Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening

Petermann, Klaudia; Vordenbäumen, Stefan; Pyun, Jae Chul; Braukmann, Achim; Bleck, Ellen; Schneider, Matthias; Jose, Joachim

doi:10.1016/j.ab.2010.07.030

Cited by 27 publications

(21 citation statements)

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“…This was facilitated by Autodisplay, an efficient and convenient surface display system in E. coli [20], [21]. By similar means, an SD-ELISA for human Ro/SS-A autoantigen has previously been established: Validation of the assay on Ro/SS-A positive patients with systemic lupus erythematosus and healthy volunteers resulted in a sensitivity and selectivity that was at least identical, if not improved in comparison to commercially available standard ELISA [16]. The advantage of SD ELISA is that protein purification for coating the ELISA microplate with the antigen is not necessary (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In order to avoid effects caused by the host E. coli , and therefore being independent of CSN1S1, the absorption values obtained from the control E. coli UT5600(DE3) cells were subtracted from the values obtained with E. coli displaying CSN1S1, as it has been done before in the Ro/SS-A SD-ELISA [16]. The difference in the mean of the absorption values measured for each cell type were supposed to represent the serum reaction against mere CSN1S1.…”

Section: Resultsmentioning

confidence: 99%

“…Bacteria were routinely grown at 37°C in Lysogeny-Broth (LB) containing 50 mg/L of ampicillin with ethylenediaminetetraacetate (EDTA, final concentration 10 µM) and β-mercaptoethanol (10 mM). SDS-PAGE, outer membrane protein preparation, surface detection of CSN1S1 and flow cytometer analysis was performed as described before [16].…”

Section: Methodsmentioning

confidence: 99%

“…For this purpose, serum of 62 healthy volunteers who were or were not breast-fed was assessed for CSN1S1 antibodies using a SD-ELISA based on E. coli displaying the protein, similar to a strategy that was established before for human autoantigen Ro/SS-A [16]. It turned out that a history of having been breast-fed was strongly associated to an IgG-antibody reaction against CSN1S1, whereas a negative reaction stood into relation with formula feeding.…”

Section: Introductionmentioning

confidence: 99%

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Autoantibodies to αS1-Casein Are Induced by Breast-Feeding

et al. 2012

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BackgroundThe generation of antibodies is impaired in newborns due to an immature immune system and reduced exposure to pathogens due to maternally derived antibodies and placental functions. During nursing, the immune system of newborns is challenged with multiple milk-derived proteins. Amongst them, caseins are the main constituent. In particular, human αS1-casein (CSN1S1) was recently shown to possess immunomodulatory properties. We were thus interested to determine if auto-antibodies to CSN1S1 are induced by breast-feeding and may be sustained into adulthood.Methods62 sera of healthy adult individuals who were (n = 37) or were not (n = 25) breast-fed against human CSN1S1 were investigated by a new SD (surface display)-ELISA. For cross-checking, these sera were tested for anti Epstein-Barr virus (EBV) antibodies by a commercial ELISA.ResultsIgG-antibodies were predominantly detected in individuals who had been nursed. At a cut-off value of 0.4, the SD-ELISA identified individuals with a history of having been breast-fed with a sensitivity of 80% and a specificity of 92%. Under these conditions, 35 out of 37 sera from healthy donors, who where breast-fed, reacted positively but only 5 sera of the 25 donors who were not breast-fed. The duration of breast-feeding was of no consequence to the antibody reaction as some healthy donors were only short term breast-fed (5 days minimum until 6 weeks maximum), but exhibited significant serum reaction against human CSN1S1 nonetheless.ConclusionWe postulate that human CSN1S1 is an autoantigen. The antigenicity is orally determined, caused by breast-feeding, and sustained into adulthood.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Autoantibodies to αS1-Casein Are Induced by Breast-Feeding

et al. 2012

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“…Li et al, 2008;Kaeßler et al, 2011), immunogenic and functional T cell epitopes (e.g. Konieczny et al, 2000;Westendorf et al, 2005;Buddenborg et al, 2008;Petermann et al, 2010), and bacterial toxin subunits (e.g. Maurer et al, 1997;Konieczny et al, 2000).…”

Section: Autotransporter-mediated Surface Presentationmentioning

confidence: 99%

Structures and functions of autotransporter proteins in microbial pathogens

Benz¹,

Schmidt²

2011

International Journal of Medical Microbiology

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Since their discovery more than 20 years ago the autotransporter protein superfamily has been growing continuously and currently represents the largest protein family in (pathogenic) Gram-negative bacteria. Autotransporter proteins (AT) adhere to a common structural principle and are composed of a C-terminal β-barrel-shaped 'translocator' domain and an N-terminal 'passenger' domain. The translocator is anchored in the outer membrane and is indispensable for the N-terminal passenger part to traverse the outer membrane. Most if not all AT harbor a chaperone segment that increases protein stability and may be located in the passenger or translocator domain. The passenger mediates the specific virulence function(s) of the particular AT. Accordingly, passenger domains of AT can be quite variable. Interestingly, AT have been identified as the first glycosylated proteins in Gram-negative bacteria. Despite the considerable efforts invested in the characterization of autotransporter biogenesis, various aspects such as the participation of accessory proteins, the fate of the translocator, or the translocation of glycosylated proteins still remain only poorly understood. In addition, recent evidence indicates that the prefix 'auto' might be slightly exaggerated. Here, we will selectively discuss novel insights at various stages of AT biogenesis.

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Autodisplay of Nitrilase from Alcaligenes faecalis in E. coli Yields a Whole Cell Biocatalyst for the Synthesis of Enantiomerically Pure (R)‐Mandelic Acid

2011

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In the present study, a whole cell biocatalyst for the synthesis of (R)‐mandelic acid from mandelonitrile was constructed. For this purpose, nitrilase from Alcaligenes faecalis subsp. faecalis ATCC 8750 was displayed on the surface of Escherichia coli by using Autodisplay. Localization of the nitrilase in the cell envelope of E. coli was monitored by SDS‐PAGE and surface exposure was verified by its accessibility to externally added protease. The whole cell biocatalyst converted up to 2.6 mM of (R)‐mandelic acid under optimum conditions at pH 7.5 and 45 °C within 24 h (1 mL culture, OD578=10). By using chiral HPLC, the ee value of the product was determined to be >99 %. The surface displayed nitrilase showed an apparent Km value of 3.6 mM and an apparent Vmax value of 1 nmol min−1 mL−1 when a bacterial suspension of OD578 3 was used. Substrate inhibition by benzaldehyde was similar to that of the free enzyme. The whole‐cell biocatalyst retained 55 % of its initial (R)‐mandelic acid production after 5 cycles of repeated use, and could be stored at −70 °C for 180 d without a substantial loss of activity. In addition the whole cell biocatalyst converted 9.3 mM phenylacetonitrile within 16 h.

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Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening

Cited by 27 publications

References 28 publications

Autoantibodies to αS1-Casein Are Induced by Breast-Feeding

Autoantibodies to αS1-Casein Are Induced by Breast-Feeding

Structures and functions of autotransporter proteins in microbial pathogens

Autodisplay of Nitrilase from Alcaligenes faecalis in E. coli Yields a Whole Cell Biocatalyst for the Synthesis of Enantiomerically Pure (R)‐Mandelic Acid

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