P R O D UC T FLYER

Key TLR Ligands
Tomorrow’s Reagents Manufactured Today®

For TLR2 & TLR6/2

For TLR5

MALP-2

Flagellin (high purity)

ALX-162-027-C050
50 µg
ALX-162-034-C500
500 µgBULK
Synthetic. Originally isolated from Mycoplasma fermentans. This MALP-2 corresponds to the originallyisolated isomer, which expresses potent endotoxinlike activity and approaches in certain experimental
systems the toxicity of LPS. MALP-2 signalling, unlikethat of LPS, is induced via TLR2 and TLR6 signalling.
LIT: Purification and partial biochemical characterization of a Mycoplasma
fermentans-derived substance that activates macrophages to release nitricoxide, tumor necrosis factor, and interleukin-6: P.F. Muhlradt and M. Frisch;
Infect. Immun. 62, 3801 (1994)  For a comprehensive bibliography please
visit our website.

For more Details see Page 3.

For TLR3
Polyinosinic-polycytidylic acid . K[poly(I:C) . K (TLRgrade™) (synthetic)]

highlight

International Edition

ALX-746-021-M005
5 mg
Specific ligand for TLR3 [1, 2] and MDA5/Helicard [3].
LIT: [1] The dsRNA binding site of human Toll-like receptor 3: J.K. Bell, et al.; PNAS
103, 8792 (2006)  [2] Subcellular localization of Toll-like receptor 3 in humandendritic cells: M. Matsumoto, et al.; J. Immunol. 171, 3154 (2003)  [3] Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses: H.
Kato, et al.; Nature 441, 101 (2006)

ALX-522-058-C010
10 µg
Isolated from Salmonella typhimurium strain 14028.
SPeCIFICITy: Binds to human and mouse TLR5. BIoLogICAL ACTIvITy: Activation of TLR5 in human epithelial cellassays based on NF-κB luciferase fusions.
LIT: Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediatedmigration of dendritic cells: F. Sierro, et al.; PNAS 98, 13722 (2001)  Pathophysiological role of Toll-like receptor 5 engagement by bacterial flagellin in colonicinflammation: S.H. Rhee, et al.; PNAS 102, 13610 (2005)

For TLR7 & 8
R-848[S 28463; Resiquimod]

ALX-420-038-M005
5 mg
ALX-420-038-M025
25 mg
Selective ligand for TLR7 in mouse and for TLR7 andTLR8 in human. Potent antitumor and antiviral compound.
LIT: The immune response modifier resiquimod mimics CD40-induced B cell
activation: G.A. Bishop, et al.; Cell. Immunol. 208, 9 (2001)  Small anti-viralcompounds activate immune cells via the TLR7 MyD88-dependent signaling
pathway: H. Hemmi, et al.; Nat. Immunol. 3, 196 (2002)  For a comprehensive
bibliography please visit our website.

Also available:
TLR4 Ligands: Ultra-pure TLRgradeTM LPS. For more information see Pages 4-5.
TLR9 Ligands: Ultra-pure TLRgradeTM oDNs. For more information see Pages 6-7.

For a complete overview on TLR Ligands see Pages 2-3.
www.alexis-biochemicals.com

I N N AT E I M M U N I T YInternational Edition

Toll-like Receptors & Their Ligands
Ligands

TLR1 / TLR2
ALX-165-066/-069

OriginTLR1 complexed with TLR2
Lipoproteins/triacylated lipopeptides: Pam3CSK4, JBT3002, OspA

Bacteria, Mycobacteria

Soluble Lipoproteins

Neisseria meningitides

Bacterial Lipoproteins (BLPs)
Lipoarabinomannan (LAM)
MALP-2 (Mycoplasmal Macrophage-activating Lipopeptide-2)
Glycosylphosphatidylinositol (GPI)
Glycolipids
PorinsBacteria
Mycobacteria
Mycoplasma
Trypanosoma cruzi
Treponema maltophilum
Neisseria sp.

TLR2
ALX-162-027

TLR2/Dectin-1 TLR2 interacts with Dectin-1
Zymosan

Fungi

Double-stranded RNA (dsRNA)
Polyinosine-polycytidylic Acid (poly(I:C))

Viral
Synthetic

mRNA
tRNA

Host
Host/fungi/?

TLR3
ALX-746-021

TLR4 / CD14

TLR4/MD-2 complexed with CD14 and/or LPS-binding Protein [LBP]

ALX-581-007 to
ALX-581-020 &ALX-581-150

S-Lipopolysaccharides (LPS) (smooth) wild-type (wt) LPS (contains repeated O-polysaccharide units)
R-Lipopolysaccharides (LPS) (rough) mutant (Ra, Rb: extended core-polysaccharide) LPS (S-LPS-like)
R-Lipopolysaccharides (LPS) (rough) mutant (Rc, Rd1, Rd2, Re: short core-polysaccharide) LPS (Lipid A-like)
Flavolipin
Paclitaxel (Taxol®) (recognizes mouse TLR4)
Acyclic Lipid A-like Analog (R-112022)
Type III Repeat Extra Domain A (EDA)(*)
LMW Oligosaccharides of Hyaluronic Acid (sHA)
Polysaccharide Fragments of Heparan Sulfate(*)
Fibrinogen(*)
Fusion Protein of RSV(*)
Envelope Proteins of MMTV(*)
Glycoinositolphospholipids (GIPLs)
Heat Shock Proteins (HSPs)(*)

ALX-351-001

TLR4

Gram-negative bacteria (e.g. E. coli, Salmonella)Gram-negative bacteria (e.g. E. coli, Salmonella)Gram-negative bacteria (e.g. E. coli, Salmonella)
Flavobacterium meningosepticum
Plant
Synthetic
Host
Host
Host (only mouse tested)
Host
Respiratory syncytial virus
Mouse mammary tumor virus
Trypanosoma cruzi
TLR2 vs TLR4

