Purification of recombinant hepatitis delta antigen expressed in E. coli cells.

Recombinant DNA technology enables the massive production of recombinant hepatitis delta antigen (recHDAg) retaining immunological properties and transport functions. However, purification procedures of the recombinant delta antigen have, to date, not been described in the literature. We present a purification procedure allowing one to obtain highly purified recHDAg from bacterial cells expressing the hepatitis delta antigen.

[The prevention of DVT after general surgical interventions for cancer pathology]. / Profilassi delle TVP dopo interventi di chirurgia generale per patologie oncologiche.

The efficacy of postoperative DVT prophylaxis of defibrotide and heparin calcium was evaluated in a group of 47 patients undergoing general surgery for oncological pathologies. A sub-group (24 patients) received defibrotide treatment, 400 mg b.i.d. i.m.; the second subgroup (23 patients) was treated with heparin calcium, 5000 IU b.i.d. s.c., from the day before operation until the 7th postoperative day. No postoperative thrombotic symptoms were observed in either group of patients.

[Treatment with indobufen of platelet hyperaggregation in a group of patients with vascular diseases]. / Il trattamento con indobufene dell'iperaggregazione piastrinica in un gruppo di pazienti affetti da patologie vascolari.

Twenty-five patients with enhanced spontaneous platelet aggregation and impaired arterial or venous circulation syndromes have been treated by indobufen. Each of them received, by oral administration, 200 mg of the drug twice a day for periods of time from fifty one to hundred eleven days. This treatment normalized, in all the cases, the platelet aggregation. In eight patients of the group, three hours after the last drug administration, the average of the maximum platelet aggregation curves deflections, induced by ADP, epinephrine, ristocetin and collagen, resulted significantly reduced, while, in seven other patients, twelve hours after the last drug administration, only the aggregation curves induced by ADP and epinephrine resulted significantly inhibited. Most on the patients with arterial impairment syndromes noticed some improvements of their symptoms. Also the patients with light subjective post-phlebitic symptoms declared to feel better. The drug has been well tolerated. Therefore, its use is recommendable whenever it is advisable to get a full antiplatelet effect whose the prompt reversion might be required.

The "soluble sandwich" approach for immunoassays: methodological and instrumental implications.

The simultaneous presence of homogeneous and heterogeneous reactions at different binding sites of a multiepitope antigen makes the description of the kinetic parameters of the so called "one step" solid phase immunometric assays complex. The authors extended the "one step" approach to the concept of the "soluble sandwich" methodology which differs from the former by the delayed solid phase capture of the biotinylated immunocomplex to a streptavidin coated solid support. Using prolactin monoclonal IEMAs as a model, the equilibria involved in the reactions have been studied on a thermodynamic basis through a description of the kinetics of the interactions between biotinylated Mabs and solid phase streptavidin both in presence and/or in absence of the antigen and HRP-conjugated antibody. A comparative evaluation of models in which the biotinylated antibody was previously insolubilized on the streptavidin solid phase has been performed as well. The experimental work was carried out by using 125I labelled McBiot and Prolactin to trace individual interactions and peroxidase/H2O2/TMB systems to develop the enzymatic analytical signals. A new instrument/data reducing system was also optimized to expand the OD reading range provided by conventional, single wavelength colorimeters. The greater flexibility theoretically expected for the "soluble sandwich" approach and the possibility to extend the analyte working range without detrimental effects on the readability of low doses responses have been experimentally confirmed.

[Hemorheological changes in retinal vein occlusions]. / Alterazioni emoreologiche nelle occlusioni venose retiniche.

Some hemorheological factors were studied in 34 patients affected by retinal vein occlusion (RVO) and in 25 healthy control subjects. Whole blood viscosity, plasma viscosity, fibrinogen and hematocrit were increased in RVO patients compared with controls. Erythrocyte filterability was reduced in RVO patients compared with controls. However, when evaluating the type of capillaropathy at fluorescein angiography, blood viscosity and plasma viscosity were altered both in patients with and without evidence of capillary non-perfusion. Reduction of red cell filterability showed statistical significance only in patients with capillary non-perfusion. This finding suggests that reduced erythrocyte filterability might be a causal factor in the development of capillary non-perfusion in RVO patients.

HBeAg/anti-HBe determination with a new monoclonal immunoradiometric assay.

