Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

BACKGROUND: Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. METHODS: Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. RESULTS: The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. CONCLUSIONS: Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen-food syndrome and to study the IgG epitope of the allergens.

The testosterone binding mechanism of an antibody derived from a naïve human scFv library.

A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10(8) clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR-L3 and CDR-H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three-dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire.

Development of hypoallergenic variants of the major horse allergen Equ c 1 for immunotherapy by rational structure based engineering.

The use of recombinant allergens is a promising approach in allergen-specific immunotherapy (AIT). Considerable limitation, however, has been the ability of recombinant allergens to activate effector cells leading to allergic reactions. Recombinant hypoallergens with preserved protein folding and capacity to induce protective IgG antibodies binding effectively to the native allergen upon sensitization would be beneficial for safer AIT. In this study, hypoallergen variants of the major horse allergen Equ c 1 were designed by introducing one point mutation on the putative IgE epitope region and two mutations on the monomer-monomer interface of Equ c 1 dimer. The recombinant Equ c 1 wild type and the variants were produced and purified to homogeneity, characterized by size-exclusion ultra-high performance liquid chromatography and ultra-high resolution mass spectrometry. The IgE-binding profiles were analyzed by a competitive immunoassay and the biological activity by a histamine release assay using sera from horse allergic individuals. Two Equ c 1 variants, Triple 2 (V47K + V110E + F112K) and Triple 3 (E21Y + V110E + F112K) showed lower allergen-specific IgE-binding capacity and decreased capability to release histamine from basophils in vitro when using sera from six allergic individuals. Triple 3 showed higher reduction than Triple 2 in IgE-binding (5.5 fold) and in histamine release (15.7 fold) compared to wild type Equ c 1. Mutations designed on the putative IgE epitope region and monomer-monomer interface of Equ c 1 resulted in decreased dimerization, a lower IgE-binding capacity and a reduced triggering of an allergic response in vitro.

Dimerization of lipocalin allergens.

Lipocalins are one of the most important groups of inhalant animal allergens. The analysis of structural features of these proteins is important to get insights into their allergenicity. We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen. The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the ß-sheet in dimer formation. According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution. Native mass spectrometry was employed to characterize quantitatively transient dimerization of two lipocalin allergens, Bos d 2 and Bos d 5, in solution.

Structural aspects of dog allergies: the crystal structure of a dog dander allergen Can f 4.

Four out of six officially recognized dog allergens are members of the lipocalin protein family. So far, a three-dimensional structure has been determined for only one dog allergen, Can f 2, which is a lipocalin protein. We present here the crystal structure of a second lipocalin allergen from dog, a variant of Can f 4. Moreover, we have compared and analyzed the structures of these two weakly homologous (amino acid identity 21%) dog allergens. The size and the amino acid composition of the ligand-binding pocket indicate that Can f 4 is capable of binding only relatively small hydrophobic molecules which are different from those that Can f 2 is able to bind. The crystal structure of Can f 4 contained both monomeric and dimeric forms of the allergen, suggesting that Can f 4 is able to form transient (weak) dimers. The existence of transient dimers in solution was confirmed by use of native mass spectrometry. The dimeric structure of Can f 4 is formed when the ends of four ß-strands are packed against the same strands from the second monomer. The residues in the interface are mainly hydrophobic and the formation of the dimer is similar to the major horse allergen Equ c 1. Interestingly, the crystal structure of dog Can f 2 has been reported to show a different type of dimer formation. The capability of these allergens to form dimers may be important for the development of immediate allergic reaction (mast cell activation) because oligomeric allergens can effectively present multivalent epitopes.

A structural insight into the molecular recognition of a (-)-Delta9-tetrahydrocannabinol and the development of a sensitive, one-step, homogeneous immunocomplex-based assay for its detection.

(-)-Delta9-tetrahydrocannabinol (THC) is the main psychoactive compound found in cannabis. In this study, an anti-THC Fab fragment, designed T3, was isolated from a display library cloned from the spleen cells of a mouse immunized with a THC-bovine serum albumin conjugate, and the crystal structures of the T3 Fab in its free form and in complex with THC were determined at 1.9 A and 2.0 A resolution, respectively. The THC binding site of the T3 Fab is a narrow cavity: the n-pentyl group of THC protrudes deep into the interface area between the variable domains and the C(10) monoterpene moiety of the hapten is partially exposed to solvent. The metabolites of THC, with modifications in the C(10) monoterpene moiety, 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol and 11-hydroxy-?(9)-tetrahydrocannabinol, are bound by the T3 Fab with a higher affinity than THC. The crystal structures suggest that Ser52H and Arg53H of the T3 Fab are able to make hydrogen bonds with the metabolites, which leads to an increased binding against these metabolites. By developing a T3 Fab-Delta(9)-THC immunocomplex binding antibody from a naïve antibody phage display library, the specificity of the Delta(9)-THC binding is highly increased, which allows a one-step, homogeneous, fluorescence resonance energy transfer-based sensitive immunoassay, with a detection limit of 20 ng/ml from saliva samples.

The contribution of polystyrene nanospheres towards the crystallization of proteins.

BACKGROUND: Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins. METHODOLOGY: The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT), and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times. CONCLUSIONS: All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.