Plasma Levels of ADAMTS13 Antigen Determined with an Enzyme Immunoassay Using a Neutralizing Monoclonal Antibody Parallel ADAMTS13 Activity LevEls

2007 ◽  
Vol 85 (5) ◽  
pp. 403-407 ◽  
Author(s):  
Hideo Yagi ◽  
Shin Ito ◽  
Seiji Kato ◽  
Hisahide Hiura ◽  
Masanori Matsumoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Plasma Levels ◽  
Activity Levels ◽  
Adamts13 Activity ◽  
Neutralizing Monoclonal Antibody

Novel monoclonal antibody-based enzyme immunoassay for determining plasma levels of ADAMTS13 activity

Transfusion ◽  
2006 ◽  
Vol 46 (8) ◽  
pp. 1444-1452 ◽  
Author(s):  
Seiji Kato ◽  
Masanori Matsumoto ◽  
Tomomi Matsuyama ◽  
Ayami Isonishi ◽  
Hisahide Hiura ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Plasma Levels ◽  
Adamts13 Activity

scholarly journals Severe Plasmodium Falciparum Malaria Is Associated with Circulating Ultra-Large Von Willebrand Multimers and ADAMTS13 Inhibition

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3918-3918
Author(s):  
Deirdre Larkin ◽  
Vince P Jenkins ◽  
James Bunn ◽  
Alister G Craig ◽  
Roger JS Preston ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cerebral Malaria ◽  
Falciparum Malaria ◽  
Plasma Levels ◽  
Platelet Adhesion ◽  
Critical Role ◽  
Activity Levels ◽  
Adamts13 Activity ◽  
Plasma Haemoglobin ◽  
Normal Plasma

Abstract Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IE) to activated endothelial cell (EC) surfaces plays a key role in the pathophysiology of cerebral malaria. Moreover, recent evidence suggests that platelet adhesion and aggregation are critical in facilitating this cytoadhesion process. We have recently reported marked increased plasma VWF and VWF propeptide levels in severe P. falciparum infection, indicative of acute EC perturbation. Furthermore, plasma VWF:Ag levels in patients with malaria inversely correlated with platelet count, and plasma VWF propeptide levels correlated with other established biochemical markers of malaria severity, including plasma lactate. Nevertheless, it remains unclear whether VWF plays any direct role in mediating IE cytoadhesion, or whether the increased plasma levels of both VWF and propeptide merely serve as a marker of acute EC activation. To address this question, we collected plasma samples from a cohort of African children presenting with cerebral malaria (CM), or non-cerebral severe malaria (SM). In children with CM and SM, plasma VWF:Ag levels were significantly elevated (medians 3.1 and 3.4 IU/ml) as before. VWF collagen binding (VWF:CB) levels were also markedly increased (medians 7.6 and 7.0 IU/ml). Furthermore, the relative rise in VWF:CB was much greater, so that the ratio of CB:Ag was consistently increased. VWF multimer analysis demonstrated abnormal circulating ULVWF multimers in children with CM and SM, consistent with acute, regulated VWF secretion. To characterize mechanisms responsible for the markedly increased VWF:CB and circulating ULVWF in malarial plasma, we investigated ADAMTS13 antigen and activity levels in CM and SM plasma. Plasma ADAMTS13 activity levels (FRETS-VWF73 assay) and antigen levels were both significantly reduced (medians = 0.63 U/ml and 0.56 U/ml; p< 0.001) in children with CM and SM compared to controls. Classical mixing studies of malaria and normal plasma demonstrated no evidence of immediate ADAMTS13 inhibition. However, ADAMTS13 activity in normal plasma was significantly reduced (~60%) following 30 min incubation with malarial plasma (75%:25% mix). This inhibitory ability of malaria plasma was further confirmed by spiking malarial plasmas with recombinant human ADAMTS13. Significant time-dependent inhibition of FRETS-VWF73 activity was again observed in the plasmas of children with severe P. falciparum, but not in normal control plasmas. Potential inhibitors of ADAMTS13 in vivo include interleukin-6 (IL-6), thrombospondin-1 (TSP-1), thrombin and plasmin, free plasma haemoglobin, and reduced FVIII levels. Although plasma IL-6 levels were significantly elevated in children with either CM (mean 240 pg/ml; p<0.001) or SM (mean 217 pg/ml; p=0.01) compared to normal controls, levels did not approach those previously reported necessary to inhibit ADAMTS13 activity. Disseminated intravascular coagulation (DIC) is associated with enhanced thrombin generation, and consumption of FVIII, both of which can inhibit VWF proteolysis by ADAMTS13. However, in children with CM or SM respectively, we observed significantly increased plasma FVIII:C levels. Although intravascular haemolysis is also a recognised complication of malarial infection, we observed only minor increased plasma haemoglobin concentrations, again well below that previously described to significantly inhibit ADAMTS13 activity. Finally, in contrast to the increased plasma levels of IL-6 and FVIII:C, we found that plasma TSP-1 levels were not significantly elevated in children with either CM or SM compared to pooled normal plasma. In conclusion, we demonstrate that the presence of ULVWF multimers in the plasma of children with severe P. falciparum malaria is the result of acute EC activation and release of ULVWF from WP bodies; significantly reduced plasma ADAMTS13 antigen levels a circulating but unidentified inhibitor of human ADAMTS13 activity. In view of the critical role played by VWF in mediating platelet adhesion/aggregation, and the accumulating evidence suggesting that platelet adhesion/aggregation also facilitate cytoadhesion of IE, we propose a novel role for ULVWF multimers in the pathophysiology of severe P. falciparum malaria.