The performance of two assays for the HBeAg/anti-HBe system associated with hepatitis B virus infection, using monoclonal (EBK-Sorin) and polyclonal (HBe-Abbott) antibodies, has been compared. The results on a random population (1,000 samples) demonstrated for the monoclonal reagent a higher sensitivity, without any loss in specificity, and with the further advantage of the use of lower radioactivity levels. The proportion of positives and negatives obtained with the two kits was found to remain unchanged in the case of HBeAg, while a markedly larger percentage of positives (7% higher) was detected for anti-HBe using the monoclonal antibodies.

Purification of hapten-enzyme conjugates for enzymoimmunoassay.

Chromatography on hydroxyapatite, including a potassium phosphate linear-gradient elution, was applied to the purification of hapten-enzyme conjugates to be used as immunoenzymatic tracers (namely, progesterone, phenobarbital, diphenylhydantoin coupled to glucose-6-phosphate dehydrogenase). The method effectively removed unreacted enzyme and separated the conjugate classes according to the number of substitutions between enzyme linking groups and hapten derivatives. The adequacy of the chromatographic procedures in improving the analytical performance of the enzymatic tracer appears to depend on direct interdependence between the degree of substitution and the immunological properties of the conjugates.

Measurement of 5-androsten-3beta,17beta-diol in spermatic and peripheral venous blood samples from the same human subjects by a radioimmunoassay method.

An original method for 5-androsten-3beta,17beta-diol (A-diol) measurement using an antiserum against A-diol-16-CMO-BSA is described. A-diol and testosterone (T) were determined by radioimmunoassay methods in spermatic and peripheral venous plasma of nine normal subjects during surgical intervention for inguinal hernia repair. In spermatic venous plasma the levels of T and A-diol were, respectively, 25.9 +/- 13.3 and 4.8 +/- 5.1 microgram/100ml (mean +/- SD) with an A-diol/T ratio of 0.19 +/- 0.15 (mean +/- SD); in peripheral plasma the levels of T and A-diol were, respectively, 269 +/- 58 and 91 +/- 25 ng/100 ml (mean +/- SD) with an A-diol/T ratio of 0.35 +/- 0.12 (mean +/- SD) significantly different from spermatic venous plasma (p less than 0.01). From these data a mean testicular A-diol secretion of about 0.70 mg/24 h can be calculated: this value corresponds approximately to the 50% of the blood production rate (BPR) of this steroid. So it can be assumed that a large amount of A-diol in systemic blood comes from sources outside the male gonad.

Digoxin radioimmunoassay: a comparison of the assay performances using tritiated and 125I radioiodinated tracers.

The preparation of 125I-labelled tracer for digoxin radioimmunoassay (125I monoiodinated 3-succinyl digoxigenin-1-tyrosine) is described, and its performance in radioimmunoassay of plasma samples is compared with that obtained with tritiated digoxin. The accuracy levels were assessed through the evaluation of different potential sources of systematic errors, such as interference from digoxin-related molecules and plasma proteins and methodological artefacts possibly associated with the immunocomplex instability, and through a series of checks including the recovery and parallelism tests and the correspondence of results obtained with the two tracers. The slope and the repeatability with time of the calibration curves and the spread of replicate estimates were taken into consideration to assess the assay precision. An essential equivalence in terms of reliability of measurement was proved for the two methodological variants, so that practical aspects and economic factors remain the main criteria to evaluate the relative merits: from this point of view, the advantages of using 125I-labelled tracer, as an alternative to tritiated digoxin, are discussed.

A rapid radioimmunoassay for determination of dehydroepiandrosterone sulphate in human plasma.

A rapid radioimmunoassay for the determination of dehydroepiandrosterone sulphate (DHEA-S) in diluted human plasma is described. The radioimmunoassay is performed using an antiserum to DHEA-3-hemisuccinate-BSA. A charcoal-dextran mixture is used for separation of the bound from the free fraction. The reliability criteria of the method in terms of precision, accuracy, sensitivity and specificity have been evaluated. The mean level of DHEA-S in plasma samples from normal men (age range 24-37) is 241-5+/-72-5 mug/dl. In normal premenopausal women the highest values are in the second third of the menstrual cycle (corresponding to the ovulatory phase). In normal old men (age range 64-86) and in post-menopausal women (age range 55-88) the mean values are, respectively, 51-9+/-33-8 and 53-8+/-34-0. The DHEA-S levels found in several cases of adreno-cortical disorders are reported. In primary adrenal insufficiency and in Cushing's syndrome due to adrenal adenoma or bilateral multinodular hyperplasia low values are found; in Cushing's syndrome due to bilateral adrenal hyperplasia normal values are usually found.