Veikko Näntö

Immunoassay by time-resolved electrogenerated luminescence

Abstract An immunoanalytical method based on electrogenerated luminescence was developed in which the labelling compound is the terbium chelate of 2,6-bis[ N,N -bis(carboxymethyl)aminomethyl]-4-benzoylphenol which is linked by a thioureido group to the antibody. The sandwich complex of the labelled antibody, antigen and antibody immobilized on the surface of an aluminium electrode is excited by an electrical pulse and, after a short delay from the end of the pulse, light emission is measured. The electroluminescence immunoassay is exemplified by the heterogeneous and homogeneous assays of human pancreatic phospholipase A 2 , for which both assay methods give a reasonably good linearity in the range 10–300 ng ml −1 .

Competitive solid-phase immunoassay of testosterone using time-resolved fluorescence

A competitive solid‐phase immunoassay for the determination of testosterone in serum samples using time‐resolved fluorescence is described. The solid phase is a testosterone‐3‐(O‐carboxymethyl)‐oxime‐ovalbumin conjugate coated to polystyrene microtiter strips. Europium‐labelled polyclonal and monoclonal antibodies against testosterone‐3‐(O‐carboxymethyl)‐oxime‐bovine serum albumin were compared. Their behavior was quite similar although the polyclonal antibody was more sensitive, giving a detection limit of 15 fmol testosterone per assay. Correlation with RIA was very good (r = 0.982 and y = −0.150 + 0.969x).

High concentrations of chromium in lung tissue from lung cancer patients.

The pulmonary chromium content was determined by plasma atomic emission spectrometer (DCP‐AES) from 53 lung cancer and 43 control patients, and compared with smoking habits, severity of emphysema and occupational history. The chromium content from the lung cancer patients was higher than that from the smoking (P < 0.025) or nonsmoking control patients (6.4 ± 4.3, 4.0 ± 4.0, and 2.2 ± 0.6 μg/g dry weight, respectively). A positive correlation between the pulmonary chromium and smoking time (P < 0.025) and the severity of emphysema (P < 0.001) was found in the control but not in the cancer patients. The difference in the pulmonary chromium content was greatest between those lung cancer and control patients who were light smokers or had mild emphysema. This group of lung cancer patients included subjects with occupational exposure to chromium. The possibility of occupational cancer should be considered especially with light smokers. The grade of emphysema and metals such as chromium accumulating from tobacco could serve as objective indicators of smoking.

A PET Study on the Acute Effect of Ethanol on Striatal D2 Dopamine Receptors with [11C]Raclopride in Healthy Males

The effect of acute oral ethanol intake (1·0 g/kg) on cerebral D2‐receptors ([11C]raclopride binding) was studied in seven healthy volunteers, using water and alcohol in two separate 59‐min PET sessions. In the alcohol experiments, the blood ethanol concentration at the beginning of the imaging was 26·4±3·8 mmol/l and remained stable during the PET session. Ethanol was not found to influence binding of [11C]raclopride to the dopamine D2 receptors in the human striatum, as indicated by the unchanged ratio Bmax/Kd of the whole (left and right) striatum. The Tmax values of the control and ethanol experiments did not differ in the whole striatum, but the right to left difference of striatal Tmax was turned from negative to positive by ethanol (P=0·02). However, the difference between hemispheres in Bmax/Kd was not significantly altered by ethanol intake. There was considerable interindividual variation in the response of all the above parameters to acute ethanol exposure. According to the present results, the acute effects of peroral ethanol exposure on striatal D2 receptor binding potential are of relatively small magnitude in man. However, the changes in Tmax suggest that ethanol may influence the right–left difference of [11C]raclopride binding.

Serum trace element concentrations and iron metabolism in allogeneic bone marrow transplant recipients.

Serum trace element concentrations, parameters of iron metabolism and serum protein concentrations were investigated in thirteen adult recipients of bone-marrow transplants receiving total parenteral nutrition. Six of the patients died during the four weeks follow-up. Serum zinc concentrations were initially low but increased during the treatment. They also tended to be lower in dying patients than in survivors. Concentrations of serum copper and selenium remained unaltered. Serum iron started to increase during the preconditioning and remained raised for three weeks. No significant changes occurred in serum transferrin levels. Transferrin saturation increased during the preconditioning and started to return to normal after day +14. Serum ferritin was greatly raised from the start and increased further during the procedure. Routine trace element substitution seemed to be sufficient during total parenteral nutrition with the possible exception of zinc. A return to normal transferrin saturation after day +14 may be an early favourable sign that the graft is taking and hematopoietic recovery commencing.

Effect of platelet count on serum and plasma potassium: evaluation using database information from two hospitals

The availability of retrospective data from potassium (K+) analyses from two hospitals, one using serum and the other plasma for electrolyte measurements, offered us the possibility to investigate the effect of blood platelet count on serum and plasma K+ concentrations. A weak correlation between plasma K+ and platelet count was observed. The in vitro increase of serum K+ in proportion to the platelet count has clinical significance in conditions, where it may impede the detection of an underlying true K+ disorder. Nomograms and correction factors, based on the correlation between platelet count and serum K+, have been suggested also in some recent reports. In the present study unselected routine patient data was used as source data. The effect of platelet count on the concentration of K+ in serum was lower than reported in previous studies, as indicated by the regression analysis. An increase of 1000 x 10(9)/l in the blood platelet count would cause an increase of about 0.7 mmol/l in the serum K+ concentration (p < 0.0001, r = 0.155). The weak correlation between platelet count and serum K+ does not support the application of platelet-count-based correction of serum K+ level in thrombocytosis. The laboratory should notify the clinician of the significance of the in vitro increase of K+ caused by increased platelet count. K+ should be measured from plasma in such cases.

