A high-sensitivity enzyme immunoassay for the quantification of soluble human semaphorin 4D in plasma.

Human semaphorin 4D (SEMA4D), a type I integral membrane glycoprotein, regulates key cellular functions (e.g. cell-cell communication, platelet activation). Its 120 kDa extracellular region can be shed from the membrane to release soluble SEMA4D (sSEMA4D). Studies on circulating sSEMA4D levels are mostly performed with poorly characterized assays and use serum and plasma as matrix. We developed and validated a sandwich ELISA utilizing two monoclonal antibodies with resolved epitopes and determined affinities. Human serum and plasma samples were analyzed, and the influence of protease activity on sSEMA4D concentration was tested by collecting samples in the presence of the protease inhibitor TAPI-1. Both antibodies recognize conformational epitopes in the sema domain. Validation for plasma (EDTA, citrate, heparin) showed valid specificity, precision, accuracy, dilution linearity, and robustness. The assay shows a calibration range from 62.5 to 2000 pmol/L with a quantification limit of 31 pmol/L. sSEMA4D was significantly higher in serum than in plasma, whereas serum and plasma levels from samples collected in the presence of TAPI-1 showed no statistical difference. This ELISA provides a reliable tool for the quantification of sSEMA4D in human plasma. Serum is not recommended as matrix due to the accumulation of shed SEMA4D during blood coagulation altering serum sSEMA4D levels.

Characterization of a sandwich ELISA for quantification of total human soluble neuropilin-1.

BACKGROUND: Neuropilin-1 (NRP1) is a highly interactive molecule that exists as transmembrane and soluble isoforms. Measurement of circulating levels of soluble NRP1 (sNRP1) in human serum and plasma has proven to be difficult due to present matrix interferences and due to the lack of a reliable technique. METHODS: We developed a highly specific and sensitive sandwich ELISA assay for total sNRP1 quantification in peripheral blood, and we validated the test according to ICH guidelines. The linear epitopes of the employed polyclonal and monoclonal anti-human NRP1 antibodies were mapped with microarray technology. We included a sample pre-treatment step with guanidine hydrochloride (GuHCl) to release sNRP1 from existing interferants. RESULTS: The ELISA assay which is calibrated with sNRP1 isoform 2 and covers a calibration range from 0.375 to 12 nmol/L detects sNRP1 in human serum and plasma (heparin, EDTA, and citrate). Multiple linear epitopes recognized by the polyclonal coating antibody are distributed over the whole sNRP1 sequence. The monoclonal detection antibody binds to a linear epitope which is in the N-terminal region of the a1 domain of human sNRP1. Assay parameters like precision (intra-assay: 6%), dilution linearity (95%-115%), specificity (98%), and spike recovery (81%-109%) meet the international standards of acceptance. CONCLUSION: Our novel sandwich ELISA provides a reliable tool for the quantitative determination of total human sNRP1. The assay detects free and previous ligand-bound total NRP1.

Characterization of a sandwich ELISA for the quantification of all human periostin isoforms.

BACKGROUND: Periostin (osteoblast-specific factor OSF-2) is a secreted protein occurring in seven known isoforms, and it is involved in a variety of biological processes in osteology, tissue repair, oncology, cardiovascular and respiratory systems or allergic manifestations. To analyze functional aspects of periostin, or the ability of periostin as potential biomarker in physiological and pathological conditions, there is the need for a precise, well-characterized assay that detects periostin in peripheral blood. METHODS: In this study the development of a sandwich ELISA using monoclonal and affinity-purified polyclonal anti-human periostin antibodies was described. Antibodies were characterized by mapping of linear epitopes with microarray technology, and by analyzing cross-reactive binding to human periostin isoforms with western blot. The assay was validated according to ICH/EMEA guidelines. RESULTS: The monoclonal coating antibody binds to a linear epitope conserved between the isoforms. The polyclonal detection antibody recognizes multiple conserved linear epitopes. Therefore, the periostin ELISA detects all known human periostin isoforms. The assay is optimized for human serum and plasma and covers a calibration range between 125 and 4000 pmol/L for isoform 1. Assay characteristics, such as precision (intra-assay: ≤3%, inter-assay: ≤6%), spike-recovery (83%-106%), dilution linearity (95%-126%), as well as sample stability meet the standards of acceptance. Periostin levels of apparently healthy individuals are 864±269 pmol/L (serum) and 817±170 pmol/L (plasma) respectively. CONCLUSION: This ELISA is a reliable and accurate tool for determination of all currently known periostin isoforms in human healthy and diseased samples.

High-density IgE recognition of the major grass pollen allergen Phl p 1 revealed with single-chain IgE antibody fragments obtained by combinatorial cloning.

The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens with a molecular mass of ∼25-30 kDa. Group 1 allergens are recognized by >95% of grass pollen allergic patients. We investigated the IgE recognition of Phl p 1 using allergen-specific IgE-derived single-chain variable Ab fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollen-allergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1-derived peptides, the binding sites of two ScFvs were mapped to the N terminus of the allergen. In surface plasmon resonance experiments they showed comparable high-affinity binding to Phl p 1 as a complete human IgE-derived Ab recognizing the allergens' C terminus. In a set of surface plasmon resonance experiments simultaneous allergen recognition of all three binders was demonstrated. Even in the presence of the three binders, allergic patients' polyclonal IgE reacted with Phl p 1, indicating high-density IgE recognition of the Phl p 1 allergen. Our results show that multiple IgE Abs can bind with high density to Phl p 1, which may explain the high allergenic activity and sensitizing capacity of this allergen.

The human IgE repertoire.

