Characterization of Bacteroides gingivalis by direct fluorescent antibody staining and cellular fatty acid profiles

Bacteroides gingivalis is a newly proposed species which includes strains isolated from the mouth. Thirteen strains of B. gingivalis isolated from three geographic locations in the United States and France were examined with direct fluorescent antibody staining and analysis of total cellular fatty acids and compared with 16 strains of B. asaccharolyticus of nonoral origin by the same methods. Bacteroides gingivalis strains reacted with the B. gingivalis conjugate (fluorescein isothiocyanate labeled antibody reagent) only, while the B, asaccharolyticus strains reacted with the B. asaccharolyticus conjugate only. The B. gingivalis strains showed negative fluorescence with fluorescein isothiocyanate conjugates for other black-pigmented Bacteroides species. The specificity of the B. gingivalis conjugate was demonstrated by its failure to stain 88 strains of aerobic and anaerobic bacteria other than B. gingivalis. The fatty acid profiles of B. gingivalis and B. asaccharolyticus were readily distinguishable. The B. gingivalis profile was also distinguishable from those of other pigmenting Bacteroides species on the basis of concentration ratios among the characteristic components. These results support the species separation of B. gingivalis and B. asaccharolyticus. Further, they indicate the usefulness of cellular fatty acid profiles as an adjunct to the use of specific fluorescent antibody conjugates for identification of Bacteroides species.     

Rapid diagnosis of cytomegalovirus infections by direct immunoperoxidase staining with human monoclonal antibody against an immediate-early antigen.

Direct immunoperoxidase technique using a horseradish peroxidase (HRP)-conjugated Fab' fragment of human monoclonal antibody (humab C7), designated HRP-C7, was evaluated as a rapid diagnosis of cytomegalovirus (CMV) infection. A total of 138 clinical specimens consisting of 124 urine samples and 14 oral swabs were examined for CMV by the direct HRP-C7 staining in comparison with conventional virus isolation. The number of CMV-positive samples by each method was 40 (29.0%) for the former and 37 (26.8%) for the latter, respectively. By HRP-C7 staining, CMV was identifiable within 24 hr after inoculation. By conventional isolation method, an average of 10.3 days had passed before cytopathic effect characteristic of CMV appeared in the cell culture. Some false-positive and false-negative cases were discussed in relation to toxicity of urine samples, storage of the samples, and amount of CMV in the sample. The sensitivity and specificity of HRP-C7 method against conventional isolation method were 89.2% and 93.1%, respectively. Thus, HRP-C7 staining is useful for a rapid diagnosis of CMV infections.     

Characterization of a monoclonal antibody against human placenta type IV collagen by immunoelectroblotting, antibody-coupled affinity chromatography, and immunohistochemical localization

We have produced four monoclonal antibodies against type IV collagen obtained from human placenta. An antibody with a high titer by ELISA, named JK-199, reacted not only with type IV collagen in the triple-helical conformation but also with thermally denatured chains. After affinity chromatography on JK-199 antibody-coupled resin, the amino acid composition and CD spectrum of the affinity-purified peptides from the crude pepsin extract of human placenta were typical of those of human type IV collagen in the triple-helical conformation. On SDS-polyacrylamide gel electrophoresis, the purified protein showed only one broad band with a molecular weight of approximately 260,000 before reduction and six smaller peptide bands after reduction. On immunoelectroblotting, JK-199 reacted with all six peptide bands. Immunohistochemically, typical basement membranes were exclusively and strongly stained with JK-199 on frozen sections of PLP-fixed human placentas without any enzymatic pretreatment in the routine immunoperoxidase method. Judging from these findings, it is concluded that the epitopes of type IV collagen that reacted with JK-199 are exposed on the surface of basement membranes. This antibody should be useful for identification of type IV collagen in normal or pathological basement membranes or other structures.     

Analysis of guanase by agarose gel electrophoresis and activity staining

A method was developed to separate guanase by agarose gel electrophoresis and to detect its activity by staining of the bands with a mixture of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NADP+, and a substrate of guanine, ethanol, phenazine methosulfate, nitrotetrazolium blue, and KCN in Tris-(hydroxymethyl)methylamine buffer (pH 8.0). Serum samples showed bands 1 (faster moving) and 2 corresponding to the positions of albumin and alpha 2-globulin, respectively, found by serum protein staining. The same bands were detected with guanase from human liver and kidney, although band 2 from the latter samples was not as distinct as that from the liver samples.     

