DEVELOPMENT OF A NEW ASSAY METHOD FOR THE DETECTION OF DD/E COMPLEX

1987 ◽  
Author(s):  
K KUROSO ◽  
S IKEMATSU ◽  
M HADA ◽  
M FUJIMAKI ◽  
K FUKUTAKE
Keyword(s):  
Degradation Products ◽  
Early Stage ◽  
High Sensitivity ◽  
Assay Method ◽  
Quantitative Detection ◽  
Horse Radish Peroxidase ◽  
Microtitre Plate ◽  
Antibody Technique ◽  
Anti Body ◽  
D Dimer

A new assay method for the detection of DD/E complex derived from crosslinked fibrin is developed. This assay is performed on a microtitre plate using capture/tag antibody technique, in which the monoclonal antibody against D dimer fragment (DD-3B6, MAbCO) is coated and anti-E fragment polyclonal F(ab)’2 conjugated with horse radish peroxidase is for a tag-anti body. Antigen dilution curve is drawn in the range of 0.01-1.0 pg/ml of purified DD/E complex. DD/E complex can be measured specifically and other high molecular weight derivatives from crosslinked fibrin show a little crossreaction, though fibrinogen and fibrinogen degradation products show no crossreactivities on this assay. D dimer fragment dissociated from DD/E complex after further plasmin digestion is less reactive in this assay, while this type of D dimer can be detected by DIMERTEST-EIA (MAbCO). These data suggest that an early stage of plasmin digestion of crosslinked fibrin can be detected by this method. A trace amount of DD/E complex curculating in plasma from a small thrombus is possibly detected, because this assay gives an excellent high sensitivity with the detection limit of 0.01 jug/ ml. Normal value of plasma DD/E complex (n=50) indicates below 0.12 pg/ml as 90 percentile. Patients with DIC (n=24) show high levels of DD/E complex between 0.6 and 40 g/ml. These elevated levels of DD/E complex may suggest consequently the existence of the plasmic digestion of crosslinked fibrin in the cases with DIC. In summary, it is concluded that the development of this assay will add one technique to discriminate between fibrinolysis and fibrinogenolysis and this assay is useful for the quantitative detection of DD/E complex produced in an early stage of fibrinolysis seen in various thrombotic disorders, and for the evaluation of the efficacy of thrombolytic therapy.

D-Dimers, Thrombin Antithrombin III Complexes and Prothrombin Fragments 1 + 2: Diagnostic value in Clinically Suspected Deep Vein Thrombosis

1991 ◽  
Vol 65 (01) ◽  
pp. 028-032 ◽  
Author(s):  
B Boneu ◽  
G Bes ◽  
H Pelzer ◽  
P Sié ◽  
H Boccalon
Keyword(s):  
Deep Vein Thrombosis ◽  
Antithrombin Iii ◽  
High Sensitivity ◽  
Diagnostic Value ◽  
Predictive Values ◽  
Vein Thrombosis ◽  
High Negative Predictive Value ◽  
Deep Vein ◽  
D Dimer ◽  
Mercury Strain Gauge

SummaryThis study was performed to determine the accuracy of D-Dimer fibrin derivatives, thrombin-antithrombin III (TAT) complexes and prothrombin fragments 1 + 2 (F 1 + 2) determinations for the diagnosis of deep vein thrombosis (DVT). One hundred and sixteen consecutive patients referred to the angiology unit of our hospital for a clinically suspected DVT were investigated. They were submitted to mercury strain gauge plethysmography and to ultrasonic duplex scanning examination; in cases of inconclusive results or of proximal DVT (n = 35), an ascending phlebography was performed. After these investigations were completed, the diagnosis of DVT was confirmed in 34 and excluded in 82. One half of the patients were already under anticoagulant therapy at the time of investigation. The 3 biological markers were assayed using commercially available ELISA techniques and the D-Dimer was also assayed with a fast latex method. The normal distribution of these markers was established in 40 healthy blood donors. The most accurate assay for the diagnosis of DVT was the D-Dimer ELISA which had both a high sensitivity (94%) and a high negative predictive value (95%). The D-Dirner latex, TAT complexes and F 1 + 2 were far less sensitive and provided negative predictive values which ranged between 78 and 85%. In spite of positive and significant correlations between the levels of ihe 3 markers, their association did not improve their overall accuracy for detecting D\/L Therefore, with the exception of the D-Dimer ELISA, these markers were of little value for the diagnosis of DVT in this specific population.

