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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The human IgE repertoire Gadermaier, Elisabeth; Levin, Mattias; Flicker, Sabine ...
International archives of allergy and immunology,
01/2014, Letnik:
163, Številka:
2
Journal Article
Recenzirano
Odprti dostop
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.
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.
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.
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.
•Development of a sandwich ELISA to quantify soluble semaphorin 4D (sSEMA4D).•Antibodies bind with high affinities to conformational epitopes in the sema domain.•Successful validation according to FDA guidelines.•Serum contains protease-shed sSEMA4D leading to 3-fold higher levels than in plasma.•Plasma is a suitable matrix for reproducible quantification of sSEMA4D.
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