Dextran, a hapten carrier in immunoassays for s-triazines. A comparison with ELISAs based on hapten-protein conjugates.

A conjugate of 2-aminohexylamino-4-ethylamino-6-isopropylamino-1,3,5-triazine (AHA), a derivative of the herbicide atrazine, with dextran as carrier has been synthesized and used as the coating antigen in ELISA procedures. The quantification of terbutryn, atrazine and prometryn in ELISA formats using monoclonal antibodies and the AHA-dextran conjugate was at least as sensitive as ELISAs using protein conjugates as immobilized antigens (sensitivity at 50% B/B0 was 0.4-0.6 micrograms/l for terbutryn). Formats with immobilized antibody and enzyme labelled AHA proved to be less sensitive (1.5 micrograms/l for terbutryn). The observed differences in sensitivity do not apparently result from structural effects of the carrier bound hapten since all conjugates were prepared with one form of the hapten, 2-aminohexylamino-atrazine, which was covalently linked via its amino function to the carriers or enzymes.

Synthesis of mono- and bifunctional peptide-dextran conjugates for the immobilization of peptide antigens on ELISA plates: properties and application.

Dextran has been used as a carrier molecule for the synthesis of monofunctional peptide-dextran conjugates. The immunodetection of such carrier immobilized peptides on ELISA plates was compared to that of peptides adsorbed directly to immunoplates. The main features observed with peptide-dextran conjugates were as follows: only small amounts of peptide (1-2 mg) were necessary for coupling via alpha- or epsilon-amino groups to NaIO4-activated dextran (4 mg); the coupling yield was up to 68%; an amino acid analysis of the conjugate enabled the amount of carrier immobilized peptide to be calculated; an estimated 15-17 peptides were bound per dextran molecule (MW 73,500); using a carbohydrate as carrier reduces the possibility of non-specific interactions because no hydrophobic or ionic sites and no protein-like epitopes exist on the carrier apart from the peptide ligand. It can be assumed that some peptide ligands provide the forces for an interaction with the plate surface whereas other remain free for the interaction with the antibody. Thus, the detection with monoclonal anti-peptide antibodies allowed peptide-dextran conjugates to be used at coating concentrations of 1-3 nM peptide, corresponding to 0.6-2.6 ng peptide-dextran per well. In contrast, concentrations of 150-500 nM were required for coating with peptides. The applicability of monofunctional peptide-dextran conjugates was demonstrated by investigating the titer and specificity of a polyclonal anti-peptide serum developed against human gastrointestinal glutathione peroxidase. The introduction of biotin as a second ligand of the dextran conjugate permitted its capture on streptavidin coated plates. This synthesis of bifunctional peptide-biotin-dextran conjugates opens up additional possibilities for applications.

A new peptide-affinity tag for the detection and affinity purification of recombinant proteins with a monoclonal antibody.

A monoclonal anti-peptide antibody (2E11) was raised against the synthetic peptide 38 (C-L-D-K-S-G-L-P-S-D-R-F-F-A) representing a part of the variable region of the Vbeta 6.2 T-cell receptor. This mAb (IgG(1), kappa light chain) bound very specifically to peptide 38 as shown by ELISA but did not recognize the corresponding native Vbeta 6.2 T-cell receptor on T-cells. For epitope analysis, overlapping peptides of 4-10 amino acids in length corresponding to the sequence of peptide 38 were synthesized and assayed by SPOT synthesis on cellulose sheets. The shortest peptide recognized was L-P-S-D-R. The specificity of mAb 2E11 was examined with 100 different peptides comprising other parts of the different variable Vbeta domains of the human T-cell receptor that do not include the epitope region L-P-S-D-R. None of these peptides were recognized. The chemical synthesis of a peptide with the sequence L-P-S-D-R on Sepharose beads allowed to efficiently purify the mAb 2E11 in a single step by affinity chromatography. An equilibrium binding constant of 4.9x10(6) l/mol was determined for mAb 2E11 by using rhodamine-green-labelled peptide 38 in fluorescence correlation spectroscopy. In order to demonstrate that peptide 38 can be used as an affinity-tag, it was fused to the carboxyl-terminus of interferon regulatory factor-1 (IRF-1). It could be shown that in vitro translated peptide 38 tagged IRF-1 was immunoprecipitated by the mAb 2E11 and that the fusion protein could be purified by immunoaffinity chromatography. Additionally peptide 38 was fused to the amino-terminus of the Taq polymerase. This recombinant protein was expressed in E. coli and specifically detected in a Dot blot and Western blot using mAb 2E11.

