Detection of antiphospholipid antibodies by immunostaining on thin layer chromatography plates

Sorice, Maurizio; Griggi, T.; Circella, A.; Garofalo, Tina; D'Agostino, F; Pittoni, V.; Pontieri, G. M.; Lenti, Luisa; Valesini, Guido

doi:10.1016/0022-1759(94)90282-8

Cited by 32 publications

(36 citation statements)

References 19 publications

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“…After extraction of phospholipids using standard protocols, 19,20 we applied nanospray mass spectrometry (nanoES-MS) analysis in positive mode, which allows for recognition of minor species present in lipid extracts of cells and can resolve the various molecular forms of natural CL and their metabolites such as monolysocardiolipin, MCL. [29][30][31] The top panel in Figure 3a shows the characteristic ES-MS spectrum of a mitochondrial lipid extract from untreated (control) cells in the 1100-1600 mass-to-charge (m/z) range and illustrates the presence of multiple forms of MCL and CL.…”

Section: Ms Analysis Of Membrane Lipids In U937 Cellsmentioning

confidence: 99%

“…Studies with recombinant Bid indicated that its proapoptotic association with mitochondria could involve interaction with CL and its metabolites like MCL 18,31 Since we observed a relocation of CL on the plasma membrane of Fas-stimulated U937 cells, 19 as well as in fraction P100 (Figures 4 and 5), we investigated whether endogenous Bid showed comparable changes in its intracellular distribution after Fas stimulation of U937 cells. Although 1 h stimulation was insufficient for producing evident caspase cleavage of Bid into p15 tBid (cf.…”

Section: Subcellular Distribution Of Bid and Other Proteins During Famentioning

confidence: 99%

“…12,19 Reactivity showed specificity for CL and MCL, since no significant reaction was detected with other negatively charged lipids like PS and phosphatidylinositol (PI), nor with LBPA or b 2 -glycoprotein I. Affinity-purified normal human IgG (from blood donors) were used as a control. Commercial antibodies for Bid, cathepsin D, EEA1, Lamp1, cytochrome c and subunit IV of cytochrome oxidase were obtained from R&D Systems Inc. (Minneapolis, MN, USA), Santa Cruz Biotechnologies (Santa Cruz, CA, USA), Upstate Biotechnologies (Lake Placid, NY, USA), BD-PharMingen and Molecular Probes (Eugene, OR, USA).…”

Section: Antibodiesmentioning

confidence: 99%

“…8 We have evaluated this possibility in light of our previous observation that CL becomes exposed on the surface of cells undergoing apoptosis, 12 which has been documented also using a-CL antibodies selected from patients with the antiphospholipid antibody syndrome (APS). 19 In particular, we studied how Bid, which is required for mediating mitochondrial membrane damage during Fas-induced apoptosis, 15,16 might be involved in the intracellular transport of CL (as well as its metabolites) and potentially account for its relocation onto the plasma membrane of Fas-stimulated cells. We thus undertook a detailed study of the cellular events occurring early after Fas stimulation of a sensitive cell line (U937), with specific attention to the intracellular redistribution of Bid and mitochondrial lipids.…”

Section: Introductionmentioning

confidence: 99%

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Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis

Sorice

Circella

Cristea³

et al. 2004

Cell Death Differ

129

130

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We previously reported that during death receptor-mediated apoptosis, cardiolipin (CL) relocates to the cell surface, where it reacts with autoantibodies from antiphospholipid syndrome sera. Here, we analysed the intracellular distribution of CL and its metabolites during the early phase of cell death signalling triggered by Fas stimulation in U937 cells and mouse liver. We found a redistribution of mitochondrial CL to the cell surface by using confocal microscopy and flow cytometry. Mass spectrometry revealed that CL and its metabolites relocated from mitochondria to other intracellular organelles during apoptosis, with a conversion into non-mitochondrial lipids. Concomitantly, cytosolic Bid relocated to the light membranes comprised in fraction P100, including the plasma membrane and associated vesicular systems. A direct Bid-CL interaction was demonstrated by the observation that CL and monolysoCL coimmunoprecipitated with Bid especially after Fas stimulation, suggesting a dynamic interaction of the protein with CL and its metabolites.