TLR4/MD-2 in the absence of CD14 and/or LPS-binding Protein [LBP]

see above
R-Lipopolysaccharides (LPS) (rough) mutant (Rc, Rd1, Rd2, Re: short core-polysaccharide) LPS (Lipid A-like) Gram-negative bacteria (e.g. E. coli, Salmonella)
ALX-581-200 to -203 Lipid A (lacks core-polysaccharide: active component of LPS)
Gram-negative bacteria (e.g. E. coli, Salmonella)

TLR5
ALX-522-058

Flagellin

TLR6 / TLR2

TLR6 complexed with TLR2

ALX-162-027

Malp-2 (Mycoplasmal Macrophage-activating Lipopeptide-2) / Diacylated Macrophage-activating
Lipopeptide-2
Diacylated Lipopeptide FSL-1
Diacylated Lipopeptide Pam2CSK4Soluble Tuberculosis Factor (STF)

Mycoplasma

Imiquimod / Gardiquimod
R-848 (Resiquimod)
S-27610Loxoribine / TOG
3M-13
Bropirimine
Single-stranded RNA (ssRNA) / Polyuridylic acid . K (Poly(U) . K)
U1snRNA
siRNA

Synthetic
Synthetic
Synthetic
Synthetic
Synthetic
Synthetic
Non-viral & viral ssRNA
Host
Synthetic

ALX-420-038

3M-2
R-848 (Resiquimod)
Single-stranded RNA (ssRNA) / Polyuridylic acid . K (Poly(U) . K)

Synthetic
Synthetic
Non-viral & viral ssRNA

TLR9

TLR9 (intracellular and extracellular)Gram-negative bacteria

Part of the lipoprotein LP44 of Mycoplasma salivarium
Synthetic
Mycobacteria

TLR7
ALX-420-039/-040
ALX-420-038
ALX-480-097

TLR8

Unmethylated CpG DNA
ALX-746-001 to -006 CpG ODNs
ALX-746-024 to -026 AT-ODNs
Hemozoin

Bacteria / Protozoa/ viral
Synthetic
Synthetic
Protozoa (Malaria piment)

TLR10
Unknown

TLR11
ALX-522-093

Profilin
Uropathogenic E. coli (UPECs)

Protozoa (Toxoplasma gondii)
Bacteria(*) See Technical Note on Page 4

2

For updated prices and additional information visit www.alexis-biochemicals.com, contact your local distributor, or call +41 61 926 89 89.

Key Toll-like Receptor Ligands
TLR1/2

TLR4

R-848

Pam3Cys-Ser-(Lys)4 . 3HCl

Paclitaxel

ALX-165-066-M002		
2 mg
Selective agonist of TLR1 complexed with
TLR2. Cell permeable, water soluble synthetic
cationic lipohexapeptide analog of the immunologically active N-terminal portion of bacterial lipoprotein that potently activates monocytes and macrophages.

ALX-351-001-M001		
ALX-351-001-M005		
ALX-351-001-M025		
Isolated from
Taxus brevifolia.
LPS mimetic inmouse but not
human involving the TLR4
signalling pathway.

ALX-420-038-M005		
5 mg
ALX-420-038-M025		
25 mg
Selective ligand for TLR7 in mouse and for
TLR7 and TLR8 in human. Potent antitumor
and antiviral compound.

[Pam3CSK4 . 3HCl]

LIT: Lipopeptide derivatives of bacterial
lipoprotein constitutepotent immune adjuvants combined withor covalently coupled
to antigen or hapten: A. Reitermann, etal.; Biol. Chem. Hoppe
Seyler 370, 343 (1989) For a comprehensive
bibliography please
visit our website.

1 mg
5 mg
25 mg

LIT: The immune response modifier resiquimod mimics CD40-induced B cell activation: G.A. Bishop, et al.; Cell. Immunol. 208, 9 (2001)§ Small anti-viral compounds activate
immune cells via the TLR7 MyD88-dependent signaling pathway: H. Hemmi, et al.; Nat. Immunol. 3, 196 (2002)
 For a comprehensive bibliography
please visit our website.

TLR11

TLR5

Profilin (Toxoplasma gondii) (rec.)Flagellin (high purity)

Pam3Cys-Ser-(Lys)4 (Aca-Aca-Biotin) .
2TFA
ALX-165-069-M001		
Selective,
biotin-labelledligand of TLR1
complexedwith TLR2.

[Taxol®][S 28463; Resiquimod]

1 mg

ALX-522-058-C010		
10 µg
Isolated from Salmonella typhimurium strain
14028. Specificity: Binds to human and mouse
TLR5. Biological Activity: Activation of TLR5 in
human epithelial cell assays based on NF-κB
luciferase fusions.
LIT: Flagellin stimulation of intestinal epithelial cells triggers CCL20mediated migration of dendritic cells: F. Sierro, et al.; PNAS 98, 13722(2001) § Pathophysiological role of Toll-like receptor 5 engagementby bacterial flagellin in colonic inflammation: S.H. Rhee, et al.; PNAS
102, 13610 (2005)

TLR7/8
TLR2 & TLR6/2
MALP-2

ALX-162-027-C050		
50 µg
ALX-162-034-C500		
500 µg	BULK
Synthetic. MALP-2 was originally isolated from
Myco-plasma fermentans. This MALP-2 corresponds to the originally isolated isomer, which
expresses potent endotoxin-like activity andapproaches in certain experimental systems
the toxicity of LPS. MALP-2 signalling, unlikethat of LPS, is not transduced via TLR4, but
is induced via TLR2 and TLR6 signalling. For
more information about MALP-2 (incl. stereochemistry) see www.malp-research.de. Specificactivity: ~2x108units/mg. One unit is definedas the dilution giving half maximal release of
nitric oxide from C3H/HeJ mouse peritoneal
exudate cells in the standard assay.
LIT: Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages
to release nitric oxide, tumor necrosis factor, and interleukin-6: P.F.
Muhlradt and M. Frisch; Infect. Immun. 62, 3801 (1994)  For a comprehensive bibliography please visit our website.