Increased cerebral blood flow in depressed patients responding to electroconvulsive therapy.

UNLABELLED: Considerable data support the existence of impaired regional cerebral blood flow (rCBF) in major depression. We compare rCBF in depressed patients before and after electroconvulsive therapy (ECT) to define whether the impairment is a "state"-related property or a trait phenomenon. METHODS: Twenty patients with a major depressive disorder were studied by 99mTc-HMPAO brain SPECT, 2-4 days before and 5-8 days after a course of ECT. Three transaxial brain slices delineating anatomically defined regions of interest at approximately 4, 6 and 7 cm above the orbitomeatal line were used, with the average number of counts for each region of interest normalized to the area of maximal cerebellar uptake. RESULTS: Technetium-99m-HMPAO uptake significantly increased in patients who responded to ECT but remained unchanged in patients who did not respond to the treatment (response defined as a reduction of at least 60% on the Hamilton Depression Rating Scale). An inverse correlation was observed between severity of depression and HMPAO uptake, and clinical improvement was positively correlated with the increase in tracer uptake. CONCLUSIONS: These findings imply that reduced rCBF in depression, as reflected in brain 99mTc-HMPAO uptake, is a "state"-related property and is reversible by successful treatment. Technetium-99m-HMPAO uptake may serve as an objective state marker for depression, an an indicator of the severity of depression and as an objective means of evaluating response to treatment.

Transient neurological events during dipyridamole stress test: an arterial steal phenomenon?

Dipyridamole-associated adverse neurological side effects have not been extensively described. We present two cases of dipyridamole-associated transient motor neurological events with no evidence of residual neurological deficits detected clinically or by head CT. The patients showed no evidence of significant extracranial (internal carotid) artery disease. We propose the presence of a regional cerebral perfusion disturbance due to an intracranial vascular steal phenomenon as the mechanism for the above side effects of dipyridamole.

Intrinsic dual-energy processing of myocardial perfusion images.

UNLABELLED: We have developed a software-based method for processing dual-energy 201TI SPECT emission projection data with the goal of calculating a spatially dependent index of the local impact of gamma-ray attenuation. We refer to this method as intrinsic dual-energy processing (IDEP). METHODS: IDEP exploits the differential attenuation of lower energy emissions (69-83 keV) and higher energy emissions (167 keV) resulting from the decay of 201TI to characterize the relative degree of low-energy gamma-ray attenuation throughout the myocardium. In particular, IDEP can be used to estimate the relative probability that a low-energy gamma-ray emitted from a particular region of the myocardium is detected during the acquisition of SPECT projection data. Studies on phantoms and healthy human volunteers were performed to determine whether the IDEP method yielded detection probability images with systematic structure visible above the noise of these images and whether the systematic structure in the detection probability images could be rationalized physically. In patient studies, the relative regional detection probabilities were applied qualitatively to determine the likely effects of attenuation on the distribution of mapped photon emissions. RESULTS: Measurements of the detection probability in uniform phantoms showed excellent agreement with those obtained from computer simulations for both 180 degrees and 360 degrees acquisitions. Additional simulations with digital phantoms showed good correlation between IDEP-estimated detection probabilities and calculated detection probabilities. In patient studies, the IDEP-derived detection probability maps showed qualitative agreement with known nonuniform attenuation characteristics of the human thorax. When IDEP data were integrated with the findings on the emission scan, the correlation with coronary anatomy (known in 6 patients and hypothesized on the basis of clinical and electrocardiographic parameters in 5 patients) was improved compared with evaluating the mapped emission image alone. CONCLUSION: The IDEP method has the potential to characterize the attenuation properties of an object without use of a separate transmission scan. Coupled with the emission data, it may aid coronary diagnosis.

Cytotoxic effect of atrazine on murine B-lymphocytes in vitro.

When a stable cell line from mouse connective tissue was used to test the cytotoxicity of atrazine no significant influence on cell proliferation was observed at concentrations of 0.003-200 microM. However, freshly isolated mouse spleen cells reacted very sensitively to the presence of atrazine. During the course of the experiment the spleen cells were pretreated with atrazine (0.05-5 microM) before the proliferation of B-lymphocytes was stimulated with LPS. A reduced number of developing B-blasts was observed in the atrazine pretreated cultures. The B-blasts were fused with myeloma cells and cultivated in HAT (hypoxantine, aminopterin,thymidine)-medium for hybridoma selection. The number of surviving clones was significantly lower on atrazine pretreatment.