The Measurement of Heparin

1973 ◽  
Vol 30 (03) ◽  
pp. 471-479 ◽  
Author(s):  
K. W. E Denson ◽  
John Bonnar
Keyword(s):  
Degradation Products ◽  
Factor Xa ◽  
Assay Method ◽  
Thrombin Time ◽  
Fibrinogen Degradation Products ◽  
Low Levels

SummaryA method for the measurement of heparin utilising the potentiating effect of heparin on the action of anti-factor Xa is described. The effect on the assay of platelet contamination of plasma, the presence of fibrinogen degradation products and low levels of anti-factor Xa have been studied. The assay method has been compared with the calcium thrombin time method and a group of obstetrical patients have been studied using both methods.

A Novel Method for the Rapid Purification of Human and Rat Fibrin(OGEN) Degradation Products in High Yields

1979 ◽  
Author(s):  
W. Nieuwenhuizen ◽  
I. A. M. van Ruijven-Vermeer ◽  
F. Haverkate ◽  
G. Timan
Keyword(s):  
Degradation Products ◽  
Complete Separation ◽  
Purification Method ◽  
Amino Terminal ◽  
Novel Method ◽  
Rapid Purification ◽  
The One ◽  
High Yields ◽  
Size Heterogeneity ◽  
D Dimer

A novel method will be described for the preparation and purification of fibrin(ogen) degradation products in high yields. The high yields are due to two factors. on the one hand an improved preparation method in which the size heterogeneity of the degradation products D is strongly reduced by plasmin digestion at well-controlled calcium concentrations. At calcium concentrations of 2mM exclusively D fragments, M.W.= 93-000 (Dcate) were formed; in the presence of 1OmM EGTA only fragments M.W.= 80.000 (D EGTA) were formed as described. on the other hand a new purification method, which includes Sephadex G-200 filtration to purify the D:E complexes and separation of the D and E fragments by a 16 hrs. preparative isoelectric focussing. The latter step gives a complete separation of D (fragments) (pH = 6.5) and E fragments (at pH = 4.5) without any overlap, thus allowing a nearly 100% recovery in this step. The overall recoveries are around 75% of the theoretical values. These recoveries are superior to those of existing procedures. Moreover the conditions of this purification procedure are very mild and probably do not affect the native configuration of the products. Amino-terminal amino acids of human Dcate, D EGTA and D-dimer are identical i.e. val, asx and ser. in the ratgly, asx and ser were found. E 1% for rat Dcate=17-8 for rat D EGTA=16.2 and for rat D- dimer=l8.3. for the corresponding human fragments, these values were all 20.0 ± 0.2.

Stability Indicating Liquid Chromatographic Method for the Determination of Fenofibrate and its Application to Kinetic Studies

2013 ◽  
Vol 5 (4) ◽  
pp. 1866-1874
Author(s):  
K. Srinivasa Rao ◽  
Keshar N K ◽  
N Jena ◽  
M.E.B Rao ◽  
A K Patnaik
Keyword(s):  
Degradation Products ◽  
Kinetic Studies ◽  
Pharmaceutical Formulations ◽  
Assay Method ◽  
Pharmaceutical Dosage Form ◽  
Stability Indicating ◽  
Zero Order Kinetics ◽  
Relative Standard ◽  
Liquid Chromatographic

A stability-indicating LC assay method was developed for the quantitative determination of fenofibrate (FFB) in pharmaceutical dosage form in the presence of its degradation products and kinetic determinations were evaluated in acidic, alkaline and peroxide degradation conditions. Chromatographic separation was achieved by use of Zorbax C18 column (250 × 4.0 mm, 5 μm). The mobile phase was established by mixing phosphate buffer (pH adjusted 3 with phosphoric acid) and acetonitrile (30:70 v/v). FFB degraded in acidic, alkaline and hydrogen peroxide conditions, while it was more stable in thermal and photolytic conditions. The described method was linear over a range of 1.0-500 μg/ml for determination of FFB (r= 0.9999). The precision was demonstrated by relative standard deviation (RSD) of intra-day (RSD= 0.56– 0.91) and inter-day studies (RSD= 1.47). The mean recovery was found to be 100.01%. The acid and alkaline degradations of FFB in 1M HCl and 1M NaOH solutions showed an apparent zero-order kinetics with rate constants 0.0736 and 0.0698  min−1 respectively and the peroxide degradation with 5% H2O2 demonstrated an apparent first-order kinetics with rate constant k = 0.0202 per min. The t1/2, t90   values are also determined for all the kinetic studies. The developed method was found to be simple, specific, robust, linear, precise, and accurate for the determination of FFB in pharmaceutical formulations.  

