Cell cycle kinetic measurements in an irradiated rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine

Cell cycle kinetics after X-irradiation were studied in a solid rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine (BrdUrd) in cells in which the DNA was labeled by BrdUrd. It could be shown that this tumor was composed of about 80% diploid host cells, and only 20% of the cells in the dissociated tumor were actually tetraploid tumor cells. When rats were injected intraperitoneally with BrdUrd to label S-phase cells in the tumor, only a fraction of both types of cells became labeled with BrdUrd during S-phase, even 24 h after injection. The diploid BrdUrd-labeled cells progressed rapidly into cycle; 4 h after injection of BrdUrd, labeled diploid G1-phase cells could be observed. Only 25% of the tetraploid S-phase cells could be labeled by a single injection of BrdUrd (160 mg/kg body weight). These labeled tetraploid cells progressed through the cell cycle with similar velocities as did labeled diploid cells. Using a "Mini Osmotic Pump" containing bromodeoxycytidine (BrdCyd) at high concentration (0.3 mol/L) that released BrdCyd continuously into the organism where it was converted to BrdUrd, it could be shown that after 2 days about 60% of cells in S-phase and 70% of cells in G2-phase were labeled. The fraction of labeled G2-phase cells in irradiated tumors (D = 10 and 20 Gy) was enhanced between 10 and 50 h after irradiation due to a radiation-induced G2 block in cycling tetraploid tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-beta-lactam

We report the first example of a monoclonal antibody-catalysed hydrolysis of a beta-lactam where the antibodies were generated by a simple transition-state analogue. A rat monoclonal antibody (1/91c/4d/26) generated by using an acyclic 4-nitrophenylphosphate immunogen catalysed the hydrolysis of corresponding 4-nitrophenyl carbonates but, more importantly, also catalysed the hydrolysis of N-(4-nitrophenyl)-azetidinone at pH 8 with k(cat)=8.7 x 10(-6)s(-1) and K(M)=35 microM. This is the first example of a rat monoclonal catalytic antibody.     

Immunohistochemical characterization of a crypt cell-specific plasma membrane protein in rat small intestine epithelium using a monoclonal antibody

Proteins of the basolateral membrane (BLM) of small intestine epithelial cells of adult rats, in the MW ranges of 50-65 KD, 85-100 KD, and over 100 KD, were obtained as follows. After isolation of the BLM and subsequent SDS-PAGE and transblotting of the proteins on nitrocellulose sheets, the bands in these MW ranges were cut out of the nitrocellulose sheet and extracted. Balb/C mice were immunized with these protein fractions and a monoclonal antibody (MAb) was then produced. MAb SI/CC1 obtained via immunization with the 50-65 KD protein fraction shows specificity for the crypt epithelium of the small intestine. It can be used to characterize, by light and electron microscopic immunohistochemical methods, a crypt cell protein (SI/CC1-Ag) with a very specific localization. Fluorescence labeling shows that the SI/CC1-Ag can be found only in the epithelium of small intestine crypts (except for the granules in eosinophilic granulocytes). The epithelium of the colon, as well as the epithelia of other organs, could not be labeled. In the small intestine crypts, SI/CC1-Ag is found only in the Paneth cells located in the basal crypt section, and in the undifferentiated cells in the middle crypt section; it is lacking in the cells of the upper crypt section. Gold labeling shows that SI/CC1-Ag in the undifferentiated cells is localized exclusively in the basolateral PM domain. On the Paneth cells, the content of the secretory granules is labeled, along with the basolateral PM domain; the labeling sometimes present on their luminal part is probably due to passively absorbed secretion from these cells. The SI/CC1-Ag in the BLM of undifferentiated and Paneth cells is found only on Days 21-23 post partum, whereas the Paneth cell granules could be labeled as early as the Day 16 post partum. With immunodetection with SI/CC1, one band at about 55 KD is specifically labeled in the protein pattern of the isolated small intestine cell BLM. In the protein pattern of the isolated crypt cells two bands were labeled, again one at 55 KD and one at about 120 KD. These findings indicate that SI/CC1-Ag is a 55 KD protein that appears on Days 21-23 post partum in the BLM of undifferentiated cells and of Paneth cells.     

Localisation of 5'-nucleotidase in a rat liver cell line using a monoclonal antibody and indirect immunofluorescent labelling

A new method of labelling anti-IgG with rhodamine while protecting the IgG binding site is described. Indirect immunofluorescence of the ectoenzyme 5'-nucleotidase using a monoclonal antibody shows that it has a punctate distribution over the whole cell surface of a cultured rat liver cell line. The density of fluorescent spots representing the surface localisation of 5'-nucleotidase and of clathrin-coated pits was analysed by a computerised counting technique. The cell surface localisation of 5'-nucleotidase differs from clathrin-coated pits and microvilli. 5'-Nucleotidase within the cell is not uniquely associated with any clearly defined organelle.     

