Monophosphoryl lipid a

Manufactured by Merck Group

Sourced in United States

Monophosphoryl lipid A is a bacterial derivative used in laboratory settings. It functions as an adjuvant, enhancing the immune response to co-administered antigens. This product is intended for research use only and its specific applications should be determined by the user.

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13 protocols using monophosphoryl lipid a

Synthesized Peptide Adjuvants for Autoimmunity

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Peptides vimentin amino acid (aa)28-42cit (cit-SYVTTST-cit-TYSLGS), aa28-42wt (RSYVTTSTRTYSLGS), aa28-49wt (RSYVTTSTRTYSLGSALRPSTS), aa28-49cit (cit-SYVTTST-cit-TYSLGSAL-cit-PSTS), aa32-46cit (TTST-cit-TYSLGSAL-cit-P), human aa415-433wt (LPNFSSLNLRETNLDSLPL), human aa415-433cit (LPNFSSLNL-cit-ETNLDSLPL), mouse aa415-433wt (LPTFSSLNLRETNLESLPL), mouse aa415-433cit (LPTFSSLNL-cit-ETNLESLPL), human aa418-431cit (FSSLNL-cit-ETNLDSL), human enolase aa241-260cit (VIGMDVAASEFF-cit-SGKYDLD), human aa241-260wt (VIGMDVAASEFFRSGKYDLD), mouse aa241-260cit (VIGMDVAASEFY-cit-SGKYDLD), mouse aa241-260wt (VIGMDVAASEFYRSGKYDLD), human aa 241-255cit (VIGMDVAASEFF-cit-SG), human aa 246-260cit (VAASEFF-cit-SGKYDLD), fibrinogen aa78-91cit (NQDFTN-cit-INKLKNS), collagen II aa1236-1249cit (LQYM-cit-ADQAAGGLR)¹⁵ and Hepatitis B surface antigen aa 181–193³⁶ were synthesized at >90% purity by Genscript (USA) and stored lyophilised at −80°C. On the day of use they were reconstituted to the appropriate concentration in phosphate buffered saline (PBS).
Adjuvants used include TLR9 agonist CpG ODN 1826 (Invivogen) and TLR4 agonist monophosphoryl lipid A (MPLA; Sigma); both used at a dose of 5µg/mouse/immunisation.

Brentville V.A., Symonds P., Cook K.W., Daniels I., Pitt T., Gijon M., Vaghela P., Xue W., Shah S., Metheringham R.L, & Durrant L.G. (2019). T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; Targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy. Oncoimmunology, 8(5), e1576490.

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Antigen-Specific T Cell Activation Protocol

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Mice were immunized intraperitoneally (i.p.) with 50 μg endotoxin‐free ovalbumin (Endograde OVA; Profos) in combination with monophosphoryl lipid A (Sigma‐Aldrich) or Imject alum (Thermo Scientific) as adjuvants. For analysis of affinity maturation, OT‐II‐cell transferred WT or Cd3e^–/– mice were immunized i.p. with 50 μg NP₁₉‐OVA (Biosearch Technologies) in alum. For SW_HEL and OT‐II co‐transfers, mice were immunized i.p. with 30–35 μg of HEL^WT‐OVA_323‐339 or the mutated form HEL^2X‐OVA_323‐339 in alum (recombinant proteins were kindly provided by Humabs BioMed).

Preite S., Baumjohann D., Foglierini M., Basso C., Ronchi F., Rodriguez B.M., Corti D., Lanzavecchia A, & Sallusto F. (2015). Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells. European Journal of Immunology, 45(11), 3010-3021.

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Synthesis and Evaluation of Novel Adjuvant MT06

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The synthetic nor-AbuMDP analogue adjuvant, MT06. was prepared as described by Ledvina and coworkers [23 ] (Fig 1). This non-pyrogenic analogue adjuvant was selected for this study from a series of such nor-AbuMDP analogues. Monophosphoryl lipid A (MPLA) was purchased from Sigma (St. Louis, MO) (Fig 1). Both Montanide PET GEL A (Seppic, Paris, France) and Alum (InvivoGene, San Diego, CA) were used as standard adjuvants for comparison.

Krupka M., Masek J., Barkocziova L., Turanek Knotigova P., Kulich P., Plockova J., Lukac R., Bartheldyova E., Koudelka S., Chaloupkova R., Sebela M., Zyka D., Droz L., Effenberg R., Ledvina M., Miller A.D., Turanek J, & Raska M. (2016). The Position of His-Tag in Recombinant OspC and Application of Various Adjuvants Affects the Intensity and Quality of Specific Antibody Response after Immunization of Experimental Mice. PLoS ONE, 11(2), e0148497.