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Section: Ms Analysis Of Membrane Lipids In U937 Cellsmentioning

confidence: 99%

Section: Subcellular Distribution Of Bid and Other Proteins During Famentioning

confidence: 99%

Section: Antibodiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis

Sorice

Circella

Cristea³

et al. 2004

Cell Death Differ

129

130

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show abstract

“…Antibodies may be directed against other phospholipids such as phosphatidylethanolamine, phosphatidylinositol, or against components of the protein C pathway or annexin V. An aPL-negative conventional test result may also be caused by a "technical" problem in that the test cannot detect aPL. In this regard, in a study investigating the possibility of detecting aPL by immunostaining on thin-layer chromatography plates, aPL were detected in the serum from ten patients with features of APS but aPL-negative by ELISA [53].…”

Section: Recurrent Thrombotic Events Despite Optimal Anticoagulationmentioning

confidence: 99%

Management of the antiphospholipid syndrome

Espinosa

Cervera

2010

Autoimmun Highlights

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Antiphospholipid syndrome (APS) is characterized by recurrent venous or arterial thromboses, fetal losses and thrombocytopenia in the presence of antiphospholipid antibodies, namely lupus anticoagulant, anticardiolipin antibodies or antibodies directed to various proteins, mainly β 2 glycoprotein I, or all three. There is consensus in treating patients with APS and first venous thrombosis with oral anticoagulation to a target international normalized ratio (INR) of 2.0–3.0. A recent systematic review recommended a target INR of >3.0 in those patients with APS and arterial thrombosis. The approach in women with obstetric manifestations of APS is based on the use of aspirin plus heparin. The best treatment for patients with the catastrophic variant of the APS is a combination of anticoagulation, corticosteroids, and plasma exchange or intravenous immunoglobulins.

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Planar Chromatography in Clinical Chemistry

Jain

Sherma

2000

Encyclopedia of Analytical Chemistry

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Planar chromatography (PC) like any other chromatographic technique is a multistage distribution process. It is a form of liquid chromatography in which the stationary phase is supported on a planar surface rather than a column. Separations in PC occur because of differential migration velocities through the sorbent layer in a fixed separation time. The sample zones are fixed in space at the completion of the separation, allowing off‐line monitoring. There are two planar techniques: paper chromatography and thin‐layer chromatography (TLC). In recent years, TLC has superseded paper chromatography owing to greater speed, versatility, and reproducibility. Improvements in techniques and equipment as well as the ready availability of high‐quality plates with uniform layers have extended and increased the use of TLC in clinical chemistry and other disciplines, offering quantitative as well as qualitative analysis. High‐performance thin‐layer chromatography (HPTLC), in particular, has developed to the extent that separation and quantitation can provide results that are comparable with other analytical methods such as high‐pressure liquid chromatography (HPLC). The widespread application of TLC is also due to the fact that it is simple, robust, and flexible, and has a large sample throughput. Furthermore, coupled spectroscopic methods (TLC/mass spectrometry (TLC/MS), TLC/Fourier transform infrared spectrometry (TLC/FTIR) etc.) have considerably enhanced the reproducibility of TLC. TLC is often critically compared with column chromatography such as HPLC. This is a matter of endless dispute. Each technique has its own advantages and limitations. TLC is only inexpensive when qualitative results are needed. When reliable quantitative data are required, TLC is as expensive as column chromatography. In the authors' opinion, complementary rather than competitive chromatography offers the best solution to analytical requirements.

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Detection of antiphospholipid antibodies by immunostaining on thin layer chromatography plates

Cited by 32 publications

References 19 publications

Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis

Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis

Management of the antiphospholipid syndrome

Planar Chromatography in Clinical Chemistry

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