter plates supplied with the PeliKine
Human IL-6 ELISA Rapid Kit. Do not mix reagents from different lots.

•

Sodium azide inactivates the horseradish peroxidase (HRP). Do
not use solutions containing sodium azide, nor add sodium azide to
the supplied materials.

•

All reagents contain merthiolate (0.001% w/v).

•

Mix all reagents thoroughly before use (avoid foaming).

•

Briefly spin all vials prior to use to prevent reagent loss (1 min at
3000 x g).

3.2.1 PeliKine Human IL-6 ELISA Rapid Set A (M2018)
The PeliKine Human IL-6 ELISA Rapid Set A is a "sandwich-type" enzyme immunoassay.
The target cytokine human IL-6 is bound to a pre-coated microtiter plate and is detected byan enzymatic reaction forming a colored product in proportion to the amount of cytokine
present in the sample.

3.2.1.1 Package content
The PeliKine Human IL-6 ELISA Rapid Set A contains material sufficient for 3 plates (288tests). The following reagents are included:
AmountComponent

1 vialSDS

ClosureVolumeConcentrate

Biotinylated antibody

Yellow

375 µL100x

1 vialStreptavidin-poly-HRP conjugate

Brown

20 µL

10,000x

1 vial
1 bottle

IL-6 standard
HPE bufferBlack
Transparent

750 µL
55 mL

4000 pg/mL
5x14Return to TOC

3.2.1.2 Storage and stability
Store the components of the PeliKine Human IL-6 ELISA Rapid Set A at -18°C to -32°C.
The expiration date is shown on the box label. Do not use reagents beyond the expiration
date.
Store the prepared HPE buffer (working strength buffer) at 2 to 8°C in a closed container
and use within 1 week. Bring the buffer to room temperature (18 to 25°C) prior to use.

3.2.1.3 Sensitivity
The limit of detection (LOD) can be calculated as follows:
Mean calculated zero signal + 3 SD: ≤ 1 pg/mL (shake or static incubation).
The assay sensitivity may be increased by longer incubation times (up to 2 hours).

3.2.1.4 Reference standard
The recombinant human IL-6 standard has been calibrated against the WHO International
Standard (IL-6 89/548)6; National Institute for Biological Standards and Control, Potter Bar,
Hertfordshire, U.K. 1 WHO Unit = 10 pg IL-6.

3.2.2 PeliKine Human IL-6 ELISA Rapid Set B (M2019)
The PeliKine Human IL-6 ELISA Rapid Set B completes the PeliKine Human IL-6 ELISA
Rapid Set A by providing the pre-coated plates, the plate seals, the washing bufferconcentrate, and the ready-to-use substrate and stop solutions.
•

Only use reagents from one set. Do not mix reagents from different
lots.

•

Do not add any preservative to the reagents. Preservatives may
impair the color development of the HRP substrate system.

•

The washing buffer contains merthiolate (0.001% w/v). Handle withappropriate care.

•

Avoid exposure of TMB substrate solution and stop solution tometals or metal ions to prevent unintended color formation.

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3.2.2.1 Package content
The PeliKine Human IL-6 ELISA Rapid Set B contains all materials sufficient for one
PeliKine compact human IL-6 kit (288 tests, 3 plates). The following reagents are included:
AmountComponent

SDS

Appearance

VolumePreparation

2 bottles Washing Buffer,
20-fold concentrate

White bottle

2 x 50 mL

2 x 1 L buffer

1 bottle

TMB substrate solution

Brown bottle

40 mL

Ready-to-use

1 bottle

Stop solution

White bottle

40 mL

Ready-to-use

3 pcs

Human IL-6 ELISA
pre-coated plates

Vacuum sealed,
desiccant3 x 96 wells

Ready-to use

12 pcsPlate seals

Transparent foil-

Ready-to use

3.2.2.2 Storage and stability•

Store the PeliKine Human IL-6 ELISA Rapid Set B at 2 to 8°C. The expiration date isshown on the box label. Do not use reagents beyond the expiration date.

•

Store the prepared washing buffer (working strength buffer) at 2 to 8°C in closed
containers and use within 2 month.

3.3 Further reagents and consumables
Reagents in the table below are recommended for use with the PyroCell® MAT HS Rapid
System. Alternative reagents may be used however, their suitability for the intended
purpose needs to be verified.
Reagent

VolumeCat. #

Reference standard endotoxin (RSE), e.g.
preparations from USP or EP (BRP)

10,000 U

E700 (USP)

Sterile, non-pyrogenic water (LAL water) forreconstitution of RSE

100 mL

W50-100

Distilled or deionized water for buffer preparation

1250 mL

N/A(optional) Two relevant non-endotoxin pyrogens

N/A

refer to CoA

Iscove's Modified Dulbecco’s Medium (IMDM)

500 mL

12-722F

The following standard laboratory consumables shall be appropriate for cell culture andendotoxin testing, i.e. they need to be sterile and non-pyrogenic:

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Consumable
Borosilicate glass tubes

VolumeCat. #

30 pcs/packN207(optional) De-pyrogenated sample container

25 pcs/box

80-507U

Reagent Reservoirs, non-pyrogenic

10 pcs/pack00190035

96-well flat bottom microplates (cell culture)

50 pcs/pack

25-340

Pipette tips, non-pyrogenic

as appropriate

See Catalog

Polystyrene disposable pipettes

as appropriate

See Catalog

96-well round bottom untreated microplates for
harvesting the cell culture supernatant

as appropriate

See Catalog

Conical polypropylene centrifuge tubes, 50 mL

as appropriate

See Catalog

3.4 Laboratory equipment needed
The following equipment is required to conduct the assay:
Equipment
Freezer (-80°C or lower, alarm setting)

Step
Storage of PyroCell® MAT HS Kit

Freezer (-20°C or lower)

Storage of PeliKine Human IL-6 ELISA
Rapid Set A

Refrigerator (2 to 8°C)

Storage of PeliKine Human IL-6 ELISA
Rapid Set B
Storage of working strength buffer

Laminar airflow cabinet with HEPA filter

Aseptic preparation of cell culture plateCO2-incubator (humidified, 37°C, 5% CO2)

Overnight incubation of cell culture plate

Water bath, 37°C

Controlled thawing of pMAT Cells

Vortex mixer

Preparation of Reference Standard
Endotoxin (RSE)

Adjustable multichannel pipettor (30-300 μL)

