Detection of lipopolysaccharides blotted on nylon membranes

Abstract

A sensitive method for the detection of Gram-negative bacterial lipopolysaccharide (LPS) blotted on nylon membranes is described. LPSs are separated by SDS-PAGE and then electrophoretically transferred to nylon membranes. Immobilized LPS is oxidized with periodate and then reacted with a hydrazide conjugated to the steroid, digoxigenin. LPS is visualized by alkaline phosphatase labelled antibodies against the steroid and the enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. LPS banding patterns of both rough (R-) and smooth (S-) type LPSs from over 15 different bacterial species are similar to those of silver stained companion gels, but without nonspecific staining of proteins. The detection of S-LPS from Pseudomonas aeruginosa F-D type 1 and R-LPS from Escherichia coli K12 is sensitive to 10-20 ng per lane. The use of this detection system in combination with antibody or lectin studies on identical blots can provide an effective tool in locating the precise position of certain epitopes or sequences in both R- and S-type LPSs on the blots.

Get full access to this article

View all access options for this article.

References

Gibson B.W. , Melaugh W. , Phillips N.J. , Apicella M.A. , Campagnari A.A. , Griffiss J.M. Investigation of the structural heterogeneity of lipooligosaccharides from pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry. J Bacteriol 1993; 175: 2702-2712.

Jennings H.J. , Johnson K.G. , Kenne L. The structure of an R-type oligosaccharide core obtained from some lipopolysaccharides of Neiserria meningitidis. Carbohydr Res 1983; 121: 233-241.

Schneider H. , Hale T L. , Zollinger W D. , Seid Jr R.C. , Hammock C A. , Griffiss J M. Heterogeneity of molecular size and antigenic expression within lipooligosaccharides of individual strains of Neisseria gonorrhoeae and Neisseria meningitidis . Infect Immun 1984; 45: 544-549.

Hitchcock P.J. , Leive L. , Makela P.H. , Rietschel E.T. , Strittmatter W. , Morrison DC Lipopolysaccharide nomenclature-past, present, and future. J Bacteriol 1986; 166: 699-705.

Tsai C.M. , Mocca L.F. , Frasch C.E. Immunotype epitopes of Neisseria meningitidis lipooligosaccharide types 1 through 8. Infect Immun 1987; 55: 1652-1656.

Jann B. , Reske K. , Jann K. Heterogeneity of lipopolysaccharides. Analysis of polysaccharide chain lengths by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Eur J Biochem 1975; 60: 239-246.

Russel R R B , Johnson KG SDS-polyacrylamide gel electrophoresis of lipopolysaccharides. Can J Microbiol 1975; 21: 2013-2018.

Tsai C.M. , Frasch C.E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 1982; 119: 115-119.

Hitchcock P.J. , Brown T.M. Morphological heterogeneity among Salmonella lipopolysaccharides chemotypes in silver-stained polyacrylamide gels. J Bacteriol 1983; 154: 269-277.

10.

Kido N. , Ohta M. , Kato N. Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Bacteriol 1990; 172:1145-1147.

11.

Appelmelk B.J. , Su D. , Verweij-van Vught A M J J, Thijs B G, MacLaren D M. Polymyxin B-horseradish peroxidase conjugates as tools in endotoxin research . Anal Biochem 1992; 207: 311-316.

12.

Haselbeck A. , Hosel W. Description and application of an immunological detection system for analyzing glycoproteins on blots. Glycoconj J 1990; 7: 63-74.

13.

Keren Z. , Berke G. , Gershoni J.M. Identification of cell surface glycoproteins by periodate-alkaline phosphatase hydrazide. Anal Biochem 1986; 155: 182-187.

14.

Gershoni J.M. , Bayer E.A. , Wilchek M. Blot analyses of glycoconjugates: enzyme-hydrazide - a novel reagent for the detection of aldehydes. Anal Biochem 1985; 146: 59-63.

15.

Stein M.A. , McAllister S.A. , Johnston K.H. , Diedrich D.L. Detection of lipopolysaccharides blotted to polyvinylidene difluoride membranes . Anal Biochem 1990; 188: 285-287.

16.

Tsai C.M. , Frasch C.E. Heterogeneity and variation among Neisseria meningitidis lipopolysaccharides . J Bacteriol 1983; 155: 498-504.

17.

Gu X.X. , Tsai C.M. Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media. Anal Biochem 1991; 196: 311-318.

18.

Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 ; 277: 680-685.

19.

Towbin H. , Staehelin T. , Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354.

20.

Davies R.L. , Wall R.A. , Borriello S.P. Comparison of methods for the analysis of outer membrane antigens of Neisseria meningitidis by Western blotting. J Immunol Meth 1990; 134: 215-225.

21.

Davies R.L. , Parton R. , Coote J G. , Gibbs H.A. , Freer J.H. Evaluation of different methods for the detection of outer membrane proteins and lipopolysaccharides of Pasteurella haemolytica by immunoblotting. J Immunol Meth 1994; 167: 35-45.

22.

O'Shannessy D.J. , Hoffman W.L. Site-directed immobilization of glycoproteins on hydrazide-containing solid supports. Biotechnol Appl Biochem 1987; 9: 488-496.

23.

Gu X. X Tsai C.M. , Karpas A.B. Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis. J Clin Microbiol 1992; 30: 2047-2053.

24.

Jovin T.M. Multiphasic zone electrophoresis. I. Steady-state moving-boundary systems formed by different electrolyte combinations. Biochemistry 1973; 12: 871-879.

25.

Jovin T.M.

Multiphasic zone electrophoresis. II. Design of integrated discontinuous buffer systems for analytical and preparative fractionation

Biochemistry 1973; 12: 879-890.

26.

Moos Jr M. , Nguyen N Y. , Liu T.Y. Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J Biol Chem 1988; 263: 6005-6008.

27.

Dunn S.D. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies. Anal Biochem 1986; 157: 144-153.

28.

Kuhn H.M. , Meier-Dieter U. , Mayer H. ECA, the enterobacterial common antigen. FEMS Microbiol Rev 1988; 54: 195-222.

29.

Satoh K. , Busch H. Silver staining of phosphoserine and phosphothreonine in nucleolar and other phosphoproteins. Cell Biol Int Rep 1981; 5: 857-866.

30.

Olert J. , Sawatzki G. , Kling H. , Gebauer J. Cytological and histochemical studies on the mechanism of the selective silver staining of nucleolus organizer regions (NORs). Histochemistry 1979; 60: 91-99.

31.

Lasfargues A. , Caroff M. , Chaby R. Structural features involved in the mitogenic activity of Bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice. FEMS Immunol Med Microbiol 1993; 7: 119-130.

32.

Lebbar S. , Caroff M. , Szabo L. , Merienne C. , Szilogyi L. Structure of a hexasaccharide proximal to the hydrophobic region of lipopolysaccharides present in Bordetella pertussis endotoxin preparations. Carbohydr Res 1994; 259: 257-275.

33.

Knibbs R.N. , Goldstein I.J. , Ratcliffe R.M. , Shibuya N. Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis. J Biol Chem 1991; 266: 83-88.

34.

Tsai C.M. , Civin C.I. Eight lipooligosaccharides of Neisseria meningitidis react with a monoclonal antibody which binds lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) . Infect Immun 1991; 59: 3604-3609.

35.

Moran E.E. , Brandt B.L. , Zollinger W.D. Expression of the L8 lipopolysaccharide determinant increases the sensitivity of Neisseria meningitidis to serum bactericidal activity. Infect Immun 1994; 62: 5290-5295.