scholarly journals Identification of BHLHE40 expression in peripheral blood mononuclear cells as a novel biomarker for diagnosis and prognosis of hepatocellular carcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pattapon Kunadirek ◽  
Chaiyaboot Ariyachet ◽  
Supachaya Sriphoosanaphan ◽  
Nutcha Pinjaroen ◽  
Pongserath Sirichindakul ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Early Stage ◽  
High Sensitivity ◽  
Peripheral Blood Mononuclear ◽  
Diagnosis And Prognosis ◽  
Transcription Profiles ◽  
Blood Mononuclear Cells

AbstractNovel and sensitive biomarkers is highly required for early detection and predicting prognosis of hepatocellular carcinoma (HCC). Here, we investigated transcription profiles from peripheral blood mononuclear cells (PBMCs) of 8 patients with HCC and PBMCs from co-culture model with HCC using RNA-Sequencing. These transcription profiles were cross compared with published microarray datasets of PBMCs in HCC to identify differentially expressed genes (DEGs). A total of commonly identified of 24 DEGs among these data were proposed as cancer-induced genes in PBMCs, including 18 upregulated and 6 downregulated DEGs. The KEGG pathway showed that these enriched genes were mainly associated with immune responses. Five up-regulated candidate genes including BHLHE40, AREG, SOCS1, CCL5, and DDIT4 were selected and further validated in PBMCs of 100 patients with HBV-related HCC, 100 patients with chronic HBV infection and 100 healthy controls. Based on ROC analysis, BHLHE40 and DDIT4 displayed better diagnostic performance than alpha-fetoprotein (AFP) in discriminating HCC from controls. Additionally, BHLHE40 and DDIT4 had high sensitivity for detecting AFP-negative and early-stage HCC. BHLHE40 was also emerged as an independent prognostic factor of overall survival of HCC. Together, our study indicated that BHLHE40 in PBMCs could be a promising diagnostic and prognostic biomarker for HBV-related HCC.

Rapid, highly sensitive and quantitative detection of interleukin 6 based on SERS magnetic immunoassay

2021 ◽  
Vol 13 (15) ◽  
pp. 1823-1831
Author(s):  
Xiaomei Wang ◽  
Li Ma ◽  
Shijiao Sun ◽  
Tingwei Liu ◽  
Hao Zhou ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
High Sensitivity ◽  
Quantitative Detection ◽  
Detection Time ◽  
Highly Sensitive ◽  
Sandwich Method

We have developed a SERS magnetic immunoassay method based on the principle of sandwich method for rapid and quantitative detection of IL-6. The developed SERS method has the advantages of high sensitivity and detection time is only 15 min.

MONOCLONAL ANTIBODIES OF THE IgM AND IgG CLASS SPECIFIC FOR CROSSLINKED FIBRIN DEGRADATION PRODUCTS

1987 ◽  
Author(s):  
P J Gaffney ◽  
L J Creighton ◽  
A Curry ◽  
B MacMahon ◽  
R Thorpe
Keyword(s):  
Monoclonal Antibodies ◽  
Thrombolytic Therapy ◽  
Epitope Mapping ◽  
Degradation Products ◽  
Subcutaneous Route ◽  
Fibrin Degradation Products ◽  
Conformational Epitopes ◽  
Immunoglobulin Class Switching ◽  
Unique Specificity ◽  
D Dimer

Monoclonal antibodies (mabs) to crosslinked fibrin degradation products (XL-FDP) having the general formula D/Y[X]nY/D (known as X-oligomer) and D-D (known as D dimer) have been raised in balb/C mice by both a novel mtrasplenic and a conventional subcutaneous route of immunisation and by combinations of both these procedures. Mabs to X-oligomers (NIBn 52 and NIBn 123) obtained by an intrasplenic procedure have been demonstrated to crossreact only with X-oligomer in a 2-site ELISA procedure and not with D dimer or whole fibrinogen and have been shown to be of value m the examination of clinical material obtained from patients with various types of thrombosis and have also been useful in monitoring the efficacy of thrombolytic therapy. The X-oligomer mabs are immunoglobulins of the M class. It was demonstrated that their unique specificity for conformational epitopes on the large X-oligomer fragments does not reside in the IgM structure since alterative immunisation procedures have been used to generate mabs of the IgG class which have the same specificity. Using immunoglobulin class switching in culture rather than during immunisation was suggested by certain cell lines which produced both IgM and IgG specific for X-oligomer. This latter point needs rigorous validation.Immunoglobulin G type mabs to highly purified D dimer were raised by conventional subcutaneous immunisation of balb/C mice. One of these, NIBn-11, was found to crossreact with PVC-immobilised X-oligomer and D dimer but not with fibrinogen. However NIBn-11 did not bind to D dimer in a 2-site ELISA procedure while crossreactmg quite avidly with X-oligomer. This suggests that the D dimer epitope to which NIBn-11 is directed is expressed in some conformations and not m others and that these conformations are always expressed in the complex X-oligomer group of fragments. These mabs, whilst of value in measuring certain unique fibrin fragments m plasma, are useful in the epitope mapping of fibrinogen/fibrin and their plasmm-mediated

