Biomedical subjects

M Vanderlaan

Publications and source records attributed to M Vanderlaan.

At least 37 records · Page 2Linked to original sources

PubMed1988

Monoclonal antibodies for the immunoassay of mutagenic compounds produced by cooking beef.

A family of 2-amino-N-methylimidazoazaarene (AIA) mutagens are produced in meats by cooking, and are of concern as potential carcinogens in the human diet. These are potent genotoxins in bacterial mutation assays, but are present only in trace quantities in cooked foods. Conventional analytical chemistry methods have allowed us to identify six members of the AIA class, but these methods are much too labor-intensive for us to quantify mutagens in large numbers of meat samples. To improve the assay of these mutagens we have developed six monoclonal antibodies (named IQ-1, IQ-2, AIA-1, AIA-2, AIA-4, and AIA-7) and demonstrated their utility in an immunoassay for cooked food mutagens. Each antibody has its own unique binding selectivity pattern; some are compound-specific, and some class-specific. A comparison was made between cooked beef extracts that differed 200-fold in mutagenic activity. The high-mutagen extract had significantly more material that was immunologically cross-reactive with the anti-AIA antibodies than did the low-mutagen extract. Taken as a set, these antibodies will allow rapid quantitation of foods for several AIA mutagens, and will aid in the isolation and characterization of other AIAs and AIA-metabolites.

Animals↗

PubMed1987

Monoclonal antibodies for dioxin: antibody characterization and assay development.

A set of 5 anti-dioxin monoclonal antibodies (mAbs), named DD-1, DD-3, DD-4, DD-5 and DD-6, have been isolated. In order to evaluate the ability of these mAbs to recognize various kinds of polychlorinated dibenzodioxins and dibenzofurans, a competition enzyme-linked immunosorbtion assay (ELISA) was developed. All 5 antibodies recognize tetrachloro- and pentachloro-dibenzodioxins and -dibenzofurans. They fail to bind either non-chlorinated, mono-, hexa-, or octa-chlorinated dibenzodioxins, nor do they recognize non-chlorinated, octachloro- or 1,2,3,4,8,9-hexachloro-dibenzofurans. Chlorine substitution on both rings appears necessary for antibody recognition. In the course of our experiments, 3 of the mAbs did not recognize any of the polychlorinated biphenyls (PCBs) tested, while 2 mAbs (DD-1 and DD-6) weakly recognized the 3,3',4,4'-tetrachloro congener. DD-4 and DD-5 are the most specific of the antibodies for the dibenzodioxin and dibenzofuran structure. They do not recognize any of a panel of chlorinated phenols, benzenes, or pesticides. Significantly, these antibodies do not react with PCBs, pentachlorophenol, 2,4-dichlorophenoxyacetic acid, trichlorophenol, or 2,4,5-trichlorophenoxyacetic acid (the latter is weakly recognized by DD-6), any or all of which might be present in large quantities in some dioxin-contaminated samples. Finally, the competition ELISA is able to easily detect 0.5 ng of the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin. It should thus prove useful as an environmental screen for contamination.

Animals↗

PubMed1986

Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin.

Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.

Amino Acid Sequence↗

PubMed1986

Improved high-affinity monoclonal antibody to iododeoxyuridine.

Three monoclonal antibodies (Mabs), IU-1, IU-4, and B-44 were evaluated in enzyme immunoassays (ELISA) and by flow cytometry for their abilities to recognize bromodeoxyuridine (BrdUrd)- and iododeoxyuridine (IdUrd)-substituted DNA's, nucleotides, and nucleosides. IU-4 is a new Mab, derived from mice immunized with 5-iodo-2'-deoxyuridine-5'-monophosphate (IdUMP) conjugated (IdUMP) conjugated through the phosphate group to albumin. This immunogen was selected to resemble IdUMP in DNA. In competition ELISA assays, IU-4 prefers IdUrd to BrdUrd and prefers halogenated nucleotides over the corresponding nucleosides. In both ELISA and flow analysis, IU-4 recognizes IdUrd in DNA at substitution frequencies at least as low as one IdUrd one per 1,000 normal bases. The high affinity of IU-4 for IdUrd-DNA contrasts with IU-1 and B-44, which show a strong binding dependency on the frequency of base substitution and require DNA that is essentially fully substituted with BrdUrd for binding in both flow and ELISA assays. The high affinity of IU-4 for IdUrd in DNA and its independence of IdUrd residue spacing make it a superior reagent for the quantitative labeling of halogenated thymidine analogues in whole cells.

Animals↗

PubMed1986

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.

Animals↗

PubMed1985

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.

Animals↗

PubMed1985

Characterization of monoclonal antibodies to bromodeoxyuridine.

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.

Animals↗

PubMed1985

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity.

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.

