Development of A Lateral Flow Highway: Ultra-Rapid Multitracking Immunosensor for Cardiac Markers.

The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.

Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA.

D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.

Plasmic degradation of crosslinked fibrin. Characterization of new macromolecular soluble complexes and a model of their structure.

Crosslinked fibrin was digested by plasmin, and three soluble complexes larger than DD/E were purified and characterized. After gel filtration chromatography, the purified complexes were shown to have molecular weights of 465,000, 703,000, and 850,000, as determined by equilibrium sedimentation. Each of the complexes was dissociated into two or more fragments by SDS-polyacrylamide gel electrophoresis. The structure of these subunit fragments was deduced from determinations of their molecular weights and polypeptide chain composition and from known sites of plasmin cleavage of fibrin. Fragments larger than DD have been identified that contain intact gammagamma crosslinks as well as fragments resulting from cleavages at or near this site. The former include DY (mol wt 247,000), YY (mol wt 285,000), DXD (mol wt 461,000), and YXD (mol wt 500,000); and the latter include fragments XD (mol wt 334,000) and XY (mol wt 391,000). A schematic model was developed to explain the structure of the large noncovalently bound complexes based on their molecular weight and observed component fragments. Our scheme supports the two-stranded half-staggered overlap model as the basic unit of fibrin structure, in which each complex consists of fragments from two adjacent complementary antiparallel fibrin strands. The smallest derivative, complex 1, is the DD/E complex; complex 2 contains apposed DY and YD fragments, and complex 3 consists of fragments DXD and YY. Complex 4 is less well-characterized, but its intact structure is projected to consist of YXD and DXY fragments from adjacent fibrin strands. Each complex is heterogeneous in subunit composition, reflecting additional plasmin cleavages within and/or adjacent to its theoretical boundaries. Since most of the protein initially released into solution from degrading fibrin is as complexes larger than DD/E, the derivatives described in this report are likely to be major circulating degradation products of crosslinked fibrin in vivo.

Age-Adjusted D-Dimer in the Prediction of Pulmonary Embolism: Does a Normal Age-Adjusted D-Dimer Rule Out PE?

Risk assessment for pulmonary embolism (PE) currently relies on physician judgment, clinical decision rules (CDR), and D-dimer testing. There is still controversy regarding the role of D-dimer testing in low or intermediate risk patients. The objective of the study was to define the role of clinical decision rules and D-dimer testing in patients suspected of having a PE. Records of 894 patients referred for computed tomography pulmonary angiography (CTPA) at a University medical center were analyzed. The clinical decision rules overall had an ROC of approximately 0.70, while signs of DVT had the highest ROC (0.80). A low probability CDR coupled with a negative age-adjusted D-dimer largely excluded PE. The negative predictive value (NPV) of an intermediate CDR was 86-89%, while the addition of a negative D-dimer resulted in NPVs of 94%. Thus, in patients suspected of having a PE, a low or intermediate CDR does not exclude PE; however, in patients with an intermediate CDR, a normal age-adjusted D-dimer increases the NPV.

Effect of fibrin degradation products on fibrinolytic process.

Fibrin clot lysis by plasminogen/plasmin system results in fibrin degradation products formation with subsequent release into bloodstream. The fragments contain specific binding sites for fibrinolytic system components and can interact with them. In this study, we investigated the way in which fibrin fragments effect fibrinolytic process. We have shown that high molecular weight products of fibrin degradation and fibrin fragments of DDE-complex and DD, but not end product Е3, stimulate plasmin formation. Additionally, components of DDE-complex mixture of fragments Е1 and Е2 have potentiation ability. The intermediate fibrin fragments hmFDPs and DDE attenuate clot lysis by plasmin and hmFDPs protect plasmin from α2-antiplasmin inhibition but under further fragmentation to endpoint fibrin fragments loose this ability. The plasma inhibitors reduce fibrinolytic system activity generated by the degradation products. Thus, fibrin fragments formed during the clot lysis can bind and move out fibrinolytic system components from clot volume and in this way result in clot resistance to hydrolysis.

Structure-activity studies on synthetic analogs to vasoactive peptides derived from human fibrinogen.