Preparation of cell culture and ELISA plate

Adjustable pipettors for accurate delivery of
liquid volumes (diverse, 1-1000 µL)

Reagent preparation, preparation of the cell
culture plate and ELISA plate

Beakers, flasks, or cylinders

Preparation and storage of working strength
buffer for the ELISA(optional) Device for delivery of washingbuffer or automated plate washer

Supports washing steps for the ELISA(optional) Microplate shaker

Increases sensitivity of the ELISA

Microplate reader (read out: 450nm;
reference wavelength: 540-590nm), e.g.
Nebula® Multimode Reader, Cat.# 25-375S
Nebula® Absorbance Reader, Cat. # 25-365S

ELISA assay read-out

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3.6 Recommended literature
1. European Directorate for the Quality of Medicines (EDQM). European Pharmacopeia
chapter 2.6.30: Monocyte Activation Test, Edition 10
2. European Centre for Validation of Alternative Methods (ECVAM), 2006: In Vitro Pyrogen
Test using Human Whole Blood/IL-6. Test method validation report (TM 2002-05)
3. Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM), 2008: Validation Status of Five In Vitro Test Methods Proposed forAssessing Potential Pyrogenicity of Pharmaceuticals and Other Products. Test method
evaluation report (NIH No. 08-6392)
4. United States Food and Drug Administration (FDA), 2012: Guidance for Industry
Pyrogen and Endotoxins Testing: Questions and Answers
5. United States Pharmacopeia (USP), 2007: Validation of Compendial Procedures<1225>. Rockville, MD. USP 41–NF:1-5.
6. RE Gaines Das, S Poole: The International Standard for interleukin-6. Evaluation in an
International Collaborative Study J Immunol Methods (1993) 160(2):147-53.
7. Helle M, Boeije L, de Groot E, de Vos A, Aarden L: Sensitive ELISA for interleukin-6.
Detection of IL-6 in biological fluids: synovial fluids and sera. J.Immunol.Methods. 1991
Apr 8;138(1):47-56.
8. Molenaar-de Backer, M. W. A., Gitz, E., Dieker, M., Doodeman, P. and ten Brinke, A.
(2021) “Performance of monocyte activation test supplemented with human serum
compared to fetal bovine serum”, ALTEX - Alternatives to animal experimentation, 38(2),pp. 307–315. doi: 10.14573/altex.2008261.
9. Solati S, Aarden L, Zeerleder S, Wouters D: An improved monocyte activation test using
cryopreserved pooled human mononuclear cells. Innate Immunity. 2015 Oct;21(7):67784.

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4. Definitions and Calculations
The terms cut-off value and LOD represent the same information but use
different units. The cut-off value is expressed in the unit of response, i.e.
optical density (OD) while the LOD is expressed as a concentration, e.g.
EU/mL.
Important! Historical data of the PyroCell® MAT HS Kit suggest an LOD≤ 0.08 EU/mL if used with the PeliKine Human IL-6 ELISA Rapid Kit.
Important! Endotoxin concentrations stated throughout this user guide,such as LOD, are calculated as concentration per sample. The
concentration in the reaction well of the cell culture plate is half of thestated concentration, e.g. half LOD. Use the LOD per sample to calculate
the MVD of the product to be tested.
Important! Consult the Certificate of Analysis for additional information.
The MAT is used to demonstrate that the amount of pyrogenic contaminants in the producttested does not exceed the contaminant limit concentration (CLC). To assure that the CLC
can be detected in the assay, it is necessary not to exceed the Maximum Valid Dilution(MVD) based on the limit of detection (LOD) of the test system.

4.1 Calculating the Maximum Valid Dilution (MVD) and the Contaminant Limit
Concentration (CLC)
The Maximum Valid Dilution (MVD) is defined as the maximum allowable dilution of a
product or test sample at which the contaminant limit can be determined.1
The MVD is calculated using the following expression:
𝑀𝑀𝑀𝑀𝑀𝑀 =CLC x CLOD

CLC = Contaminant Limit Concentration (e.g. EU/mg).
C = Concentration of test solution (e.g. mg/mL).
LOD = Limit of Detection (e.g. EU/mL) for the sample.

The CLC is calculated using the following expression:
𝐶𝐶𝐶𝐶𝐶𝐶 =KM

K = Threshold pyrogenic dose per kilogram of bodymass (e.g. EU/kg).
M = Maximum recommended bolus dose of productper kilogram of body mass (e.g. mg/kg).

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4.2 Calculating the Limit of Detection (LOD)
The LOD is determined by the mean value of the replicates of the responses to the blankand the endotoxin standard curve. The LOD is the concentration in Endotoxin Units per
milliliter (EU/mL), corresponding to the cut-off value of the measured response (opticaldensity, OD). The cut-off value is calculated using the following expression1:
𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑓𝑓𝑓𝑓 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 = 𝑥𝑥 + (3𝑠𝑠)𝑥𝑥 = mean of the four replicates of the response of the blank𝑠𝑠 = standard deviation of the four replicates of the responses to the blankAccording to the European Pharmacopeia each user has to establish the LOD in theirlaboratory based on the current experiment or historical data1. Using the PyroCell® MAT
HS Kit combined with the PeliKine Human IL-6 ELISA Rapid Kit an LOD of ≤ 0.08 EU/mL
per sample can be expected. Consult the Certificate of Analysis (CoA) for additional
information.

5. Test Procedure
5.1 Use of qualified cells
pMAT Cells contained in the PyroCell® MAT HS Kit consist of a cryopreserved pool ofhuman peripheral blood mononuclear cells (PBMC) serving as the monocytic cell source1
for the MAT. PBMCs are isolated from blood donations of healthy volunteers. Blood donors
are qualified to satisfy the criteria described in Ph. Eur. chapter 2.6.30, section 5.3 prior toblood donation. All donations are tested negative for common infectious disease marker.
On the day of donation equal amounts of PBMC from four donors are pooled andcryopreserved in a controlled-rate freezer within a validated time frame as of the blood
donation. After thawing, the pMAT Cells are qualified with the PeliKine Human IL-6 ELISA
Rapid Kit for conformance to section 5-5 of Ph. Eur. chapter 2.6.30 and to meet the criteria
outlined in section 6-1 and 6-3 regarding the reactivity towards reference standard
endotoxin (RSE) and non-endotoxin pyrogens (NEP). In addition, the averaging effect ofpooling is considered by comparing the reactivity of the pool and each of the four individual
donors towards endotoxin. For details on the individual cell lot please consult the respective
certificate of analysis (CoA).
On the day of the experiment the pMAT Cells are thawed and immediately diluted incomplete medium to achieve the optimal cell concentration per reaction well.