Animals↗

PubMed1985

Monoclonal antibodies specific for sickle cell hemoglobin.

Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility.

Amino Acid Sequence↗

PubMed1985

Measurement of sister chromatid exchanges at very low bromodeoxyuridine substitution levels using a monoclonal antibody in Chinese hamster ovary cells.

A monoclonal antibody to bromodeoxyuridine (BrdUrd) incorporated into DNA allowed visualization of sister chromatid exchanges (SCE) when as little as 0.6% of the thymine in a single DNA strand has been substituted. Measurement of the SCE frequency as a function of BrdUrd substitution in a normal Chinese hamster ovary cell line showed a plateau of six SCEs per cell for substitution levels up to at least 20%. A clear elevation in frequencies was noted at 60% substitution. However, in the mutant line EM9, previously shown to have a highly elevated frequency of SCE, the level of exchanges declined continuously as the percentage of BrdUrd substitution decreased. At 0.6% substitution, the frequency of SCE was still 4-fold higher than that of the parental cells. The antibody procedure described here should be useful in evaluating the extent to which SCEs induced by mutagenic agents result from interactions between the DNA damage caused by the agent and the BrdUrd routinely used for measuring SCE.

Animals↗

PubMed1984

Morphologic changes in rat urothelial cells during carcinogenesis: I. Histologic and cytologic changes.

The methods of either image or flow cytometry applied to developing bladder tumors in rats requires satisfactory cell samples and a system for cell classification that is related to the lesions from which the cells are derived. Seven- to eight-week-old male Fisher 344 rats were fed 0.05% of the carcinogen N-butyl-4-N-hydroxybutylnitrosamine (BBN) in drinking water for 10 wk and then returned to tap water. Animals were killed at 14, 26, 34, 45 and 62 wk after the start of carcinogen feeding. Age-matched untreated animals were controls. Bladders were fixed, embedded, sectioned, and histologically evaluated, or were dissociated with a trypsin/EDTA solution into single cells that were Papanicolaou stained and evaluated for cytopathologic changes. Overnight urines were collected before killing; urine sediments were Papanicolaou stained and evaluated. Histologic features were hyperplasia at 14 wk, followed by slowly progressing papillary transitional cell tumors that eventually led to invasive carcinoma and were similar to those reported for this animal model. Treated animals had cytologic features of repair at 14 and 26 wk and neoplastic features at 45 and 62 wk. Both reparative and neoplastic changes were found at 34 wk. Cells were much more numerous in urines from treated rats (greater than 1,000 per sample) than in urines from controls (less than 1,000 per sample). Although exfoliated cells in urine samples were generally of poor quality, as many as 11% of cells were adequately preserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Morphologic changes in rat urothelial cells during carcinogenesis: II. Image cytometry.

Improved early detection of neoplasia by screening of urothelial cells requires an understanding of the features distinguishing normal and neoplastic cell populations. We have begun a program of study based upon a rat model system for the controlled observation of early-stage lesions produced by the carcinogen N-butyl-N-(4-hydroxybutyl)- nitrosamine. Cells dissociated directly from normal and malignant urothelium were characterized by conventional cytopathology techniques and by quantitative microscopy (for nuclear texture and nuclear and cytoplasmic size, shape, and stain content) to derive a comprehensive picture of bladder tumor development. By following the changes that occur in the dissociated urothelial cells we have found that the nuclear area, total nuclear stain, nuclear shape, and the nuclear chromatin change significantly over a 48-wk interval as the lesions progress toward malignancy.

Animals↗

PubMed1984

Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry.

A monoclonal antibody against histone 2B (anti-H2B) was used as a reagent to stain isolated chromosomes for analysis using flow cytometry. Chromosome suspensions were treated with a mouse monoclonal antibody specific for the histone 2B (clone HBC-7) and then with a fluorescein-labeled goat anti-mouse-IgM antibody. The chromosomes were also stained for DNA content with either Hoechst 33258 or propidium iodide. The amount of antibody and the amount of DNA-specific stain bound to each chromosome were measured simultaneously using flow cytometry. The order of the steps in the staining protocol is important. Propidium iodide prevents anti-H2B from binding to chromosomes, and therefore must be added only after antibody labeling is completed. In contrast, the addition of Hoechst 33258 before antibody labeling reduces antibody binding by only 20%-30%. Binding of anti-H2B was proportional to the DNA content of both human and Chinese hamster chromosomes. Human chromosomes bind an average of three to four times more anti-H2B than do Chinese hamster or mouse chromosomes of the same DNA content. This was determined by analyzing mixtures of human and Chinese hamster chromosomes and human and mouse chromosomes. The results demonstrate that it is possible to label the proteins of chromosomes in suspension with fluorescent antibodies and to use these reagents for the analysis of chromosome structure by flow cytometry.

