An enzyme-linked immunoassay for direct measurement of the gelatin-binding capacity of human plasma fibronectin.

A new solid-phase enzyme immunoassay measuring the gelatin-binding capacity of plasma fibronectin has been developed. This assay is based on the direct and high-affinity interaction between fibronectin and gelatin coated to polyvinyl chloride plates. The amount of fibronectin bound to gelatin is then measured by sequential incubation with a specific rabbit anti-human fibronectin antiserum, with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies and with substrate. The final degradation of the substrate is read at 492-650 nm in an ELISA processor. The assay allows the accurate detection of fibronectin concentrations ranging from 1 to 20 micrograms/ml, is inhibited by the addition of gelatin to plasma, is highly reproducible (interplate CV less than 10%), requires 100 microliter of plasma only and has been fully automated. Significant linear correlations were noted between total antigenic fibronectin (measured by laser nephelometry) and fibronectin gelatin-binding capacity in plasma from 310 blood donors. Both parameters were higher in men than in women and significantly increased according to age. Dissociation between immunoreactive fibronectin and fibronectin gelatin-binding capacity was observed in two polytraumatized patients. This enzyme immunoassay therefore provides a new method to investigate functional alterations of the gelatin-binding domain of fibronectin in various pathological conditions.

Quantification of rat T-kininogen using immunological methods. Application to inflammatory processes.

Antibodies raised in rabbits against rat T-kininogen (alpha 1-cysteine proteinase inhibitor) were used to develop a radioimmunoassay and a nephelometric quantification for T-kininogen. These assays were specific and analytically reliable. We also described a radioimmunoassay for kinin measurement. These immunological methods have been used to study the behaviour of T-kininogen during inflammatory processes and specify the two properties of this kind of kininogen: its inhibitory capacity towards cysteine proteinases and its activity as precursor of T-kinin. Control plasma level of T-kininogen in male rats was lower than that of female rats. The maximum level was observed in plasma, liver, kidney and uterus of female rats during metestrus. After turpentine injection, T-kininogen level increased not only in plasma but also in liver and kidney. In carrageenan-induced peritoneal exudates, we found a large accumulation of T-kininogen and of immunoreactive kinins, these latter being identified by HPLC as bradykinin.

Plasma prokallikrein and kininogens in burned patients.

Using specific immunological (1) and enzymatic (2) methods, we have measured prokallikrein, total, high, and low molecular weight kininogens in 36 severely burned patients. At admission to the intensive care unit, all constituents were significantly decreased when compared to previously defined reference intervals. The values remained low during the three first days after burn. The changes affecting total and low molecular weight kininogens were significantly correlated (p less than 0.05) with the severity of the burn area. Prokallikrein and kininogens levels were also closely related to the concentrations of C3c and C4 complement factors.

[Relation between the vectorcardiogram and mean pulmonary arterial pressure in pulmonary pathology]. / Contribution à la recherche de relations entre vectocardiogramme et pression artérielle pulmonaire moyenne en pathologie pulmonaire.

Mean pulmonary artery pressure (PAP) is considered to be a valuable indicator of the severity of disease in patients with chronic lung damage. Its measurement requires minicatheterisation, an invasive technique associated with some morbidity. This study was undertaken to establish a relationship between PAP and vectorcardiography (VCG) in 76 patients with chronic lung disease. The patients were divided into three groups: Group I, PAP less than 19 mmHg; Group II, PAP between 19 and 30 mmHg; Group III, PAP greater than 30 mmHg. The patients presented with an obstructive pulmonary syndrome in 67,1% of cases, and an emphysematous syndrome in 32,9% of cases. The ECG tracing was suggestive of chronic cor pulmonale in 20 patients. The three VCG planes were analysed. The direction and amplitude of the 0,01 sec, 0,02 sec, 0,04 sec, 0,06 sec and maximum vector over each QRS loop were measured; the direction of rotation of the QRS loop and, in the horizontal plane, the duration of the terminal forces were also determined. Treatment of the angular data by Down's centre of gravity method allowed definition of the preferential direction of the vectors and of an index of precision. The statistical significance of the results was checked by the usual methods (chi-square, F test). The results confirmed the importance of the right posterior quadrant of the horizontal plane in assessing the degree of electrical overload and for evaluating the repercussions on the pulmonary circulation. In this plane, all angular positions of the 0,06 sec vector beyond -125 degrees corresponded in 9 out of 10 cases to higher than normal PAP (greater than 19 mmHg). A voltage of over 0,80 MV was always associated with higher than normal PAP. The predictive value of this method was 92%, with a specificity of 88% and a sensitivity of 39%. The VCG was also more sensitive than the ECG in assessing PAP of 19 to 30 mmHg; 25,6% of patients had deviation of the 0,06 sec vector of beyond -125 degrees in the horizontal plane whilst only 6,3% of cases had ECG criteria of chronic cor pulmonale. This study shows that simple and reliable non-invasive VCG criteria may be used to assess mean pulmonary artery pressure.