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5.2 Preparatory testing - Choosing the right method
Before proceeding to routine testing of a product with MAT, preparatory testing (= productspecific validation) should be performed (Fig. 3).1 Preparatory testing is required toestablish the preparation of the endotoxin standard curve in the laboratory, to confirm thatthe specific product to be examined does not interfere with the assay, or that interference
can be overcome by product dilutions not exceeding the MVD of the product. It further
assures that both bacterial endotoxin and non-endotoxin pyrogens (NEP) are detected.
Finally, testing of three manufactured product lots using the optimal product dilutions
determined during product validation confirms the choice of the appropriate MAT method(method A – Quantitative test, method B – Semi-quantitative test, or method C – Reference
lot comparison test).

Fig. 3: Preparatory testing requirements.

An endotoxin standard curve prepared from reference standard endotoxin (RSE, # E700) is
required for preparatory testing and for method A1. It is used to convert the OD signal
measured for product samples into endotoxin equivalent units (EEU). Establish theendotoxin standard curve by performing the MAT on 7 ascending RSE dilutions, and a blank(negative control). Prepare each dilution in 4 replicates on a 96-well microtiter plate.
For data analysis, chose an appropriate regression model for curve fitting. For the PyroCell®
MAT HS Kit a 5-parameter logistic model (5-PLM) is recommended. Lonza is offering an
excel based analysis template as an easy-to-use tool that automatically performs allnecessary calculations. For more information on data analysis please also refer to section 6.
Once the endotoxin standard curve is established or if method B is used during routine
testing, we recommend to use 5 endotoxin reference dilutions in order to fit more test
samples on the same microtiter plate.
The test for interfering factors requires a positive product control (PPC, endotoxin spike at aconcentration at the middle of the standard curve) to be recovered from product dilutions
within the range of 50-200% of the spike.1 All product dilutions shall not exceed the MVD forthe product in order to ensure that the contaminant can still be detected at the CLC. If
product interferences cannot be overcome by dilution, method C is recommended overmethods A and B.

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Each product dilution, the endotoxin dilutions, and the blank are prepared in 4 replicates on
a 96-well microtiter plate. Determine the appropriate product dilution as follows:•

Prepare dilutions for the endotoxin standard curve.

•

Prepare geometric dilutions of the product with all dilutions not exceeding the MVD.

•

For all product dilutions, generate a PPC by spiking with an endotoxin concentrationnear the estimated middle of the standard curve, i.e. 0.32 EU/mL (method A) or twice
the LOD, i.e. 0.16 EU/mL (method B).

•

Test the dilution series with and without PPC in the same experiment on the same
microtiter plate.

•

Calculate the mean recovery of the spiked endotoxin by subtracting the mean
concentration of dilutions without endotoxin from PPC dilutions. Compare to the
expected values by using the endotoxin standard curve.

•

Determine the highest product sample concentration with a valid spike recovery.

Within the same experiment, evaluate the dilution series of the product to be parallel to theendotoxin standard curve. If this is not the case, use of method B is recommended.

Is the Product free ofinterfering factors?

Yes

No

Is the dose response
curve parallel to theendotoxin standard
curve?

Yes

Method A:
Quantitative Test

Method C
Reference Lot
Comparison Test

No

Method B:
Semi-Quantitative Test

Fig 4: Simplified decision tree to support the choice of the MAT method.

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Test the system reactivity towards NEP by using either a historic product batch with NEP
contaminants (or elicited a positive response in the rabbit pyrogen test) or use at least two
different NEP ligands for toll-like receptors that may reflect the highest risk for the product.
At least one relevant NEP ligand should be spiked into the highest valid concentration of thetest product that was determined during interference testing, and must be recovered within
the range of 50-200% of the spike. It is recommended to test commercial NEPs for absenceof bacterial endotoxin, e.g. with a BET prior to use.
Finally, use the highest valid test product concentration to exclude interference of the productwith the chosen read-out system.1 OD values obtained in the IL-6 ELISA for an IL-6 standard
dilution series in the presence and absence of the product preparation should not differ bymore than 20%.
Based on the results from preparatory testing, select the suitable method for routine testing:•••

Method A: Quantitative test
Method B: Semi-quantitative test
Method C: Reference lot comparison test

For Method C, the dilution of the product and the reference lot depends on the type ofanalysis used to make the comparison between the two and is determined by the user.

5.3 Routine testing - Monocyte activation• Important! Ensure aseptic handling of cells. Use sterile, endotoxin-freematerials only.
• Important! Adhere to GCCP (good cell culture practices). Work in alaminar airflow cabinet, wear gloves, and clean the exterior of equipment
and materials with disinfectant, e.g. 70% isopropyl alcohol (IPA) prior touse.
• Refer to Ph. Eur., chapter 2.6.30 for description of MAT test methods.
Stimulation of pMAT Cells with product is carried out by an overnight cell culture step at
37 ± 1°C in an appropriate atmosphere (5% CO2, humidified air). The stimulation period
shall allow for sufficient accumulation of the IL-6 cytokine and is typically achieved within
18 – 24h.

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5.3.1 Preparation of complete medium•

Ensure sterility of the IMDM, e.g. by using an unopened bottle.

•

Important! A minimum of 1 mL IMDM without MAT Human Serum
Supplement is needed for the first RSE dilution. Preparing the first
RSE dilutions with complete medium may result in an inaccurate
endotoxin standard curve.

•

Equilibrate the complete medium to room temperature (18 to 25 °C).

The complete medium used for the pMAT cell incubation consists of IMDM with added MAT
Human Serum Supplement (M2017LS) that is included in the PyroCell® MAT HS Kit.
1. Thaw the MAT Human Serum Supplement vial in a water bath at 37°C.
2. Add the entire vial content (2 mL) to a sterile, non-pyrogenic 50 mL tube containing
38 mL IMDM.
3. Mix gently by inverting the closed tube. Use the complete medium within 8 hours.

5.3.2 Preparation of reference endotoxin dilutions•

Important! Prepare all endotoxin dilutions shortly before theexperiment.

•

Important! Do not vortex samples containing complete medium toprevent the formation of air bubbles.

•

Vortex the RSE stock solution (2,000 EU/mL) for 30 min before use.

