Biomedical subjects

H I Elsner

Publications and source records attributed to H I Elsner.

9 recordsLinked to original sources

PubMed1999

A novel microtiter plate based method for identification of B-cell epitopes.

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B-cell epitopes in two different proteins using a peptide-based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15-mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor-alpha, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non-covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor-alpha were added to identify potential linear B-cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non-covalent binding although the peptides were in fact present on the non-covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B-cell epitopes in protein antigens.

Amino Acid Sequence↗

PubMed1999

Therapeutic antibodies elicited by immunization against TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in the pathogenesis of several chronic inflammatory diseases. Monoclonal antibodies against TNF-alpha are currently used for the treatment of rheumatoid arthritis and Crohn's disease. This report describes a simple and effective method for active immunization against self TNF-alpha. This vaccination approach leads to a T-cell-dependent polyclonal and sustainable anti-TNF-alpha autoantibody response that declines upon discontinuation of booster injections. The autoantibodies are elicited by injecting modified recombinant TNF-alpha molecules containing foreign immunodominant T-helper epitopes. In mice immunized with such molecules, the symptoms of experimental cachexia and type II collagen-induced arthritis are ameliorated. These results suggest that vaccination against TNF-alpha may be a useful approach for the treatment of rheumatoid arthritis and other chronic inflammatory diseases.

Animals↗

PubMed1996

Breaking of B cell tolerance toward a highly conserved self protein.

Self proteins are processed and presented by APCs in the same way as foreign proteins. Presentation of fragments derived from self proteins does not, however, lead to Th cell stimulation because of T cell tolerance. In this study, a novel approach was used to investigate whether B cell tolerance toward a self Ag could be due to the absence of this Th cell recognition. The highly conserved nonimmunogenic protein ubiquitin was used as a model protein. Two modified ubiquitin molecules were constructed with ubiquitin segments exchanged either with the T cell epitope, OVA(325-336), which binds to the mouse A(d) MHC class II molecule, or with the T cell epitope, hen egg lysozyme(50-61), which binds to the A(k) molecule. Mice were immunized with the resulting proteins. Both modified proteins elicited strong autoantibody responses toward soluble native ubiquitin, demonstrating that insertion of a single foreign T cell epitope can overcome the B cell nonresponsiveness. The T cell regulatory role of one of the inserted foreign T cell epitopes in ubiquitin was studied, and at least two different Th cell specificities were found to operate in the response. The T cells were directed against: 1) the inserted epitope, and 2) a combination of the inserted epitope and parts of the neighboring ubiquitin regions. Therefore, the absence of T cell help seems to be an important reason for B cell tolerance toward self proteins.

Amino Acid Sequence↗

PubMed1995

Hydrocoating: a new method for coupling biomolecules to solid phases.

Solid-phase immunoassays such as enzyme-linked immunosorbent assays require one of the assay components to be immobilized. Most frequently this is achieved by passive adsorption of the antigen or antibody to a hydrophobic polymer surface composed of, e.g., polystyrene. Alternatively the biomolecule can be bound indirectly via passively adsorbed carrier proteins or directly via functional groups on the solid phase using cross-linking agents. Here we describe a new technique--hydrocoating--for covalent immobilization of biomolecules, such as peptides, in highly hydrophilic surroundings. Peptides were immobilized on microtiter plates via covalent bonds to an activated hydrophilic polymer. Soluble dextran was activated using 2,2,2-triflouroethanesulphonyl chloride (tresyl chloride) leading to activation of hydroxyl groups on the dextran polymer. This activated dextran molecule was immobilized on a surface containing amino groups leaving a sufficient number of active groups for secondary binding of other biomolecules. Peptides, that were either undetectable or poorly recognized when adsorbed on polystyrene, were readily recognized when immobilized by the hydrocoating technique. Furthermore, peptides immobilized by this method were recognized 5-10-fold better compared to the same peptides immobilized covalently on a surface containing secondary amino groups. The technique appears to provide an alternative to passive adsorption of biomolecules on solid phases and may be useful in the future development of immunoassays.

Amino Acid Sequence↗

PubMed1989

Ultrasonic degradation of DNA.

Different results are obtained when DNA in aqueous solution and DNA in biological tissue are exposed to ultrasound. At intensities of ultrasound comparable to those applied clinically, ultrasonication is able to degrade purified DNA in aqueous solution, making ultrasonication a useful tool for preparing DNA fragments in vitro. Ultrasonic degradation of DNA in solution occurs by breaking hydrogen bonds and by single-strand and double-strand ruptures of the DNA helix. Two mechanisms are mainly responsible: cavitation and a thermal or mechanical effect. Stable cavitation is seen at low intensities of ultrasound. Increasing the intensity of the ultrasound above 2 W/cm2 is followed by increases in single-strand ruptures due to the creation of free radicals by transient cavitation. Following sonication, the distribution of the resulting DNA fragments approaches a lower size limit of 100-500 bp. Breaks in the DNA helix occur mainly between oxygen and carbon atoms, resulting in DNA fragments with a phosphorylated 5' end and a free alcohol at the 3' end. The relative lack of specificity in degrading the DNA helix makes ultrasonication a complementary alternative to the highly specific fragmentation obtained by restriction endonucleases.

DNA↗

PubMed1986

Protein crosslinking reagents containing a selenoethylene linker are cleaved by mild oxidation.

