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Lipopolysaccharide is the major constituent of the outer membrane of gram-negative bacteria and, once released from the bacterial surface into the bloodstream, is a potent activator of the host immune system, which can lead to septic shock. LPS has a hydrophilic region consisting of a repeating oligosaccharide that is strain-specific (O-antigen) and a core polysaccharide, which is covalently linked to a hydrophobic lipid moiety (lipid A). Lipid A is the most conserved part and is responsible for the toxicity of LPS. Therefore, finding molecules able to bind to this region and neutralize LPS toxicity is of relevant interest as it may provide new therapies to prevent septic shock (Chen et al., 2006). Several proteins and peptides were reported to bind LPS and alter its toxicity towards reduction and even enhancement (Brandenburg et al., 1998), such as serum albumin (Ohno and Morrison, 1989), lipopolysaccharide binding protein (LBP) (de Haas et al., 1999), casein (López-Expósito et al., 2008), lysozyme, the antibiotic polymyxin B and antimicrobial peptides (Chen et al., 2006). Although some of these proteins are neutral and even anionic/acidic (pI<7) (Jang et al., 2009), due to the amphipathic structure of LPS and the presence of negatively charged phosphate groups on the lipid A, the most important factors that are considered for optimal binding to LPS are a cationic/basic (pI>7) and amphipathic nature (Chen et al., 2006).
Here we describe a competitive ELISA that can be used to identify proteins or peptides that bind LPS, as a first approach before analyzing the possible activity in vitro and in vivo. In this ELISA, serial dilutions of the protein or peptide to be tested are preincubated with a fixed concentration of fluorescein isothiocyanate (FITC)-labeled LPS from Escherichia coli serotype O111:B4 and then added to wells of a microtitre plate which are blocked with a casein hydrolysate that binds LPS (Martínez-Sernández et al., 2014). Binding of the protein to LPS displaces LPS from binding to the casein, which is revealed using a horseradish peroxidase (HRP)-labeled anti-FITC polyclonal conjugate. This method allows simultaneous analysis of several proteins or peptides in a short period of time and no recognizing molecules (e.g., antibodies) to a specific protein or peptide are needed.

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Competitive ELISA for Protein-lipopolysaccharide (LPS) Binding
竞争性酶联免疫吸附试验检测蛋白和脂多糖(LPS) 的结合

微生物学 > 微生物生物化学 > 蛋白质 > 相互作用
作者: Victoria Martínez-Sernández
Victoria Martínez-SernándezAffiliation: Departamento de Microbiología y Parasitología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
Bio-protocol author page: a1814
 and Florencio M. Ubeira
Florencio M. UbeiraAffiliation: Departamento de Microbiología y Parasitología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
For correspondence: fm.ubeira@usc.es
Bio-protocol author page: a1815
Vol 4, Iss 22, 11/20/2014, 3524 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1298

[Abstract] Lipopolysaccharide is the major constituent of the outer membrane of gram-negative bacteria and, once released from the bacterial surface into the bloodstream, is a potent activator of the host immune system, which can lead to septic shock. LPS has a hydrophilic region consisting of a repeating oligosaccharide that is strain-specific (O-antigen) and a core polysaccharide, which is covalently linked to a hydrophobic lipid moiety (lipid A). Lipid A is the most conserved part and is responsible for the toxicity of LPS. Therefore, finding molecules able to bind to this region and neutralize LPS toxicity is of relevant interest as it may provide new therapies to prevent septic shock (Chen et al., 2006). Several proteins and peptides were reported to bind LPS and alter its toxicity towards reduction and even enhancement (Brandenburg et al., 1998), such as serum albumin (Ohno and Morrison, 1989), lipopolysaccharide binding protein (LBP) (de Haas et al., 1999), casein (López-Expósito et al., 2008), lysozyme, the antibiotic polymyxin B and antimicrobial peptides (Chen et al., 2006). Although some of these proteins are neutral and even anionic/acidic (pI<7) (Jang et al., 2009), due to the amphipathic structure of LPS and the presence of negatively charged phosphate groups on the lipid A, the most important factors that are considered for optimal binding to LPS are a cationic/basic (pI>7) and amphipathic nature (Chen et al., 2006).
Here we describe a competitive ELISA that can be used to identify proteins or peptides that bind LPS, as a first approach before analyzing the possible activity in vitro and in vivo. In this ELISA, serial dilutions of the protein or peptide to be tested are preincubated with a fixed concentration of fluorescein isothiocyanate (FITC)-labeled LPS from Escherichia coli serotype O111:B4 and then added to wells of a microtitre plate which are blocked with a casein hydrolysate that binds LPS (Martínez-Sernández et al., 2014). Binding of the protein to LPS displaces LPS from binding to the casein, which is revealed using a horseradish peroxidase (HRP)-labeled anti-FITC polyclonal conjugate. This method allows simultaneous analysis of several proteins or peptides in a short period of time and no recognizing molecules (e.g., antibodies) to a specific protein or peptide are needed.