Reference endotoxin dilutions are used to determine the LOD of the test system and to
estimate the amount of pyrogen in test sample. Include reference endotoxin dilutions, and ablank on each microtiter plate. Each dilution is prepared in 4 replicates.
When working with the PyroCell® MAT HS kit we recommend to prepare a standard curvewith 7 endotoxin dilutions, and a blank for the quantitative test method A, and 5 endotoxin
dilutions, and a blank for the semi-quantitative test method B. Based on the label sensitivityof ≤ 0.08 EU/mL for the PyroCell® MAT HS system prepare geometric dilutions from 0.04 (1/2
LOD) to 0.64 EU/mL (5-point standard curve) or 0.02 to 1.28 EU/ml (7-point standard curve)
respectively. Example plate layout are illustrated in Fig 5.

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A

B

Fig. 5: (A) Suggested plate layout for method A (R0-R7). (B) Suggested plate layout for method B(R0-R5). Concentrations are based on a historical LOD of ≤ 0.08 EU/mL.

Prepare an endotoxin stock solution of 2,000 Endotoxin Unit (EU)/mL by following the user
instructions:
1. Add 5 mL of endotoxin-free LAL water to a vial of RSE (Lonza # E700).
2. Reconstitute RSE by vortexing for 30 min at maximum speed. The resulting solution(2,000 EU/mL) is the stock solution.
Use 50 µL stock solution to prepare endotoxin dilutions for one 96-well microtiter plate.
Aliquots of the stock solution can be stored frozen at -80°C for future use. Upon thawing
vortex the aliquot for 3 min before use.

5.3.2.1 Method A
Prepare a serial dilution starting at 1.28 EU/mL with two-fold dilutions down to 0.02 EU/mL(1.28 – 0.64 – 0.32 – 0.16 – 0.08 – 0.04 – 0.02 EU/mL), and a blank in sterile, nonpyrogenic borosilicated glass tubes (# N207) as described below (also refer to Fig 6A):
3. Add 50 μL of RSE stock solution (2000 EU/ mL) to 950 μL IMDM (WITHOUT MAT
CULTURE MEDIUM SUPPLEMENT). The final concentration is 100 EU/mL. Vortex
thoroughly for 3 min.
4. Add 80 μL of 100 EU/mL RSE dilution to 920 μL complete medium. The final
concentration is 8 EU/mL. Mix by pipetting up and down 10x, with a volume of atleast 500 μL.
Do not vortex samples containing complete medium and prevent the formationof air bubbles as this may decrease the cells’ reactivity towards endotoxin.
5. Add 160 μL of 8 EU/mL RSE dilution to 840 μL complete medium. The final
concentration is 1.28 EU/mL. Mix by pipetting up and down 10x, with a volume of atleast 500 μL.
6. Add 200 μL of 1.28 EU/mL endotoxin to wells A1 to D1 (see Fig 5).

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7. Add 100 μL of complete medium to the other wells used for the standard curve (E1H1, A2-H2, A3-H3, and A4-H4).
8. Make a two-fold dilution by pipetting 100 μL from R7 (1.28 EU/mL, A1-D1) to R6(0.64 EU/mL, A2-D2) using a multi-channel pipettor with 4 lines. Mix thoroughly by
pipetting up and down 10x. Prevent the formation of air bubbles.
9. Repeat dilution as described in point 6 for transferring 100 µL from R6 to R5 (A3-D3),
from R5 to R4 (A4-D4), from R4 to R3 (E1-H1), from R3 to R2 (E2-H2), and from R2
to R1 (E3-H3). Discard the final 100 μL from R1.
10. Save the remaining 8 EU/mL RSE solution prepared at step 4 for preparation ofendotoxin spiked samples (see section 5.3.3 and Fig. 6A).

Aliquot RSE,
2,000 EU/ml
in LAL water
Upon thawing,
vortex for 3 min

50 µL RSE,
(2,000 EU/mL) in
950µL IMDM
Vortex 3 min

80 µL RSE,(100 EU/mL) in 920µL
complete medium
Mix by pipetting up
and down 10x

160 µL RSE,
(8 EU/mL) in 840µL
complete medium
Mix by pipetting up
and down 10x

160 µL RSE,
(8 EU/mL) in 1840 µL
complete medium
Mix by pipetting up
and down 10x2,000 EU/mL

100 EU/mL

8 EU/mL

1.28 EU/mL

0.64 EU/mL

Fig. 6A: Serial dilution for method A. Prepare 1.28 EU/mL endotoxin dilution and 0.64 EU/mL spikesolution.

5.3.2.2 Method B
Prepare a dilution series starting at 0.64 EU/mL with two-fold dilutions down to 0.04 EU/mL(0.64 – 0.32 – 0.16 – 0.08 – 0.04 EU/mL), and a blank in sterile, non-pyrogenic
borosilicated glass tubes (# N207). An example for a dilution series is shown in Fig 6B
below:
Proceed with the preparation of endotoxin dilutions:
1. Add 50 μL of RSE stock solution (2000 EU/ mL) to 950 μL of IMDM (WITHOUT MAT
CULTURE MEDIUM SUPPLEMENT). The final concentration is 100 EU/mL. Vortex
thoroughly for 3 min.
2.
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3. Add 40 μL of 100 EU/mL RSE dilution to 960 μL of complete medium. The final
concentration of 4 EU/mL. Mix thoroughly by pipetting up and down 10x, with avolume of at least 500 μL.
Do not vortex samples containing complete medium and prevent the formationof air bubbles as this may decrease the cells’ reactivity towards endotoxin.
4. Add 160 μL of 4 EU/mL RSE dilution to 840 μL of complete medium. The final
concentration of 0.64 EU/mL. Mix thoroughly by pipetting up and down 10x, with avolume of at least 500 μL.
5. Add 200 μL of 0.64 EU/mL endotoxin to wells A1 to D1 (see Fig 5).
6. Add 100 μL of complete medium to the other wells used for the standard curve (E1H1, A2-H2 and A3-H3).
7. Make a two-fold dilution by pipetting 100 μL from R5 (0.64 EU/mL, A1-D1) to R4(0.32 EU/mL, A2-D2) using a multi-channel pipettor with 4 lines. Mix thoroughly by
pipetting up and down 10x. Prevent the formation of air bubbles.
8. Repeat dilution as described in point 6 for transferring 100 µL from R4 to R3 (A2-D2),
from R3 to R2 (E1-H1) and R2 to R1 (E2-H2). Discard the final 100 μL from R1.
9. Save the remaining 4 EU/mL RSE solution prepared at step 2 for preparation ofendotoxin spiked samples (see section 5.3.3 and Fig. 6B).

