Jarkko Eskola

Bile salt-dependent oxygenation of polyunsaturated phosphatidylcholines by soybean lipoxygenase-1

Abstract Aqueous solutions of purified bile salts and phosphatidylcholine (PC) were susceptible to oxygenation by soybean lipoxygenase-1. The bile salts could not be replaced by e.g., Tween 20 or 80, octylglucoside, Triton X-100, SDS or sulphobetaine based ampholytic detergents. The liberation of the fatty acid moieties of PC prior to oxygenation did not occur. The rate of oxygenation showed complex dependency on the concentration of bile salts whereas a linear increment with increasing PC concentrations could be demonstrated by using constant bile salt: fatty acid ratios. We expected, therefore, that a specific bile salt-PC complex instead of monomer phospholipid was required in the catalysis by lipoxygenase-1. Ultraviolet spectroscopic and luminometric monitoring of PC oxygenation supported this view: (i) The formation of conjugated dienes corresponded to only about 50% of the O 2 consumed as calculated on molar basis, (ii) Total absence of luminol chemiluminescence suggested that the other half of the consumed oxygen was not utilized to luminol oxidation, but either caused the cooxidation of bile salts in a coupled reaction with the oxygenation of PC or was incorporated into nonconjugated double bond structures of PC. Special attention was called to the applicability of soybean lipoxy genase-1 in the preparation of peroxidized phospholipid liposomes.

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 .

Heterogeneous and homogeneous electrochemiluminoimmunoassays of hTSH at disposable oxide-covered aluminum electrodes

Heterogeneous and homogeneous immunoassays of human thyroid stimulating hormone (hTSH) were developed on immunometric basis using aromatic Tb(III) chelates as electrochemiluminescent labels and varied types of disposable oxide-covered aluminum electrodes as the solid phase of the immunoassays. The long luminescence lifetime of the present labels allows the use of time-resolved electrochemiluminescence detection and provide the low detection limits of these labels and, thus, sensitive immunoassays. The primary antibody of immunometric immunoassays was coated upon aluminum oxide surface by physical absorption. In homogeneous immunoassays using 66l cell and 15 min incubation time, a linear calibration range of 0.25-324U/ml was obtained by applying only a single cathodic excitation pulse in the detection step of the assay.

Clara-cell secretory protein in preterm infants' tracheal aspirates correlates with maturity and increases in infection.

Clara‐cell secretory protein (CCSP), produced primarily by Clara cells in the conducting airways, is the most abundant soluble protein in pulmonary lavage fluid. CCSP is thought to be an immunosuppressive or anti‐inflammatory protein with protective functions in the respiratory tract against exaggerated inflammatory reactions. CCSP was measured in 98 tracheoalveolar fluid (TAF) samples from 24 preterm infants (gestational age, 27.9 ± 2.3 weeks, birth weight 1,020 ± 305 g) with respiratory distress syndrome during the first 2 postnatal weeks. The ratio of urea‐N in serum and in TAF was used to correct for dilution of TAF samples.

Application of a New Monoclonal Antibody for Time-resolved Fluoroimmunoassay of Human Pancreatic Phospholipase A2

A monoclonal antibody, designated 2E1, against human pancreatic phospholipase A2 was produced by hybridization of myeloma cells with spleen cells of immunized BALB/c mice. The hybridomas were screened for antibody production by time-resolved fluoroimmunoassay (TR-FIA). The antibody was found to belong to subclass I of murine IgG. The specificity of the antibody was confirmed by immunohistochemistry of pancreatic and other tissues, by immunoblotting of a crude aqueous extract of human pancreas and purified human pancreatic phospholipase A2 and by TR-FIA. A solid-phase time-resolved fluoroimmunoassay was developed by using the monoclonal anti-phospholipase A2 antibody as the catching antibody and a polyclonal sheep anti-phospholipase A2 antibody labelled with europium as the detecting antibody. The validity of the new TR-FIA of human pancreatic phospholipase A2 was confirmed by using it to measure the phospholipase A2 concentrations in serum samples from healthy subjects and from patients suffering from acute pancreatitis.