Keywords: LPS(LPS), Lipopolysaccharide(内毒素), ELISA(ELISA), Casein(酪), LPS-binding(LPS结合)

[Abstract]

Materials and Reagents

  1. Casein (Hammarsten grade) (BDH Prolabo, VWR International, catalog number: 440203H )
  2. Lipopolysaccharides from Escherichia coli O111: B4-FITC conjugate (Sigma-Aldrich, catalog number: F3665 )
  3. Polymyxin B sulfate salt (Sigma-Aldrich, catalog number: P4119 )
  4. Proteins that were used for the example below (see Representative data) but which are not necessary for the assay:
    1. LBP human recombinant (Sigma-Aldrich, catalog number: SRP6033 )
    2. Bovine serum albumin (Sigma-Aldrich, catalog number: A7906 )
    3. Myoglobin from equine skeletal muscle (Sigma-Aldrich, catalog number: M0630 )
  5. Salts
    1. Na2HPO4 anhydrous (Merck KGaA, Merck Millipore, catalog number: 106586 )
    2. NaH2PO4.H2O (Merck KGaA, Merck Millipore, catalog number: 106346 )
    3. NaCl (Merck KGaA, Merck Millipore, catalog number: 106404 )
    4. EDTA disodium salt 2-hydrate (AppliChem GmbH, PanReac AppliChem, catalog number: 131669 )
  6. Sheep anti-FITC: HRP (Bio-Rad Laboratories, AbD Serotec, catalog number: 640005 )
  7. SIGMAFASTTM OPD (Sigma-Aldrich, catalog number: P9187 )
  8. Tween® 20 (Merck KGaA, Merck Millipore, catalog number: 822184 )
  9. 0.3 M NaOH (prepared from 50% w/w NaOH solution) (18.94 M) (Sigma-Aldrich, catalog number: 415413 )
  10. 7.4% HCl (37% commercial solution diluted 1/5 with distilled water) (Merck KGaA, Merck Millipore, catalog number: 100317 )
  11. Phosphate buffered saline (PBS) containing 0.05% Tween® 20 (PBS-T)
  12. 3 N H2SO4 (1.5 M)
  13. Casein hydrolysate solution (see Recipes)
  14. Concentrated PB (see Recipes)
  15. PBS (see Recipes)
  16. PBS-EDTA (see Recipes)

Equipment

  1. 96-well microplate (polystyrene with clear flat bottom wells) (Greiner Bio-One GmbH, catalog number: 762071 )
  2. Automatic (mono- and multichannel) pipettes and pipette tips
  3. Calibrated beakers, graduated cylinders and conical flasks
  4. Magnetic stir bars and magnetic stirrer
  5. Manual or automatic microplate washer (e.g., DAS plate washer)
  6. Microcentrifuge tubes (e.g., Eppendorf; Kartell, catalog numbers: 297, for 1.5 ml , and 1298 , for 0.5 ml tubes)
  7. Microplate adhesive sealing films (e.g., AlumaSeal® II film; Sigma-Aldrich, catalog number: A2350 )
  8. Microplate reader (equipped for measuring absorbance at 492 nm) (e.g., Beckman Coulter, model: Biomek® plate reader )
  9. Microtitre plate shaker with a small orbit diameter (preferably 1.5 mm) set to 750 rpm (e.g., Stuart, model: SSM5 )
  10. pH meter
  11. Reagent reservoir for multichannel pipettes
  12. Scales
  13. Shaking incubator set to 37 °C and 150 rpm (e.g., Panasonic Corporation, SANYO Electric, model: Orbi-Safe TM)

