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Biomedical subjects

W Schramm

Publications and source records attributed to W Schramm.

At least 19 recordsLinked to original sources
PubMed1992

Antibody-antigen complex formation with immobilized immunoglobulins.

We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays.

Antibodies, Monoclonal↗
PubMed1992

Cotinine in an ultrafiltrate of saliva.

BACKGROUND: We have developed a device for the simplified collection of a prepurified sample of saliva in the mouth. METHOD: The device is based on the principle of an osmotic pump and accumulated about 1.2 ml of an ultrafiltrate of saliva within 8 min. We have investigated the ultrafiltrate for its utility as a biological medium in the evaluation of cigarette smoking status. RESULTS: (a) In 58 matched samples from 13 subjects, the correlation coefficient for the cotinine concentration in the saliva and the ultrafiltrate was 0.95; (b) in matched plasma and ultrafiltrate samples from 27 smokers, the correlation coefficient for the cotinine concentrations was 0.96 with plasma containing 1.2 times the ultrafiltrate mean; (c) in a nonsmoker, elevated cotinine levels could be detected in the ultrafiltrate more than 24 hr after smoking 2 cigarettes, and the pattern of rise and decrease reflected that in whole saliva; and (d) in a habitual smoker; the mean cotinine concentration in the ultrafiltrate was 157 ng/ml (SD +/- 25.7 ng/ml) during a period of smoking 15 cigarettes per day and dropped to a mean of 47 ng/ml (SD +/- 10.5) when smoking was reduced to 5 cigarettes per day; after cessation of smoking, detectable concentrations of cotinine persisted for up to 5 days. CONCLUSION: The device facilitated the aesthetic, noninvasive collection of a biological sample useful in the validation of smoking status.

Adult↗
PubMed1992

Continuous monitoring of analyte concentrations.

We have investigated the application of a modified, heterogeneous, competitive enzyme immunoassay for the continuous measurement of small analytes in a medium stream. The analytical system contains two antibodies that are immobilized on spatially separated areas, one binding the analyte (Ab1) and the other binding the enzyme (Ab2). An analyte-enzyme conjugate serves as signal generator. The analyte-enzyme conjugate functions as a heterobifunctional shuttle that can bind to either antibody. A semipermeable membrane retains the enzyme shuttle in the internal volume of the sensor but permits the passage of small analytes from the medium stream. The amount of enzyme bound to Ab1 is inversely proportional and the amount of enzyme bound to Ab2 is directly proportional to the analyte concentration. We have demonstrated that this analytical system (1) can provide a larger total signal; (2) has a sensitivity comparable with conventional competitive immunoassays; (3) does not require the separation of bound from free antigens; and (4) is therefore suitable for the continuous measurement of analytes in a medium stream. With a model system, an increase from 0 ng ml-1 to 20 ng ml-1 of the steroid hormone progesterone and the subsequent fall to 0 ng ml-1 could be monitored.

Antibodies, Monoclonal↗
PubMed1992

Effect of treatment with methylprednisolone on duration of pseudopregnancy and on macrophages and T lymphocytes in rabbit corpora lutea.

The potential role of macrophages and T lymphocytes in the destruction of the corpus luteum at the end of the luteal phase was investigated by treating pseudopregnant rabbits with the immunosuppressant glucocorticoid methylprednisolone. Eleven specific pathogen-free New Zealand White rabbits were injected with pregnant mares' serum gonadotrophin (40 iu, i.m.), followed 2 days later by human chorionic gonadotrophin (40 iu, i.v.) to stimulate ovulation. The following day (day 1 of pseudo-pregnancy) all animals had an oestradiol-filled Silastic capsule implanted s.c., to ensure that oestradiol, the luteotrophic hormone in this species, would not be limiting. From day 10 of pseudopregnancy, three animals were injected with a low dose of methylprednisolone (2 mg kg-1 per day) until day 20. Three other animals were injected with a higher dose of methylprednisolone (20 mg kg-1 per day) from day 13 of pseudopregnancy until day 19. Five animals served as control, vehicle-injected animals. Blood samples were taken at intervals and assayed for progesterone. Immunofluorescence was used to stain luteal tissue for macrophages, T lymphocytes and class II antigens, and positive cells were counted under high-power magnification. Methylprednisolone treatment reduced (by about 70%), but did not eliminate, the macrophages in the regressing corpora lutea. In contrast, the high dose of methylprednisolone essentially eliminated T lymphocytes, and reduced (by about 90%) the number of cells expressing class II antigen in the luteal tissue. Despite the effects of methylprednisolone on these cells, serum progesterone profiles were not altered by treatment with methylprednisolone, and pseudopregnancy was of normal duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

[Biochemical factors as objective parameters for assessing the prognosis in polytrauma].