TLR3 –
Specific Ligand for TLR3
Polyinosinic-polycytidylic acid . K[poly(I:C) . K (TLRgrade™) (synthetic)]

ALX-746-021-M005		
5 mg
Specific ligand for TLR3 [1, 2] and MDA5/Helicard [3].
LIT: [1] The dsRNA binding site of human Toll-like receptor 3: J.K.
Bell, et al.; PNAS 103, 8792 (2006)  [2] Subcellular localization ofToll-like receptor 3 in human dendritic cells: M. Matsumoto, et al.;J. Immunol. 171, 3154 (2003)  [3] Differential roles of MDA5 and
RIG-I helicases in the recognition of RNA viruses: H. Kato, et al.; Nature 441, 101 (2006)

Gardiquimod

ALX-420-040-M025		
25 mg
ALX-420-040-M100		
100 mgSelective ligand for human or mouse TLR7. Induces the activation of NF-κB in HEK 293 cells
expressing TLR7. At highconcentrations (3µg/ml)
slightly activates TLR8.
More active than imiquimod (Prod. No. ALX-420039).

Imiquimod[R-837]

ALX-420-039-M100		
100 mg
ALX-420-039-M250		
250 mg
Topical immune response modifier that inhibits angiogenesis. Up-regulates IL-18 anddown-regulates MMP-9 through recognition
of TLR7 and subsequent activation of MyD88dependent pathway.
LIT: Imiquimod applied topically: a novel immune response modifier and new class of
drug: R.L. Miller, et al.; Int. J. Immunopharmacol. 21, 1 (1999)  For a comprehensive bibliography please visit our website.

Loxoribine[7-Allyl-8-oxoguanosine]

ALX-480-097-M025		
25 mg
ALX-480-097-M100		
100 mgSelective ligand for TLR7 via IFN. Activates natural killer cells and primes cytolytic precursor
cells for activation by IL-2.
LIT: Loxoribine (7-allyl-8-oxoguanosine) activates natural killer cells
and primes cytolytic precursor cells
for activation by IL-2: B.L. Pope, etal.; J. Immunol. 151, 3007 (1993)
For a comprehensive bibliography
please visit our website.

I N N AT E I M M U N I T YInternational Edition

ALX-522-093-C010		
10 µg
Produced in E. coli. Full length profilin (aa1-163) from Toxoplasma gondii is fused to a
N-terminal tag. Specificity: Binds to mouse
TLR11.

TLR Ligand Sets
TLR Ligands Set I

APO-54N-018-KI01		
1 Set
KIT/SET CONTAINS: Sterile, ready-to-use solutionsof ligands to TLRs 1 to 9: 25µg of Pam3Cys-Ser(Lys)4 . 3HCl (TLR1/2) / 1mg Polyinosinic-polycytidylic acid . potassium salt (TLR3) / 20µg
LPS from E. coli, Serotype R515 (Re) (TLR4) /2µg Flagellin (purified) (TLR5) / 2µg MALP-2(TLR6/2) / 100µg Polyuridylic acid . potassium
salt (TLR7/8) / 100µg CpG ODN 2395 (TLR9).
For activation of TLRs 1 to 9.
LIT: A soluble form of lymphocyte activation gene-3 (IMP321) induces activation of a large range of human effector cytotoxic cells:
C. Brignone, et al.; J. Immunol. 179, 4202 (2007)

TLR (human) Ligands Set II

APO-54N-030-KI01		
1 Set
KIT/SET CONTAINS: Sterile, ready-to-use solutions of ligands to TLRs: 25µg of Pam3Cys-Ser(Lys)4 . 3HCl (TLR1/2) / 1mg Polyinosinic-polycytidylic acid . potassium salt (TLR3) / 50µg
LPS from E. coli, Serotype R515 (Re) (TLR4) /10µg Flagellin (purified) (TLR5) / 2µg MALP2 (TLR6/2) / 100µg Imiquimod (TLR7) / 100µg
R848 (TLR7/8) / 100µg CpG ODN 2216 (Type A)(TLR9). For activation of human TLRs.

TLR (mouse) Ligands Set III

APO-54N-031-KI01		
1 Set
KIT/SET CONTAINS: Sterile, ready-to-use solutionsof ligands to TLRs: 25µg of Pam3Cys-Ser-(Lys)4 .
3HCl (TLR1/2) / 1mg Polyinosinic-polycytidylicacid . potassium salt (TLR3) / 50µg LPS from E.
coli, Serotype R515 (Re) (TLR4) / 10µg Flagellin (purified) (TLR5) / 2µg MALP-2 (TLR6/2) /100µg Imiquimod (TLR7) / 100µg CpG ODN1585 (Type A) (TLR9) / 10µg Profilin (from
Toxoplasma gondii) (TLR11). For activation ofmouse TLRs 1 to 11.

3I N N AT E I M M U N I T YInternational Edition

TLRgrade™ – LPS & Lipid A Reagents
Sterile, ready-to-use liquid formulation – No hazardous handling! No further purification required! Potent and selective activators of TLR4
(10–1000 ng/ml). Ultrapure (≥99.9%) – Concentration: 1mg/ml
Prod. No.

Product

Size

ALX-581-015-L002

LPS from Salmonella minnesota R345 (Rb) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-017-L002

LPS from Salmonella minnesota R5 (Rc) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-018-L002

LPS from Salmonella minnesota R7 (Rd) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-008-L002

LPS from Salmonella minnesota R595 (Re) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-010-L002

LPS from E. coli, Serotype EH100 (Ra) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-014-L002

LPS from E. coli, Serotype J5 (Rc) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-007-L002

LPS from E. coli, Serotype R515 (Re) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-012-L002

LPS from E. coli, Serotype O111:B4 (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-013-L002

LPS from E. coli, Serotype O55:B5 (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-009-L002

LPS from Salmonella abortus equi S-form (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-150-R500
ALX-581-011-L002

LPS from Salmonella abortus equi S-form (TLRgrade™) (Ready-to-Use) (Biotin)
LPS from Salmonella typhimurium S-form (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-019-L002

LPS from Salmonella enteritidis S-form (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-020-L002

LPS from Salmonella minnesota S-form (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-200-L002

Lipid A from E. coli, Serotype R515 (Re) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-201-L002

Lipid A from Salmonella minnesota R595 (Re) (TLRgrade™) (Ready-to-Use)

2 ml

ALX-581-203-L001

Monophosphoryl Lipid A [MPL-A] from E. coli, Serotype R515 (Re) (TLRgrade™) (Ready-to-Use)

1 ml

ALX-581-202-L001

Monophosphoryl Lipid A [MPL-A] from Salmonella minnesota R595 (Re) (TLRgrade™) (Ready-to-Use)

1 ml

All the TLR grade™ LPS and Lipid A preparations are specific activators of Toll-likereceptor (TLR) 4 and do not activate TLR2 orother TLRs as determined with splenocytes
and macrophages from TLR4 deficient mice(see Figure 1).