Detection of Philadelphia chromosome using PCR and europium-labeled DNA probes.

More than 95% of the patients with chronic myelogenous leukemia (CML) carry translocations between protooncogene abl of chromosome 9 and bcr gene of chromosome 22, resulting in the Philadelphia chromosome (Ph1). After allogeneic bone marrow transplantation (BMT) it is important to detect possible residual malignant cells in CML patients. A new sensitive hybridization method combined with polymerase chain reaction (PCR), based on the detection of the europium (Eu3+) label by time-resolved fluorescence, was applied for the detection of Ph1 chromosome. Total RNA from 10(6) peripheral blood leukocytes was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction. After cDNA synthesis by reverse transcriptase, the PCR amplification (30 cycles) was carried out. In the detection phase two oligonucleotide probes were used in the hybridization reaction, one biotinylated (bcr gene, exon 2) and one (abl gene) labeled with Eu3+. The hybrids were collected in a streptavidin-coated microtitration well and the bound Eu3+ was measured in a time-resolved fluorometer. To assess the sensitivity of the method, different numbers of CML cell line K562 cells were mixed with 10(5) apparently normal human leukocytes. Five K562 cells/10(5) leukocytes could be detected. Six patients with CML confirmed by clinical and cytogenetic criteria were studied. Three of the patients underwent an allogeneic BTM 6-18 months before the investigation and all of them were Ph1-negative. The other three patients who were nontransplanted were positive as expected.

The Effect of Postoperative Radiotherapy on Leukocyte Zinc, Serum Trace Elements and Nutritional Status of Breast Cancer Patients

Mononuclear (MNC) and polymorphonuclear cell (PMNC) zinc content was determined together with serum zinc, copper, selenium and iron concentrations in 24 operable breast cancer patients during and after postoperative radiotherapy. Anthropometric and biochemical indices of nutritional status were measured as background data. The measurements were carried out in the years 1987-1988. Nine patients used unconventional multivitamin or trace element preparations. A steady but statistically insignificant decrease in PMNC zinc was seen during treatment. No changes occurred in MNC zinc. Serum copper levels increased in five patients possibly due to tamoxifen treatment, but no other alterations occurred in serum trace element levels. Appetite was well maintained and nutritional status remained unaltered. Postoperative radiotherapy for breast carcinoma had thus no effect on either trace element or nutritional status. Patient-initiated alternative treatments did not significantly affect their trace element levels. This was probably due to small supplementation doses or irregular use of the preparations.

Investigations of dye binding in the sequential staining of mucosaccharides by Alcian Yellow-Alcian Blue. 1. Histochemical experiments on different animal and human mucosaccharides

SynopsisIt may be assumed that, histochemically, carboxyl groups and sulphate half-ester groups of muco electrolyte concentration of the dye baths in the two steps of a sequential Alcian Yellow-Alcian Blue method. In the present study the specificity and reliability of this method has been investigated.When the staining conditions were the same in both steps, the second dye (Alcian Blue) was found to stain mucosubstances in spite of the prior staining with Alcian Yellow. Binding of Alcian Blue was observed in all but very dilute Alcian Blue solutions. The degree of Alcian Blue binding depended on the dye concentration and staining time of the second step (Alcian Blue), and it varied widely for different mucosubstances. Although an incomplete saturation of anionic groups with dye molecules in the first step cannot be completely excluded, it is thought that Alcian Blue displaces Alcian Yellow from the carboxyl and sulphate groups of mucosubstances in tissue sections.It seems that the sequential Alcian Yellow-Alcian Blue method, under the conditions investigated, does not provide a reliable means for differentiating carboxyl and sulphate groups of mucosbstances in tissue sections simultaneously, because the second dye obviously is capable of displacing the first dye from sulphate groups. However, it is possible to distinguish non-sulphated acid mucosaccharides from sulphated mucosaccharides.

The effect of intermaxillary fixation on leukocyte zinc, serum trace elements and nutritional status of patients undergoing maxillofacial surgery

Mononuclear cell (MNC), polymorphonuclear cell (PMNC) and serum zinc levels were studied in 17 oral surgical patients with intermaxillary fixation. Serum copper, iron, selenium and bromide concentrations were also measured together with common indices of nutritional status. Nine patients received nutritional counselling. Eight patients had, in addition, oral supplementation with a commercial formula. No changes in intracellular or serum zinc levels were seen during the study period. A statistically significant decrease was seen in mean body weight with subsequent changes in anthropometry. Maximal mean weight loss was 6.0 +/- 3.8% in control group and 3.8 +/- 2.7% in supplemental group. The impaired oral intake due to intermaxillary fixation does not interfere significantly with zinc status as estimated by MNC, PMNC or serum zinc levels. The reduction in body weight and anthropometric indices in the relatively short fixation period may be clinically significant in some patients. Supplementation with a commercial formula helps to maintain the nutritional status of these patients.