Aliquot RSE,
2,000 EU/mL
in LAL water
Upon thawing,
vortex for 3 min

50 µL RSE,
(2,000 EU/mL) in
950µL IMDM
Vortex 3 min

40 µL RSE,(100 EU/mL) in 960µL
complete medium
Mix by pipetting up
and down 10x

160 µL RSE,
(4 EU/mL) in 840µL
complete medium
Mix by pipetting up
and down 10x

160 µL RSE,
(4 EU/mL) in 1840 µL
complete medium
Mix by pipetting up
and down 10x2,000 EU/mL

100 EU/mL

4 EU/mL

0.64 EU/mL

0.32 EU/mL

Fig. 6B: Serial dilution, prepare 0.64 EU/mL endotoxin dilution and 0.32 EU/mL spike solution.

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5.3.3 Preparation of product sample dilutions• Important! Prepare the product sample on the day of the experiment.
• Important! Prepare RSE dilutions in borosilicated glass tubes• Using the example layout below, 2-3 product preparations can betested on a single plate
For routine application of Method A (quantitative) and Method B (semi-quantitative) it isrequested to fit the following preparations on the same 96-well plate: A fresh preparation ofan endotoxin standard curve (method A) or endotoxin dilutions (method B) and three
dilutions of a product preparation, each measured with and without an endotoxin spike.
Each dilution is tested in 4 replicates suggesting 2 different samples fitting on a plate if
method A is used and 3 different samples for method B. Optional, an NEP control can beprepared. Examples for a plate layout for method A and B with one product sample is given
below (Fig. 7):
A

B

Fig. 7: Suggested plate layout for the quantitative (A) or semi-quantitative (B) MAT method.
Endotoxin reference standards with concentrations in EU/mL (A, R0 – R7) or x LOD (B, R0 – R5).
Sample (S1) in 2 successive two-fold dilutions (S1-1/2 and S1-1/4) without and with spiked
endotoxin (PPC, row E-H, indicated by darker color). The spike concentration should be near the
middle or the standard curve for method A (0.32 EU/mL) or at 2x LOD for method B (0.16 EU/mL).

1. Add 200 μL of the product test sample S1-1 (highest concentration with valid PPC
recovery, determined during preparatory testing) in wells A5 to H5 (Method A), or A4 to
H4 (Method B).
2. Add 100 μL of complete medium into the other wells used for the sample dilutions.
3. Prepare a two-fold dilution by pipetting 100 μL from S1 to S1-1/2 and mix by pipetting up
and down 10x. Prevent formation of air bubbles.
4. Repeat for transfer from S1-1/2 to S1-1/4 and discard the final 100 μL.
5. If a further samples are to be tested: repeat step 1-4 for each additional sample.

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6. Add 50 μL of complete medium to endotoxin dilutions and product test samples without
an endotoxin spike.
7. Prepare the endotoxin spike solution (PPC) from the saved dilutions of step 5.3.2.1 or
5.3.2.2, respectively.
Method A: Add 160 μL of 8 EU/mL endotoxin to 1840 μL complete medium. The final
concentration is 0.64 EU/mL.
Method B: Add 160 μL of 4 EU/mL endotoxin to 1840 µL complete medium. The final
concentration is 0.32 EU/mL.
Mix by pipetting up and down 10x, with a volume of at least 1 mL.
Do not vortex! Prevent formation of air bubbles.
8. Add 50 μL of the PPC preparation to wells for spiked samples (Ex to Hx, marked with +RSE spike).
Method A: The endotoxin spike concentration in the sample corresponds to 0.32 EU/mL
value from the standard curve.
Method B: The endotoxin spike concentration in the sample corresponds to the endotoxin
dilution with 0.16 EU/mL.

5.3.4 Incubation of test samples with pMAT Cells••

Important! Appropriate handling of cryopreserved cells is critical tomaintain full functionality of the cells.
Important! Ensure aseptic handling of cells. Adhere to GCCP (goodcell culture practices). Work in a laminar airflow cabinet, wear gloves,and clean the exterior of equipment and materials with disinfectant, e.g.
70% isopropyl alcohol (IPA) prior to use.

1. Take one vial of pMAT Cells from the -80°C freezer. Immediately thaw the vial in awater bath at 37ºC until a small clump of ice remains visible (< 5 min).
2. Transfer the entire contents of the vial (1 mL) into a 50 mL conical tube and immediately
but slowly add 5 mL complete medium (equilibrated to room temperature) while gently
swirling the tube (<5 min). Do not vortex or vigorously pipet, take care not to form
air bubbles.
3. Transfer mixture to a sterile reservoir and transfer 50 μL of the suspended pMAT Cells
to each well containing prepared test dilutions using a multi-channel pipette with 8 lines.
4. Incubate the microplate with lid in a CO2-incubator at 37°C (humidified air, 5% CO2) for
18-24 hours.

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5.4 Routine testing – Human IL-6 ELISA•

Adhere to GLP (good laboratory practices) for ELISA. An ELISA can be
performed on a regular laboratory bench.

•

Important! The Pelikine Human IL-6 ELISA Rapid Kit has been validatedwith the PyroCell® MAT Kit and the PyroCell® MAT HS Kit.

The Pelikine Human IL-6 ELISA Rapid Kit is a "sandwich-type" Enzyme-linked Immunosorbent Assay (ELISA) for fast and specific quantification of the human IL-6 cytokines (IL-6)
in MAT assays. Secreted IL-6 contained in the cell culture supernatants are simultaneously
bound to anti-IL-6 capture antibodies coated to the polystyrene microtiter wells of a 96-wellplate, and biotinylated anti-IL-6 antibodies added in the same incubation step. Unboundmaterial is removed by washing. Subsequently, a horseradish peroxidase (HRP) conjugatedstreptavidin is added that binds onto the biotinylated side of the cytokine sandwich. Afterremoval of unbound material by washing, a substrate solution is added.
A colored product is formed in proportion to the amount of IL-6 from supernatants, or IL-6
reference dilutions. After the reaction is terminated by adding a stop solution, the absorbance(optical density, OD) is measured in a microtiter plate reader at 450nm and a reference
wavelength (540 to 590nm). The OD of the endotoxin standard dilutions is then plottedagainst their concentration (EU/ mL) and the resulting standard curve is used to calculate theconcentration of a contaminant in the product samples (see section 6). The contaminantconcentration is expressed in endotoxin equivalent units (EEU).
For more information about the PeliKine human IL-6 ELISA Rapid Kit you may also refer tothe additional package inserts of the kit.