Immune functions in inflammatory bowel and coeliac diseases

Different immune functions of 10 patients with glutein‐sensitive enteropathy (GSE), 9 with Crohns disease (CD), 11 with ulcerative colitis (UC) and 13 healthy controls were characterized. The numbers of suppressor T cells in GSE were comparable to those of the controls; otherwise, the lymphocyte subpopulations were decreased in these bowel diseases. In the whole‐blood cultures, the lymphocyte proliferative responses to PHA were normal in the bowel diseases, but the responses to Con A were decreased in CD. In cultures with D‐penicillamine, the inhibition of the helper effect of CD patients was more pronounced in PHA‐stimulated cultures than in Con A‐stimulated cultures. The total Ig and IgA production did not markedly differ among the groups. PWM‐induced IgM secretion was significantly decreased in GSE, CD and UC, and IgG secretion in CD and UC, as compared to controls. In GSE, an increased Con A inducible suppressor cell activity was observed in the IgM production. Altogether, no clear‐cut immunological imbalance was detected in any of the bowel diseases; this in agreement with previous works. However, there are some differences in the regulatory cell balance among the patients with GSE, CD and UC. The determination of lymphocyte proliferative responses to PHA and Con A together with D‐penicillamine seems to provide a new immunological criterium for distinguishing between Chrohns disease and ulcerative colitis.

Inorganic pyrophosphatase-labelled enzyme immunoassay for β2-microglobulin

Abstract β2-Microglobulin was purified from human peritoneal dialysate by ultrafiltration, gel chromatography and DEAE-high performance chromatography. Anti-β2-monoclonal antibodies were developed in mice and a pair of the antibodies was utilized to develop an enzyme-linked immunosorbent assay (ELISA) kit for the analyte with utilizing a new enzyme label, inorganic pyrophosphatase (EC 3.6.1.1). The sensitivity of the assay was 0.6 μg/l (3×SD) and the assay was linear up to absorbance values of around 2.0. No hook effect occurred in any putative concentrations of β2-microglobulin in serum. The precision of the assay of one run varied within 5–7% CV and the interassay precision was 2.8–8.6% CV, while the recovery was 99.2±6.0%. Excellent correlation occurred against an established radioimmunoassay method. All the used reagents were storable for a minimum of 1 year at +4°C. It was decided that inorganic pyrophosphatase is a label of choice for ELISA.

RT-PCR from Eosinophil-Depleted Leukocytes without RNA Extraction: Cell Selection Using Streptavidin PCR Tubes

OBJECTIVES The major RNase activity of leukocytes has been attributed to eosinophil-derived neurotoxin EDN. Depletion of eosinophils enables RT-PCR from 10(5) leukocytes without RNA extraction. In this study we introduced streptavidin-coated PCR tube strips for the selection of eosinophil-free leukocytes for RT-PCR analysis. DESIGN AND METHODS Polypropylene 0.2 ml PCR tube strips were coated with streptavidin and biotinylated antibodies against cell surface antigens were attached to the tubes. CD7-positive T-lymphocytes, CD19-positive B-lymphocytes and CD16-positive cells (mainly neutrophils and monocytes) were positively selected by incubating of 1-2 x 10(5) leukocytes in the antibody-coated PCR tubes for 30 min at 23 degrees C. RESULTS The mean amount of cells bound into a tube was 31,500 (CV25%) T-cells and 8,600 (CV61%) B-cells from 12 blood samples, and 23,600 (CV22%) CD16+ cells from 17 samples. The influence of selected cell lysate on the RT-PCR analysis of Philadelphia chromosome (bcr/abl translocation) from 100 K562 cells was small: 78% (CV28%) of the leukocyte-free signal was obtained in the presence of CD16+ cells or 89% (CV15%) and 99% (CV11%) and in the presence of T-cells and B-cells, respectively. CONCLUSIONS These results suggest that through the introduction of eosinophil-free cell population into RT-PCR a reproducible method with reasonable leukocyte yield and avoiding RNA extraction was developed.