Procedure

  1. Determine the number of wells needed for the assay. The test should be performed at least in duplicate. Following the ELISA worksheet example below, if you test in duplicate 8 serial dilutions of an unknown protein/peptide plus the same dilutions of polymyxin B (positive inhibition control) and a known protein/peptide with no binding ability to LPS (negative inhibition control) you need 52 wells: 3 x 8 x 2 = 48 wells for protein/peptide dilutions, two wells with LPS-FITC (reference control) and two wells without LPS-FITC (negative control).
  2. Add 200 µl of casein hydrolysate solution to the wells of a 96-well microplate. Seal the microplate with the adhesive film and incubate overnight at 4 °C without shaking.
  3. Separately, prepare a 2x solution of LPS O111: B4-FITC conjugate (5 µg/ml) and 2x serial dilutions of the protein/peptide to be tested (starting dilution 80 µg/ml) in PBS-EDTA. Prepare the same dilutions of polymyxin B (positive inhibition control) and a negative inhibition control (e.g., ovalbumin). Eight serial ¼ dilutions are optimal for most proteins. For the ELISA worksheet example prepare:
    1. 2x solution of LPS-FITC: 3.5 ml of LPS-FITC at 5 µg/ml in PBS-EDTA.
    2. 2x serial dilutions of the proteins/peptides in PBS-EDTA:

      Table 1. Preparation of serial dilutions
      Dilution (dil) number
      Volume and source of proteins/peptides
      Volume of PBS-EDTA
      Protein concentration
      1
      3.2 μl from a 5 mg/ml stock
      196.8 μl
      80 µg/ml
      2
      50 μl of dil 1
      150 μl
      20 µg/ml
      3
      50 μl of dil 2
      150 μl
      5 µg/ml
      4
      50 μl of dil 3
      150 μl
      1.25 µg/ml
      5
      50 μl of dil 4
      150 μl
      0.312 µg/ml
      6
      50 μl of dil 5
      150 μl
      0.078 µg/ml
      7
      50 μl of dil 6
      150 μl
      0.020 µg/ml
      8
      50 μl of dil 7 150 μl
      0.005 µg/ml

  4. Mix 120 µl of LPS-FITC solution (2x) with 120 µl of each dilution (2x) or PBS-EDTA (reference control) in individual microcentrifuge tubes, mix well, and incubate the samples for 1 h at RT.
  5. Aspirate the content of the wells coated with the casein hydrolysate and wash 3 times with 200 µl of PBS (no soak) at RT without shaking.
  6. Immediately, add 100 µl of the preincubated solutions or PBS-EDTA (negative control) to each well in duplicate. Seal the microplate and incubate for 30 min at RT under shaking at 750 rpm.
  7. Wash the plate 5 times with 200 µl of PBS-T (no soak) at RT without shaking.
  8. Add 100 µl of sheep anti-FITC: HRP diluted 1/4,000 in PBS-T. Seal the microplate and incubate for 30 min at RT under shaking at 750 rpm.
  9. Wash the plate as described in step 7.
  10. Prepare the chromogen [o-phenylenediamine dihydrochloride (OPD)] solution following the manufacturer’s instructions.
  11. Add 100 µl of the OPD solution to each well and incubate for 20 min at RT, without shaking and in the dark.
  12. Add 25 µl of 3 N H2SO4 to each well to stop the reaction.
  13. Read the absorbance of the wells at 492 nm within 30 min.
  14. The percentage of inhibition for each protein dilution is calculated according to the formula: [(average OD reference control) - (average OD protein dil + LPS-FITC)]/ (average OD reference control) x 100. OD: Optical density.
    *Alternatively, incubations in steps 6 and 8 can be performed in an incubator for 1 h at 37 °C without shaking, after sealing the plate with the adhesive film.

    Table 2. ELISA worksheet example

    1
    2
    3
    4
    5
    6
    7
    A
    100 µl LPS-FITC (reference control)
    100 µl
    Protein dil 1 + LPS-FITC
    100 µl
    Polymyxin B dil 1 + LPS-FITC
    100 µl
    Ovalbumin dil 1 + LPS-FITC
    B

    100 µl
    Protein dil 2 + LPS-FITC
    100 µl 
    Polymyxin B dil 2 + LPS-FITC
    100 µl 
    Ovalbumin dil 2 + LPS-FITC
    C
    100 µl
    PBS-EDTA
    (negative control)
    100 µl
    Protein dil 3 + LPS-FITC
    100 µl
    Polymyxin B dil 3 + LPS-FITC
    100 µl
    Ovalbumin dil 3 + LPS-FITC
    D
    100 µl
    Protein dil 4 + LPS-FITC
    100 µl
    Pol ymyxin B dil 4 + LPS-FITC
    100 µl
    Ovalbumin dil 4 + LPS-FITC
    E