FIgURe 1: Activation of macrophages from TLR4 wild type compared to TLR4 deficient mice by TLR grade™ LPS and TLR grade™
Lipid A from ALEXIS® Biochemicals. Lipid A or LPS concentrations,which induced maximal activation of TLR4 wild type mouse macrophages, were also applied to TLR4 deficient mouse macrophages(105) as follows: 80ng Salmonella LPS (S. minnesota R595 Re), 80ng
E. coli LPS (R515 Re), 400ng E. coli Lipid A (R515 Re) and 400ng
Salmonella LPS (S. abortus equi S-form). 10 units of IL-6 correspondto the detection limit of the IL-6 ELISA.

4500 µlTechnical Note
Contaminated TLR Ligands
The delicate and specific recognition of different PAMPs by TLRs revealed that only thepurest ligands, free of any other immunostimulatory contamination, allow to successfully elucidate the role of each TLR. While
LPS was thought to not only activate TLR4
but also TLR2, repurification of commercial
preparations of both E. coli and Salmonella
minnesota showed that LPS no longer induces cellular activation through TLR2 [1-5].
On the other hand highly purified HSP60 [6-7]and HSP70 [8] do not stimulate TLR4 as previously reported [9]. Furthermore it has beenshown that purified peptidoglycans activate
Nod1 and does not involve TLR2 or TLR4 [10,11]. Even synthetic CpG ODNs show different activation of certain immune cell subsets
when highly purified (TLRgrade™ CpG ODNs,
see next chapter).

LIT: [1] Lipopolysaccharides (LPS) of oral black-pigmented bacteria
induce tumor necrosis factor production by LPS-refractory C3H/
HeJ macrophages in a way different from that of Salmonella LPS:
T. Kirikae, et al.; Infect. Immun. 67, 1736 (1999)  [2] Repurificationof lipopolysaccharide eliminates signalling through both humanand murine toll-like receptor 2: M. Hirschfeld, et al.; J. Immunol.
165, 618 (2000)  [3] Toll-like receptor 4, but not toll-like receptor
2, is a signalling receptor for Escherichia and Salmonella lipopolysaccharides: R.I. Tapping, et al.; J. Immunol. 165, 5780 (2000) [4] Two lipoproteins extracted from Escherichia coli K-12 LCD25
lipopolysaccharide are the major components responsible for Tolllike receptor 2-mediated signalling: H.K. Lee, et al.; J. Immunol. 168,
4012 (2002)  [5] Murein lipoprotein, peptidoglycan-associated lipoprotein, and outer membrane protein A are present in purified
rough and smooth lipopolysaccharides: J. Hellman, et al.; J. Infect.
Dis. 188, 286 (2003)  [6] Recombinant human heat shock protein
60 does not induce the release of tumor necrosis factor alpha frommurine macrophages: B. Gao & M.F. Tsan; J. Biol. Chem. 278, 22523(2003)  [7] Endotoxin-free heat-shock protein 70 fails to induce
APC activation: H. Bausinger, et al.; Eur. J. Immunol. 32, 3708 (2002)
 [8] Endotoxin contamination in recombinant human heat shockprotein 70 (Hsp70) preparation is responsible for the induction oftumor necrosis factor alpha release by murine macrophages: B.
Gao & M.F. Tsan; J. Biol. Chem. 278, 174 (2003)  [9] Interaction of
TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies innate and adaptive immune responses: T. Warger, et al.; J. Biol.
Chem. 281, 22545 (2006)  [10] Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan: S.E. Girardin, etal.; Science 300, 1584 (2003)  [11] Toll-like receptor 2-dependent
bacterial sensing does not occur via peptidoglycan recognition:
L. H. Travassos, et al.; EMBO Rep. 5, 1000 (2004)

For updated prices and additional information visit www.alexis-biochemicals.com, contact your local distributor, or call +41 61 926 89 89.

overview on LPS-forms – “R versus S”
Colony morphology is indicative of the Oglycosylation status. Wild-type bacteria
form smooth colonies, synthesize “smooth”
or S-form LPS that contain O-polysaccharide
chains. S-form LPS also contain R-form LPS
molecules in variable proportion depending
on culture conditions. So-called “rough-mutants” of gram-negative bacteria synthesize“rough” or R-form LPS. These R-forms lack theO-polysaccharide chains and the core saccharide chain may be present in different stagesof completion, giving rise to defined R-classes (e.g. Ra, Rb, Rc, Rd, Re). All R-form LPS are
devoid of any S-form LPS [1].
The S-form LPS are commonly the preferredchoice for whole animal studies, activating
TLR4-positive cells strictly dependent on thepresence of membrane-anchored or soluble
CD14. They also activate the TLR4/MyD88independent pathway (TRAM/TRIF: type I
IFN) and are therefore selective for classical
APC expressing CD14 (e.g. monocytes, macrophages, DC) in vivo. The in vivo activity ofS-form LPS may be modulated by increased
levels of LBP (LPS-binding protein) during inflammation [2].
The R-form LPS and Lipid A are primarily usedin cellular in vitro activation studies. They activate TLR4-positive cells independent of thepresence of membrane-anchored or soluble
CD14. They do not activate the TLR4/MyD88independent pathway (TRAM/TRIF: type I

IFN), but they also activate non-classical APC(PMN/mast cells) in vivo. They are very usefulfor in vitro cellular activation assays, where
CD14/LBP may be absent or only available
in limited amounts. A nontoxic nonpyrogenic derivative of Lipid A is Monophosphoryl
Lipid A (MPL-A) exhibiting adjuvant properties that may be used in vaccine development [3].

