Martin Vanderlaan

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.

One-step purification of mouse monoclonal antibodies from ascites fluid by hydroxylapatite chromatography

A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or IgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ascites proteins, as determined by SDS-polyacrylamide gel electrophoresis. Hydroxylapatite chromatography can also separate monoclonal IgG antibodies from contaminating IgG antibodies found in ascites fluid of animals that have been immunosuppressed prior to ascites induction. Furthermore, the evidence presented here suggests that some hybridomas of SP2/0 origin synthesize an extraneous light chain resulting in the secretion of hybrid antibody molecules.

Monoclonal antibodies specific for the M- and N-forms of human glycophorin A.

Four mouse monoclonal antibodies directed against the red cell membrane protein glycophorin A have been isolated and characterized. They are produced by hybridomas derived from SP2/0 myeloma cells and spleen cells from Biozzi mice immunized with a mixture of human erythrocytes from homozygous blood group M and N individuals. These antibodies recognize and bind to purified glycophorin A and to glycophorin on the red cell surface. All are of the IgGl, kappa light chain subclass and bind to determinants presented on the 39 amino acid, trypsin-sensitive, N-terminal peptide of glycophorin A. Three display differential specificities for the two allelic forms of glycophorin A; two are exquisitely specific for the M-form and one preferentially binds the N-form. Treatment of red cells with neuraminidase, which removes N-acetylneuraminic acid from glycophorin A, abolishes the binding of these three antibodies. The binding of the N-specific antibody is also sensitive to modification of the amino-terminal residue of the antigen. The fourth antibody binds equally well to both the M- and N-forms as well as to neuraminidase-treated red cells; thus it recognizes a public, N-acetylneuraminic acid independent glycophorin A determinant.

γ-Glutamyltranspeptidase: A tumour cell marker with a pharmacological function

SummaryThe distribution of γ-glutamyltranspeptidase activity (γGT) in adult, foetal, and neoplastic tissues in rats and humans is reviewed. The normal adult kidney, pancreas, and jejunum are high in this activity. Around the time of birth, γGT activity increases transiently in the liver, colon, and skin; many tumours of these organs often have elevated activity as well. Although this activity in tumour tissue may reflect a general expression of onco-foetal genes, subpopulations of cells having abnormally high γGT may have a selective growth advantage over their normal counterparts. We explore this latter possibility by considering the pharmacological function this enzyme may play. γGT may facilitate the detoxification of electrophiles by glutathione conjugation, so that cells can survive in an otherwise toxic environment. This growth advantage may be particularly important to the development of liver tumours in rats during chemical carcinogenesis.

Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.

Chapter 21 Using Monoclonal Antibodies in Bromodeoxyuridine—DNA Analysis

Publisher Summary The utility of bromodeoxyuridine (BrdUrd) as a marker for cell cycle traverse studies has been substantially increased by the introduction of monoclonal antibodies (mAb) against BrdUrd incorporated into cellular DNA. These antibodies are useful as immunological reagents to stain cells containing BrdUrd fluorescently so that the intensity of fluorescence is proportional to the amount of incorporated BrdUrd. The intensity of fluorescence is great enough to permit easy microscopic or flow cytometric (FCM) analysis of BrdUrd incorporation. The cytokinetic utility of the BrdUrd labeling has been further increased by the technique of simultaneous measurement of DNA content and the amount of incorporated BrdUrd. The BrdUrd–DNA assay is based on a procedure for simultaneously staining cells with dyes that fluoresce at different wavelengths. The procedure requires that the DNA is partially denatured to expose incorporated BrdUrd to a specific antibody. Denaturation is necessary because antibodies developed so far bind only to BrdUrd in single-stranded DNA. Green fluorescence from the fluorescein-conjugated antibody is a measure of BrdUrd incorporation.

A computer-based data analysis system for enzyme-linked immunosorbent assays☆

A computerized system is presented for automating the data collection, processing, and displaying tasks involved in enzyme-linked immunosorbent assays. This system uses a through-the-well absorbance reader of microtiter plates interfaced to a minicomputer running the UNIX operating system. Optical density in each well of a 96-well microtiter plate is recorded as a function of time for up to 10 time points. These data are automatically transmitted to the remote computer. The rate of product formation is then calculated for each well, and a battery of analysis, display, and comparison programs can then be used by the researcher for data presentation. Using the initial rate of reaction as the basis for quantifying enzyme-linked immunosorbent assays focuses on the catalytic property of the enzyme and allows a large dynamic range of the assay on any plate. These programs can be adapted to virtually any mini- or microcomputer with a graphics display or a plotting device. Assuming moderately powerful computing hardware, throughputs of 50 plates per day are easily achieved. The programs work equally well with peroxidase, beta-galactosidase, or alkaline phosphatase conjugated second antibodies, and with whole cell or soluble antigens.

Split-dose Recovery for Radiation-induced Tumours in Rat Skin

Tumour-related recovery in rat skin was estimated from the dependence of tumour yield on time between split doses of electron radiation. Tumour yield versus dose was established at nine dose points, and at three points the dose was split into two equal fractions spaced 0-25, 3-2 or 6-3 hours apart. After irradiation the rats were observed periodically for at least 64 weeks, and at death the tumours were examined histologically. The dependence of yield on dose for single doses was consistent with a quadratic function up to a peak yield at about 1600 rad. The effect of split doses on tumour yield depended on the position on the dose--response curve. At the lowest split dose, the yield declined with a half-time of about 1-8 hours. At the intermediate split dose, an initial increase was followed by a decline with a half-time of about 3-9 hours. At the highest split dose, the tumour yield increased with time between exposures. Fractionation-induced increases in tumour yield were explained as a sparing effect on cell lethality, whereas tumour-related recovery per se was indicated at the lower two doses.

The Effect of a 24-Hour Fractionation Interval on the Induction of Rat Skin Tumors by Electron Radiation

Tumor incidence and hair follicle lethality in rat skin were determined after various single and split doses of monoenergetic electrons produced by a Van de Graaff accelerator. In the split-dose gr...

Development of an immunoassay for chlorinated dioxins based on a monoclonal antibody and an enzyme linked immunosorbent assay (ELISA)

Abstract A set of monoclonal antibodies to dioxin have been developed. These form the bases for a competition enzyme-linked immunosorbent assay capable of detecting 0.5 ng of 2,3,7,8-tetrachlorodibenzodioxin.