Distribution of glutamylated alpha and beta-tubulin in mouse tissues using a specific monoclonal antibody, GT335.

A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.     

Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology

Summary The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated 187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular cell density of 1.3 to 1.5×10⁷ cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period. Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated that the principal contaminant associated with the unpurified antibody was bovine serum albumin.     

Definition of the T-lymphocyte inducer of suppression in primates using a monoclonal antibody

Since some of the conserved antigens between man and phylogenetically lower primate species may be more immunodominant on lymphocytes of the lower primate species, we reasoned that immunization of mice with lymphocytes from lower primates might prove a useful strategy for developing monoclonal antibodies which recognize functionally important structures on both human and nonhuman primate lymphocytes. In employing this approach for the development of monoclonal antibodies, we have developed the antibody anti-2H4 which recognizes a structure on both T on non-T mononuclear cells of a wide array of primate species. 2H4+ rhesus monkey T lymphocytes exhibited a greater proliferative response to lectin and alloantigenic stimulation than 2H4- cells, suggesting that anti-2H4 might separate primate T lymphocytes into functionally distinct cell populations. In fact, helper activity for antibody production by rhesus monkey B lymphocytes in response to pokeweed mitogen (PWM) resided in the 2H4- T-cell population. Furthermore, the 2H4+ T-lymphocyte population activated the suppressor function of T8+ rhesus monkey cells. The fact that the surface antigen which defines this T-cell subset is widely conserved in nonhuman primates suggests that anti-2H4 recognizes a functionally important structure.     

The use of a monoclonal antibody to measure plasma atriopeptins in rat

A monoclonal antibody with specificity for atrial natriuretic peptides (ANP) was produced, that can be used for the radioimmunological determination of ANP-immunoreactivity (ANP-IR) in rat plasma. The antibody recognizes atriopeptin I, II, III, as well as alpha-hANP and alpha-hANP fragment (7-28) and does not crossreact with ANP-fragments (13-28) and (18-28). Plasma levels of ANP-IR in conscious Wistar rats were determined before and after volume-loading. Basal plasma levels of ANP-IR were 108 +/- 12 pg/ml, and after volume-loading increased to 800 +/- 59 pg/ml.     

Enzyme immunoassay for plasma estradiol using a monoclonal antibody

A microtitre plate enzyme immunoassay (EIA) for plasma estradiol is described, involving competition between sample estradiol and an immobilized estradiol-bovine serum albumin complex for a monoclonal anti-estradiol antibody, followed by immobilized antibody quantitation using enzyme-labelled antiglobulins. The assay dose-response curve covered a range of 6-1500 fmol/well. The intra- and inter-assay coefficient of variation for the assay of three plasma pools ranged from 3.1 to 4.7% and from 4.7 to 10.6% respectively. The assay showed satisfactory correlation with a standard estradiol radioimmunoassay. Pre-coated microtitre plates were stable, dried, at 4 degrees C for up to 3 months and the anti-estradiol was stable to lyophilization and also was stable in solution at 4 degrees C for up to 1 month.     

Measurement of PGH synthase activity using a pan-specific monoclonal antibody

A pan-specific monoclonal antibody that recognizes a variety of prostaglandin moieties and does not recognize arachidonic acid or the hydroxy-eicosatetraenoic acids was used to assess the general ability of a tissue to produce prostaglandins. Although this assay does not give a quantitative measure of PGH synthase activity, it does provide a sensitive and convenient means of screening a large number of samples for prostaglandin production.     

Binding site of the rat liver specific monoclonal antibody

A rat liver-specific antigen (RLSA) lost its binding ability to the corresponding monoclonal antibody after treatment with N-glycanase or sialidase, which suggested that the specific binding site might be in a portion of the sugar chain containing sialic acid. The specific antigen reacted with wheat germ agglutinin, lentil lectin, erythroagglutinating phytohemagglutinin and Ricinus communis agglutinin, but not with concanavalin A or peanut agglutinin. These results suggest that the specific antigen has asparagine-linked complex-type sugar chains which might be the binding sites of the monoclonal antibody.     