A homobifunctional cleavable crosslinking reagent containing a selenoethylene group in the linker, and related reagents, have been synthesized and tested in a model system involving formation of a complex between albumin and cytochrome c. Functionally, complex formation was suggested by albumin inhibition of the ascorbate reduction of cytochrome c. Structurally, complex formation was demonstrated by crosslinking and subsequent separation of crosslinked complex from non-crosslinked proteins by SDS-polyacrylamide gel electrophoresis. The crosslinks were found to be cleavable by mild oxidation with low concentrations of periodate or with N-chlorobenzenesulfonamide immobilized on polystyrene beads (Iodo-Beads).

Albumins↗

PubMedDate not supplied

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids.

Peptides consisting solely of D-amino acids (D-peptides) as opposed to their L-counterparts (L-peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L- and D-peptides, capable of binding to both avidin and streptavidin, were found. The L-peptides contained the previously described HPQ/M motifs, and among the D-peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position. At the third position in this motif several different amino acid residues were found, although N was the most frequent. Peptides representing two of the D-motifs were synthesized as well as peptides containing the HPQ/M motifs, and their binding properties were examined. Although the D-peptides were originally selected using avidin they also inhibited binding between immobilized biotin and soluble streptavidin as well as avidin. The IC50 of some of the peptides were approximately 10(5) times higher than the IC50 for biotin but some had a lower IC50 than iminobiotin. The D-peptides, which were originally selected from the library using avidin, could also inhibit the binding between streptavidin and biotin. Likewise, L-peptides selected from a library screened with streptavidin, could inhibit the binding of both streptavidin and avidin to immobilized biotin. Furthermore, the D-peptide, VFSVQSGS, as well as biotin could inhibit binding of streptavidin to an immobilized L-peptide (RYHPQSGS). This indicates that the biotin-like structure mimicked by these two seemingly very different peptides may react with the same binding sites in the streptavidin molecule.

Amino Acid Sequence↗

PubMedDate not supplied

Induction of cross-reactive antibodies against a self protein by immunization with a modified self protein containing a foreign T helper epitope.

Self proteins are handled in the same way as foreign proteins by antigen presenting cells, but because of T-cell tolerance the presentation of self peptides does not normally lead to T cell activation. By providing physically linked T-cell help it is possible to overcome the B cell non-responsiveness toward self antigens. We have shown previously that a very potent antibody response, cross-reactive with a self protein, can be rapidly induced by immunizing with a recombinant immunogen consisting of the self protein with a foreign immunodominant T helper epitope inserted into its sequence (Dalum, I., Jensen, M. R., Hindersson, P., Elsner, H. I. and Mouritsen, S. (1996) J. Immnunol. 157, 4796). In this study we compare this approach for inducing autoantibodies against a self protein with the traditional method of conjugating the self antigen to a foreign carrier protein. The highly conserved self protein ubiquitin with an inserted epitope from ovalbumin (UbiOVA) is used as a model protein and compared to two traditionally conjugated immunogens consisting of ubiquitin chemically conjugated to a peptidic T helper epitope or to ovalbumin. The traditionally conjugated immunogens induce much slower and low titered ubiquitin specific antibody responses than the recombinant construct which also is capable of inducing antibodies directed against a much broader range of potential ubiquitin B cell determinants than the chemically conjugated immunogens. All three constructs are processed by antigen presenting cells and ovalbumin derived T cell epitopes are presented to T helper cells. From these observations it seems likely that the presence of non-shielded autologous B cell determinants on the immunogen is critical for the ability to induce a strong autoantibody response with a diverse fine specificity. Furthermore, the ubiquitin specific antibodies induced by UbiOVA contain higher levels of IgG2a/b relative to IgG1 compared to the conjugates. We therefore speculate that the insertion of a T cell epitope directly into the self antigen could possibly induce an immune response with a different Th1/Th2 balance than a response induced with traditional conjugates.

Amino Acid Sequence↗

PubMedDate not supplied

Use of psoralens for covalent immobilization of biomolecules in solid phase assays.

The ability of compounds to adsorb passively to hydrophobic polymer surfaces composed of, e.g., polystyrene generally is restricted to limited types of molecules such as proteins. Some proteins, many peptides, polysaccharides, oligonucleotides, and small molecules as well as pro- and eucaryotic cells cannot adsorb directly to such surfaces. Also, solid phase adsorbed antigens, antibodies, or gene probes may not be recognized by its corresponding ligand due to denaturation or steric hindrance of the molecular tertiary structure. Covalent binding, on the other hand, orientates all immobilized compounds in a defined way on the solid phase, thereby exposing the interacting sites on the enzymes, antibodies, gene probes, etc. Here we describe a method for modifying a polymer surface by contacting the polymer with derivatives of psoralen under irradiation with long-wavelength UV light. The psoralen derivatives were immobilized covalently on the polymer surface by this process. The psoralen molecules was conjugated to appropriate chemical linkers, incubated in aqueous solutions, and irradiated with UV light. This resulted in solid phase introduction of functional groups such as, e.g., amino groups on the polystyrene surface. The functional groups could subsequently be used for immobilization of biomolecules using conventional cross-linker technology. The method only involved premodification of the psoralens to be immobilized whereas no pretreatment of the polymer was required. Psoralen modified microtiter plates seems to have future application for the development of solid phase hybridization and immunoassays.

Biotin↗