In situ deprotection: a method for covalent immobilization of peptides with well-defined orientation for use in solid phase immunoassays such as enzyme-linked immunosorbent assay.

The orientation of an immobilized antigen is important for recognition by, e.g., an antibody. When noncovalent passive adsorption is used for immobilization, the number of ways that the antigen can attach to the surface is numerous and control of how the antigen orientates on the surface is limited. Covalent immobilization restricts the number of the ways that the antigen can be immobilized to the number of reactive groups on the antigen and, hence, the orientation of the immobilized antigen is more predictable. Peptide antigens were synthesized and purified with protection groups on the lysine and cysteine side chains. These peptides, which have only one good nucleophilic group (the N-terminal alpha-amino group), were immobilized covalently in microtiter plates supplied with tresyl groups on the surface and the protection groups were cleaved off in situ after immobilization. The controlled orientation of these peptides resulted in enhanced recognition by antibodies in general. An enzyme-linked immunosorbent assay for detection of antibodies against a peptide derived from outer surface protein C from Borrelia burgdorferi, found in Lyme borreliosis patients, was established using this strategy. Lyme borreliosis suspect patient sera showed up to a 10-fold increase in the signal when the orientation of the peptide antigen was controlled by the in situ deprotection strategy.

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.

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.

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.

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.