A measurement of pulmonary blood volume increase during systole in humans.

Pulmonary blood volume increase during systole was measured in patients by analysing chest images obtained during ECG gated radionuclide angiography examination. The difference in the total radiation counts between systole and diastole in regions of interest, which included the lungs and the left ventricle, was measured and the relative pulmonary systolic blood volume increase (SBVI)-the ratio between the pulmonary SBVI and the cardiac stroke volume-was calculated. The relative pulmonary SBVI, which is a measure for the compliance of the pulmonary blood vessels, was found to be 0.26-0.85, and the average value was 0.57 +/- 0.15. The relative pulmonary SBVI was inversely correlated with the patient age (r = 0.27, p < 0.05) and with the left ventricular ejection fraction and stroke volume (r = 0.36, p < 0.01) due to decreased arterial compliance for older patients and for increased pulmonary blood volume respectively. The correlation coefficients were not high, indicating that the compliance of the patients is determined mainly by other individual factors. Radionuclide plethysmography enables qualitative assessment of pulmonary arterial compliance.

Value of dobutamine/nitrate radionuclide angiography in predicting revascularization effects on ventricular function.

OBJECTIVE: Dobutamine and Nitrate Radionuclide Angiography (RNA) may help to distinguish viable from nonviable myocardium and to predict revascularization effects on LV function. SETTING: Ambulatory. EXPERIMENTAL DESIGN: Prospective. PATIENTS AND METHODS: We studied 12 patients (11 male, 1 female; mean age 56 +/- 10 years) with three-vessel disease, severe LV dysfunction, and prior MI for a mean follow-up time of 10 +/- 3.4 months. Thallium-201 scintigraphy favored potential viability in all patients: inducible ischemia with satisfactory perfusion in more than one coronary arterial territory. Global and regional LVEF's were calculated from preoperative RNA (baseline rest study and with continuous dobutamine infusion with gradual rate increase plus oral nitrates) and postoperative RNA at 1 and 6 months. RESULTS: There was no operative mortality, but two late deaths occurred. Symptomatically, most patients showed improvement. Global LVEF increased during dobutamine and nitrates preoperatively (p < 0.01), but not at 1 and 6 months postoperatively (without pharmacological intervention). Mean LVED volume was not significantly changed postoperatively. Regional EF improvement was found in 4 out of 9 LV wall segments under dobutamine and nitrates, and this increase persisted postoperatively at 1 and 6 months. CONCLUSIONS: Mild but significant increase in global LVEF during dobutamine and nitrates administration is not predictive of postoperative LVEF improvement. However, regional EF improvement appears to be predictive of post-revascularization LV functional improvement.

Diagnostic dilemmas in parathyroid scintigraphy.

PURPOSE: Tc-99m MIBI scintigraphy can be useful for preoperative localization of parathyroid adenomas, despite its limited sensitivity. To refine interpretation, the authors conducted prospective reviews of the scintigraphic studies of patients with concomitant thyroid nodular disease and revised the false-negative and false-positive results. MATERIALS AND METHODS: Seventy-seven patients with primary hyperparathyroidism, associated with a solitary parathyroid adenoma, underwent dual-phase MIBI scintigraphy with subsequent injection of Tc-99m pertechnetate before surgery. The false-negative and false-positive scintigraphic findings were identified and revised. RESULTS: After surgery, revision of 19 false-negative scans of the total 77 studies led to the demonstration of an adenoma in seven patients. In five of the seven studies, the adenoma had been overlooked as a result of rapid tracer washout. Two additional small focal lesions would have also been identified if subtraction had been used for differences in contour of the MIBI and pertechnetate images. Four studies were interpreted falsely as abnormal, leading to a positive predictive value of 91%; three were associated with thyroid nodular disease. CONCLUSIONS: Differential washout of MIBI from thyroid and parathyroid tissue is not universal. When MIBI is washed out rapidly from parathyroid adenomas, subtraction of a thyroid image should be performed and differences in contour delineated to localize the adenoma accurately. Some, but not all, thyroid lesions account for the false-positive findings.

Liver hemangioma. A perioperative Tc-99m RBC SPECT correlation.

Scintigraphic diagnosis of a liver hemangioma may be difficult when its size approaches the spatial resolution of a gamma camera, and it may not be visualized on a Tc-99m tin-colloid liver SPECT study. The contribution of Tc-99m labeled RBC scintigraphy is shown in a patient with a liver hemangioma discovered during laparoscopic cholecystectomy for chronic cholecystitis. Despite negative CT, US, and Tc-99m tin-colloid SPECT of the liver, Tc-99m labeled RBC SPECT disclosed a focus of increased activity consistent with the hemangioma.

