Improved bromodeoxyuridine/DNA analysis by anti-BudR monoclonal antibody versus right angle light scatter

Dynamic cell cycle analysis is based on the incorporation of labelled precursors into DNA. Although antibodies to BrdU are very useful for analysing in flow cells which synthesize DNA, this approach has two main limitations. First, the detection of low incorporating cells is often difficult; second, four parameter flow cytometry is not able to correlate cell cycle to any other cellular marker. We have developed a methodology that, employing an IgGH + L as a second antibody and side scatter instead of propidium iodide fluorescence, allows a better discrimination of BudR+ cells. This approach allows the collection of an extra-fluorescent signal, and the analysis of specific cellular markers within the cell cycle.     

In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Thin viable slices of normal or pathological human tissues were incubated in vitro with bromodeoxyuridine (BrdU). Later, cryostatic sections and histological sections from the same samples embedded in paraffin were examined by an immunohistochemical method using a monoclonal antibody anti-bromodeoxyuridine (anti-BrdU-MAb): on both cryostatic and histological sections, the nuclei of the S-phase cells proved positive. The optimization of the technique depends on the concentration of bromodeoxyuridine in the culture medium (160 microM), the duration of incubation (not less than two h), the method of DNA denaturation (2N or 4N HCl) and the dilution of the anti-BrdU-MAb (1:50). In vitro, immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections.     

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.     

Detection of bromodeoxyuridine incorporation in mammalian chromosomes by a bromodeoxyuridine antibody

A commercially available bromodeoxyuridine (BrdUrd) antibody was used to demonstrate sister chromatid differentiation (SCD) and to evaluate sister chromatid exchanges (SCEs) in V79 Chinese hamster cells. V79 cells were cultivated for one cell cycle in the presence of BrdUrd, followed by a second cell cycle in the absence of BrdUrd. Chromosome preparations were stained by a common immunologic staining technique. The staining pattern observed is similar to that after FPG (fluorescent plus Giemsa) staining, though with reverse staining specificity. The sensitivity of BrdUrd detection is enhanced by a factor of 20 compared to the FPG technique and thus allows the evaluation of SCEs at very low BrdUrd concentrations. The application of the antibody technique gives information about the origin and localization of SCEs and produces further evidence for the spontaneous occurrence of SCEs.     

Improved monoclonal antibody tumor/background ratios with exchange transfusions

Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both ¹³¹I-5G6.4 specific and ¹²⁵I-UPC-10 non-specific MoAb. Twenty-four hours later 65–80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P < 0.05) decreased normal tissue activities of ¹³¹I (except for muscle) by 63–85%. while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1–0.8 to 2.3–6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.     

A non-radioactive method to detect RNA or DNA using an oligonucleotide probe with bromodeoxyuridine free ends, a monoclonal antibody against bromodeoxyuridine and immunogold silver staining

A non radioactive method for probing RNA or DNA on dot and Northern blots using a synthetic oligonucleotide with bromodeoxyuridine free ends is described. The present experiment was carried out with human testis and placental RNA's. The probe was the 21 base long sequence coding for the amino acids 18 to 24 of the insulin-like growth factor I (IGF-I) with two bromodeoxyuridine dinucleotides added at the 5' and 3' ends. The probe was detected with a monoclonal antibody against bromodeoxyuridine and immunogold silver staining (IGSS). Our method was compared to the peroxydase (HRP) revelation of the same probe. The results obtained show a lower background with IGSS than with HRP revelation. A sensitivity similar to that of 32P labelling was found with the advantages of an increase in the rapidity of the procedure (24 hours instead of 9 days exposure) and the absence of handling radioactive substances. Moreover, as the monoclonal antibody against BrdU detects single stranded DNA only, the use of BrdU free ends-labelled oligonucleotide allows the development of the revelation procedure without any previous denaturation of the hybrid. This particular point is an indisputable advantage for detecting hybridization in situ.     

Islet cell analysis and purification by light scatter and autofluorescence

Rat pancreatic A- and B-cells differ in light scatter and flavin-adenine-dinucleotide (FAD)-related fluorescence and are thus represented by two easily distinguishable populations in a fluorescence-activated cell sorter (FACS). Sorting of dissociated islet cells yields highly purified single A- and B-cell preparations. FACS-analysis of islet cells also indicated that FAD-fluorescence in 3-cells is reduced within a 5 minute exposure to 20 mM glucose, whereas no variations were observed in A-cell fluorescence nor with 3-0-methylglucose or fructose. FACS-analysis of blood cells and of dissociated liver, parotid, pituitary and pancreatic exocrine cells demonstrated a wide variation in the respective FAD-fluorescence intensities, which could be used for their purification as viable single cells as well as in studying their metabolic redox state.     

Thermodynamic analysis of monoclonal antibody binding to duplex DNA.

