Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-14
2002-08-20
Peselev, Elli (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007320, C436S529000
Reexamination Certificate
active
06436653
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a method for immobilising special classes of polysaccharides to solid surfaces. Such a method is highly valuable in the construction of reliable assays for the detection of an antibody corresponding to the polysaccharide antigen. The present invention also relates to modified solid surfaces and to the use of such surfaces in various diagnostic assays. Furthermore, the present invention relates to novel KDO derivatives which are valuable intermediates in the construction of such modified solid surfaces.
BACKGROUND OF INVENTION
Bacterial lipopolysaccharides (LPSs) are characteristic outer membrane constituents of Gram-negative bacteria. LPSs are widely used as antigens in diagnostic assays specially designed for the specific detection of antibodies in serum, plasma, meat juice, saliva or other body fluids, originating from bacterial infections in humans and animals. LPSs are highly immunogenic and comprise one of the epitope characteristics for a given bacterial strain. In fact, the definition of a serotype is often based on the LPS and/or capsular polysaccharide (CPS) antigenicity. The antigenic specificity of the LPS molecule resides in the polysaccharide part of the LPS, the O-antigen, whereas the toxicity of the LPS is caused by residues contained in the lipid part of the LPS, called the lipid A. LPSs are highly amphiphilic compounds because of the joint presence of a hydrophilic O-polysaccharide group and a hydrophobic lipid group in the LPS molecule. Most of the characterised LPSs have the same principal structure which is especially conserved in the lipid A and in the inner core parts of the LPSs. The core is the part of the polysaccharide that comprises the bond between the O-antigen and the lipid A. This bond is invariably comprised of a ketosidic bond between the hemiketal function of the innermost KDO-residue and a hydroxyl-group of a GlcNAc-residue of the lipid A. The O-antigen of a specific bacterial serotype varies with respect to numbers of repeating units and may contain non-stoicheometrical substitutions with acetyl, phosphate, glycosyl or other groups. Generally, LPS-molecules without O-antigens, that is carrying only (parts of) the core saccharides in addition to the lipid A are called “rough” LPS, while LPS-molecules carrying O-antigens are called “smooth” (Raetz, C. R. H. in
Escherichia coli
and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C. E. A., ed.) Vol. 1, 2nd Ed., pp. 1035-1063, American Society for Microbiology, Washington D.C., 1996; Hitchcock et al, 1986, J. Bacteriol. 166, 699-705).
ELISA, enzyme-linked immunosorbent assay, is a well known method for detection of antibodies. In this assay, the LPSs are coated (or immobilised) on a solid surface (e.g. a plastic surface) by passive adsorption, where they serve as probes for specific antibodies. The method consists of incubation of the LPS-coated surface with the biological sample being assayed for the presence of antibodies, followed by incubation of the LPS-antibody complex with a labelled secondary antibody.
Previously, LPSs have generally been immobilised onto a solid surface without any modification of the molecules since the hydrophobic lipid A part of the molecules functions as a fairly efficient “anchor” binding the LPSs to the surface via non-covalent hydrophobic interactions leaving the hydrophilic O-polysaccharides pointing outward accessible for interactions with binding components, e.g. antibodies. However, it has been shown that the efficiency by which the LPSs are immobilised onto hydrophobic surfaces depends on both the nature of the surface and the equilibrium between free LPSs and formed LPS micelles. The equilibrium between free LPSs and formed LPS micelles depends on the amphiphilic nature of the LPSs and varies between LPSs from different bacteria strains as well as between different LPS serotypes. Certain types of LPSs have shown to be very difficult to immobilise onto solid surfaces by non-covalent bonds without addition of various micelle-dispersing agents (detergents) to the coating solution.