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5.4.1 Preparation of working strength buffer•

Working strength dilutions of the HPE buffer and the washing buffer
may be be prepared prior to the day of experiment. For storage
information refer to section 3, “storage information”.

•

Before preparing the working-strength buffer, briefly warm the bufferconcentrates to 37°C in a water bath to dissolve any precipitates.

•

All stated volumes refer to one ELISA plate.

•

The substrate solution contains a mixture of hydrogen peroxide and3,3',5,5'-tetramethylbenzidine (TMB). Protect the substrate solution
from prolonged exposure to light.

•

Protect TMB substrate solution and stop solution from contact withmetal or metal ions. This may lead to unspecific color formation.

•

The TMB substrate solution should be color-less. Do not use TMB
substrate solution that appears blue. Replace solution.

Stop solution contains sulfuric acid solution (0.18 M) in water.
Consult the SDS for Product Safety Information.

Reagent

Buffer Preparation

Washing buffer

Dissolve 50 mL of the concentrate in 950 mL of distilledwater.

HPE buffer

Dissolve 15 mL of the concentrate in 60 mL of distilledwater, mix contents.

TMB Substrate Solution

Ready-to-use.

Stop Solution

Ready-to-use.

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5.4.2 Harvest the cell culture supernatant and prepare dilutions•

Only part of cell culture supernatants is used for one routine ELISA.
Remaining supernatant may be stored frozen at ≤ 20°C for future
experiments.

•

The storage time of the frozen supernatant may vary between products
and needs to be determined by the user.

•

Frozen supernatants/ samples should be thawed as quickly as possible
in a water bath at 18-25°C. Do not use temperatures above 25°C.

•

Important! The protocol refers to a 1:5 dilution of cell culture
supernatants.

Following a 18-24 hour stimulation of pMAT Cells, harvest 100-150 μL of the supernatant bycarefully placing the pipette tip at the side of the well. Avoid turbulence in order to leave thepMAT Cells at the bottom of the plate. Transfer the supernatant to a fresh 96-well roundbottom, untreated microplate. Keep the original plate layout.
The optimal dilution of cell culture supernatants may vary between products. The below
ELISA protocol is suggesting a 1:5 dilution that is providing the largest dynamic range. Less
dilution, e.g. 3-fold may increase the accuracy for the lowest reference endotoxin dilutions
and may eventually lead to a lower experimental LOD. At the same time however, the moreconcentrated endotoxin reference dilutions may reside outside the reader’s detection range.
When stimulating monocytes with inherently pyrogenic test samples, higher dilution of thesupernatants is often required. Here, we recommend pre-dilution in a fresh, untreatedmicrotiter plate.
Examples:Supernatant
Dilution

DilutionfactorVolumesupernatantVolumeHPE buffer (1x)Volume applied
to ELISA

Pre-coated plate

3x

-

-

33,3 µL

5x

-

-

20 µL

30 µL

20 µL

90 µL

20 µL

270 µL

20 µL

Pre-dilution,
untreated plate

10x
20x

30 µL

50x

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5.4.3 (Optional) Preparation of an IL-6 standard curve• An IL-6 standard curve may be included to control the IL-6 ELISA, and as
reference for method C. It is optional for routine MAT methods A or B.
• Adding an IL-6 standard curve requires 8-16 “empty” wells (fig. 8) on the
microtiter plate layout.
• It is recommended to prepare two separate dilution series (duplicate) per
assay.
• Mix each dilution well before proceeding to the next step, e.g. by
pipetting up and down 10x with at least 75% of volume.
The IL-6 standard curve will contain 450, 150, 50, 16.7, 5.6, 1.9, 0.6 and 0 pg/mL IL-6 in
HPE buffer:•

Label 7 tubes, one tube for each dilution: 450, 150, 50, 16.7, 5.6, 1.9 and 0.6 pg/mL
representing the final IL-6 standard concentration in the ELISA plate.

•

Pipette 49 μL of working-strength HPE buffer into the tube labelled 450 pg/mL and80 μL into all other tubes.

•

Transfer 63 μL of the IL-6 standard (4000 pg/mL, black cap) into the tube labeled
450 pg/mL. Mix well.

•

Serial dilution: Transfer 40 μL of the 450 pg/mL dilution into the second tube labelled
150 pg/mL. Mix well. Repeat the dilution step 5 more times by adding 40 μL of theprevious diluted standard to the next tube. Always mix well before proceeding to thenext serial dilution step.

•

Use the HPE buffer as a blank (negative control).

Fig. 8: Suggested plate layout for the IL-6 standard curve. Preparation of a duplicate series
is recommended. Ensure availability of 8-16 “empty wells” (1-2 columns) on the plate layout.

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5.4.4 ELISA incubation and wash steps•

All 96 wells of each plate are pre-coated with anti-human IL-6 capture
antibodies. Use pre-coated plates immediately after opening the vacuum
sealed pouch.

•

Always empty wells completely before adding a new solution. Do notallow wells to stand uncovered or dry for extended periods of time
between incubation steps. Cover plate with an adhesive seal.

•

Equilibrate all buffers and antibody dilutions to 18 to 25°C before use.

•

Avoid repeated freeze-thawing of the IL-6 standard. Up to 3 freeze-thaw
cycles have no effect on the IL-6 levels of the IL-6 standard.

•

Keep the streptavidin-HRP conjugate at -18 to -32°C to ensure stability.
Thaw shortly before use.

•

Mix contents in wells by tapping the edge of the microtiter plate for a few
seconds.

•

The protocol below is for static incubation.

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Preparation (1 plate)•

Homogenize the harvested
supernatant by pipetting up
and down 5 times.

Biotinylated IL-6 antibody• Dilute 120 µL biotinylated
antibody (yellow cap) in 9.6
mL HPE buffer (working
strength).
• Mix by inverting tube 10
times.
Wash with washing buffer
Streptavidin-HRP conjugate• Dilute 2 µL streptavidin-HRP(brown cap) in 20 mL of HPE
buffer (working-strength).
• Mix by inverting tube 10
times.