    100 µl
    Protein dil 5 + LPS-FITC
    100 µl
    Polymyx in B dil 5 + LPS-FITC
    100 µl
    Ovalbumin dil 5 + LPS-FITC
    F
    100 µl
    Protein dil 6 + LPS-FITC
    100 µl
    Polymyxin B dil 6 + LPS-FITC
    100 µl
    Ovalbumin dil 6 + LPS-FITC
    G
    100 µl
    Protein dil 7 + LPS-FITC
    100 µl
    Polymyxin B dil 7 + LPS-FITC
    100 µl
    Ovalbumin dil 7 + LPS-FITC
    H
    100 µl
    Protein dil 8 + LPS-FITC
    100 µl
    Polymyxin B dil 8 + LPS-FITC
    100 µl
    Ovalbumin dil 8 + LPS-FITC
     

Representative data



Figure 1. Example of inhibition curve obtained with several molecules using the reported method to analyze LPS binding. LPS-FITC (0.25 µg/well) was incubated with four-fold dilutions of polymyxin B (circles), LBP human recombinant (squares), bovine serum albumin (BSA) (inverted triangles) and myoglobin from equine skeletal muscle (triangles). It is noteworthy that an excess of protein/peptide might have a “zone effect” in the assay, as occurs with polymyxin B.

Notes

  1. The ideal OD range is 0.6-1.2 for the reference control, which was obtained using LPS-FITC at 0.25 µg/well. As the FITC content may vary between batches this concentration might need to be adjusted before performing the inhibition assay. The extent of labeling of the LPS-FITC used to develop this method was of 7.20 µg FITC/mg LPS.
  2. Binding of a protein to LPS does not imply that it is linked to a biological effect. Further experiments should be done to test the activity in vitro and in vivo.
  3. Depending on several factors (amino acid composition, type of interaction with LPS, number of binding sites, etc.) each protein or peptide needs a different optimal concentration to obtain the maximal inhibition. Hence, several dilutions of the proteins or peptides need to be assayed.
  4. The main advantage of using a competitive ELISA instead of an indirect ELISA is that in the latter, the protein/peptide to be tested is coated to the wells and blocking agents may interfere with LPS binding (e.g., binding LPS per se, causing steric hindrance). Casein hydrolysate is commonly used as a blocking agent in ELISA and also serves as LPS binding agent, enabling its use in the competitive system reported.
  5. Inhibition will occur if the protein or peptide to be tested binds to the same or a close LPS region as the casein hydrolysate. Although casein hydrolysate is composed of several derived peptides and therefore the mechanism of binding to LPS is poorly understood, as polymyxin B inhibits LPS binding to casein, this suggests that the casein hydrolysate binds at least to the lipid A region of LPS.
  6. LPS is incubated in PBS-EDTA in the absence of detergents as both LPS and detergents have an amphipathic nature and may give unexpected results difficult to interpret when incubated together.

Recipes

  1. Casein hydrolysate solution
    Note: The preparation is based on the method described by Pearce-Pratt and Roser (2010) with some modifications.
    1. Dissolve 2.5 g of casein in 80 ml of 0.3 M NaOH and incubate overnight at 37 °C under shaking at 150 rpm
    2. Adjust pH to 8 slowly with 7.4% HCl
      Note: Aggregation of casein fragments occurs transiently while the pH is adjusted. Keep the pH meter electrode out of the solution when you add HCl. Read the pH or add more HCl dropwise always after the aggregates are dissolved.
    3. Add 2 ml of concentrated PB (the pH lowers to approximately 7.5)
    4. Adjust pH to 7.2 with 7.4% HCl
    5. Made up to 200 ml with distilled water and readjust the pH if necessary
  2. Concentrated PB (0.75 M, pH 7.2)
    For a 50 ml solution:
    Dissolve 4.08 g of Na2HPO4 anhydrous (Mw: 141.96) and 1.22 g of NaH2PO4.H2O (Mw: 137.99) up to 50 ml with distilled water
  3. PBS (150 mM, pH 7.2)
    For a 1 L solution:
    Dissolve 1.63 g of Na2HPO4 anhydrous (Mw: 141.96), 0.49 g of NaH2PO4.H2O (Mw: 137.99) and 7.89 g of NaCl (Mw: 58.44) up to 1 L with distilled water
  4. PBS (150 mM, pH 7.2)-EDTA (1 mM)
    Add EDTA disodium salt 2-hydrate at a concentration of 1 mM in PBS
    As EDTA lowers the pH, readjust the pH to 7.2 with NaOH

Acknowledgments

This work was supported by grants AGL2011-30563-C03 (Ministerio de Ciencia e Innovación, Spain) and CN 2012/155 (Xunta de Galicia, Spain). Victoria Martínez-Sernández holds a predoctoral fellowship (Programa de Formación del Profesorado Universitario) from the Spanish Ministerio de Educación, Cultura y Deporte. This protocol was modified from Martínez-Sernández et al. (2014).