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Generation of Monoclonal Antibodies Against Purified GPCRs

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Purified GPCR-BRIL proteins were reconstituted into liposomes consisting of 20:1 (w/w) egg phosphatidylcholine (Sigma-Aldrich)/monophosphoryl lipid A (Sigma-Aldrich). MRL/lpr mice were immunised with liposomal GPCR-BRIL. After immunisation, the mouse spleens were removed, and spleen cells were fused with myeloma cells. Hybridoma cells were screened by liposome-ELISA, denatured dot blot analysis, and fluorescence size exclusion chromatography (FSEC). To screen antibodies that specifically bind to the antigen with correct structure, but not the denatured one, liposome-ELISA positive and denatured dot blot clones were pooled, and then we confirmed the binding capacity in aqueous solution by FSEC. Hybridoma cells producing SRP2070 were intraperitoneally administered to mice, and ascites were collected. SRP2070 was purified by ammonium sulphate precipitation and protein G column chromatography. The subclass of SRP2070 was mouse IgG2a. After cleaving SRP2070 with papain, SRP2070Fab was purified by protein A column chromatography to remove the Fc portion and further purified by size exclusion chromatography (Superdex200 10/300 GL, GE Healthcare) by the changing buffer into PBS.

Miyagi H., Asada H., Suzuki M., Takahashi Y., Yasunaga M., Suno C., Iwata S, & Saito J.I. (2020). The discovery of a new antibody for BRIL-fused GPCR structure determination. Scientific Reports, 10, 11669.

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Formulation of Adjuvanted Vaccines

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l-histidine, 3-(N-morpholino)propanesulfonic acid (MOPS), triethylamine, and monophosphoryl lipid A (MPL) were purchased from Sigma-Aldrich (St. Louis, MO). Squalene was purchased from Echelon Biosciences (Salt Lake City, UT). Sucrose was purchased from Pfanstiehl Laboratories (Waukegan, IL). Polysorbate 80 was purchased from Fluka. Alhydrogel 2% was purchased from Brenntag Biosector A/S (Frederikssund, Denmark). The synthetic monophosphoryl lipid A analogue PHAD was purchased from Avanti Polar Lipids.

Hu G., Varisco D.J., Das S., Middaugh C.R., Gardner F., Ernst R.K., Picking W.L, & Picking W.D. (2023). Physicochemical characterization of biological and synthetic forms of two lipid A-based TLR4 agonists. Heliyon, 9(7), e18119.

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IRBP-induced Experimental Autoimmune Uveitis

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IRBP (2 mg/ml) will be emulsified with synthetic adjuvant, Monophosphoryl-lipidA + Trehalose dicorynomycolate adjuvant (0.5 mg monophosphoryl lipid A + 0.5 mg trehalose dicorynomycolatesynthetic) (Sigma, St Louis, MO) for the second immunization. At day 90–120 after the initial immunization to induce EAU, the mice were injected with 100 μl of IRBP emulsified with monophosphoryl lipid A-trehalose dicorynomycolatesynthetic adjuvant subcutaneously into two different sites on their back from initial immunization sites. Mice were monitored for retinal inflammation as described above and images were taken as described below.

Peters K., McDonald T., Muhammad F., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2023). A2Ar-dependent PD-1+ and TIGIT+ Treg cells have distinct homing requirements to suppress autoimmune uveitis in mice. Mucosal immunology, 16(4), 422-431.

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Intradermal Immunization with DNA Vaccine

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Adult mice received two doses of immunization by intradermal (id) route at a 20-days interval with 50 μg of the DNA plasmids in a total volume of 50 μL of PBS delivered at the base of the tail. PBS solution was used as negative control. For adjuvant-associated immunization, mice received two doses of 50 μg of ZK_ΔSTP vaccine in the presence of commercial adjuvant formulations, such as Alum (i.e., aluminum hydroxide, 10 mg/mouse, Thermo Scientific, USA), MPLA (monophosphoryl lipid A, 10 μg/mouse, Sigma Aldrich, USA) or a combination of Alum (10 mg/mouse) + MPLA (10 μg/mouse). The solutions comprising the DNA vaccine along with each commercial adjuvants were freshly prepared immediately before immunization according to the manufacturers’ recommendation. Briefly, the plasmid DNA (50 μg) was diluted in each adjuvant suspension and gently homogenized by pipetting. Immunogenicity evaluation was performed at different time points after boost. Sera, inguinal lymph nodes (iLN) and spleens were also obtained at different time points.