Background staining and sensitivity of the unlabelled antibody-enzyme (PAP) method. Comparison with the peroxidase labelled antibody sandwich method using formalin fixed paraffin embedded material.

The PAP procedure was compared with the peroxidase labelled antibody sandwich method and was found to be at least 20 times more sensitive. Background staining was reduced by the addition of normal swine serum to all the immune sera or by pretreating sections with it at the beginning of either method. The PAP procedure could be effectively reduced to a period of 1 hour or less.     

Characterization of a rabbit polyclonal antibody against threonine-AMPylation

An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.     

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of ^99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed ^99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the ^99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The ^99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the ^99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between ^99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N₂S₂) compound. The Fab′ with IgG2a isotype displayed tighter binding to ^99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N₂S₂ challenge and incubation with human plasma. The in vivo biodistribution of five ^99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable ^99mTc labeling of antibody fragments from three of the major murine isotypes.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

A method for enzyme- and immunohistochemical staining of large frozen specimens

Summary The extracellular presence of adenosine polyphosphatase was investigated in a number of rat tissues. The enzyme was demonstrated in basement membranes of epithelial cells of duodenum, urinary bladder, tongue, choroid plexus, submandibular salivary gland, lung and kidney, as well as in basement membranes of capillaries in these tissues. Furthermore adenosine polyphosphatase was demonstrated on collagen fibrils and in the cytoplasm of fibroblasts of all investigated tissues. It appears that the presence of adenosine polyphosphatase in basement membranes is a widespread phenomenon. Since extracellular ADP and ATP are known to promote respectively platelet aggregation and inflammation, the presence of extracellular ADP and ATP-hydrolyzing activity might contribute to inhibit these processes.     

Various keratin antibodies produce immunohistochemical staining of human myocardium and myometrium

Summary Various polyclonal and monoclonal antibodies to keratins were used to stain different human muscle tissues by paired immunofluorescence and the unlabelled antibody peroxidase-anti-peroxidase method. In the myocardium, distinct coloration of the intercalated discs was produced by two polyclonal reagents to human epidermal keratins but not by two monoclonal antibodies to cytokeratins from pig renal tubular cells. In the myometrium — mainly in the middle layer of the uterine wall — cytoplasmic coloration of a varying fraction of the smooth muscle bundles was produced, especially by one of the polyclonal and by both monoclonal reagents. The staining was often confined to the perinuclear region. The keratin-positive myometrial cells usually coexpressed vimentin and actin in various proportions. These findings indicated that intermediate filaments of the keratin type, or antigenically similar elements, are not restricted to cells of epithelial origin. Other types of muscle cells did not react with keratin antibodies, but keratin-positive macrophages were occasionally found in tongue musculature and in inflamed epicardium. Altogether, our observations emphasize that keratin reactivity cannot be considered specific for epithelial (or mesothelial) cells without reservation.Supported by the Norwegian Cancer Society, Jahre's Fund, and the Norwegian Research Council for Science and the Humanities     

Various keratin antibodies produce immunohistochemical staining of human myocardium and myometrium

Various polyclonal and monoclonal antibodies to keratins were used to stain different human muscle tissues by paired immunofluorescence and the unlabelled antibody peroxidase-anti-peroxidase method. In the myocardium, distinct coloration of the intercalated discs was produced by two polyclonal reagents to human epidermal keratins but not by two monoclonal antibodies to cytokeratins from pig renal tubular cells. In the myometrium--mainly in the middle layer of the uterine wall--cytoplasmic coloration of a varying fraction of the smooth muscle bundles was produced, especially by one of the polyclonal and by both monoclonal reagents. The staining was often confined to the perinuclear region. The keratin-positive myometrial cells usually coexpressed vimentin and actin in various proportions. These findings indicated that intermediate filaments of the keratin type, or antigenically similar elements, are not restricted to cells of epithelial origin. Other types of muscle cells did not react with keratin antibodies, but keratin-positive macrophages were occasionally found in tongue musculature and in inflamed epicardium. Altogether, our observations emphasize that keratin reactivity cannot be considered specific for epithelial (or mesothelial) cells without reservation.     

