125I-labelling = 125I-Labelling.

Erythropoietin, the hormone that regulates erythropoiesis in mammals, was 125I-labelled by using the catalytic properties of lactoperoxidase and the H2O2-generating properties of glucose oxidase. This methodology, both rapid and simple, not only produced hormone preparations with high specific radioactivity but also did not substantially alter the biological integrity of erythropoietin when it was assayed in vivo.     

CA 125 in biological fluids

CA 125 is not a specific tumor marker, and is synthesized by normal and malignant cells of different origin (mainly in tissues derived from the müllerian epithelia) in a similar proportion. Abnormal CA 125 levels may be found in fluids of different origin (ascites, pleura, pericardium, amniotic fluid, cyst fluid, bronchoalveolar fluid, etc.) and in serum from patients with these fluids. Differences in serum CA 125 found in malignant or benign diseases may be related to the number of cells that synthesize the marker, and are highly dependent on the access to serum, where the marker is normally determined. Moreover, CA 125 is a very good tumor marker in ovarian and lung cancer. The sensitivity of CA 125 in ovarian cancer is related to stage (40-95%), histological type (lower levels in mucinous adenocarcinoma), and the marker is useful in the early detection of recurrence (sensitivity 80%) and in therapy monitoring. It's sensitivity in lung cancer is lower than in ovarian cancer, 39% in locoregional malignancies and 69% in metastatic disease, but clearly related to stage and histology (mainly in adenocarcinomas and large cell lung cancer) and it is useful in prognosis and disease monitoring.     

Somatostatin radioimmunoassay with 125I-Nalpha-tyrosyl-somatostatin

Nalpha-Tyrosyl-somatostatin was synthesized and proved to be homogeneous. Radioiodination of this tyrosine-containing somatostatin analogue by either the lactoperoxidase method or the chloramine T method led to the formation of crude iodinated compound, which was purified by ion exchange chromatography on CM-Sephadex C-25 using a linear ammonium acetate buffer gradient. This purification process was found to be satisfactorily reproducible and suitable for the preparation of 125I-Nalpha-tyrosyl-somatostatin. Using the purified 125I-somatostatin analogue, radioimmunoassay for somatostatin was performed and the assay system was proved to be sensitive and specific for somatostatin. Immunoassays of hot-water extracts of porcine and tupaia brain, pancreas, stomach and various regions of the intestine in the system revealed that those tissues contained immunoreactive somatostatin at various concentrations. Of the results, it was remarkable that somatostatin immunoreactivity was found in the ileum, middle colon and rectum in both animals, although the concentration were lower when compared with those in the stomach, duodenum and jejunum.     

Ligand blotting with 125I-fluoresceinamine-heparin

A highly sensitive method for ligand blotting with heparin has been developed. This ligand-blotting method is successful largely due to the ability to prepare heparin derivatives of high radiospecific activity. Heparin was modified with fluoresceinamine according to the method of C.G. Glabe, P.K. Harty, and S.D. Rosen [1983) Anal. Biochem. 130, 287-294), and this fluoresceinamine-derivatized heparin can be radioiodinated to a specific activity of 100,000 cmp/ng of uronic acid. This is a 500-fold increase in specific activity over Bolton-Hunter-modified heparin, as prepared by A.D. Cardin, K.R. Witt, and R.L. Jackson [1984) Anal. Biochem. 137, 368-373). 125I-Fluoresceinamine-derivatized heparin retains its ability to interact specifically with heparin-binding proteins such as human protease nexin-I and antithrombin III. 125I-Fluoresceinamine-derivatized heparin can be used to visualize and quantify heparin binding proteins on nitrocellulose. Protease nexin-I can be visualized at the nanogram level. In addition, ligand blotting with 125I-fluoresceinamine heparin can be combined with Cleveland digestion (D.W. Cleveland, S. Fisher, M.W. Kirschner, and U.K. Laemmli (1977) J. Biol. Chem. 252, 1102-1106) in order to identify heparin binding fragments of proteins with heparin binding domains.     

