Measurement of Native and Degraded Forms of Plasminogen in Human Plasma

1979 ◽  
Author(s):  
M.F. Scully ◽  
V.V. Kakkar
Keyword(s):  
Human Plasma ◽  
Final Concentration ◽  
Hexanoic Acid ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
In Vitro Degradation ◽  
Presence And Absence ◽  
Rate Of Activation

A method has been devised to measure the levels of native and degraded forms of plasminogen either alone or in mixtures using the known differences in the rate of activation as measured using the chromogenic substrate, S2251. Preliminary studies using the purified zymogens ascertained the conditions under which maximum differences in the rate of activation are observed. Blood is taken into citrate and trasylol (final concentration 1000K IU/ml of blood to prevent in-vitro degradation of plasminogen) and plasma prepared. Plasminogen is precipitated at 13% Na2SO4 and washed 5 times with 17% NA2SO4 to remove trasylol and antiplasmins. The fractions are reconstituted in saline and the rate of activation by urokinase measured in the presence and absence of 6-amino-hexanoic acid (final concentration 2.5mM), the ratio of these activities being linearly related to the percentage of degraded plasminogen in the mixture. The observed rate is not dependent an the concentration of plasminogen in the mixture. The behaviour of degraded and native plasminogen by this procedure was shown to be the same as that of the purified zymogens by the assay of plasma samples in which the original plasminogen was replaced by purified degraded and native plasminogen although the recovery of native plasminogen was less than the degraded forms. Various patient plasma samples have been assayed by this method

Factor XII Determinations in the Presence and Absence of Phospholipid Antibodies

1998 ◽  
Vol 79 (01) ◽  
pp. 87-90 ◽  
Author(s):  
D. W. Jones ◽  
M. Winter ◽  
M. J. Gallimore
Keyword(s):  
Lower Limit ◽  
Lupus Anticoagulant ◽  
Factor Xii ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Presence And Absence ◽  
Lower Limit Of Normal

SummaryFactor XII (FXII) levels were determined in plasma samples from 29 normal donors, 10 patients with inherited FXII deficiency (all lupus anticoagulant [LA] negative) and 67 LA positive patients, using clotting (FXIIct), chromogenic substrate (FXIIcs) and immunochemical (FXIIag) assays. Excellent correlations were obtained in the three FXII assays with the LA negative samples and between the FXIIcs and FXIIag assays in the LA positive samples. Correlations between both the FXIIcs and FXIIag with FXIIct in the LA positive patients were poor. Of 67 LA positive samples studied, 25 (37.3%) showed lower values in the FXIIct assay; 13 (19.4%) of these patients were pseudo FXII deficient with values of FXII below the lower limit of normal.These results indicate that a diagnosis of FXII deficiency can be made inappropriately in the presence of phospholipid antibodies and that such a diagnosis should not be made by FXIIct assay alone.

scholarly journals ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250265
Author(s):  
Hubert Hayden ◽  
Nahla Ibrahim ◽  
Johannes Klopf ◽  
Branislav Zagrapan ◽  
Lisa-Marie Mauracher ◽  
...  
Keyword(s):  
Human Plasma ◽  
Dynamic Range ◽  
Neutrophil Extracellular Traps ◽  
Plasma Samples ◽  
Dna Complexes ◽  
High Background ◽  
Isotype Control ◽  
Extracellular Traps

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.

AN INSULIN ASSAY BASED ON THE INCORPORATION OF LABELLED GLYCINE INTO PROTEIN OF ISOLATED RAT DIAPHRAGM

1959 ◽  
Vol 19 (3) ◽  
pp. 259-262 ◽  
Author(s):  
K. L. MANCHESTER ◽  
P. J. RANDLE ◽  
F. G. YOUNG
Keyword(s):  
Human Plasma ◽  
Plasma Samples ◽  
Rat Diaphragm ◽  
Normal Human Plasma ◽  
Advantages And Disadvantages ◽  
Straight Line ◽  
Normal Human ◽  
Isolated Rat

SUMMARY Insulin increases the incorporation of [14C]glycine into the protein of isolated rat diaphragm in vitro. This effect of the hormone can be used as the basis of a straight-line assay for insulin. The advantages and disadvantages of such a method of assay are discussed. Normal human plasma also stimulates incorporation of glycine into diaphragm protein, and the activities of two plasma samples were equivalent to 2 and 10 mu. insulin/ml. plasma, respectively.

scholarly journals IN VITRO DEGRADATION OF ATRACURIUM IN HUMAN PLASMA

1985 ◽  
Vol 57 (11) ◽  
pp. 1085-1088 ◽  
Author(s):  
R.L. STILLER ◽  
D. RYAN COOK ◽  
S. CHAKRAVORTI
Keyword(s):  
Human Plasma ◽  
In Vitro Degradation

scholarly journals IN VITRO DEGRADATION OF ATRACURIUM IN HUMAN PLASMA

1987 ◽  
Vol 59 (6) ◽  
pp. 806-807 ◽  
Author(s):  
J.B. STENLAKE ◽  
R. HUGHES
Keyword(s):  
Human Plasma ◽  
In Vitro Degradation

In vitro degradation of neurotensin in human plasma

Peptides ◽  
1986 ◽  
Vol 7 (3) ◽  
pp. 383-387 ◽  
Author(s):  
Y.C. Lee ◽  
L.O. Uttenthal ◽  
H.A. Smith ◽  
S.R. Bloom
Keyword(s):  
Human Plasma ◽  
In Vitro Degradation

scholarly journals Creatine Kinase Isoforms: Investigation of Inhibitors of in Vitro Degradation and Establishment of a Reference Range