[Short- and medium-term hemodynamic effects of prazosin in chronic refractory cardiac failure]. / Etude de effets hémodynamiques à court et moyen termes de la prazosine dans l'insuffisance cardiaque chronique réfractaire.

The hemodynamic effects of prazosin, a post-synaptic alpha blocker, were studied in 10 patients with chronic stable cardiac failure, in Classes III and IV of the NYHA classification, resistant to digitalis and diuretic therapy. The measurements of pressure and cardiac output were carried out by right heart catheterisation and thermodilution. The administration of the first dose (1 to 3 mg) led to a 38% reduction in mean right atrial pressure, a 22% reduction in mean pulmonary artery pressure, a 22 ù reduction of wedge pulmonary artery pressure and a 15% reduction of systemic vascular resistance. The systolic index rose by 20%, systemic blood pressure and heart rate remained unchanged. After 7 days, treatment with 3 to 12 ng/day of prazosin mean right atrial pressure had fallen by 54%, mean pulmonary artery pressure by 37%, and wedge pulmonary artery pressure by 48%. The systolic index increased by 27%. The blood pressure was unchanged, but the heart rate decreased by 10%. The patients lost an average of 2,7 Kg in weight. These results show that in the short term, prazosin had a mixed vasodilator effect on both arteries and veins. In the medium term the arterial vasodilator action appears to be significantly smaller but remains detectable with doses greater than 8 mg/day, whilst the venous vasodilator effect persists, unchanged.

[Specificity of the radioimmunoassay for low molecular weight human kininogen]. / Spécificité du dosage radio-immunologique du kininogène humain de faible poids moléculaire.

The authors have studied the crossing immunoreactivity of various antigens in the radioimmunoassay of Low Molecular Weight Kininogen: serum and plasma proteins, human albumin, Bradykinin, High Molecular Weight Kininogen and the products of enzymatic degradation of Low Molecular Weight Kininogen.

[Formation of human immunoreactive LMW and HMW-kininogens during coagulation. Comparison with bradykinin activity]. / Evolution du LMW et du HMW-kininogènes humains immunoreactifs au cours de la coagulation. Comparaison avec l'activité bradykinine.

Variations of the levels of human HMW and LMW kininogens have been studied in serum and in plasma incubated in vitro during 30 hours, at three different temperatures: 4 degrees, 22 degrees and 37 degrees C. There is a small difference between the level of LMW kininogen in plasma and in serum, but the serum and plasma level of LMW kininogen are almost stable during the time of incubation at the three temperatures. However, HMW kininogen is reduced in serum to about fifty percent of its plasma level. It decreases in plasma and in serum during incubation, overall at 4 degrees C. A strict parallelism stays between the radioimmunoassay and the bioassay.

Purification and characterization of plasma T-kininogen from Wistar and brown Norway rats.

A rapid and convenient three-step purification scheme has been developed for the purification of T-kininogen (alpha 1-cysteine proteinase inhibitor) from rat plasma. The purification process includes chromatography on hydroxyapatite, immunoaffinity chromatography and gel filtration. This procedure is applied to plasma from the brown Norway rat which is known to be deficient in high and low molecular weight kininogens. The method furnished large amounts of T-kininogen from turpentine-treated Wistar rats as well as from untreated and turpentine-treated deficient brown Norway rats. The amino acid and hexose content of the three T-kininogens has been determined. While the composition of the molecules isolated from both injured rats was similar, the neutral sugar content of T-kininogen purified from untreated brown Norway rats was lower and its amino acid composition showed slight differences. The three molecules have identical behaviour and similar physicochemical and immunological properties when analysed by SDS electrophoresis, isoelectrofocusing and two-dimensional immunoelectrophoresis.

[Radioimmunologic determination of human kininogen of high molecular weight]. / Le dosage radioimmunologique du kininogène humain de haut poids moléculaire.

HHW-kininogen has been purified from human plasma. We have then studied the optimal conditions for labeling this protein with 125I. This molecule has been used for the preparation of a radioimmunoassay. We have studied the analytical performances of our dosage : sensitivity, precision, accuracy and specificity.

Human kininogens of low and high molecular mass: quantification by radioimmunoassay and determination of reference values.

Kininogens of low and high molecular mass, highly purified from human plasma, were used to raise antisera in rabbits. To obtain immunologically intact tracers, we labeled the kininogens by the lodogen method, followed by double chromatography, first on Sephadex G50 and then on Sephadex G200 for high-molecular-mass kininogen or on G100 for low-molecular-mass kininogen. Both assays are sensitive, accurate, and reproducible. On an equimolar basis, the high-molecular-mass kininogen cross reacted completely in the determination of low-molecular-mass analyte. Moreover, the radioimmunoassay for the former was highly specific. After optimizing the time and temperature of incubation to provide rapid and reliable results, we determined 95% reference intervals from a large sample of healthy subjects (250 men, 200 women): 109-272 and 69-116 mg/L for the low- and high-molecular-mass kininogens, respectively.