Counterparts to two vasoactive peptides previously isolated from fibrin(ogen) degraded by plasmin (EC 3.4.21.7) were synthesized by the solid phase procedure. The synthetic undecapeptide (Ser-Gln-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys) was isolated in a homogeneous state by chromatography on Sephadex G-25 and DEAE-Sepharose CL-6B and the pentapeptide (Ala-Arg-Pro-Ala-Lys) by chromatography on BioGel P-6 and column zone electrophoresis. The effect of these two peptides and of fifteen analogs to the pentapeptide on microvascular permeability in rat skin was investigated. The two synthetic counterparts were as potent as the natural peptides. With respect to the analogs, the influence of different functional groups was first studied. This was followed by attempts to minimize the active structure, induce or relieve rigidity of the peptide back-bone or otherwise accomplish modifications by a change in chirality at critical positions. Our results show that the tetrapeptide Arg-Pro-Ala-Lys has the same effect on microvascular permeability as the pentapeptide in the assay system used. Basic amino acids at both ends, as well as a proline residue adjacent to the N-terminal amino acid appear important for full or essentially full activity. On the other hand, substitution of the Ala at position 4 with several other amino acids did not result in a significant loss in biological potency.

Purification and partial characterization of a D-like fragment from human fibrinogen, produced by human leukocyte elastase.

Digestion of human fibrinogen with human leukocyte elastase in the presence of Ca2+ yields a D-like fragment of Mr 93000. This fragment was purified by gel filtration on Sephacryl S-200 followed by chromatofocusing. The purified fragment was partially characterized and compared with a fragment termed D-cate, which is produced by plasmin digestion of fibrinogen in the presence of Ca2+. The molecular weights of the constituent chains of the D-like fragment and D-cate were similar. The D-like fragment precipitated with antisera directed against D-cate, but not with antisera against fragment E. The name D-elastase for the fragment is suggested. Differences between the D-elastase and D-cate fragments were found in amino-terminal amino acids, in isoelectric point and in the expression of D antigenic determinants. Two major functional differences were demonstrated: fragment D-elastase had a much stronger anticlotting potency than D-cate and the binding of Ca2+ by D-elastase and D-cate differed qualitatively and quantitatively. Since it has been suggested that the calcium-binding and anticlotting properties of D-cate are related to a carboxyl-terminal 13000 stretch of the gamma-chain, the present findings for D-elastase indicate that the differences in these properties between D-cate and D-elastase are due to differences in this area of the molecule.

Binding properties of 111In-diethylenetriaminepentaacetic acid (DTPA)-fragment E1,2, a fibrin-specific probe, are dependent on use of protecting complex during attachment of DTPA groups.

Fragments E1 and E2, plasmic degradation products of crosslinked fibrin, bind specifically to polymers of fibrin. A mixture of these fragments, denoted as fragment E1,2, was radiolabeled with 111In after covalently attaching metal chelating groups (diethylenetriaminepentaacetic acid, DTPA) to the fragment, using two approaches. In the first approach, DTPA groups were attached directly to purified fragment E1,2. In the second approach, attachment sites of DTPA groups were directed away from the active region of the molecule by having fragment E1,2 bound in complex, with its active sites protected during the derivatization. Direct attachment of DTPA groups to fragment E1,2 resulted in complete loss of binding to fibrin in vitro. When derivatized in complex, 111In-DTPA-fragment E1,2 retained a higher degree of binding to human fragment DD and human plasma clots in vitro than did radioiodinated fragment E1, even when up to eight DTPA groups were attached per molecule of fragment E1,2.

Crystallization of fragment D from human fibrinogen.

Fragment D from human fibrinogen has been crystallized. The fragment, which is composed of three disulfide-linked chains (alpha' beta' gamma' = 88,000), was generated with either plasmin or mild trypsin digestion. The crystals diffracted out to 3.5 A; the space group is P2(1), unit cell dimensions a = 108 A, b = 48 A, c = 167 A, beta = 106 degrees. Fragment D was also co-crystallized with the ligand GPRP-amide, in which case the space group is consistent with P212121, unit cell dimensions a = 476 A, b = 82 A, c = 432 A.

Separation of fibrinogen degradation products (FDP) by means of adsorption to insolubilized fibrinmonomer (FM-ag).

The phenomenon of complex formation between fibrinmonomer and fibrinogen degradation products was investigated by means of adsorption of FDP to insolubilized thrombin-modified fibrinogen (FM-ag). Since it could be demonstrated that there are different adsorption characteristics for early FDP and late FDP, the possibility of separation of FDP by means of affinity chromatography on FM-ag columns was evaluated using plasmic digests of 3H-Ac-labelled fibrinogen. The identification of FDP was performed by disc-electrophoresis. The results indicate that the adsorption of early FDP is comparable to the behaviour of fibrinogen, whereas late FDP show essential difference in the affinity towards FM-ag, evident by the result that fragment E adsorbs only to a minimal extents. Fragments D and E derived from fibrinogen as well as from non-crosslinked fibrin, revealed identical adsorption characteristics. Under specified conditions the procedure is suitable as a preparative method for the separation of fragments D and E.