Add to each well(Supernatant dilutions: 1:5)
• Add 20 µL supernatant to
the pre-coated plate. Keep
the plate layout.
• (Optional) add 20 µL of
IL-6 standard dilutions to“empty” wells.
• Add 80 µL of biotinylated
IL-6 antibody dilution. Mix
3 times by gently pipetting
up and down.
• Cover plate with adhesiveseal.
≥ 300 μL washing buffer(working strength).
•••

Add 100 µL of
Streptavidin-HRP
conjugate dilution.
Cover plate with adhesive
seal.
Gently agitate the plate bytapping the edge for a few
seconds

Wash with washing buffer≥ 300 μL washing buffer(working strength).

TMB substrate solution• Ready to use.

••

Add 100 µL TMB
substrate solution
Gently agitate the plate bytapping the edge for a few
seconds

Incubation/ Wash
1 hour at 18 to 25°C

Prepare streptavidinHRP conjugate dilution
immediately before
wash step.
5 times.

30 min at 18 to 25°C.

Equilibrate TMB
substrate solution andstop solution to 18 to
25°C before wash step.
5 times.

Approximately 10 min at
18 to 25°C in the dark,e.g. use a drawer.

Do not cover plate withProtect TMB substrate fromaluminum foil!
exposure to light!
Stop solution• Ready to use.

Add 100 µL of stop solution.

35

The color is stable formax. 30 min.

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5.5 Microtiter plate reading• The microtiter plate reader must be equipped with filters at wavelength
450 nm and a reference wavelength filter between 540 and 590 nm (e.g.
ELx808™ Reader, Lonza # 25-315S). Alternatively, a multimode reader
may be used (e.g. Nebula® Multimode Reader, Lonza # 25-375S)• If the substrate reaction appears too intense, reduce the incubation time.
Do not dilute the TMB substrate.
• If WinKQCL® Software is used, perform a “quick-read” at each
recommended wavelength. Export the data into the MAT Analysis Tool.
• Place the microtiter plate into an ELISA reader and record absorbance (OD) at 450 nm,
and at a reference wavelength (e.g. 550 nm).
• Subtract the reference wavelength measurement at 550 nm from the measurement at
450 nm before proceeding with further analysis of the ELISA data. For some software
packages this may be performed automatically.

6 Analysis of Results• The analysis and interpretation of MAT data depend on the chosen MAT
method. Refer to Ph. Eur. chapter 2.6.30 for a detailed description1.
• Any appropriate software available in-house or on-line may be used forthe analysis of MAT data.
• The software CombiStats, developed by the European Directorate for
the Quality of Medicines and Healthcare (EDQM, www.edqm.eu) may
assist you in statistical analysis according to the Ph. Eur. chapter 5.3.
• Lonza offers an Excel-based MAT Analysis Tool to assist in the analysisof MAT data. Please contact Lonza scientific support for more
information.
Product-specific validation is providing the basis for choosing the appropriate MAT method
for testing and data analysis (see section 5.2). It ensures that the validity criteria for therespective test method are met. An overview on how to assess the pyrogen content in a
product sample is given below:
36Return to TOC

1. For each sample dilution, calculate the net absorbance values by subtracting thereference OD550 nm reading from the OD450 nm reading.
2. (optional) Remove outliers caused by documented deviations to work instructions,e.g. pipetting errors.
3. (optional) Apply a sound statistical method to identify outliers within each group of
replicates (e.g. Dixon method with 90 to 99% Q-critical value).
4. Calculate the average OD value for each test replicate.
5. Perform a log transformation of the OD values.
6. Plot the OD values on the y-axis and the RSE values on the x-axis and perform aregression.
7. With the regression equation calculate the concentration of each product dilution.
8. Multiply the obtained endotoxin concentration with the dilution factor of the productsample to obtain the contaminant concentration in the sample.
9. Determine the LOD (see section 4).
It is recommended to set a limit for the acceptable relative standard deviation (max. %CV,
e.g. CV ≤ 25%) of the replicates of each dilution in order to identify potential issues during
data acquisition that lead to variability in results.
For statistical evaluation of the MAT, logistic regression models like the 4-parameter logistic
model (4-PML) or 5-parameter logistic model (5-PML) are most frequently used. Bioassays,e.g. ELISA, usually start as an asymptote at low doses, increase linear into an “S”-shaped
curve, and end up at another asymptote at high doses meaning that they are only linear
across a specific range of concentration magnitudes. While the 4-PLM is used fora symmetrical curve, the 5PLM is used if the curve from one asymptote to the other is not
symmetrical. For statistical data analysis with the PyroCell® MAT HS system, a 5-PML modelis recommended.

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6.1 Method A – Quantitative test
• Important! If the basal release of the IL-6 cytokine measured in theblank is OD ≥ 0.1, inappropriate handling of the pMAT Cells or anassay contamination with a pyrogen is indicated. Make sure to usematerials tested free of detectable endotoxin (i.e. endotoxin content
lower than the assay sensitivity). Critically assess procedures and theenvironment when preparing the assay. Repeat the experiment withfresh, endotoxin-free materials.
• The “historical LOD” is defined as the endotoxin concentration that
reproducibly shows an OD reading above the cut-off value withdifferent batches of pMAT Cells. Initially, it is recommended to use thehistorical LOD for calculating, e.g. the MVD of the product. The LOD
may be adjusted based on laboratory data or the current experiment1.
• When applying method A, the analysis software used should becapable to demonstrate that (1) the regression of response on log10
dose is statistically significant (p < 0.01), and (2) the regression of
response on log10 dose does not deviate significantly from linearity (p> 0.05 (see Ph. Eur. chapter 2.6.30 and 5.3 “Statistical analysis” formore information).
Method A is based on the conversion of the OD signals for the product dilutions tested into a
contaminant concentration (EEU/ mL) using the endotoxin standard curve. After applying thedilution factor correction, the contaminant concentration of the undiluted product (EEU/mL) is
compared to the CLC for that product. Routine testing of products with method A is
performed with the highest product concentration that was demonstrated for a valid spikerecovery in preparatory testing (see section 5) and two additional 2-fold dilutions of thisdilution not exceeding the MVD. Each dilution is tested on the same microtiter plate with andwithout an endotoxin spike at or near the middle of the standard curve.
First data inspection of the average OD of the endotoxin standard curve dilutions should
confirm ascending OD values with increasing endotoxin concentrations. All OD values ofendotoxin dilutions must be greater than the OD value of the blank.
•

Perform a log transformation and plot the average net absorbance (mean OD450nm –OD550nm) of the endotoxin dilutions against their concentrations.