One hundred patients with multiple injuries (mean ISS 37 patients) were prospectively evaluated over a period of 14 days following trauma. Significant differences in the blood levels of PMN elastase, cathepsin B, lactate, neopterin, C-reactive protein (CRP) and antithrombin III (ATIII) were found in non-survivors and in survivors with and without organ failure. On admission, a prediction of organ failure was possible with an accuracy of 63% to 69% (PMN elastase, cathepsin B, ATIII). Death was predictable with an 80% to 90% accuracy within the first 4 days (PMN elastase, lactate, CRP, neopterin). The prognostic value of these factors was comparable to trauma scores regarding organ failure and better with respect to death. Biochemical parameters may be helpful in estimating the severity of the injury and prognosis and in monitoring the ICU course of such patients.

Acute-Phase Proteins↗
PubMed1992

[Distal biceps tendon rupture. Therapy and forensic insurance evaluation].

Compared with proximal ruptures of the biceps sinew, distal ruptures of the biceps sinew are extremely rare and generally need operative treatment. However, the large number of different operation techniques described in literature clearly shows that a safe method of treatment has still not been found. In the "Bergmannsheil" Hospital in Gelsenkirchen-Buer, 11 distal ruptures of the biceps sinew in 10 patients were operatively treated during 1985 and 1989. The average age of the patients was 44.6 years. All accidents occurred during physical work. The account of how the accident had happened suggested that accident and injury were related in 8 cases, especially when the findings at operation and the course were also taken into consideration. In 3 cases the rupture of the biceps sinew was not caused by any outward influence. The operative treatment took the form of transosseous reinsertion of the biceps sinew in a modification of Platt's technique in 6 patients; in 1 case the sinew was restored by means of a fascia late strip even though 49 days had passed since the accident. In the course of follow-up examinations we noted a considerable deficit of supination power remaining in the affected lower arm, although the clinical results were good otherwise; we have therefore developed our own method, taking particular account of the essentially more important function of the biceps brachii muscle as a supinator in addition to its flexion function. The method applied is described. At the follow-up examination distinctly better supination power was noted in the 4 patients operated on by this method; the flexibility of the cubital joint was not reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

[Attempt of preventive treatment of striae gravidarum using preventive massage ointment administration].

Striae distensae are an appreciable cosmetic problem for many pregnant women. Preventive application of a water/oil massage cream was tested in a group of 24 gravidae (control group: 26 patients). In the untreated control group striae distensae were observed in two-thirds of the patients, whereas the prophylactically-treated gravidae showed development of striae in only one third of the group given the test preparation. Better results were obtained in women with a favourable constitutional predisposition than in patients with a tendency to overweight. The massage cream was well tolerated by all gravidae.

Adult↗
PubMed1991

HIV-1 reproduction is inhibited by peptides derived frm the N- and C-termini of HIV-1 protease.

Two octapeptides derived from the sequence of the N- and C-termini of HIV-1 protease were tested for their ability to inhibit HIV-1 reproduction. Weak inhibitory activity was found with each of the two peptides. It is assumed that HIV-1 protease is the target of the inhibitory action. In spite of the moderate inhibitory activity the results are encouraging since they may be improved by various means.

Amino Acid Sequence↗
PubMed1991

Modeling of immunosensors under nonequilibrium conditions. I. Mathematic modeling of performance characteristics.