References

  1. Brandenburg, K., Koch, M. H. and Seydel, U. (1998). Biophysical characterisation of lysozyme binding to LPS Re and lipid A. Eur J Biochem 258(2): 686-695.
  2. Chen, X., Dings, R. P., Nesmelova, I., Debbert, S., Haseman, J. R., Maxwell, J., Hoye, T. R. and Mayo, K. H. (2006). Topomimetics of amphipathic beta-sheet and helix-forming bactericidal peptides neutralize lipopolysaccharide endotoxins. J Med Chem 49(26): 7754-7765.
  3. de Haas, C. J., van der Zee, R., Benaissa-Trouw, B., van Kessel, K. P., Verhoef, J. and van Strijp, J. A. (1999). Lipopolysaccharide (LPS)-binding synthetic peptides derived from serum amyloid P component neutralize LPS. Infect Immun 67(6): 2790-2796.
  4. Jang, H., Kim, H. S., Moon, S. C., Lee, Y. R., Yu, K. Y., Lee, B. K., Youn, H. Z., Jeong, Y. J., Kim, B. S., Lee, S. H. and Kim, J. S. (2009). Effects of protein concentration and detergent on endotoxin reduction by ultrafiltration. BMB Rep 42(7): 462-466.
  5. Lopez-Exposito, I., Amigo, L. and Recio, I. (2008). Identification of the initial binding sites of alphas2-casein f(183-207) and effect on bacterial membranes and cell morphology. Biochim Biophys Acta 1778(10): 2444-2449.
  6. Martinez-Sernandez, V., Mezo, M., Gonzalez-Warleta, M., Perteguer, M. J., Muino, L., Guitian, E., Garate, T. and Ubeira, F. M. (2014). The MF6p/FhHDM-1 major antigen secreted by the trematode parasite Fasciola hepatica is a heme-binding protein. J Biol Chem 289(3): 1441-1456.
  7. Ohno, N. and Morrison, D. C. (1989). Lipopolysaccharide interaction with lysozyme. Binding of lipopolysaccharide to lysozyme and inhibition of lysozyme enzymatic activity. J Biol Chem 264(8): 4434-4441.
  8. Pearce-Pratt, R. and Roser, B. (2010). Comparison of Blocking Agents for ELISA. Thermo Fisher Scientific Technical Bulletin: 07a.

材料和试剂

  1. 酪蛋白(Hammarsten等级)(BDH Prolabo,VWR International,目录号:440203H)
  2. 来自大肠杆菌O111:B4-FITC缀合物(Sigma-Aldrich,目录号:F3665)的脂多糖
  3. 多粘菌素B硫酸盐(Sigma-Aldrich,目录号:P4119)
  4. 用于以下实施例(见代表性数据)但对于测定不是必需的蛋白质:
    1. LBP人重组体(Sigma-Aldrich,目录号:SRP6033)
    2. 牛血清白蛋白(Sigma-Aldrich,目录号:A7906)
    3. 来自马骨骼肌的肌红蛋白(Sigma-Aldrich,目录号:M0630)
    1. Na 2 HPO 4无水物(Merck KGaA,Merck Millipore,目录号:106586)
    2. (Merck KGaA,Merck Millipore,目录号:106346)
      />
    3. NaCl(Merck KGaA,Merck Millipore,目录号:106404)
    4. EDTA二钠盐2-水合物(AppliChem GmbH,PanReac AppliChem,目录号:131669)
  5. 羊抗FITC:HRP(Bio-Rad Laboratories,AbD Serotec,目录号:640005)
  6. SIGMAFAST TM opD(Sigma-Aldrich,目录号:P9187)
  7. Tween 20(Merck KGaA,Merck Millipore,目录号:822184)
  8. 0.3M NaOH(由50%w/w NaOH溶液制备)(18.94M)(Sigma-Aldrich,目录号:415413)
  9. 7.4%HCl(37%市售溶液,用蒸馏水稀释1/5)(Merck KGaA,Merck Millipore,目录号:100317)
  10. 含有0.05%Tween 20(PBS-T)的磷酸盐缓冲盐水(PBS)
  11. 3 H H 2 SO 4(1.5M)
  12. 酪蛋白水解产物溶液(参见配方)
  13. 浓缩PB(参见配方)
  14. PBS(请参阅配方)
  15. PBS-EDTA(参见配方)