A technique based on fluorescence polarization (anisotropy) was used to measure the binding of antibodies to DNA under a variety of conditions. Fluorescein-labeled duplexes of 20 bp in length were employed as the standard because they are stable even at low ionic strength yet sufficiently short so that both arms of an IgG cannot bind to the same duplex. IgG Jel 274 binds duplexes in preference to single-stranded DNA; in 80 mM NaCl Kobs for (dG)20.(dC)20 is 4.1x10(7) M-1 compared with 6.4x10(5) M-1 for d(A5C10A5). There is little sequence specificity, but the interaction is very dependent on ionic strength. From plots of log Kobs against log[Na+] it was deduced that five or six ion pairs are involved in complex formation. At low ionic strength,Kobs is independent of temperature and complex formation is entropy driven with DeltaH degrees obs and DeltaC degrees p,obs both zero. In contrast, in 80 mM NaCl DeltaC degrees p,obs is -630 and -580 cal mol-1K-1 for [d(TG)]10.[d(CA)]10 and (dG)20.(dC)20 respectively. IgG Jel 241 also binds more tightly to duplexes than single-stranded DNA, but sequence preferences were apparent. The values for Kobs to [d(AT)]20 and [d(GC)]20 are 2.7x10(8) and 1.3x10(8) M-1 respectively compared with 5.7x10(6) M-1 for both (dA)20. (dT)20 and (dG)20.(dC)20. As with Jel 274, the binding of Jel 241 is very dependent on ionic strength and four or five ionic bonds are involved in complex formation with all the duplex DNAs which were tested. DeltaC degrees p,obs for Jel 241 binding to [d(AT)]20 was negative (-87 cal mol-1K-1) in 80 mM NaCl but was zero at high ionic strength (130 mM NaCl). Therefore, for duplex-specific DNA binding antibodies DeltaC degrees p,obs is dependent on [Na+] and a large negative value does not correlate with sequence-specific interactions.     

Flow cytometric estimation of cell cycle parameters using a monoclonal antibody to bromodeoxyuridine

An estimation of cell kinetic parameters was made by simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdUrd) contents of cells. The procedure described in this paper involves the incorporation of BrdUrd by S phase cells, labeling the BrdUrd with an indirect immunofluorescent technique using a monoclonal anti-BrdUrd antibody, and staining DNA with propidium iodide (PI). The amount of incorporated BrdUrd in HeLa cells was proportional to that of synthesized DNA through S phase. For all cell lines examined, the pattern of BrdUrd incorporation was essentially the same and the rate of DNA synthesis during S phase was not constant. The bivariate BrdUrd/DNA distributions showed a horse-shoe pattern, maximum in the mid S phase and minimum in the early and late S phases. Furthermore, the durations of cell cycle (Tc) and S phase (Ts) were estimated from a FLSm (fraction of labeled cells in mid S phase) curve that was generated by plotting the percentage of BrdUrd pulse-labeled cells in a narrow window defined in the mid S phase of the DNA histogram. The values of these parameters in NIH 3T3, HeLa S3, and HL-60 cells were in good accordance with the reported data. This FCM method using the monoclonal anti-BrdUrd antibody allows rapid determination of both cell cycle compartments and also Ts and Tc without the use of radioactive DNA precursors.     

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.     

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.     

Cell cycle analysis using numerical simulation of bivariate DNA/bromodeoxyuridine distributions

This report describes a mathematical model of cell proliferation for simulation of bivariate DNA/bromodeoxyuridine (BrdUrd) distributions. The model formulates the change with time in the frequency of cells with any DNA content and in the amount of incorporated BrdUrd, according to given cytokinetic parameters, i.e., durations and dispersions of cell cycle phases and DNA synthesis rate during S-phase. We have applied this model to sequential DNA/BrdUrd distributions measured for Chinese hamster ovary cells asynchronously grown in vitro, 1) for 30 min in 10 microM BrdUrd followed by growth in BrdUrd-free medium for 0 to 24 h, or 2) during continuous incubation in 3 microM BrdUrd plus 30 microM thymidine for 2 to 24 h. The matches between the experimental and simulated distributions give the G1, S, G2M, and total cell cycle durations (and coefficients of variation) of 5.6 h (0.08), 7.0 h (0.07), 1.4 h (0.16), and 14.0 h (0.05), respectively. The model is shown to be useful for quantitative interpretation of the bivariate distributions.     

Optical characterization of liposomes by right angle light scattering and turbidity measurement

Liposomes have frequently been used as models of biomembranes or vehicles for drug delivery. However, the systematic characterization of lipid vesicles by right angle light scattering and turbidity has not been carried out despite the usefulness of such studies for size estimation. In this study, liposomes of various sizes were prepared by sonication and extrusion. The mean cumulant radii of the vesicles were determined by dynamic light scattering. The lamellarities were estimated based on fluorescence quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha-phosph ati dylethanolamine by sodium dithionite. Right angle light scattering intensity and optical density at 436 nm per unit lipid concentration were measured as a function of vesicle radius. With a vesicle radius < or =100 nm, the optical parameters could be well explained by the Rayleigh-Gans-Debye theory in which the liposomes were modeled as homogeneous spheres with mean refractive indices determined by the volume fractions of lipids in vesicles.     