Animals↗

PubMed1984

Binding specificities of eight monoclonal antibodies to human glycophorin A--studies with McM, and MkEn(UK) variant human erythrocytes and M- and MNV-type chimpanzee erythrocytes.

Four newly derived mouse monoclonal antibodies to human glycophorin A are described. Three of these antibodies bind preferentially to the N form of glycophorin A; the fourth recognizes a shared determinant of the M and N forms. All four antibodies are directed toward the 39 amino acid, amino-terminal portion of the protein, and the N-specific antibodies require for binding the presence of N-acetyl-neuraminic acid on the glycosidically linked oligosaccharides. Cross-reaction of the N-specific antibodies to homozygous MM erythrocytes appears to result from binding to glycophorin B. In addition, these antibodies together with four previously reported glycophorin monoclonal antibodies, including two that specifically recognize the M form of glycophorin A, were tested for binding to McM and MkEn(UK) variant human erythrocytes and M- and MNV-type chimpanzee erythrocytes. Results obtained for five of the six M- or N-specific monoclonal antibodies point to the general immunodominance of the amino-terminal serine-leucine polymorphism and the requirement for sialic acid. One of the two M-specific monoclonal antibodies, 9A3, discriminates between the M, N, and Mc forms of glycophorin A solely on the basis of the amino-terminal serine-leucine polymorphism. The other M-specific antibody, 6A7, requires a more complex determinant involving the glycine-glutamic acid polymorphism at the fifth position in the sequence as well. The epitopes for all three N-specific monoclonal antibodies include the amino terminal leucine that occurs in the N form of glycophorin A and may also include the glutamic acid that occurs at position five. Our studies support the proposed Lepore-type glycophorin A-B hybrid gene rearrangement for the En(UK) allele found in the English En(a-) family. The data also confirm the expression of the M-like glycoprotein on chimpanzee erythrocytes and the presence of a human glycophorin B-like antigen on the MNV-type cells.

Amino Acid Sequence↗

PubMed1983

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.

Antibodies, Monoclonal↗

PubMed1983

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.

Agglutination Tests↗

PubMed1983

Increased DNA content as an early marker of transformation in carcinogen-exposed rat tracheal cell cultures.

To determine if abnormal cellular DNA content, suggestive of aneuploidy, is an early indicator of transformation by chemical carcinogens, we exposed primary cultures of rat tracheal epithelial cells to N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), 12-O-tetradecanoylphorbol-13-acetate (TPA), MNNG followed by TPA, or solvent as a control. After 40 and 60 days in culture suspensions of the cells were made, fixed, stained with DNA-dye Hoechst 33342, and the fluorescence per cell measured with a flow cytometer. The DNA histogram showed that freshly isolated rat tracheal epithelial cells, and control cells at days 40 and 60 had predominantly diploid DNA content values. The late primary (day 40) and early passaged (day 60) control cells commonly show increased numbers of cells with tetraploid DNA contents. TPA induced aneuploidy in few cultures by day 40, but by day 60 all samples tested had significant numbers of cells with aneuploid DNA content. In contrast a single MNNG treatment or MNNG followed by TPA regularly caused extensive aneuploidization by day 40. Multiple cycling subpopulations with aberrant DNA contents appear. These dramatic changes in cellular DNA content suggestive of drastic ploidy changes in MNNG and MNNG + TPA exposed cultures are early events, preceding other evidence of neoplastic transformation by many cell generations. Our results suggest that aneuploidy is an early event in the transformation of rat tracheal epithelial cell cultures by chemical carcinogens.

Aneuploidy↗

PubMed1982

Histochemistry of NADH diaphorase and gamma-glutamyltranspeptidase in rat bladder tumors.

To improve identification of preneoplastic bladder cancer cells, we have studied two enzyme histochemical changes in bladder tumors induced in male Fisher 344 rats by the carcinogen N-butyl-N-(4-hydroxybutyl)-nitrosamine. In early areas of focal nodular hyperplasia there was a dramatic increase in staining for NADH:menadione oxidoreductase (diaphorase)activity. In nonfocal areas as well, there were many individual cells with intense staining, while the controls were of uniform moderate staining. Large papillomas and carcinomas often showed heterogeneous staining. gamma-Glutamyltranspeptidase (GGT) was absent from normal urothelium and from all tumors except the most advanced carcinomas and large papillomas. In old, carcinogen-exposed animals, GGT activity was seen in the luminal surface of tumors and in the interlesion urothelium. In newborn rats and in rats with regenerative hyperplasia following wounding of the urothelium, the diaphorase staining was less than that in the untreated adult. Our findings suggest that increased diaphorase activity may serve to identify early islands of carcinogen-induced, enzymatically altered bladder cells, while GGT will not.

Animals↗