设备

  1. 96孔微板(具有透明平底孔的聚苯乙烯)(Greiner Bio-One GmbH,目录号:762071)
  2. 自动(单通道和多通道)移液器和移液器吸头
  3. 校准烧杯,量筒和锥形瓶
  4. 磁力搅拌棒和磁力搅拌器
  5. 手动或自动微孔板清洗机(如,DAS板清洗机)
  6. 微离心管(例如,Eppendorf; Kartell,目录号:297,对于1.5ml,和1298,对于0.5ml管)
  7. 微板粘合剂密封膜(例如,AlumaSeal II膜; Sigma-Aldrich,目录号:A2350)
  8. 微板读数器(装备用于测量492nm处的吸光度)(例如,Beckman Coulter,型号:Biomek 读板器)
  9. 具有设定为750rpm(例如,Stuart,型号:SSM5)的小轨道直径(优选1.5mm)的微量滴定板振荡器
  10. pH计
  11. 多通道移液器的试剂储液器
  12. 刻度
  13. 将设定为37℃和150rpm(例如,Panasonic Corporation,SANYO Electric,型号:Orbi-Safe TM )的培养箱摇动

程序

  1. 确定测定所需的孔数。测试应至少进行一式两份。在下面的ELISA工作表实施例中,如果您在未知蛋白质/肽加上相同稀释度的多粘菌素B(阳性抑制对照)和已知不具有LPS结合能力的蛋白质/肽(阴性抑制对照)的8个系列稀释液中进行测试,您需要52个孔:3×8×2 = 48个用于蛋白质/肽稀释的孔,具有LPS-FITC(参考对照)的两个孔和不具有LPS-FITC(阴性对照)的两个孔。
  2. 加入200微升酪蛋白水解产物溶液到96孔微孔板的孔中。用粘合剂膜密封微板,并在4℃下振荡孵育过夜。
  3. 单独地,制备LPS O111:B4-FITC缀合物(5μg/ml)的2x溶液和在PBS-EDTA中的待测试的蛋白质/肽(起始稀释度80μg/ml)的2x系列稀释液。制备相同稀释度的多粘菌素B(阳性抑制对照)和阴性抑制对照(例如卵白蛋白)。八个系列¼稀释是大多数蛋白质的最佳选择。对于ELISA工作表示例准备:
    1. 2x LPS-FITC的溶液:在PBS-EDTA中的5μg/ml的3.5ml LPS-FITC
    2. 在PBS-EDTA中的蛋白质/肽的2x系列稀释液:

      表1.制备连续稀释
      稀释(稀释)编号
      蛋白质/肽的量和来源
      PBS-EDTA的体积
      蛋白质浓度
      1
      从5mg/ml储液中取出3.2μl 196.8微升
      80μg/ml
      2
      50微升稀1
      150μl
      20μg/ml
      3
      50μldil 2
      150μl
      5μg/ml
      4
      50μldil 3
      150μl
      1.25μg/ml
      5
      50μl稀释液4
      150μl
      0.312μg/ml
      6
      50μldil 5
      150μl
      0.078μg/ml
      7
      50μldil 6
      150μl
      0.020μg/ml
      8
      50μl的稀释液7 150μl
      0.005μg/ml

  4. 在各个微量离心管中混合120μlLPS-FITC溶液(2x)与120μl每种稀释液(2x)或PBS-EDTA(参考对照),混匀,并在室温下孵育样品1小时。
  5. 吸出涂覆有酪蛋白水解产物的孔的内容物,并在室温下用200μlPBS(无浸泡)洗涤3次,无振荡。
  6. 立即,每孔加入100μl预孵育的溶液或PBS-EDTA(阴性对照)一式两份。 密封微量培养板,并在室温下以750rpm振荡孵育30分钟
  7. 在室温下用200μlPBS-T(无浸泡)洗涤板5次,无振荡。
  8. 加入100μl在PBS-T中稀释1/4,000的绵羊抗-FITC:HRP。 密封微量培养板,并在室温下以750rpm振荡孵育30分钟
  9. 按照步骤7所述清洗平板。
  10. 按照制造商的说明准备色原体[邻苯二胺二盐酸盐(OPD)]溶液。
  11. 加入100微升OPD溶液到每个孔,孵育20分钟,在室温,不摇动和在黑暗中。
  12. 向每个孔中加入25μl3N H 2 SO 4以终止反应。
  13. 在30分钟内读取492nm处的孔的吸光度。
  14. 根据下式计算每种蛋白质稀释的抑制百分比:[(平均OD参考对照) - (平均OD蛋白稀释+ LPS-FITC)] /(平均OD参考对照)×100。
    *或者,在用粘合剂膜密封板之后,可以在37℃下在振荡器中在不摇动的情况下在孵育器中进行步骤6和8中的孵育。