Glc

Gal

Glc

GlcNac

Hep

Gal

KDO
KDO

O-Polysaccharide(repeating units)

core
Polysaccharide

LIT: [1] R-form LPS, the master key to the activation ofTLR4/MD-2positive cells: M. Huber, et al.; Eur. J. Immunol. 36, 701 (2006)  [2]CD14 is required for MyD88-independent LPS signaling: Z. Jiang,
et al.; Nat. Immunol. 6, 565 (2005)  [3] Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A: M. Martin,
et al.; Infect. Immun. 71, 2498 (2003)

Technical Note
LPS are amphipatic molecules whose hydrophobicity increases with decreasing
length of the polysaccharide chain. Therefore Re class LPS and Lipid A are morehydrophobic than Ra class or S-form LPS.
The use of Ca2+ and Mg2+-free buffers is
recommended for the preparation of diluted solutions in order to avoid precipitation and loss of activity for Re-LPS, Lipid A and MLP-A. For in vivo applications
prepare solutions in glucose instead of
PBS.

KDO

Hep

S-LPS

Lipid A

(complete)

Lipid A(semi-rough)Lipid ARa

Lipid A

Rb

Lipid A

Rc

Lipid A

Rd1

Lipid A

Rd2

Lipid ARe

I N N AT E I M M U N I T YInternational Edition

SR-LPS

R-LPS(incomplete)

FIgURe 2: Schematic representation of the different LPS chemotypes:
GlcNAc = N-Acetylglucosamine; Glc = Glucose; Gal = Galactose;
Hep = Heptose; KDO = 2-Keto-3-desoxyoctonate.
Adapted from M. Huber, et al.; Eur. J. Immunol. 36, 701 (2006) [1].

In vivo & in vitro Biological Properties ofSalmonella minnesota & E. coli LPS
Salmonella minnesota
Product No.

LPS-form

Constituents

Mac

Dend

B Cells

PMN

Mast

ALX-581-009
ALX-581-011
ALX-581-016
ALX-581-015
ALX-581-017
ALX-581-018
ALX-581-008
ALX-581-201
ALX-581-202

S-formS-formR-form (Ra)R-form (Rb)
R-form (Rc)
R-form (Rd)
R-form (Re)––O-polysaccharides-Core-Lipid A
O-polysaccharides-Core-Lipid A
GlcNAc-Glc-Gal-(Glc-Gal)-(Hep)1-2-Hep-(KDO)2-3-Lipid A
Glc-Gal-(Glc-Gal)-(Hep)1-2-Hep-(KDO)2-3-Lipid A
Glc-(Hep)1-2-Hep-(KDO)2-3-Lipid A
Hep-(KDO)2-3-Lipid A(KDO)2-3-Lipid A
Lipid A
Monophosphoryl Lipid A (MPL-A)

++
++
++
++
++
++
++
++
++

++
++
++
++
++
++
++
++
++

++
++
+++
+++
+++
+++
+++
+++
+++

+/+/+++
+++
+++
+++
+++
+++
+++

+/+/+++
+++
+++
+++
+++
+++
+++

Product No.

LPS-form

Constituents

Mac

Dend

B Cells

PMN

Mast

ALX-581-012
ALX-581-013
ALX-581-010
ALX-581-014
ALX-581-007
ALX-581-200

S-formS-formR-form (Ra)R-form (Rc)
R-form (Re)–O-polysaccharides-Core-Lipid A
O-polysaccharides-Core-Lipid A
Complete E.coli-core (type II)-Lipid A
Glc-(Hep)1-2-Hep-(KDO)2-3-Lipid A(KDO)2-3-Lipid A
Lipid A

++
++
++
++
++
++

++
++
++
++
++
++

++
++
+++
+++
+++
+++

+/+/+++
+++
+++
+++

+/+/+++
+++
+++
+++

E. coli

CHART LegeND: Mac = Macrophages, Monocytes  Dend = Dendritic Cells (monocyte-derived)  PMN = Polymorphonuclear Leukocytes (PMN)  B Cells (mouse only)  Mast = Mast Cells
 +/- = Weak/Absent activation  ++ = Strong activation  +++ = Very strong activation

5

I N N AT E I M M U N I T YInternational Edition

Cpg oDNs & ioDNs – Ligands of Toll-like Receptor• Activity tested• Potent (0.5-5 µg/ml), endotoxin-free, and selective activators for TLR9 as confirmed with
TLR9-/- macrophages and splenocytes• Ultrapure• Easy to handle
Toll-like receptors (TLRs) are widely expressed
recognition receptors of the innate immune
system. Four out of ten TLRs identified in humans today recognize nucleic acids, which
demonstrates the fundamental importance of
microbial DNA and RNA in response to pathogenic microorganisms.
Toll-like receptor 9 (TLR9) recognizes unmethylated CpG motifs in viral and bacterial DNA.
Signalling through TLR9 can be accomplishediODNs – Potent Inhibitors of
TLR9 Signalling20
18

Il-12-p40 [ng/ml]

16
14
12
10
8
6
4
2
0

ODN1668
TLRgrade TM

plus iODN
3:1

plus iODN
10:1

iODN only

medium only

SampleFIGURE 3: TLRgrade™ CpG ODN 1668 (Prod. No. ALX-746-001) was
added at 50pM/ml to macrophages in a 96-well plate simultaneously
together with TLRgrade™ iODN 2088 (Prod. No. ALX-746-250) at theindicated molar excess, cell supernatants harvested after 24 hours
and IL-12-p40 analyzed by cytokine ELISA. Similar inhibition rates
of >90 (3:1 molar excess) or >95% (10:1) were observed with TLRgrade™ iODN 2088 and CpG ODN 1668, when analyzed for IL-6.