The distribution of fibronectin in rat tooth and periodontal tissues: an immunofluorescence study using a monoclonal antibody

The distribution of fibronectin (FN) in longitudinal, buccolingual sections of decalcified adult rat periodontium and teeth was studied by indirect immunofluorescence using a monoclonal antibody. FN was present in virtually all regions of the periodontium, including the gingiva, periodontal ligament, many blood vessel walls, alveolar bone, incisor and molar predentine and dentine, and molar acellular and cellular cementum. The cementum of the incisor, ameloblasts, stratum intermedium and stellate reticulum, and the connective tissue of the pulp and the surface of ondontoblasts facing the pulp in the incisor and molar were not labeled for FN. FN distribution was not always uniform either within a given connective tissue or between different connective tissues of the same organ.     

Reaction of the chromatoid body with a monoclonal antibody to a rat histocompatibility antigen

The monoclonal antibody OX3 against a polymorphic class II antigen encoded by the major histocompatibility locus of the rat has been shown to cross-react with the chromatoid body during spermatogenesis. Using an indirect immunofluorescence assay on frozen, fixed testis sections, the antibody revealed a pattern of fluorescent speckling that correlated with specific stages of spermatogenesis. The positive material first appeared in late pachytene spermatocytes as multiple small spots. Larger dots appeared in all regions containing round spermatids, but, as the spermatids matured, only fine dots were seen. Mature spermatids were negative, as were all early cells (spermatogonia to early pachytene spermatocytes). When suspension of fixed testicular cells were tested, the activity was clearly associated with the chromatoid body adjacent to the nucleus in round spermatids and with multiple smaller structures encircling the nucleus in primary spermatocytes. These associations were confirmed in observations on immature testes at various ages. No reactivity was seen in testes of animals whose testes had previously been irradiated to render them aspermatogenic, nor in grc/grc rats in which spermatogenesis is arrested at the primary spermatocyte stage. Because the expression of this reactivity was seen even in rats that do not express the OX3 antigen on their somatic cells, this antibody should prove useful in determining the structure of this body, its origin and fate, and any possible role it may have in spermiogenesis.     

Studies of mammalian ornithine decarboxylase using a monoclonal antibody.

A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Detection of estrogen receptor (ER) in the rat brain using rat anti-ER monoclonal IgG with the unlabeled antibody method

Application of Sternberger's unlabeled antibody enzyme method for detection of the estrogen receptor (ER) using a rat primary antibody with rat tissues has been discouraged, presumably because nonspecific staining of endogenous IgG was expected with the required anti-rat IgG bridging antibody. Because the blood-brain barrier greatly reduces immunoglobulin infiltration into the brain, we hypothesized that rat brain tissue could be specifically immunostained using rat IgG primary antibodies. A rat monoclonal anti-ER antibody (H222) specifically stained ERs in the brains of ovariectomized but not in ovariectomized estrogen-treated rats. In contrast, the uterus, a well-perfused target organ stained intensely in a nonspecific fashion. Dense populations of estrogen receptors were observed in the medial preoptic area, the bed nucleus of the stria terminalis, and the arcuate and ventromedial nuclei of the hypothalamus. A monoclonal rat IgG directed against alpha-tubulin labeled primarily cortical dendrites quite distinct from the neuronal nuclei that are the primary antigenic sites for the estrogen receptor antibody. These results confirm that the sensitive unlabeled antibody method can be applied to rat brain tissues, even when the primary antibody is rat IgG and that labeling of endogenous IgG may be used as a simple method to evaluate the integrity of the blood brain barrier.     

Detection of estrogen receptor (ER) in the rat brain using rat anti-ER monoclonal IgG with the unlabeled antibody method

Summary Application of Sternberger's unlabeled antibody enzyme method for detection of the estrogen receptor (ER) using a rat primary antibody with rat tissues has been discouraged, presumably because nonspecific staining of endogenous IgG was expected with the required anti-rat IgG bridging antibody. Because the blood-brain barrier greatly reduces immunoglobulin infiltration into the brain, we hypothesized that rat brain tissue could be specifically immunostained using rat IgG primary antibodies. A rat monoclonal anti-ER antibody (H222) specifically stained ERs in the brains of ovariectomized but not in ovariectomized estrogen-treated rats. In contrast, the uterus, a well-perfused target organ stained intensely in a nonspecific fashion. Dense populations of estrogen receptors were observed in the medial preoptic area, the bed nucleus of the stria terminalis, and the arcuate and ventromedial nuclei of the hypothalamus. A monoclonal rat IgG directed against alpha-tubulin labeled primarily cortical dendrites quite distinct from the neuronal nuclei that are the primary antigenic sites for the estrogen receptor antibody. These results confirm that the sensitive unlabeled antibody method can be applied to rat brain tissues, even when the primary antibody is rat IgG and that labeling of endogenous IgG may be used as a simple method to evaluate the integrity of the blood brain barrier.