Progressive exposure of E-neoantigen associated with degradation of crosslinked fibrin by plasmin in vitro.

Fragment E-neoantigen (Eneo) is a specific marker of structural and conformational changes associated with the degradation of fibrinogen and its related proteins. In this study Eneo expression was followed during the plasmin degradation of crosslinked (XL) fibrin in vitro, utilizing double antibody radioimmunoassay. Eneo was progressively exposed as degradation proceeded and its expression was associated with the liberation of high molecular weight fragments from XL-fibrin, their degradation into fragments DD and E, and the partial degradation of fragment E itself. An isolated protein fraction containing early high molecular weight fragments (MW greater than 360 000 d) and a fraction containing fragments YY and DY expressed approximately 170 times and 15 times less Eneo per protein respectively compared to the fragment E standard. XL-fibrin fragment E isolated from 1-hr plasmin digest expressed approximately 8 times less Eneo than fragment E isolated from 24-hr digest, indicating increased exposure of Eneo during fragment E degradation. Eneo expression of this terminal fragment E was comparable to fibrinogen fragment E. As expected, fragment DD had no Eneo immunoreactivity.

Soluble fibrin complexes: separation as a function of pH and characterization.

1. The influence of the pH on the separation of high molecular weight derivatives obtained by a limited action of thrombin on fibrinogen was studied by agarose gel chromatography. When the pH of the elution buffer was 8.5, non crosslinked associations were easily separated in two peaks eluted prior to the fibrinogen peak: one contained a dimer, the other several high polymers. At pH 6.5, only the fibrinogen peak appeared: the fibrinogen molecule proteolysed by thrombin formed no stable associations at this pH and was eluted with the intact fibrinogen molecule. In the presence of factor XIII and Ca++, numerous associations were obtained which are independant of the pH. 2. The polypeptide chain composition of the different species separated at pH 8.5 was studied by SDS-polyacrylamide gel electrophoresis. This technic showed Aalpha, Bbeta and gamma chains in the fibrinogen peak, whereas in the chromatographic fractions containing the dimer four bands corresponding to Aalpha, alpha, Bbeta and gamma chains were found. In the peak containing the high polymers, only the presence of alpha, Bbeta and gamma chains was demonstrated. 3. These experimental results concerning the effect of pH on the formation of soluble complexes showed that the presence of fibrin monomers in fibrinogen solution was not sufficient to promote any associations. The formation of such derivatives is strongly dependent on the pH of the solution. This obviously can be explained by an influence of the pH either on the ionization of polymerisation sites and the intermolecular bonds between the complex units or on the unmasking of the polymerisation sites by a hypothetical pH induced conformational change of the fibrinogen molecule.

Preparation and characterization of NH2-terminal fibrinogen B beta fragments from N-DSK of human fibrinogen.

In order to investigate the early release of NH2-terminal plasmic fragments from the B beta chain of fibrinogen, substantial quantities of B beta 1-42 and B beta 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and B beta 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. B beta 1-42 and B beta 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of B beta 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

A new genetic fibrinogen variant (fibrinogen Erfurt I). Structurally characterized by an abnormal B beta-chain and present both in plasma and platelets.

An abnormal fibrinogen was discovered in the plasma of a clinically asymptomatic woman. This fibrinogen variant was analyzed by high resolution two-dimensional gel electrophoresis and its molecular abnormality established consisting in a slight decrease in molecular mass of the B beta-chains. Analysis of fibrin revealed that cleavage of fibrinopeptide B by thrombin is normal, the molecular defect being confined to the beta-portion of the B beta-chain. The same fibrinogen variant was detected in the blood platelets of the proposita. This finding supports the assumption of a common origin of plasma and platelet fibrinogen pools. Family studies revealed the presence of the abnormal fibrinogen in a brother of the proposita, thus confirming the genetic nature of the observed variant. The underlying mutant gene occurs in both carriers in heterozygous state.

Radioimmunoassay of an early plasmin degradation product of human fibrinogen, "fragment A", and its clinical application.