with high efficiency with small, synthetic oligodeoxynucleotides (ODNs) containing optimized CpG motifs, which show great promise
as potent vaccine adjuvants and inhibitors of
Th2-mediated allergic responses.
Yet uncontrolled TLR9 activation can have deleterious consequences, exacerbating inflammatory tissue damage and increasing sensitivity to
toxic shock. Inappropriate TLR9-signalling may
also be associated with the promotion of autoimmune diseases like B cell hyperreactivity
and anti-DNA antibody production in lupus.
Natural and synthetic DNA sequences havebeen identified that are able to inhibit immune
activation through TLR9. These sequences arederived from diverse sources, including viral sequences, mutated CpG sequences, and repeatsof the TTAGGG motif present in mammalian telomeres.
Inhibitory oligodeoxynucleotides (iODNs) composed of TTAGGG multimers reproduce this suppressive activity and block the colocalization of
CpG DNA with TLR9 within endosomal vesicles.
The mechanism of action is not known, but evidence suggests that inhibitory ODNs do not interfere with cellular uptake of CpG ODNs, nor
do they simply compete for binding of TLR9. It
was shown that iODNs can affect Th1 priming
through the inhibition of STAT1, -3, and-4.
Specific inhibitors of TLR9 would represent a
valuable tool for understanding TLR9-mediatedresponses and might serve as potential therapeutics for some autoimmune diseases.

• Sterile and pyrogen-free water (1vial) is now included with every
order of CpG ODNs, ODN Controls, and iODNs!
• Sterile and pyrogen-free PBS is
also available (to be ordered separately).

Technical Note
Ultrapure TLRgrade™ ODN preparations are
selective activators of TLR9 as confirmed withmouse TLR9-/- macrophages and splenocytes.
To guarantee consistent and authentic results,
the use of endotoxin-free ddWater or PBS is
recommended for solubilization. These products have been subjected to multiple roundsof LPS removal by adsorption with activatedcharcoal.
LIT: The removal of 14C labeled endotoxin by activated charcoal:
A.S. Pegues, et al.; Int. J. Artif. Organs 2, 153 (1979)

PBS (endotoxin-free)

ALX-505-007-LD15
1.5 mlSterile and endotoxin-free PBS for use
with TLRgrade™ or endotoxin-free grade
reagents.

ddWater (endotoxin-free)

ALX-505-008-LD15
1.5 mlSterile, double distilled and endotoxin-freewater for use with TLRgrade™ or endotoxinfree grade reagents.

Selected Literature References
Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells: L.L. Stunz, et al.; Eur. J.
Immunol. 32, 1212 (2002)  Inhibitory oligonucleotides block
the induction of AP-1 transcription factor by stimulatory CpG
oligonucleotides in B cells: P. Lenert, et al.; Antisense Nucleic
Acid Drug Dev. 13, 143 (2003)  Suppressive oligodeoxynucleotides inhibit Th1 differentiation by blocking IFN-gammaand IL-12-mediated signaling: H. Shirota, et al.; J. Immunol. 173,
5002 (2004)  Inhibitors of TLR-9 act on multiple cell subsets inmouse and man in vitro and prevent death in vivo from systemic inflammation: O. Duramad, et al.: J. Immunol. 174, 5193
(2005)  Suppressive oligodeoxynucleotides protect mice fromlethal endotoxic shock: H. Shirota, et al.; J. Immunol. 174, 4579
(2005)  Therapeutic potential of oligonucleotides expressing
immunosuppressive TTAGGG motifs: D.M. Klinman, et al.; Ann.
NY Acad. Sci. 1058, 87 (2005)

Wide Panel of oDNs
Stimulatory ODNs (CpG ODNs)

Three types of stimulatory ODNs have been described, they differ in their sequences and in
the type and magnitude of immune responses induced:• Type A are characterized by poly-G motifs
with phosphorothioate (PS) linkages at the
5’ and 3’ ends and a phosphodiester (PO)palindromic CpG-containing sequence in
the ODN center. They are very strong inducers of interferon-α (IFN-α) by plasmacytoid
dendritic cells (pDC) and especially potent
NK cell activators.
• Type B are characterized by a full phosphorothioate (PS) backbone with one or more
CpG motifs without poly-G motifs. They are
weaker inducers of IFN-α, but are potent activators of B cells.
• Type C are characterized by a complete phosphorothioate (PS) backbone without poly-G
motifs, but also contain palindromic sequences combined with stimulatory CpG motifs.

6

They offer combined features, i.e. inductionof INF-α by pDC and activation of B cells.
Optimal sequences in ODNs responsible for activating TLR9 vary among species. Recent studies suggest that the species-selectivity attributed to some CpG motifs compared to GpC
motifs in control ODNs may only be observedwith phosphorothioate bond linkage (Type B
and Type C) rather than ODNs containing „natural“ phosphodiester linkages (Type A). The useof TLRgrade™ reagents shows that relative species selectivity for active CpG ODNs often depends on the concentration used.

Control ODNs (GpC ODNs)

Inactive control compounds for CpG ODNs
do not stimulate TLR9. They are composed ofsame sequence as their stimulatory counterparts, but instead of CpG they contain GpC dinucleotides.

Selected Literature References
Characterization of three CpG oligodeoxynucleotide classes
with distinct immunostimulatory activities: J. Vollmer, et al.;Eur. J. Immunol. 34, 251 (2004)  Cutting edge: species-specific TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified oligonucleotides: T.L. Roberts, et al.; J. Immunol. 174, 605 (2005)

Inhibitory/Suppressive ODNs
(iODNs)Inhibitory or suppressive ODNs have been described to neutralize the stimulatory effect ofCpG ODNs when added in excess. Two classes of inhibitory ODNs (iODNs) have been described:• Class One is characterized by the presenceof methylated CG or unmethylated GC sequences that selectively block CpG-induced
immune activation.
• Class Two of iODNs contains repetitive TTAGGG motifs patterned after those present inmammalian telomeres.

For updated prices and additional information visit www.alexis-biochemicals.com, contact your local distributor, or call +41 61 926 89 89.

Now available: BULK for in vivo studies!

Stimulatory CpG ODNs & Control GpC ODNs
Product/SequenceType

Recommended
Species

Prod. No.