Teixeira F.M., Oliveira L.D., Branco A.C., Alberca R.W., de Sousa E.S., Leite B.H., Adan W.C., Duarte A.J., Lins R.D., Sato M.N, & Viana I.F. (2024). Enhanced immunogenicity and protective efficacy in mice following a Zika DNA vaccine designed by modulation of membrane-anchoring regions and its association to adjuvants. Frontiers in Immunology, 15, 1307546.

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Molecular Mechanisms of Innate Immunity

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Mouse monoclonal antibodies against Flag (Sigma), HA (Origene), β-actin (Sigma), p-IκBα (CST), p-IRF3 (CST) and p-TBK1 (Abcam); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (PeproTech), Bafilomycin (Sigma), Monophosphoryl lipid A (Sigma), Z-KAD-FMK (MCE) were purchased from the indicated companies. Luminescent cell viability assay kit (G7570) was purchased from Promega. Mouse antisera to TRIM32 and TRIF were raised against recombinant human TRIM32 and murine TRIF(1–475) respectively. Rabbit antisera to TAX1BP1 and NDP52 were raised against recombinant murine TAX1BP1 and NDP52(1–160) respectively.

Yang Q., Liu T.T., Lin H., Zhang M., Wei J., Luo W.W., Hu Y.H., Zhong B., Hu M.M, & Shu H.B. (2017). TRIM32-TAX1BP1-dependent selective autophagic degradation of TRIF negatively regulates TLR3/4-mediated innate immune responses. PLoS Pathogens, 13(9), e1006600.

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Evaluating Inflammatory Cytokine Response to LPS

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PBMC were isolated using gradient centrifugation in Ficoll-Paque solution (PanEco). PBMC and the human pre-monocytic leukemia cell line U937 were cultured in RPMI 1640 (Gibco) supplemented with heat inactivated 10% fetal bovine serum (Sigma), 2 mM L-glutamine (Sigma) and 25 μg mL-1 gentamicin (Gibco) in a humidified atmosphere (5% CO2) at 37°C.
200 μl aliquots containing 2×10⁴ U937 cells or 2×10⁵ PBMC were put in 96-well plates (Nunc) and incubated for 24 hours in the presence of various types of LPS: Ac3-S-LPS S. flexneri 2а, intact S-LPS of S. flexneri 2а, LPS E.coli O55 (Sigma-Aldrich), Monophosphoryl lipid A (Sigma-Aldrich). Final concentration of LPS preparations was 1 μg mL⁻¹ in U937 and 100 ng mL⁻¹ in PBMC culture.
Cell-free supernatants were collected and TNFα, IL-6 and IL-8 levels were measured by commercially available ELISA kits (Vector-Best) according to the manufacturer’s instructions.

Ledov V.A., Golovina M.E., Markina A.A., Knirel Y.A., L’vov V.L., Kovalchuk A.L, & Aparin P.G. (2019). Highly homogenous tri-acylated S-LPS acts as a novel clinically applicable vaccine against Shigella flexneri 2a infection.. Vaccine, 37(8), 1062-1072.

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Liposomal Vaccine Formulation with LMPLA and Galactose

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Liposomal-monophosphoryl lipid A (LMPLA) were prepared as described.^{65 (link)} Briefly, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (Avanti Polar Lipids, Alabaster, AL) were dissolved in freshly distilled chloroform stabilized with 0.75% ethanol at a molar ratio of (9:7.5:1) along with monophosphoryl lipid A (Sigma-Aldrich, St. Louis, MO) for a final concentration of 100 μg/mL. Liposomes were dried under nitrogen stream and placed O/N in a desiccator containing silica-gel desiccant beads (Thermo Fisher Scientific). Liposomes were then resuspended for a final concentration of 10 μg/200 μL of sterile PBS, pH 7.4, containing 20 μg Galα3LN-HSA (3-atom spacer, NGP2334, Dextra Laboratories, Reading, UK) or 20 μg recombinant HSA (Thermo Fisher Scientific), then filtered using a 0.2-μm disc filter (Thermo Fisher Scientific). For immunizations in the absence of LMPLA, a stock solution of 1 mg/mL Galα3LN-HSA in sterile, deionized milli-Q water was prepared and kept in 200-μL aliquots at −20 °C until use.

Portillo S., Zepeda B.G., Iniguez E., Olivas J.J., Karimi N.H., Moreira O.C., Marques A.F., Michael K., Maldonado R.A, & Almeida I.C. (2019). A prophylactic α-Gal-based glycovaccine effectively protects against murine acute Chagas disease. NPJ Vaccines, 4, 13.

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