•

Using an appropriate analysis tool, e.g. the Lonza MAT Analysis template, a doseresponse curve is fitted through the data points.

•

Determine the LOD (see section 4).

38

Return to TOC

Fig. 9A: Typical sigmoidal 7-point endotoxin standard curve obtained with the PyroCell® MAT HS Kit.

6.1.1 Acceptance criteria
Applying method A requires that the endotoxin standard curve meets certain statistical fit
criteria that are used as curve acceptance specifications (system suitability). Furthermore,method A should be applied only upon demonstration that the dose-response curve of the
product is parallel to the dose-response curve of the endotoxin reference standard (sample
suitability).
•
•

•
•

Blank: the average of blank should be ≤ 0.1 OD.
LOD: the experimental LOD ≤ historical LOD.
Example: If the historical LOD of the PyroCell® MAT HS Kit (0.08 EU/mL) is used to
calculate the MVD of the product, the test is valid if LOD ≤ 0.08 EU/mL.
Dose response: the dose-response of the product dilutions must be parallel to theEndotoxin Standard Curve.
Goodness of fit (statistical evaluation of the fitted curve):
Confirm the quality of the standard curve by statistical analysis:o Regression of responses (appropriately transformed if necessary) on log10
dose shall be statistically significant (p < 0.01).
o Regression of responses on log10 dose must not deviate significantly fromlinearity (p > 0.05).

6.1.2. Calculation of the pyrogen content•

For each sample dilution not exceeding the MVD, calculate the endotoxin spikerecovery. Dilutions with an invalid spike recovery (outside the range of 50-200%) areexcluded from further analysis.

•

For each valid sample dilution, the OD value is converted in EEU/mL, corrected withthe dilution factor and compared to the CLC.
-

If value in EEU/ml in the pure sample is < CLC  PASS.
If value in EEU/mL in the pure sample is > CLC  FAIL.
If an NEP PPC is used it has to be detected above the cut off value.
39Return to TOC

6.2 Method B - Semi-quantitative test
• Important! If the basal release of the IL-6 cytokine measured in theblank is OD ≥ 0.1, inappropriate handling of the pMAT Cells or anassay contamination with a pyrogen is indicated. Make sure to usematerials tested for low endotoxin content, critically assess preparation
procedures and repeat the experiment with endotoxin-free materials.
• The term “historical LOD” is defined as the endotoxin concentrationthat reproducibly demonstrates an OD reading above the cut-off value
with different batches of pMAT Cells. It is recommended to use thehistorical LOD for necessary calculations, e.g. the MVD of the product.
Method B compares the OD signal of the product sample with the CLC of the product.
Sample data below the cut-off value is considered negative (= pass, meaning a pyrogenlevel below CLC). Routine testing of products with method B is performed with highest
concentration that was been demonstrated for a valid spike recovery in preparatory testing(see section 5) and two additional 2-fold dilution of this dilution not exceeding the MVD. Eachdilution is tested on the same plate with and without an endotoxin spike at 2x LOD.
First data inspection of the averages of the reference endotoxin dilutions (0.5x, 1x, 2x, and4x historical LOD) should confirm increasing OD values with increasing endotoxin
concentrations. OD values found for endotoxin dilutions with concentrations equal to or
higher than the historical LOD must be above the blank. The 0.5x LOD concentration can behigher than or equal to the blank.
•

Plot the average net absorbance (mean OD450nm – OD550nm) of the endotoxin
dilutions against endotoxin concentrations.

•

Read the endotoxin content of product samples and confirm a valid spike recovery.

•

Determine the LOD (see section 4).

Fig. 9B: Typical sigmoidal 5-point endotoxin standard curve obtained with the PyroCell® MAT
HS Kit and the PeliKine compact human IL-6 ELISA as a read-out system.

40

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6.2.1 Acceptance criteria
The following criteria must be valid:•••

Blank: the average of blank should be ≤ 0.1 OD.
LOD: The experimental LOD ≤ historical LOD.
Example: the historical LOD of the PyroCell® MAT HS Kit is used to calculate the
MVD of the product. The test is valid if LOD ≤ 0.08 EU/mL. The mean OD signal forthe endotoxin standard solutions at 1x LOD should be above the cut-off value.
Dose response: the mean response of 4 endotoxin standard dilutions should
increase progressively with increasing concentrations.

6.2.2 Calculation of pyrogen content••

For each product dilution not exceeding the MVD, the endotoxin spike recovery (PPC)
is calculated. Dilutions with an invalid spike recovery (outside the range of 50-200%)
are excluded from further analysis.
For each valid product dilution the OD value is compared to the cut-off value. If the
OD is below the cut-off value the pyrogen concentration is considered to be below theLOD of the detection system.
-

OD value (product dilution) < Cut-off value  Pass (pyrogen level of theundiluted sample is below CLC).

-

OD value (product dilution) > Cut-off value  not conclusive (a contamination
is present in the undiluted sample but the pyrogen level cannot be evaluatedwith respect to the CLC).

-

OD value (product dilution at MVD) > Cut-off value  Fail (Pyrogen level isabove CLC).

6.3 Method C - Lot comparison test
Method C is comparing the OD signals or IL-6 contents from 3 dilutions of the product to be
examined to a reference lot of the same formulation or the same class of formulation. A
reference lot must be carefully justified. Ideally, clinical data is available. The OD ratio of the
product should not exceed a justified acceptance criterion which is defined by the user.
For a lot comparison samples of the reference lot and the lot to be evaluated must be testedon the same plate.
6.3.1 Acceptance criteria
The positive control and at least one dilution of the reference lot should be above the mean
OD value of the blank. Ph. Eur. 2.6.30 recommends that a lot difference should not be higher
or lower than 2.5-fold to pass the test.

41

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6.3.2 Calculation of pyrogenicity
For each sample an OD ratio is calculated, corresponding to the sum of the mean responseof the 3 dilutions of the lot being examined divided by the sum of the mean response of the
3 dilutions of the reference lot.
•

OD ratio < acceptance criterion  pass (lot examined not pyrogenic as compared to
the reference lot).

•

OD ratio > acceptance criterion  fail (lot examined pyrogenic as compared to thereference lot).

42

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