Size

ODN 1668 (TLRgrade™) (synthetic)

Type B

Mouse

ALX-746-001-T100

100 tests

ODN 1668 (TLRgrade™) (synthetic) (BULK)Type B

Mouse

ALX-746-051-M001

1 mg

ODN 1720 (TLRgrade™) (synthetic) (Control)
5’-tccatgagcttcctgatgct-3’

Type B

Mouse

ALX-746-200-T100

100 tests

ODN 1826 (TLRgrade™) (synthetic)

Type B

Mouse

ALX-746-002-T100

100 tests

ODN 1826 (TLRgrade™) (synthetic) (BULK)Type B

Mouse

ALX-746-052-M001

1 mg

ODN 1982 (TLRgrade™) (synthetic) (Control)
5’-tccatgagcttcctgagctt-3’

Type B

Mouse

ALX-746-201-T100

100 tests

ODN 1585 (TLRgrade™) (synthetic)

Type A

Mouse

ALX-746-003-T100

100 tests

ODN 2118 (TLRgrade™) (synthetic) (Control)
5’-ggGGTCAAGCTTGAgggggg-3’

Type A

Mouse

ALX-746-203-T100

100 tests

ODN M362 (TLRgrade™) (synthetic)

Type C

Human/Mouse

ALX-746-004-T100

100 tests

ODN M383 (TLRgrade™) (synthetic) (Control)
5’-tgctgctgcttgcaagcagcttgat-3’

Type C

Human/Mouse

ALX-746-204-T100

100 tests

ODN 2216 (TLRgrade™) (synthetic)

Type A

Human

ALX-746-005-T100

100 tests

ODN 2216 (TLRgrade™) (synthetic) (BULK)Type A

Human

ALX-746-055-M001

1 mg

ODN 2243 (TLRgrade™) (synthetic) (Control)
5’-ggGGGAGCATGCTGgggggg-3’

Type A

Human

ALX-746-205-T100

100 tests

ODN 2006 (TLRgrade™) (synthetic)

Type B

Human/Mouse

ALX-746-006-T100

100 tests

ODN 2006 (TLRgrade™) (synthetic) (BULK)Type B

Human/Mouse

ALX-746-056-M001

1 mg

ODN 2137 (TLRgrade™) (synthetic) (Control)
5’-tgctgcttttgtgcttttgtgctt-3’

Type B

Human/Mouse

ALX-746-206-T100

100 tests

ODN 2395 (TLRgrade™) (synthetic)

Type C

Human/Mouse

ALX-746-020-T100

100 tests

Product/SequenceType

Recommended
Species

Prod. No.

Size

iODN 2088 (TLRgrade™) (synthetic)

Class One

Mouse

ALX-746-250-T050

50 testsiODN (ttaggg)4 (TLRgrade™) (synthetic)

Class Two

Human

ALX-746-251-T050

50 tests

5’-tccatgacgttcctgatgct-3’
5’-tccatgacgttcctgatgct-3’

I N N AT E I M M U N I T YInternational Edition

5’-tccatgacgttcctgacgtt-3’
5’-tccatgacgttcctgacgtt-3’

5’-ggGGTCAACGTTGAgggggg-3’

5’-tcgtcgtcgttcgaacgacgttgat-3’

5’-ggGGGACGATCGTCgggggg-3’
5’-ggGGGACGATCGTCgggggg-3’

5’-tcgtcgttttgtcgttttgtcgtt-3’

5’-tcgtcgttttgtcgttttgtcgtt-3’

5’-tcgtcgttttcggcgcgcgccg-3’

NoTe: Lower case letters indicate phosphorothioate linkage.

Inhibitory ODNs (iODNs)

5’-tcctggcggggaagt-3’

5’-tttagggttagggttagggttaggg-3’

NeW AT-oDNs see Backcover.
7

Latest Additions

Coming Soon

NEW AT-ODNs!

NEW Endogenous Danger Signal
Molecules for TLR4!

AT-oDN-1 (endotoxin-free) (synthetic)

ALX-746-024-C100
100 μgSynthetic. IFN-g inducing non-CpG ODN of the ATtype, found in the Malaria genome. TLR9-dependent
immune activation. SeQUeNCe: TATAATTTTAATTTCCAAGA. Nucleotides depicted in italics show the corresponding AT-ODN sequence. Includes 1 vial of ddWater (endotoxin-free) (Prod. No. ALX-505-008).

AT-oDN-2 (endotoxin-free) (synthetic)

ALX-746-025-C100
100 μgSynthetic. Lactobacillus gasseri-derived non-CpG
ODN of the AT-type. TLR9-dependent immune activation. SeQUeNCe: TATAATTTTTACCAACTAGC. Nucleotides depicted in italics show the corresponding
AT-ODN sequence. Includes 1 vial of ddWater (endotoxin-free) (Prod. No. ALX-505-008).
LIT: AT oligonucleotides inducing B lymphocyte activation exist in probiotic
Lactobacillus gasseri: H. Kitazawa, et al.; Int. J. Food Microbiol. 65, 149 (2001)
 Augmentation of T(H)-1 type response by immunoactive AT oligonucleotide from lactic acid bacteria via Toll-like receptor 9 signaling: T. Shimosato,
et al.; BBRC 326, 782 (2005)  Strong immunostimulatory activity of AT-oligodeoxynucleotide requires a six-base loop with a self-stabilized 5’-C...G-3’ stem
structure: T. Shimosato, et al.; Cell. Microbiol. 8, 485 (2006)

AT-oDN-3 (endotoxin-free) (synthetic)

ALX-746-026-C100
100 μgSynthetic. IFN-γ inducing non-CpG ODN of the ATtype, found in the Malaria genome. TLR9-dependent immune activation. SeQUeNCe: TTAACAATTTTTACCCAAGA Nucleotides depicted in italics show thecorresponding AT-ODN sequence. Includes 1 vial of
ddWater (endotoxin-free) (Prod. No. ALX-505-008).