1992 ◽  
Vol 29 (2) ◽  
pp. 202-205 ◽  
Author(s):  
J Davies ◽  
T Reynolds ◽  
M D Penney
Keyword(s):  
Creatine Kinase ◽  
High Voltage ◽  
Reference Range ◽  
Reference Intervals ◽  
Final Concentration ◽  
In Vitro Degradation ◽  
In Vitro Stability ◽  
Effect Of Inhibitors ◽  
High Voltage Electrophoresis

The in vitro stability of creatine kinase isoforms was examined by separation with high voltage electrophoresis. The effect of inhibitors of carboxypeptidase was evaluated. Preservation of samples is essential to inhibit in vitro changes in isoform pattern. EDTA at a final concentration of 15 mmol/L is recommended. Using appropriately preserved samples, normal reference intervals for the MM isoforms have been established.

Measurement of Endotoxin Levels in Plasma Using Limulus Amoebocyte Lysate and Chromogenic Substrate, S2222

1979 ◽  
Author(s):  
S Clark ◽  
M Scully ◽  
P Webb ◽  
V Kakkar
Keyword(s):  
Trypsin Inhibitor ◽  
Final Concentration ◽  
Soya Bean ◽  
Lag Phase ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Linear Calibration ◽  
Limulus Amoebocyte Lysate ◽  
Bean Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor

A method has been devised for the measurement of endotoxin in plasma using the chromogenic substrate, S2222, a substrate which has been shown to be particularly sensitive to the Limulus Lysate. Time curves of the rate of release of the chromogen in mixtures in which procoagulase activation was concurrent, were complex with a lag phase which was shortened by increasing endotoxin concentrations. At a final concentration of 0.5ng/ml and 370 activation was complete within 60 minutes. The enzyme was inhibited by soya bean trypsin inhibitor but not by pancreatic trypsin inhibitor or hirudin. In the method finally adopted the lysate (25µl) was incubated with endotoxin (E.coli 026.86 Difco) and magnesium chloride (final concentration 33mM) in a total volume of 225µl. After 12 minutes preincubation 165µl of S2222(0.4mM) was added and the increase in abdorbance at 405nm over two minutes measured using an Abbott Biochromatic Analyser 100. Linear assay curves were obtained with final concentration of 0.2 to 2.0ngs endotoxin/ml with ΔOD 405/min of 0.35 at 2.0ng endotoxin/ml of final incubation mixture. ΔOD /min in control tubes were of the order of 0.02. For measurement from plasma samples, endotoxin was first extracted with chloroform. Linear calibration curves were achieved at a concentration of endotoxin of 1 to 5ng/ml of whole blood with a net OD/min at the highest concentration of 0.25.

scholarly journals Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 455-462 ◽  
Author(s):  
M Berrettini ◽  
B Lammle ◽  
T White ◽  
MJ Heeb ◽  
HP Schwarz ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Heavy Chain ◽  
Light Chain ◽  
Plasma Samples ◽  
Single Chain ◽  
Sds Page ◽  
Immunoblotting Technique

Abstract Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti- light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.

scholarly journals Characterization of Molecular Forms of N-Terminal B-Type Natriuretic Peptide In Vitro

2010 ◽  
Vol 56 (12) ◽  
pp. 1822-1829 ◽  
Author(s):  
Minna Ala-Kopsala ◽  
Anne-Mari Moilanen ◽  
Jaana Rysä ◽  
Heikki Ruskoaho ◽  
Olli Vuolteenaho
Keyword(s):  
Human Plasma ◽  
Ventricular Myocardium ◽  
Storage Conditions ◽  
Molecular Forms ◽  
Plasma Samples ◽  
Multiple Components ◽  
Rat Hearts ◽  
Adenoviral Transfer ◽  
Antibody Epitopes

BACKGROUND The heterogeneity of circulating peptides may influence the interpretation of results from N-terminal profragment of BNP (NT-proBNP) assays. Our objective was to characterize the heterogeneity for better usability of the assays. METHODS Endogenous proBNP was purified from patient samples and treated with trifluoromethanesulfonic acid (chemical deglycosylation). The human proBNP gene was introduced into rat hearts by adenoviral transfer. Cell lysates and plasma samples containing proBNP-derived peptides were analyzed by chromatography. The fate of exogenous recombinant NT-proBNP added to fresh whole blood samples was followed by immunoassays and chromatography. The main NT-proBNP components were isolated and identified by mass spectrometry. RESULTS Immunoreactive NT-proBNP in human plasma comprised several molecular forms, as did circulating immunoreactive human NT-proBNP after adenoviral transfer of human proBNP cDNA into rat ventricular myocardium. Incubation of recombinant NT-proBNP1–76 in human plasma or serum resulted in multiple components with the 2 major components identified as NT-proBNP1–36 and NT-proBNP1–62/64. Profiling by different antisera and chromatography indicated masking of the non–mid-region epitopes likely due to formation of oligomers. More than 75% of the original immunoreactivity in the mid-region epitope was retained after 3-week storage of plasma samples at room temperature. CONCLUSIONS There is marked heterogeneity in immunoreactive NT-proBNP in plasma not related to glycosylation. The mid-region epitope of NT-proBNP is stable even in harsh storage conditions. Careful choice of antibody epitopes can yield extraordinarily robust assays.

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