Tc-99m mercaptoacetyltriglycine clearance: comparison of camera-assisted methods.

PURPOSE: To compare the accuracy of three camera-assisted methods for the measurement of Tc-99m mercaptoacetyltriglycine (MAG3) clearance. MATERIALS AND METHODS: MAG3 renal scintigraphy was performed in 21 adults with different degrees of renal function. Posterior views were obtained that included the heart and the kidneys in the camera field of view. The syringe for injection was imaged before and after injection. Blood samples were drawn 24 and 43 minutes after injection and plasma radioactivity was measured. Three camera-assisted techniques to measure MAG3 clearance were tested: 1) Biexponential fitting of the left ventricular curve, normalized to plasma activity at 24 minutes; 2) calculation of clearance by relating the integral of the plasma curve (normalized to plasma activity) to the kidney activity using the Rutland-Patlak space method; and 3) a regression equation measuring clearance from the percentage of the injected dose accumulating in the kidneys during the 1- to 2.5-minute period. The camera-assisted clearances were compared with the single-sample MAG3 clearances calculated using the Russell equation. Linear regression analysis was used to measure the correlation between the camera-based methods and the single-sample techniques. RESULTS: Correlation with r > 0.900 was found for all three techniques. The difference in correlation coefficients between the three methods was not significant; however, the regression line of method 3 was significantly closer to the line of identity (P = 0.005). CONCLUSION: Method 3 most closely fits the line of identity and is probably the most practical because no blood sample is needed.

[Myocardial protection during ischemic cardiac arrest. Hemodynamic study of the effects of allopurinol in a cardioplegic solution]. / Protection myocardique pendant l'arrêt cardique ischémique. Etude hémodynamique des effets de l'allopurinol dans un soluté cardioplégique.

The effects of the addition of 20 mg sodium allopurinate to a litre of ionic cardioplegic solution were studied. The experimental model was the working isolated perfused rat heart in oxygenated Krebs-Henseleit solution. The cardiac outputs were compared after 1 hour of ischaemia with three different methods of myocardial protection; group I: protection by hypothermia at 4 degrees C, group II: protection by ionic cardioplegic solution A, group III: by ionic cardioplegic solution B (with 20 mg Allopurinol). The results were expressed in percentage of the preischemic cardiac output. The best results were obtained in group III, especially after 15 minutes recovery (80.7 +/- 4.5 p. 100 at 15 minutes, 90.6 +/- 3.1 p. 100 at 30 minutes., 88.3 +/- 4.6 p. 100 at 1 hour). The results in group II were significantly better than in group I after 10 minutes recovery. Myocardial protection bu ionic cardioplegia associated with hypothermia at 4 degrees C gives a better post ischaemic recovery than hypothermia alone. When allopurinol is added to the cardioplegic solution, the protection is increased. These effects are similar to the haemodynamic effects of allopurinol on ischaemic myocardium together with the possible limitation of experimental myocardial infarction with regression of the oedema that has been observed with this drug.

Biomechanical analysis of the effect of changing patient-handling technique.

The objective of the study was to assess the changes in the mechanical load on the low-back when shifting from a self-chosen to a recommended patient-handling technique. Nine female health care workers without formal education in patient-handling carried out 8 different tasks involving moving, turning and lifting situations. By means of a dynamic 3D biomechanical model of the lower part of the body, peak torque, compression and shear forces at the L4/L5 joint were compared using the two different patient-handling techniques. In 5 of the 8 tasks, a significant reduction was observed in spinal loading. Application of the recommended technique decreased the compression value significantly for all tasks with a mean value above 3000 N. For the two tasks with the highest compression values when using the self-chosen technique (4223, 4446 N), the loading was reduced with 36% and 25%, respectively. If the principles behind the recommended technique are implemented and maintained, a decrease in the risk of low-back disorders during patient-handling should thus be expected.

MATRIX HCV--a newly developed semiautomated dot-immunoassay.

MATRIX HCV, a newly developed semiautomated assay for the detection of antibodies against individual antigens of the hepatitis C virus, was evaluated on blood donor specimens and compared with the results of another recombinant immuno-blot assay. 399 samples derived from a low-prevalence population revealed an assay specificity of 99.2% for MATRIX HCV. Investigation of 133 samples with repeat reactive screening results showed that the MATRIX HCV assay had a higher ability to discriminate between a clear reactive and nonreactive result compared to the recombinant immuno-blot assay.