Simultaneous detection of B-cells and T-cells by a double immunohistochemical technique using immunogold-silver staining and the avidin-biotin-peroxidase complex method

A new double immunohistochemical technique for the simultaneous detection of B-cells and T-cells was investigated, using tissue preparations obtained from human axillary lymph nodes and rejected renal allografts. The specimens were immunostained first for the demonstration of B-cells, by the immunogold-silver staining (IGSS) method using Leu-12 monoclonal antibody, and then for T-cells by the avidin-biotin-peroxidase complex (ABC) method using Leu-1 monoclonal antibody. With the present methods, both B-cells and T-cells were clearly detected and distinctively identified without cross-linking of antibodies or double reaction of enzymes.     

A monoclonal antibody against human kidney gamma-glutamyl transpeptidase: preparation, immunochemical, and immunohistochemical characterization

To perform immunohistochemical study of the distribution of gamma-glutamyl transpeptidase in human organs, a highly specific antibody against the human enzyme is required. We prepared monoclonal antibody against gamma-glutamyl transpeptidase from human kidney, using the hybridoma technique. The antibody was of the IgG1 type and the light chain belonged to the kappa class. The antibody reacted specifically with the 63 KD heavy subunit of the enzyme. Examination of the specificity of the antibody performed by immunohistochemical staining of human kidney sections revealed that the antigen was localized on the brush border and along the basolateral membrane of the epithelial cells of both the convoluted and the straight portions of the proximal tubule. This antibody was also reactive in several human organs other than kidney, including epididymis, prostate, seminal vesicle, pancreas, and normal liver, and in human hepatoma. These findings indicate the existence of an antigenic determinant common to human kidney and other organs. The monoclonal antibody did not crossreact with mouse, rat, guinea pig, rabbit, or pig kidney.     

Cytochrome P-450 localization in normal human adult and foetal liver by immunocytochemistry using a monoclonal antibody against human cytochrome P-450

Immunocytochemical studies with a monoclonal antibody (MAb-HL3), which recognises a major isozyme of human hepatic cytochrome P-450, have demonstrated this cytochrome in both cryostat and formalin-fixed paraffin-embedded sections of normal human adult liver. Prior trypsin digestion of the formalin-fixed sections prevented staining. There was a zonal distribution of immunoreactive cytochrome P-450, with localization predominantly in the hepatocytes of zone 3 of the hepatic acinus (the centrilobular region). Cytochrome P-450 was also demonstrated in foetal liver, but all foetal hepatocytes contained immunoreactive cytochrome P-450 and there was no zonal distribution of the protein. The biliary epithelium of adult liver contained a small amount of immunoreactive cytochrome P-450 whereas there was no immunoreactivity in the epithelium of foetal bile ducts.     

Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG_2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.     

RNA recognition by a human antibody against brain cytoplasmic 200 RNA

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation.     

A monoclonal antibody against COOH-terminal peptide of human liver manganese superoxide dismutase

Three stable hybridoma cell lines producing monoclonal antibodies specific for human liver manganese superoxide dismutase were established, and one monoclonal antibody, PG 11, was chosen for immunochemical studies. Immunoblotting demonstrated that the monoclonal antibody binds exclusively to the manganese superoxide dismutase. Immunohistochemical studies indicated that the enzyme is localized in the matrix of human liver mitochondria. To localize antibody-binding epitope, synthetic peptides of the NH2-terminal (residues 1-16) and COOH-terminal (residues 182-189, 190-196, and 182-196) parts of the enzyme were synthesized, and then their effects on the binding were studied using an enzyme-linked immunosorbent assay method. All of the above COOH-terminal peptides inhibited the binding whereas the NH2-terminal ones did not, indicating that PG 11 recognizes several peptides of COOH termini of manganese superoxide dismutase. This is the first report of monoclonal antibodies against human manganese superoxide dismutase with a distinct epitope and of the immunocytochemical demonstration of manganese superoxide dismutase.     

Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting

DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and N-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 A and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase delta preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptide component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase delta with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase alpha inhibited placental polymerase alpha but did not inhibit DNA polymerase delta, while the murine polyclonal antisera to polymerase delta inhibited delta but not alpha. These findings establish the existence of DNA polymerase delta in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.