Thus, the optimal coating conditions vary among LPS from different bacteria strains as well as between serotypes of the same bacteria, making simultaneous immobilisation of two or more different LPSs onto the same surface very difficult. This is envisaged to be due to the ability of a well-coating LPS type to compete out the less well-coating type. This phenomenon was illustrated with
Salmonella Infantis
LPS which was shown to coat inefficiently to plastic. In an assay for detection of
Salmonella Typhimurium
and
Salmonella Infantis
specific antibodies this lead to the substitution of
Salmonella Infantis
LPS with
Salmonella Choleraesuis
LPS which was found to coat much better and which carries the same antigenic serotypes (O-antigens) (Neilsen, B. et al., 1995, Vet. Microbiol. 47, 205-218)
The tendency of LPS to form micelles (or aggregates) is furthermore believed to reduce the stability of the LPS coating as the interactions between bimolecular (or aggregated or cluster) LPS and the surface are believed to be weaker then the interactions between a single molecule of LPS and the surface. In other words, the potential tendency of LPS to coat in clusters may lead to a decreased and unpredictable coating stability and reduce the long-term stability of the coating.
EP 0 101 119 describes the immobilisation of a lipopolysaccharide to an insoluble carrier by the condensation of the lipopolysaccharide and either an amino or carboxyl group of the insoluble carrier.
JP-A-2-242448 (Patent Abstract of Japan) describes the immobilisation of a lipid A glycoside of 3-deoxy-D-manno-2-octurosonic acid to the surface of an insoluble carrier through an amide bond.
Highly hydrophilic antigens like e.g. bacterial polysaccharide (PS) are often very difficult to adsorb (immobilise) onto the most commonly used surfaces used in serological assays, such as plastics used in ELISA and RIA (radio-immuno assay), latex particles used in agglutination techniques and PVDF (polyvinylidenedifluoride) as well as nitrocellulose and other materials used for dip-stick, blotting or other fast assays. Accordingly, PS as such coat inefficiently and demand the use of large quantities of polysaccharide antigen or extremes of pH and can not be used with mixtures of polysaccharides (Elkins et al., 1990, J. Immunol. Meth. 130, 123-131). On the contrary, in spite of the drawbacks mentioned above LPSs are almost exclusively used as coating antigens as the lipid A provides the required hydrophobicity needed for adequate coating of the surface with antigen.
PSs have previously been isolated from LPSs with the purpose of preparing conjugates of PS and carrier substances, most often carrier proteins for vaccine purposes (Aron et al., 1993, J. Clin. Microbiol. 31, 975-978; Lambden and Heckels, 1982, J. Immunol. Meth. 48, 233-240; Gupta et al., 1995, Inf. Immun. 63, 2805-2810).
Previously, it has been shown that the polysaccharide part of
Salmonella Typhimurium
lipopolysaccharide (LPS) could be derivatised with biotin and immobilised onto a streptavidin-coated ELISA-plate where it could be recognised by antibodies against PS. By using the hydrazide derivative of biotin, it was possible to react the PS directly with biotin-hydrazide without a prior oxidation step; the hydrazide was shown to react with the hemiketal of the reducing end KDO of the PS. This procedure while leading to antigenically intact biotinylated PS-derivatives, however resulted in derivatives that were not stable, and an avidin or streptavidin coating had to be introduced in the assay (Wiuff, C., Lind, P., Heegaard, P., 1997, Regioselective coupling of reducing carbohydrates to hydrazides for derivatization of bacterial polysaccharides and application to immunoassays, Proc. 2
nd
. Carbohydrate Engineering Meeting, La Rochelle, France, p. 66).
In Meikle et al. (Glycoconjugate J. 7, 207-218, 1990) a PS is used in an ELISA by directly coupling the PS to a detection enzyme in a competitive set-up.
Boas Ulrik
Heegaard Peter M. H.
Jakobsen Mogens Havsteen
Stenbaek Jauho Eva Irene
Corless Peter F.
Edwards & Angell LLP
Exiqon A/S
Peselev Elli
LandOfFree
Method for introduction of reporter groups into bacterial... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for introduction of reporter groups into bacterial..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for introduction of reporter groups into bacterial... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2959017