Quantitative 125J-Autoradiographie einzelner Zellen

Zusammenfassung Das nach der Energie seiner -Zerfälle zwischen Tritium und Kohlenstoff-14 liegende Isotop ¹²⁵J wurde auf seine Eignung zur quantitativen Autoradiographie geprüft. Absorption und Geometrie-Faktoren der radioaktiven Strahlung wurden untersucht. Hieraus ließen sich geeignete Meßbedingungen entwickeln. Durch gleichzeitige Exposition von radioaktiven Referenzquellen können absolute Radioaktivitätsmengen ermittelt werden. Als Referenzquellen sind membranmarkierte Standardzellen geeignet, die den physikalischen Eigenschaften des Isotopes Rechnung tragen. Hierzu wurden enzymatisch radiojodinierte Schaferythrozyten auf ihre Eignung geprüft. Die absolute Zahl von antigenen Stoffen auf den Oberflächen einzelner Zellen erhält man, wenn man die spezifische Aktivität der markierten Antikörpermoleküle bestimmt und die Silberkorndichte der zu untersuchenden Zellen und der Standardzellen mißt. Die Radioaktivität pro Standardzelle wird in herkömmlicher Weise ermittelt.Die neue Methode wurde zur Quantifizierung membrangebundener Immunglobulinmoleküle vom IgG-Typ auf einzelnen menschlichen Lymphozyten angewendet. Hierbei ist die Ermittlung einer immunologischen Sättigung des markierten Antikörpers wesentlich. Auf Lymphozyten von Normalpersonen und von chronisch-lymphatischen Leukämiepatienten konnten sehr unterschiedliche absolute Immunglobulinmengen bestimmt werden.

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Quantitative ¹²⁵I-autoradiography of individual cells

Summary Iodine 125, an emitter of -radiation with an energy lying between that of tritium and carbon-14, is investigated for its applicability in quantitative autoradiography. Absorption and geometric factors of radiation are elucidated. From this, appropriate measuring conditions are derived. The simultaneous exposure of radioactive standard sources permits the evaluation of absolute amounts of radioactivity. Standard cells with labelled membranes are a suitable source of reference taking into account the physical properties of the isotope. Sheep red blood cells are examined for their suitability as standard cells after enzymatic radioiodination. The absolute number of antigenic substances on the surface of single cells is obtained by determining the specific activity of the labelled antibody molecules, and by measuring the silver grain densities of the cells under investigation and of the standard cells. The radioactivity per standard cell can be assessed by conventional procedures.The new method is applied to the quantification of membrane-bound immunoglobulin molecules of the IgG-type on single human lymphocytes. The determination of an immunologic saturation of the labelled antibody is essential for this purpose. On the lymphocytes of a normal person and of a patient with chronic lymphatic leukaemia quite different amounts of immunoglobulins have been evaluated.

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Assoziation mit EURATOM 031-64 I BIAD.     

125I-UdR Induced Division Delay

The interaction of Ca++ with acidic phospholipids in black lipid films and lipid bilayers formed from two monolayers was studied by measuring their physical stability and conductance. It was found that the addition of CaCl2 to only one side of lipid bilayers formed from phosphatidylserine or cardiolipin does not appreciably change these parameters. In contrast, black films are unstable to the asymmetric addition of CaCl2. Therefore, the destabilizing effect of Ca++ cannot be attributed to a surface charge difference. The only variation in composition between both bilayer membranes, namely the solvent content of the bilayer, seems to be responsible for the distinctive effect of Ca++. A tentative explanation is presented.     

Superior immunoreactivity of 125I (Des-Tyr-betaAla)-secretin with rabbit anti-secretin sera compared to 125I-secretin and 125 I-6-Tyrosyl secretin.

A secretin analogue in which the normal amino acid sequence had been elongated by a (Des-Tyr-betaAla)-residue was studied as tracer for secretin radioimmunoassay. 125I-(DATA)-secretin exhibited superior immunoreactivity with several rabbit anti-secretin sera compared to 125I-6-Tyr-secretin and also to secretin iodinated at its N-terminal histidyl residue. This may be due, at least in part, to higher conformational integrity of the secretin moiety in the 125I-(DATA)-secretin molecule. Thus, at present, 125I-(DATA)-secretin appears to be most suitable as tracer for sensitive secretin radioimmunoassay.