Immunosensors for the detection of small analytes that use analyte-enzyme conjugates as signal generators require special attention if operated under nonequilibrium conditions. If the size of the analyte and the analyte-enzyme conjugate differ substantially, the two antigens do not diffuse at the same rate. This can cause time-dependent shifts in the sensitivity of competitive immunoassays. Therefore, immunosensors operating at short incubation times require precise timing that meets closely the specifications for which the sensors were calibrated. As an example, we have analyzed kinetic binding curves for the quantitative determination of progesterone with an immobilized monoclonal antibody and a conjugate between horseradish peroxidase and progesterone as signal generator. Mathematical paradigms have been developed to simulate the diffusion, antigen-antibody complex formation, and competitive binding processes in this analytical system. Dose-response curves obtained under nonequilibrium conditions can vary substantially from those obtained at equilibrium of antigen-antibody interaction. The degree of this variation depends on the performance characteristics of the major components of the immunosensor. The developed mathematical solutions reflect experimental results and can be used to model optimal conditions for immunosensors operating under nonequilibrium conditions. In this paper (Part I), we report on the mathematical modeling of the interaction between analyte, analyte-enzyme conjugate, and an immobilized antibody. In Part II (W. Schramm and S.-H. Paek (1991) Anal. Biochem. 196), we present experimental results and compare them with the theoretical models.

Antibodies↗
PubMed1991

Modeling of immunosensors under nonequilibrium conditions. II. Experimental determination of performance characteristics.

In an attempt to optimize immunosensors operating with an immobilized antibody as binding protein and an analyte-enzyme conjugate as signal generator that is significantly larger in molecular size than the analyte, in a previous communication (Part I) (S.-H. Paek and W. Schramm (1991) Anal. Biochem. 196) we developed mathematical models for the prediction of performance characteristics. These models are compared in this contribution with experimentally obtained results. As an example, a monoclonal antibody to the steroid hormone progesterone has been used as binding protein, an 125I-progesterone derivative, and a progesterone-horseradish peroxidase derivative as tracers for signal generation. A minimum of parameters needs to be experimentally determined to calculate the performance: the amount of immobilized antibody, the diffusion coefficient of antigens, the thickness of the penetration layer, and the on- and off-rates for binding of the antigen to the antibody. We have described simple methods to obtain these data for the labeled antigen and for the unlabeled analyte that does not provide a signal per se. Kinetic binding curves for antigen-antibody complex formation obtained with the mathematical models correlated well with experimentally obtained results for antigens of different sizes. Although equilibrium of the antigen-antibody complex for the enzyme-labeled analyte conjugate requires about 4 h in the absence of free analyte, dose-response curves can be obtained after 5 min and the relative position of these curves does not change significantly after 30 min. Using a total volume of 200 microliters for the analytical procedure in microtiter wells, agitation as a means to accelerate convective diffusion during an incubation period of 30 min is not necessary with the analyte-enzyme conjugate. However, immunosensors using large analyte-enzyme conjugates as signal generators for the detection of small analytes require strict control of the incubation time if operated within short periods of time (less than 30 min).

Antibodies, Monoclonal↗
PubMed1991

A purified antithrombin III--heparin complex as a potent inhibitor of thrombin in porcine endotoxin shock.

Inhibition of activated clotting factors is an important therapeutic approach in disseminated intravascular coagulation (DIC). We examined the possible protective effect of a purified complex of human antithrombin III (AT III) and heparin in endotoxin-induced DIC in pigs. Two groups of endotoxemic pigs were studied. AT III-heparin group pigs (n = 8) were pretreated with a bolus injection of 500 units AT III-heparin complex, followed by a continuous infusion of 1000 units of the complex for 6 hours given simultaneously with the infusion of 10 micrograms/kgh of S. abortus equi endotoxin. Controls (n = 9) were given saline in addition to the continuous infusion of endotoxin. AT III activity, prothrombin and soluble fibrin in plasma were determined by chromogenic substrate methods. Fibrinogen was measured turbidimetrically. Human AT III antigen in the treated group was 64 +/- 3% at 2 hours and increased to 84 +/- 4% until the end of the experiment. AT III activity in the AT III-heparin group was elevated throughout the whole observation period (greater than 100%), whereas it was significantly lower in the controls. Prothrombin decreased similarly in both groups by approximately 35% until the end of the experiment. AT III-heparin treatment significantly attenuated the endotoxin-induced consumption of fibrinogen and completely prevented the increase in soluble fibrin in plasma. However, no significant effect of AT III-heparin was observed on endotoxin-induced mortality and dysfunction in pulmonary gas exchange. Therefore we conclude that the purified AT III-heparin complex inhibits thrombin effects and prevents development of DIC, but fails to significantly influence clinical outcome in endotoxin shock of the pig.

