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Biomedical subjects

F Jilek

Publications and source records attributed to F Jilek.

16 recordsLinked to original sources
PubMed1986

Clinical evaluation of a new digitoxin enzyme-immunoassay.

A solid-phase enzyme-immunoassay for the determination of the digitoxin concentration in human serum (Enzymun-Test Digitoxin) was developed, and subsequently evaluated in seven laboratories. The test is based on the competition principle. Polystyrene tubes coated with anti-digitoxin antibodies (from sheep) were used as the solid phase. In the concentration range 10-40 micrograms/l, the coefficient of variation for the majority of laboratories was between 2 and 7%. The day-to-day precision only slightly differed from the within-run precision. The measuring range was between 4 and 60 micrograms/l. Enzymun-Test Digitoxin showed good agreement with three known methods for digitoxin determination. No influence on the values was observed in lipaemic, uraemic and icteric samples, dysproteinaemia sera and in the presence of various digoxin derivatives. The new enzyme-immunoassay permits the practical and reliable determination of serum digitoxin and is suited for use in routine analysis.

Cross Reactions↗
PubMed1984

Automated prothrombin-time test with use of a chromogenic peptide substrate and a centrifugal analyzer.

We modified a test for prothrombin time, to automate its performance by centrifugal analysis. The sample is activated with calcium-thromboplastin in the presence of a thrombin-specific chromogenic peptide substrate, Tos-Gly-Pro-Arg-p-nitroaniline (Chromozym TH). The endpoint of the reaction is set as the time after onset until a defined amount of substrate has been cleaved, as reflected in a fixed change in absorbance. The assay is as sensitive as clotting-type methods are to the clotting factors of the extrinsic pathway--except for fibrinogen, which can be estimated from the maximum absorbance difference. Substrate concentration has little effect on the result, and varying it does not significantly change the sensitivity to factor X, which indicates little interference with the enzymes of the coagulation cascade. The method was applied to the Cobas Bio centrifugal analyzer. An external calculator must be used to convert absorbance readings into reaction times, which can be expressed as percent-of-normal values or as "ratios" by use of reagent-specific factors. CVs were less than 2% within run, less than 6% day to day. Overall correlation with the clotting test (r = 0.95) was good. Using the Cobas Bio, we achieved a throughput rate of 200 samples per hour.

Autoanalysis↗
PubMed1980

Interaction of fibrinogen/fibrin and fibronectin with collagen.

Interaction of fibrinogen, soluble fibrin and fibronection with native collagen, type I and III, was investigated by means of affinity chromatography and radio binding assay. Among collagens type III was most active. It associated stronger with fibronectin than with fibrinogen and fibrin suggesting that fibronectin might form the nuclei for deposition of plasma proteins on exposed collagen structures.

Adsorption↗
PubMed1979

Fibronectin (cold-insoluble globulin), VI. Influence of heparin and hyaluronic acid on the binding of native collagen.

Fibronectin of human plasma associated readily with denatured collagen but gave only a weak reaction with the native protein. In the presence of heparin, however, solutions of native collagen type III, and fibronectin produced precipitates at an ionic strength of 0.2. In the presence of fibronectin and optimal additions of heparin, up to 60% of soluble native 125I-collagen type III, but only about 10% of native 125I-collagen type I, were insolubilized. Heparin also enhanced the formation of insoluble complexes from fibronectin and denatured collagen type I and type III. In the absence of collagen 125I-fibronectin was partially precipitated by heparin. Electron micrographs showed filamentous structures. Collagen did not increase the amount of 125I-fibronectin precipitated by heparin unless a critical collagen concentration was exceeded. It is suggested that heparin induced the transition of fibronectin from a globular to an elongated form, capable of forming filamentous precipitates which adsorb native collagen. Hyaluronic acid and putrescine prevented the insolubilization of native collagen type III, by fibronectin and heparin.

Collagen↗
PubMed1978

Similarity of antigelatin factor and cold insoluble globulin.

Antigelatin factor, a protein capable of complexing denatured collagen, was separated from human serum by adsorption onto immobilized collagen. Antiserum raised against the material binding to denatured collagen permitted the development of a radioassay for the determination of antigelatin factor in which the complex of antigelatin factor and denatured 125I-labeled collagen is precipitated with this antiserum. Further purification of antigelatin factor was achieved by chromatography on DEAE-cellulose yielding an electrophoretically homogeneous protein. Its migration rate in dodecyl sulfate-polyacrylamide gel electrophoresis was identical with that of cold insoluble globulin (molecular weight approx. 440 000) prepared from human plasma by a published procedure amended by DEAE-cellulose chromatography. Reduction of disulfide bonds yielded subunits of molecular weight approx. 220 000, indistinguishable from those of cold insoluble globulin. The amino acid composition of both proteins was very similar. Immunological identity of both proteins was demonstrated by gel diffusion against monospecific anti-cold insoluble globulin antiserum. Closely related binding curves were obtained if denatured 125I-labeled collagen was reacted with increasing amounts of either cold insoluble globulin or antigelatin factor and the complexes formed were precipitated with anti-cold insoluble globulin antiserum. In addition, antigelatin factor and cold insoluble globulin mediated the fixation of denatured 125I-labeled collagen to trypsinized macrophages in the same way. Therefore, it is concluded that antigelatin factor and cold insoluble globulin are identical or very closely related proteins.

Amino Acids↗
PubMed1978

[Characterization of a serum protein with selective affinity for denatured collagen (author's transl)].

A protein known as antigelatin factor (AGF) was isolated from human serum by affinity chromatography with immobilized denatured collagen. In biochemical and immunological assays AGF showed specificity to denatured, but not to native collagen of the types I, II and III. A close relationship to Cell Attachment Protein and Cold Insoluble Globulin was found in comparative studies.

Blood Proteins↗
PubMed1978

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix.

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.

Cartilage↗
PubMed1977

[Functional reaction of a fibroblast surface protein (author's transl)].

Cold-insoluble globulin (CIG) of plasma represents the soluble form of a fibroblast surface protein. As shown by affinity chromatography it is selectively adsorbed from plasma on immobilized collagen of various types. The following sequence of binding strength between CIG and collagen was evaluated by means of fractionated elution: collagen type III native less than type I native less than type I denatured. The affinity to native collagen is important for the capability of CIG to mediate the attachement of fibroplasts on collagen fibrils. In addition, CIG is a substrate of activated coagulation factor XIII which plays an important, but as yet unclarified role in wound healing.

Chromatography, Affinity↗