IgE is a key mediator in allergic diseases. However, in strong contrast to other antibody isotypes, many details of the composition of the human IgE repertoire are poorly defined. The low levels of human IgE in the circulation and the rarity of IgE-producing B cells are important reasons for this lack of knowledge. In this review, we summarize the current knowledge on these repertoires both in terms of their complexity and activity, i.e. knowledge which despite the difficulties encountered when studying the molecular details of human IgE has been acquired in recent years. We also take a look at likely future developments, for instance through improvements in sequencing technology and methodology that allow the isolation of additional allergen-specific human antibodies mimicking IgE, as this certainly will support our understanding of human IgE in the context of human disease in the years to come.

Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

BACKGROUND: Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. OBJECTIVE: We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. METHODS: Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. RESULTS: The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. CONCLUSION: Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens.

Passive immunization with allergen-specific antibodies.

The induction of allergen-specific IgG antibodies has been identified as a major mechanism responsible for the reduction of allergic inflammation in allergic patients treated by allergen-specific immunotherapy. Several studies suggest that allergen-specific IgG antibodies induced by vaccination with allergens block mast cell and basophil degranulation, IgE-facilitated allergen presentation to T cells and IgE production. The availability of recombinant allergens and technologies for the production of recombinant human antibodies allows engineering of allergen-specific antibodies which can be used for passive immunization (i.e., therapy) and eventually for the prevention of allergy (i.e., prophylaxis). This chapter summarizes data supporting the possible use of allergen-specific antibodies for treatment and prophylaxis. Finally, concrete approaches for the treatment and prevention of allergy based on blocking antibodies are envisioned.

Efficient production of recombinant secretory IgA against Clostridium difficile toxins in CHO-K1 cells.

Despite the essential role secretory IgAs play in the defense against pathogenic invasion and the proposed value of recombinant secretory IgAs as novel therapeutics, currently there are no IgA-based therapies in clinics. Secretory IgAs are complex molecules and the major bottleneck limiting their therapeutic potential is a reliable recombinant production system. In this report, we addressed this issue and established a fast and robust production method for secretory IgAs in CHO-K1 cells using BAC-based expression vectors. As a proof of principle, we produced IgAs against Clostridium difficile toxins TcdA and TcdB. Recombinant secretory IgAs produced using our expression system showed comparable titers to IgGs, widely used as therapeutic biologicals. Importantly, secretory IgAs produced using our method were functional and could efficiently neutralize Clostridium difficile toxins TcdA and TcdB. These results show that recombinant secretory IgAs can be efficiently produced, thus opening the possibility to use them as therapeutic agents in clinics.

Validation of an enzyme-linked immunosorbent assay (ELISA) for quantification of endostatin levels in mice as a biomarker of developing glomerulonephritis.

Endostatin, the C-terminal fragment of type XVIII collagen, was shown to be one of the most potent endothelial cell-specific inhibitors of angiogenesis. As altered circulating endostatin concentration is associated with impaired kidney function, new tools for measuring endostatin in rodents may be helpful to further investigate and understand its role within kidney disease progression. A novel and commercially available ELISA for the quantification of mouse and rat endostatin was developed and validated according to international quality guidelines including the parameters specificity, robustness, accuracy, dilution linearity, precision, limit of detection (LOD) and lower limit of quantification (LLOQ). Endostatin and blood urea nitrogen (BUN) concentration were measured in mice with a glomerulonephritis phenotype. The validation revealed that within the range of 0.5-32 nmol/L the immunoassay is robust and highly specific for the measurement of rodent endostatin with high sensitivity (LOD 0.24 nmol/L, LLOQ 0.5 nmol/L) and good reproducibility (intra- and inter-assay CV <10%). Also accuracy and dilution linearity were within the range of acceptance. BCL2 transgenic and ETV6/RUNX1;BCL2 double transgenic mice develop a glomerulonephritis phenotype over time, which was displayed by staining of kidney sections. Even before full manifestation of disease serum endostatin concentration rises significantly, whereas BUN levels just slightly increase. This newly developed and commercially available ELISA provides a reliable and accurate tool for the quantification of mouse and rat endostatin and may give new perspectives in the investigation of the role of endostatin as an important and early biomarker for reduced kidney function. Measurement of endostatin concentration is recommended to be used as a superior biomarker for chronic kidney disease compared to BUN.

Analysis of epitope-specific immune responses induced by vaccination with structurally folded and unfolded recombinant Bet v 1 allergen derivatives in man.

Previously, we have constructed recombinant derivatives of the major birch pollen allergen, Bet v 1, with a more than 100-fold reduced ability to induce IgE-mediated allergic reactions. These derivatives differed from each other because the two recombinant Bet v 1 fragments represented unfolded molecules whereas the recombinant trimer resembled most of the structural fold of the Bet v 1 allergen. In this study, we analyzed the Ab (IgE, IgG subclass, IgA, IgM) response to Bet v 1, recombinant and synthetic Bet v 1-derived peptides in birch pollen allergic patients who had been vaccinated with the derivatives or adjuvant alone. Furthermore, we studied the induction of IgE-mediated skin responses in these patients using Bet v 1 and Bet v 1 fragments. Both types of vaccines induced a comparable IgG1 and IgG4 response against new sequential epitopes which overlap with the conformational IgE epitopes of Bet v 1. This response was 4- to 5-fold higher than that induced by immunotherapy with birch pollen extract. Trimer more than fragments induced also IgE responses against new epitopes and a transient increase in skin sensitivity to the fragments at the beginning of therapy. However, skin reactions to Bet v 1 tended to decrease one year after treatment in both actively treated groups. We demonstrate that vaccination with folded and unfolded recombinant allergen derivatives induces IgG Abs against new epitopes. These data may be important for the development of therapeutic as well as prophylactic vaccines based on recombinant allergens.