Animals↗
PubMed1991

Development of a chromogenic substrate assay for the determination of hirudin in plasma.

Hirudin is a potent and specific thrombin inhibitor. Since recombinant hirudin is being considered for anticoagulant and antithrombotic therapy we developed a fast and sensitive chromogenic substrate assay for its determination in plasma. The plasma samples (20 microliters) were incubated with 1 ml reagent mixture (0.2 M Tris buffer, 0.025 M NaCl, pH 8.1, containing 0.833 M urea, 0.7 trypsin inhibitor U/ml aprotinin, 100 ng/ml Polybrene and 0.31 NIH U/ml bovine thrombin) for 1 min. Thereafter 100 microliters Chromozym TH (Tos-Gly-Pro-Arg-pNA, 1.9 mM) was added. The change in absorbance/min (delta A/min) was recorded at 405 nm. delta A/min was linear for at least 3 min. The calibration curve was linear at least up to 800 ng hirudin/ml plasma. Intra-assay and inter-assay coefficients of variation were 2.8-3.1% and 5.3-5.8% respectively. The influence of progressive thrombin inhibitors can be neglected because of the short incubation time. Plasma samples can be assayed directly if aprotinin, polybrene and urea are added to the reagent mixture.

Calibration↗
PubMed1991

Plasmatic factors of haemostasis remain essentially unchanged except for PAI activity during n-3 fatty acid intake in type I diabetes mellitus.

Diabetic patients are prone to develop vascular complications. Increased procoagulatory factors and a reduced fibrinolytic potential are considered as thrombogenic risk factors, although controversy remains. In epidemiological and dietary intervention studies fish or fish oil, rich in the two n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have demonstrated a potential to reduce cardiovascular disease. We compared the plasmatic coagulatory and fibrinolytic profile of 13 near normoglycaemic type I diabetics almost free of cardiovascular disease with healthy volunteers, matched for age and sex. Except for fibrinogen levels and tPA activity being elevated and soluble fibrin and fibrinopeptide A being reduced, no differences could be discerned between type I diabetics and controls in all investigated plasmatic parameters. In a dietary intervention study we investigated the effects of 5.4 g EPA and 2.7 g DHA per day during and after a 4-week dietary supplementation in the diabetic patients. The factors, inhibitors and activation products of coagulation and fibrinolysis measured were at best transiently affected by the diet. Only plasminogen activator inhibitory activity in plasma significantly increased during the dietary supplementation and returned to prediet values after cessation of n-3 fatty acids. Changes in PAI activity were negatively correlated to changes in serum triglycerides. We conclude that well adjusted type I diabetics show an almost unchanged haemostatic profile compared to matched healthy controls. A dietary intervention with n-3 fatty acids in these patients does not affect the plasmatic haemostatic pattern except for an increase in PAI activity.

Adult↗
PubMed1991

Time dependent release of tissue-type plasminogen activator and plasminogen activator inhibitor into the circulation of pigs during shock.

To investigate short-term activation and inhibition of fibrinolysis during shock, we studied plasma levels of tissue-type plasminogen activator (t-PA) and t-PA inhibition capacity (PAI) in anaesthetized pigs. t-PA in euglobulin fractions of plasma was measured by the conversion of plasminogen to plasmin in the presence of fibrin split products. Plasmin thus generated was measured in a chromogenic substrate assay. PAI was measured as plasma inhibition capacity for human melanoma t-PA. Controls (n = 8) had constant t-PA and PAI for 6 h. Lipopolysaccharide from Salmonella abortus equi in four different doses (n = 9 - 11), or live Escherichia coli (n = 3) induced a transient t-PA increase with peak values at 2 h. PAI decreased to 50% at 2 h and increased to 250% at 6 h. Phorbol myristate acetate (n = 7) induced no change of t-PA or PAI. Dextran sulphate (n = 4) produced a t-PA rise at 30 min, followed by a rapid decline. Endotoxin was an appropriate stimulus for activation and inhibition of fibrinolysis whereas phorbol ester failed to elicit this response.

Animals↗