scholarly journals Evaluation of Markers of Intravascular Hemolysis and Endothelial Dysfunction in Sickle Cell Anemia Patients with and without Hydroxyurea Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4826-4826
Author(s):  
Francine Chenou ◽  
Bidossessi Wilfried Hounkpe ◽  
Igor de Farias Domingos ◽  
Thais Helena Chaves Batista ◽  
Rodrigo Marcionilo Santana ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Plasma Levels ◽  
Activity Levels ◽  
Adamts13 Activity ◽  
Intravascular Hemolysis ◽  
Endothelin 1 ◽  
Thrombospondin 1 ◽  
Hydroxyurea Therapy

Background: Sickle cell anemia (SCA) is characterized by chronic hemolysis and endothelial dysfunction (ED). Plasma hemoglobin (pHb) and its heme component released from intravascular hemolysis (IH) are among the most important factors contributing to ED. Unfortunately, the importance of IH to the development of an ED and the effects of hydroxyurea therapy on IH and ED in SCA remains unclear. Aims: We evaluated plasma levels of IH and ED markers among Brazilian SCA patients not receiving hydroxyurea therapy (HbSS), and compared with those of hydroxyurea-treated SCA patients (HbSS_HU) and healthy controls (HbAA). Methods: A cross-sectional study of 60 SCA consenting patients (32 HbSS and 28 HbSS_HU; 19-42 years) in steady state, who are being followed up at the Blood Center, Pernambuco (HEMOPE) and 32 HbAA controls. The IH markers were serum Lactate Dehydrogenase [LDH] and total heme measured by enzymatic colorimetric tests, and pHb measured by enzyme-linked immunosorbent assay (ELISA). The ED markers were plasma von Willebrand factor (vWF:Ag) and vWF ristocetin cofactor activity (vWF:Rco) levels measured by the latex enhanced immunoassay. The other ED markers were the antigen of vWF-cleaving protease (ADAMTS13:Ag), thrombospondin-1 and endothelin-1 levels measured by ELISA, and ADAMTS13 Activity (ADAMTS13:Act) measured by FRETS-VWF73 method. The study was approved by the Ethics Committee of the State University of Campinas and HEMOPE under protocol No: 1.863.428. Data were analyzed using GraphPad Prism 6. Results: The pHb and LDH were significantly increased in HbSS than HbSS_HU patients (Figures 1A and 1B). Plasma levels of vWF:Ag, vWF:Rco, serum levels of total heme, thrombospondin-1 and endothelin-1 were significantly increased in HbSS and HbSS_HU patients compared to HbAA controls (Figures 1C and 2A, 2B, 2E, 2F), while serum level of thrombospondin-1 level was elevated in HbSS than HbSS_HU patients (Figure 2E). Similarly, ADAMTS13:Ag levels and ADAMTS13 activity were significantly lower in HbSS and HbSS_HU patients than HbAA controls, while ADAMTS13 activity levels were significantly elevated in HbSS_HU patients compared to HbSS patients (Figure 2D). In HbSS_HU patients, the ADAMTS13:Act was negatively correlated with heme and LDH [r = -0.47, p = 0.013; and r = -0.44, p = 0.023 respectively]. In additional, heme was positively correlated with vWF:Ag and LDH [(r = 0.47, p = 0.017) and (r = 0.56, p = 0.003) respectively]. In HbSS patients, LDH and pHb were positively correlated (r = 0.44, p = 0.014). Conclusions: Hydroxyurea therapy was associated with a reduced levels of LDH, pHb and thrombospondin-1 levels, and increased levels of ADAMTS13 activity in SCA patients. Increased ADAMTS13 activity levels may be attributed to reduction of pHb and thrombospondin-1 levels because previous invitro studies have shown that thrombospondin-1 or pHb are bound to vWF. Thus, vWF is restrained from ADAMTS13 activity and cleavage, and hyperreactive vWF might accumulate as a consequence of inhibition of ADAMTS13 activity. Increased thrombospondin-1, pHb and hyperreactive vWF levels are proposed to participate in sickle cell adhesion and promote thrombotic complications. Therefore, our results demonstrate an additional clinical benefit for the use of hydroxyurea in these patients. Disclosures No relevant conflicts of interest to declare.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

Development of a Monoclonal Antibody-Based Enzyme Immunoassay for the Pyrethroid Insecticide Deltamethrin

2010 ◽  
Vol 58 (14) ◽  
pp. 8189-8195 ◽  
Author(s):  
Ye Kong ◽  
Qi Zhang ◽  
Wen Zhang ◽  
Shirley J. Gee ◽  
Peiwu Li
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Pyrethroid Insecticide

scholarly journals Structural elements of primary CCR5-using HIV-1 gp120 proteins influencing sensitivity and resistance to the broadly neutralizing monoclonal antibody b12

Virology ◽  
2012 ◽  
Vol 432 (2) ◽  
pp. 394-404
Author(s):  
Jasminka Sterjovski ◽  
Melissa J. Churchill ◽  
Anne Ellett ◽  
Steve L. Wesselingh ◽  
Paul A. Ramsland ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Structural Elements ◽  
Neutralizing Monoclonal Antibody ◽  
Hiv 1

scholarly journals Bound/free separation methods in steroid enzyme immunoassay with monoclonal antibody.

1988 ◽  
Vol 36 (9) ◽  
pp. 3525-3531 ◽  
Author(s):  
HIROSHI HOSODA ◽  
REIKO TSUKAMOTO ◽  
SAKIKO TAMURA ◽  
TOSHIO NAMBARA
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay
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