蛋白A亲和层析法纯化单克隆抗体工艺的优化

治疗性单克隆抗体药物已成为生物医药领域市场最主要的产品类别。蛋白A亲和层析作为第一步捕获抗体蛋白最为有效的手段仍然在现有单克隆抗体纯化平台中占据主导地位。在本研究中,首先开发了一种基于低p H处理抗体细胞回收液的新型细胞液回收技术,该技术能有效去除宿主相关污染物(非组蛋白宿主杂质蛋白、组蛋白、DNA、蛋白聚合物等),同时保证较高的抗体回收率。通过该技术有效预处理后,蛋白A纯化效率可提高10倍左右,并且有效避免了抗体洗脱液中和后浊度的上升,大大减轻了后续蛋白纯化的压力。同时我们也对酸性处理中各种宿主杂质去除机制进行了研究。然后,预处理的洗脱液再经一步Capto adhere色谱纯化,非组蛋白宿主杂质蛋白降低至5 ppm、DNA小于1 ppb、组蛋白降低至检测限以下、蛋白聚合物小于0.01%。总过程抗体蛋白收率87%。该两步法抗体纯化技术可有效集成至当前主流抗体纯化平台,具有良好的大规模应用价值。     

Measurement of cell proliferation by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to bromodeoxyuridine

An ELISA was developed and optimized to measure cell proliferation using a monoclonal antibody to bromodeoxyuridine (BrdUrd). Incorporation of BrdUrd into myoblast monolayers, measured as the optical density at 492 nm, increased in response to fetal calf serum, IGF-I and EGF, the ELISA data correlated closely with data obtained by BrdUrd immunocytochemistry (r = 0.984), cell counting (r = 0.972) and tritiated thymidine uptake by liquid scintillation counting (r = 0.990). The BrdUrd ELISA is a useful alternative to measurement of tritiated thymidine uptake by scintillation counting, and has the added advantages of dispensing with the use of radioactivity and of being less labour intensive.     

Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody

The ability to measure cell proliferation is important in the study of cancer biology. The usual technique for quantitating proliferating cells in tissue explant and organ culture by detection of [3H]-thymidine incorporation into DNA by autoradiography is tedious and time-consuming. We have developed a technique for identification and quantitation of bromodeoxyuridine (an analogue of thymidine) in cultured tissue explants. Fetal mouse colon explants were exposed in vitro to bromodeoxyuridine (BUdR) or [3H]-thymidine for 3 to 72 hr and then for various periods to unlabeled thymidine. The tissues were stained with a monoclonal anti-bromodeoxyuridine antibody and in parallel [3H]-thymidine incorporation was detected by autoradiography. Incorporation of BUdR was measured by quantitating the amount of pigment deposited over nuclei after immunohistochemical staining, using an optical data digitizer. It was found that both techniques identified proliferating cells. Dividing cells were present both in crypts and in the surrounding stroma in Day 14 fetal mouse colon cultures. The immunohistochemical technique was more rapid and less cumbersome than autoradiography.     

Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.     

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Monoclonal antibody F7-26 was obtained by fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with DNA treated by nitrogen mustard (HN2). Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. Two-parameter analysis for the antibody binding and DNA content showed no binding of antibody to replicating DNA in control cells. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Binding of monoclonal antibody specific for the determinants expressed on ssDNA to the cells treated with alkylating agents may be attributed to local DNA denaturation. Potentiation of L-phenylalanine mustard cytotoxicity by buthionine sulfoximine or hyperthermia was accompanied by increased antibody binding to cellular DNA. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.     

Recombinant DNA approach for defining the primary structure of monoclonal antibody epitopes. The analysis of a conformation-specific antibody to myosin light chain 2

A monoclonal antibody (MF5), capable of recognizing a divalent cation-induced conformational change in myosin light chain 2 (LC2f), has been used to screen a cDNA library constructed in the expression vector lambda gt11. A clone has been isolated that contains the whole coding sequence of this myosin subunit. The light chain was synthesized as a fusion peptide linked to beta-galactosidase by ten amino acids encoded in the 5' untranslated region of its mRNA. Seven imperfect repeats were identified in the 3' untranslated region of the mRNA. The amino acids conferring specificity on the MF 5 epitope were established by first determining the nucleotide sequence of shorter subclones that expressed the epitope and then eliminating those amino acid residues shared by cardiac myosin LC2, which was unreactive with this antibody. The epitope, which becomes accessible to MF 5 upon removal of bound divalent cations, resides at the junction between the first alpha-helical domain and the metal binding site. Theoretically, this approach can be used to define the primary structure of most protein epitopes.