Performance of five D-dimer assays for the exclusion of symptomatic distal leg vein thrombosis

2012 ◽  
Vol 107 (02) ◽  
pp. 369-378 ◽  
Author(s):  
Jan Schwonberg ◽  
Carola Hecking ◽  
Marc Schindewolf ◽  
Dimitrios Zgouras ◽  
Susanne Lehmeyer ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Diagnostic Value ◽  
Vein Thrombosis ◽  
Clinical Probability ◽  
Wells Score ◽  
Deep Vein ◽  
Test Result ◽  
D Dimer ◽  
Rule Out ◽  
Compression Ultrasonography

SummaryThe diagnostic value of D-dimer (DD) in the exclusion of proximal deep-vein thrombosis (DVT) is well-established but is less well-known in the exclusion of distal (infrapopliteal) DVT. Therefore, we evaluated the diagnostic abilities of five DD assays (Vidas-DD, Liatest-DD, HemosIL-DD, HemosIL-DDHS, Innovance-DD) for excluding symptomatic proximal and distal leg DVT. A total of 243 outpatients whose symptoms were suggestive of DVT received complete compression ultrasonography (cCUS) of the symptomatic leg(s). The clinical probability of DVT (PTP) was assessed by Wells score. Thirty-eight proximal and 31 distal DVTs (17 tibial/fibular DVTs, 14 muscle DVTs) were diagnosed by cCUS. Although all assays showed high sensitivity for proximal DVT (range 97–100%), the sensitivity was poor for distal DVT (range 78–93%). None of the assays were individually able to rule out all DVTs as a stand-alone test (negative predictive value [NPV] 91–96%). However, a negative DD test result combined with a low PTP exhibited a NPV of 100% for all DVTs (including proximal, tibial/fibular, and muscle DVTs) with the HemosIL-DDHS and Innovance-DD. All proximal and tibial/fibular DVTs, but not all muscle DVTs, could be ruled out with this strategy using the Liatest-DD and Vidas-DD. The HemosIL-DD could not exclude distal leg DVT, even in combination with a low PTP. The combination of a negative DD with a low PTP showed a specificity of 32–35% for all DVTs. In conclusion, our study shows that when used in conjunction with a low PTP some DD assays are useful tools for the exclusion of distal leg DVT.

False-Positive D-Dimer Result in a Patient With Castleman Disease

2004 ◽  
Vol 128 (3) ◽  
pp. 328-331
Author(s):  
Kimberly Mugler ◽  
Jerry B. Lefkowitz
Keyword(s):  
Western Blot ◽  
Disseminated Intravascular Coagulation ◽  
False Positive ◽  
Degradation Products ◽  
False Negative ◽  
Castleman Disease ◽  
Laboratory Findings ◽  
Intravascular Coagulation ◽  
Immunodeficiency Virus ◽  
D Dimer

Abstract In suspected cases of disseminated intravascular coagulation, concurrent elevation of both fibrin(ogen) degradation products (FDPs) and D-dimer levels aids in confirming the diagnosis. This pattern of results reflects the action of plasmin proteolysis of cross-linked fibrin polymers as well as fibrinogen. We report the case of a patient with human immunodeficiency virus (HIV) and Castleman disease who presented with a high-positive D-dimer level and a negative FDP level in the course of a workup for disseminated intravascular coagulation. This finding suggested the possibility of either a false-positive D-dimer or a false-negative FDP level. To investigate the former, a Western blot was performed on the patient's serum to determine the presence of the D-dimer. No D-dimer band was visualized on the Western blot, confirming the false-positive nature of the D-dimer result. Insufficient quantity of patient serum, however, prevented further investigation into the etiology of this result. The false-positive D-dimer result is likely attributable to interference caused by the patient's Castleman disease–associated monoclonal gammopathy, a phenomenon that has been reported in other immunoassays. As the development of lymphoproliferative disorders is especially common within the HIV population, and hypergammaglobulinemia in Castleman disease is particularly common, clinicians should be aware of this phenomenon when the laboratory findings do not fit the clinical picture. Although it is rare, recognition of potential paraprotein interference in immunoassays will help avoid undertreatment or overtreatment of patients based on erroneous laboratory results.

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