Upon the plasmin digestion of human fibrinogen, an early cleavage product, which has been designated as fragment A, was isolated, and to study the action of plasmin in the circulation, radioimmunoassay for fragment A was carried out. This assay used rabbit immune serum obtained by injection of fragment A mixed with complete Freund's adjuvant, and fragment A was labeled with 125I using the Chloramin-T method. In 20 normal healthy donors its serum level was 3.57 +/- 1.62 microgram/ml (mean +/- SD), and it was increased significantly in certain diseases, such as acute leukemias, cardiovascular disorders, malignancies, renal failure, systemic lupus erythematosus and sepsis.

Reactivity of fibrinogen derivatives with antisera to human fibrin D-dimer and its gamma-gamma chain remnant.

Highly purified D-dimer was obtained from plasmin digest of human cross-linked fibrin. After reduction of its disulfide bonds, the gamma-gamma chain remnant, containing cross-linking site, was then isolated by ion-exchange chromatography on CM-cellulose. Antisera obtained by immunizing rabbits with D-dimer and its gamma-gamma chain remnant contained a small population of antibodies which specifically reacted with D-dimer. Thus, a specific radioimmunoassay system allowing detection and quantitation of D-dimer in the presence of fibrinogen and monomeric fragment D was made possible.

Heterozygous abnormal fibrinogen Osaka III with the replacement of gamma arginine-275 by histidine has an apparently higher molecular weight gamma-chain variant.

Congenitally abnormal fibrinogen Osaka III with the replacement of gamma Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of alpha- and gamma-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal gamma-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 gamma remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Characterization of a mode of specific binding of fibrin monomer through its amino-terminal domain by macrophages and macrophage cell-lines.

The studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (alpha-fibrin) or both fibrinopeptides A and B (alpha beta-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX congruent 200-800 molecules/cell, KD congruent to 10(-12) M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX greater than or equal to 10(5) molecules/cell, KD greater than or equal to 10(-6) M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD congruent to 10(-10) M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (beta-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the alpha- and the beta-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with electrophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Streptokinase-dependent potentiating factor (SK-potentiator) for plasminogen activation from human plasma: its identification as a fibrinogen degradation product.

A factor, named SK-potentiator, which is known to potentiate the activation of plasminogen by streptokinase (SK), was isolated from human plasma. The procedures consisted of column chromatographies on DEAE-Sephadex A-50 and heparin-agarose, followed with gel-filtration on a Sepharose 6B column and affinity chromatography on a lysine-Sepharose 4B column. The isolated SK-potentiator markedly potentiated the rate of activation of plasminogen by streptokinase. However, it did not show a potentiating effect on the activation of plasminogen by urokinase or on the plasmin activity. SK-potentiator showed a similar mobility to that of fibrinogen on both SDS-polyacrylamide gel and agarose gel electrophoresis, and cross-reacted with anti-fibrinogen antiserum. The amino acid composition of SK-potentiator was very close to that of human fibrinogen, although the content of serine and threonine was significantly lower. SK-potentiator showed a single band with a molecular weight of 300,000 on SDS-gel electrophoresis in the absence of 2-mercaptoethanol. In the presence of 2-mercaptoethanol, it showed two major bands with molecular weights of 53,000 and 48,000, respectively, which corresponded to the B beta chain and gamma chain of fibrinogen. To establish further that the isolated SK-potentiator may be one of the fibrinogen degradation products (FDP), human fibrinogen was digested with plasmin and the SK-potentiator activity generated in the course of the digestion was measured. As a result, the SK-potentiator activity was found to initially increase and then decrease with incubation time, suggesting that an early FDP has the ability to potentiate the SK-mediated activation of plasminogen. The early FDP was then isolated by gel-filtration on a column of Sepharose 6B and it was found that the SK-potentiator activity was associated with the early FDP. Moreover, the early FDP showed the same electrophoretic mobility as the isolated SK-potentiator on SDS-gel electrophoresis and their amino acid compositions were quite similar to each other. From these results, the SK-potentiator was identified as the early FDP.

A simple one-step HPLC procedure for the purification of the NH2-terminal plasmin-derived B beta 1-42 peptide of human fibrinogen.

Simplified procedures were developed to produce and isolate the plasmin-derived B beta 1-42 fragment from human fibrinogen. Peptides generated from fibrinogen by plasmin were separated by reverse phase HPLC chromatography. Two distinct peaks were identified as having amino acid compositions identical to B beta 1-42. The average final yield of HPLC-purified B beta 1-42 was 1.2 mg per 100 mg of fibrinogen. Recovery of the peptide (40-45% when compared to the theoretical yield) was significantly higher than yields obtained by open column techniques demonstrating the advantages of this simple one-step HPLC procedure.