Hyaluronic Acid Fragments (<1,500 Da)(TLRgrade™) (Ready-to-Use)[sHA (<1,500 Da) (TLRgrade™) (Ready-to-Use); Hyaluronan
Oligosaccharide (<1,500 Da) (TLRgrade™) (Ready-to-Use)]
ALX-580-004-C250
250 μgINQUIRE!
Isolated from bacteria. PURITy: ≥ 95%; Activity andendotoxin tested: TLRgrade™. The purity and size
range of HA oligomer mixture has been confirmed
by HPLC analysis and mass spectrometry. eNDoToXIN CoNTeNT: <0.02EU/μg. QUANTITy: Sufficient for ~100
tests in 100μl assays.
LIT: Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4: C. Termeer, et al.; J. Exp. Med. 195, 99 (2002)

Hyaluronic Acid Fragments (1,500-3,000 Da)(TLRgrade™) (Ready-to-Use)[sHA (1,500-3,000 Da) (TLRgrade™) (Ready-to-Use); Hyaluronan
Oligosaccharide (1,500-3,000 Da) (TLRgrade™) (Ready-to-Use)]
ALX-580-005-C250
250 μgINQUIRE!
Isolated from bacteria. PURITy: ≥ 95%; Activity andendotoxin tested: TLRgrade™. The purity and size
range of HA oligomer mixture has been confirmed
by HPLC analysis and mass spectrometry. eNDoToXIN CoNTeNT: <0.02EU/μg. QUANTITy: Sufficient for ~100
tests in 100μl assays.
LIT: Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4: C. Termeer, et al.; J. Exp. Med. 195, 99 (2002)

LIT: see Product No. ALX-746-025.

NEW Ligands for Intracellular DNA
Sensors!

www.alexis-biochemicals.com

order your FRee Copy of the
NeW NLR Flyer today atwww. alexis-biochemicals.com!

Poly(deoxyadenosine:deoxythymidine)(endotoxin-free) (synthetic)

SWITZERLAND/REST OF EUROPE
ALEXIS CORPORATION
T +41 61 926 89 89
F +41 61 926 89 79
E alexis-ch@alexis-corp.com

NORTH AMERICAAXXORA, LLC
T (858) 658-0065/1-800-900-0065
F (858) 550-8825/1-800-550-8825
E axxora-usa@axxora.com

BENELUX
10P’s AXXORA BVBA
T +32 (0) 3 466 04 20
F +32 (0) 3 466 04 29
E info@10Ps.com[Poly(dA:dT) (endotoxin-free) (synthetic)]

ALX-746-022-C050
50 μg
Synthetic. Strong IFN inducer, independent of
IRF3.

oligo(deoxyadenosine:deoxythymidine)(endotoxin-free) (synthetic)

GERMANYAXXORA DEUTSCHLAND GmbHT 07621 5500 522
Toll Free 0800 253 94 72
F 07621 5500 523
E axxora-de@axxora.com[Oligo(dA:dT) (endotoxin-free) (synthetic)]

International Distributors: Australia Sapphire Bioscience (02) 9698 2022 Austria Eubio (01) 8950145 Bangladesh Future Business Vision (0)2 863 1173 Bosnia & Herzegovina A-Z Consulting +386 1 433 63 22 / +386 1 230
18 84 Brazil Bioagency (0)11 3666 3565 / Sellex (0)11 5506 4646 Canada Cedarlane Laboratories (289) 288-0001/1-800-268-5058 Chile Biocant (2) 683 2437 China Beijing Bitab Biotech (010) 8201 5225 / Boppard 21 6288 4751
(Shanghai) 20 8732 6381 (Guangzhon) / ITS China (0)21 6481 4428/98 Colombia Lab Scientific +1 305 716 9922 Czech Republic Genetica (02) 7270 1055 Denmark Medinova Scientific 3956 2000 Ecuador Lab Scientific+1 305 716 9922 Egypt New Test For Scientific Service 03-358-3543 Estonia In vitro Eesti 630 65 20 Finland Nuppulinnan Laboratoriopalvelu (02) 07920350 France CovalAb 0437 654 236 / Coger (01) 45 33 67 17 Greece SB Biotechnology Suppliers (210) 823 3373 Hong Kong Boppard 2799 9019 Hungary Biomarker 28 419 986 India Hysel India 011-2622 78 01/02/03/04 / Imgenex India (0)674 274 3265/(0)674 329 6544 Indonesia ITS Indonesia (021)451 6222 Iran Hormoz Pajohan Lab. Equipment (021) 888 3444 Iraq Alwojoh (0)1 515 8350 Ireland Alpha Technologies 045 865 440 / 0149 62 422 Israel Almog Diagnostic (03) 977 3390 Italy Vinci-Biochem 0571 568147
Japan BioLinks 03 5443 6891 Korea Chun Yang Tech (02) 929 8071 Lithuania & Latvia In vitro Eesti +372 630 65 20 Malaysia Interscience (03) 57 40 9888 Mexico Consultoria de Laboratorios (0)55 1163 8840 The Netherlands
10P‘s Axxora 076 5425 184 New Zealand Sapphire Bioscience +61 2 9698 2022 Northern Ireland Alpha Technologies 028 28260558 Norway AH Diagnostics (23) 23 32 60 Pakistan The Worldwide Scientific (0)42-755-2355
Poland Biomibo (022) 872 0797 Portugal Baptista Marques (21) 722 06 60 Romania Medist (21) 411 5003 Russia Chimmed 095 728 4192 Singapore ITS Science & Medical (06) 273 0898 Slovenia A-Z Consulting (0)1 433 63 22/ (0)1 230 18 84 South Africa Southern Cross Biotechnology (021) 671 51 66 Spain Grupo Taper 916 596 520 Sweden Immunkemi 08-58361500 / In vitro 08-306010 Syria New-Med Technology (11) 8827 1717 Taiwan Cashmere
Scientific Company 0800 222 095/02-2567 5682 Thailand ITS Thailand (02) 308 0611 / Theera Trading (02) 412 5672 / (02) 418 1068 / S.M. Chemical Supplies (02) 542 1037 Turkey Tokra (312) 395 6009 Uruguay Lab Scientific+1 305 716 9922 Venezuela Lab Scientific +1 305 716 9922 Vietnam ITS Vietnam (08) 9255 232

UK
AXXORA (UK) Ltd.
T 01949 836111
F 01949 836222
E axxora-uk@axxora.com

OCT-08

ALX-746-023-C050
50 μg
Synthetic. Strong IFN inducer, dependent of IRF3.