    表2. ELISA工作表示例

    1
    2
    3
    4
    5
    6
    7
    A
    100μlLPS-FITC(参考对照)
    100μl
    蛋白 dil 1 + LPS-FITC
    100μl
    多粘菌素 B dil 1 + LPS-FITC
    100μl
    卵白蛋白 dil 1 + LPS-FITC
    B

    100μl
    蛋白 dil 2 + LPS-FITC
    100μl 
    多粘菌素B dil 2 + LPS-FITC
    100μl 
    卵白蛋白 dil 2 + LPS-FITC
    C
    100μl
    PBS-EDTA
    (阴性对照)
    100μl
    蛋白 dil 3 + LPS-FITC
    100μl
    多粘菌素B dil 3 + LPS-FITC
    100μl
    卵白蛋白 dil 3 + LPS-FITC
    D
    100μl
    蛋白 dil 4 + LPS-FITC
    100μl
    Pol ymyxin B dil 4 + LPS-FITC
    100μl
    卵白蛋白 dil 4 + LPS-FITC
    E

    100μl
    蛋白 dil 5 + LPS-FITC
    100μl
    Polymyx in B dil 5 + LPS-FITC
    100μl
    卵白蛋白 dil 5 + LPS-FITC
    F
    100μl
    蛋白 dil 6 + LPS-FITC
    100μl
    多粘菌素B dil 6 + LPS-FITC
    100μl
    卵白蛋白 dil 6 + LPS-FITC
    G
    100μl
    蛋白 dil 7 + LPS-FITC
    100μl
    多粘菌素B dil 7 + LPS-FITC
    100μl
    卵白蛋白 dil 7 + LPS-FITC
    H
    100μl
    蛋白 dil 8 + LPS-FITC
    100μl
    多粘菌素B dil 8 + LPS-FITC
    100μl
    卵白蛋白 dil 8 + LPS-FITC
     

代表数据



图1.使用报道的方法分析LPS结合用几个分子获得的抑制曲线的实施例。将LPS-FITC(0.25μg/孔)与多粘菌素B(圆圈)的四倍稀释液一起温育, LBP人重组(正方形),牛血清白蛋白(BSA)(倒三角形)和来自马骨骼肌的肌红蛋白(三角形)。 值得注意的是,过量的蛋白质/肽在测定中可能具有"区域效应",如发生的那样 多粘菌素B.

笔记

  1. 参考对照的理想OD范围为0.6-1.2,其使用0.25μg/孔的LPS-FITC获得。由于FITC含量可能在批次之间变化,在进行抑制测定之前可能需要调整该浓度。用于开发该方法的LPS-FITC的标记程度为7.20μgFITC/mg LPS
  2. 蛋白质与LPS的结合不意味着其与生物效应相关。应进行进一步的实验以体外测试活性和在体内 。
  3. 根据几个因素(氨基酸组成,与LPS相互作用的类型,结合位点数目等),每种蛋白质或肽需要不同的最佳浓度以获得最大抑制。因此,需要测定蛋白质或肽的几种稀释液。
  4. 使用竞争性ELISA代替间接ELISA的主要优点是在后者中,待测试的蛋白质/肽被包被到孔中,并且封闭剂可能干扰LPS结合 (例如,结合LPS 本身,造成位阻)。酪蛋白水解产物通常在ELISA中用作封闭剂,也用作LPS结合剂,使其可用于所报道的竞争性系统中。
  5. 如果待测试的蛋白质或肽结合与酪蛋白水解产物相同或接近的LPS区域,则会发生抑制。虽然酪蛋白水解物由几种衍生肽组成,因此很少了解与LPS结合的机制,因为多粘菌素B抑制LPS与酪蛋白的结合,这表明酪蛋白水解物至少结合LPS的脂质A区。
  6. LPS在不存在去污剂的情况下在PBS-EDTA中孵育,因为LPS和去污剂都具有两亲性质,并且可能给出当一起温育时难以解释的意想不到的结果。

食谱

  1. 酪蛋白水解产物
    注意:准备基于由Pearce-Pratt和Roser(2010)描述的方法,并进行一些修改。
    1. 将2.5g酪蛋白溶解在80ml的0.3M NaOH中并在37℃下以150rpm振荡孵育过夜。
    2. 用7.4%HCl缓慢调节pH至8 注意:酪蛋白片段的聚集在pH值瞬时发生 被调整。 当你保持pH计电极在解决方案之外 加入HCl。 读取pH或添加更多HCl滴后总是 聚集体溶解。
    3. 加入2ml浓缩的PB(pH降低至约7.5)
    4. 用7.4%HCl调节pH至7.2
    5. 用蒸馏水补足至200毫升,必要时重新调整pH值
  2. 浓缩的PB(0.75M,pH7.2)
    对于50ml溶液:
    将4.08g的无水Na 2 HPO 4(Mw:141.96)和1.22g的NaH 2 PO 4水溶液溶解, H 2 O(Mw:137.99)直至50ml,用蒸馏水洗涤。
  3. PBS(150mM,pH7.2) 对于1 L溶液:
    将1.63g无水的Na 2 HPO 4(Mw:141.96),0.49g的NaH 2 PO 4水溶液, 用蒸馏水将H 2 O(Mw:137.99)和7.89g NaCl(Mw:58.44)升至1L。
  4. PBS(150mM,pH7.2)-EDTA(1mM) 在PBS中加入浓度为1mM的EDTA二钠盐2-水合物
    当EDTA降低pH时,用NaOH
    重新调节pH至7.2

致谢

这项工作得到了赠款AGL2011-30563-C03(西班牙部长会议)和CN 2012/155(西班牙Xunta de Galicia)的支持。维多利亚Martínez-Sernández从西班牙文化部长教育部获得了一个前教授奖学金(大学教授项目)。该协议修改自Martínez-Sernández等人 。 (2014年)。

参考文献

  1. Brandenburg,K.,Koch,M.H。和Seydel,U。(1998)。 溶菌酶与LPS Re和脂质A结合的生物物理表征。 Eur J Biochem。 258(2):686-695。
  2. Chen,X.,Dings,R.P.,Nesmelova,I.,Debbert,S.,Haseman,J.R.,Maxwell,J.,Hoye,T.R.and Mayo,K.H。(2006)。 两亲性β-折叠和螺旋形成型杀菌肽的Topomimetics中和脂多糖内毒素 < em Med J Med Chem 49(26):7754-7765。
  3. de Haas,C.J.,van der Zee,R.,Benaissa-Trouw,B.,van Kessel,K.P.,Verhoef,J。和van Strijp,J.A。(1999)。 脂多糖(LPS)结合 来自血清淀粉样蛋白P成分的合成肽中和LPS。 Infect Immun 67(6):2790-2796。
  4. Jang,H.,Kim,H.S.,Moon,S.C.,Lee,Y.R.,Yu,K.Y.,Lee,B.K.,Youn,H.Z.,Jeong,Y.J.,Kim,B.S.,Lee,S.H.and Kim, 蛋白质浓度和去污剂对通过超滤的内毒素还原的影响。 BMB Re 42(7):462-466。
  5. Lopez-Exposito,I.,Amigo,L。和Recio,I。(2008)。 alphas2-酪蛋白f(183-207)的初始结合位点的鉴定和对细菌膜的影响和细胞形态。 Biochim Biophys Acta 1778(10):2444-2449。
  6. Martinez-Sernandez,V.,Mezo,M.,Gonzalez-Warleta,M.,Perteguer,M.J.,Muino,L.,Guitian,E.,Garate,T.and Ubeira,F.M。 由吸虫寄生虫Fasciola hepatica分泌的MF6p/FhHDM-1主要抗原是血红素结合蛋白。 J Biol Chem 289(3):1441-1456。
  7. Ohno,N。和Morrison,D.C。(1989)。 脂多糖与溶菌酶的相互作用。脂多糖与溶菌酶的结合和溶菌酶酶活性的抑制。 J Biol Chem <264(8):4434-4441。
  8. Pearce-Pratt,R。和Roser,B。(2010)。 ELISA的封闭剂比较。 Thermo Fisher Scientific技术公告:07a。
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How to cite this protocol: Martínez-Sernández, V. and Ubeira, F. M. (2014). Competitive ELISA for Protein-lipopolysaccharide (LPS) Binding. Bio-protocol 4(22): e1298. DOI: 10.21769/BioProtoc.1298; Full Text



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11/4/2016 8:54:34 AM  

Florencio M Ubeira (Author)
Departamento de Microbiología y Parasitología, Universidad de Santiago de Compostela, Spain

New ELISA methods related with this issue were recently reported by the same authors (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156530).

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