Enzyme-labeled Antibody Method Research Articles

Acquired Fanconi syndrome due to long-term adefovir administration in a patient with IgG-kappa monoclonal gammopathy and kappa Bence-Jones protein.

A 77-year-old man was admitted to our hospital for a right femoral neck fracture. He had been prescribed lamivudine for chronic hepatitis B infection for 11years, and adefovir was added 5years ago. After hospitalization, a right femoral head prosthesis was performed successfully, but an unknown hypokalemia was revealed. Hypophosphatemia, hypouricemia, glucosuria, and panaminoaciduria were also revealed, and multiple microfractures were detected by bone scintigraphy. We diagnosed him as 'osteomalacia associated with Fanconi syndrome,' which was likely due to the adefovir. Moreover, a monoclonal IgG-kappa and a kappa Bence-Jones protein were detected in his serum and urine, respectively. We switched from adefovir plus lamivudine to entecavir and started calcitriol. His excessive urinary β2-microglobulin excretion and glucosuria had decreased dramatically at 10weeks after the modification of drugs; those of the phosphate, uric acid and total protein, however, continued. Renal biopsy specimens obtained at 10weeks after discontinuation of adefovir revealed focal tubular atrophic changes with/without inflammatory cells, which were predominantly observed next to glomeruli. Kappa-dominant staining was not observed in either glomeruli or tubules with immunostaining by the enzyme-labeled antibody method. Electron microscopy revealed neither crystalline structures in the cytoplasm of proximal tubules nor electron-dense deposits. Because of the remarkable proportional reduction of other urinary protein fractions, urinary M-peak appeared 26weeks after discontinuation of adefovir, but the net amounts of the fraction decreased gradually.

Localisation of RA175 (Cadm1), a cell adhesion molecule of the immunoglobulin superfamily, in the mouse testis, and analysis of male infertility in the RA175-deficient mouse

RA175, a member of the immunoglobulin superfamily, plays an important role in cell adhesion, and RA175 gene-deficient mice (RA175(-/-) ) show oligoastheno-teratozoospermia. To understand the function of RA175, location in the testis and the morphological features of its spermatogenic cells in RA175(-/-) mice were investigated. Immunohistochemical studies revealed that RA175 immunoreactivity was observed on the cell surface of the spermatogenic cells at specific stages. A strong reaction was detected from type A spermatogonia to pachytene spermatocytes at stage IV and from step 6 to step 16 spermatids during spermatogenesis. From pachytene spermatocytes at stage VI to step 4 spermatids, the reaction was not detected by the enzyme-labelled antibody method and was faintly detected by the indirect immunofluorescence method. Abnormal vacuoles in the seminiferous epithelium, showing exfoliation of germ cells, and ultrastructural abnormality of the elongate spermatids were revealed in the RA175(-/-) testes. Other members of the immunoglobulin superfamily such as basigin, nectin-2 and nectin-3, which have an important role in spermatogenesis, were immunohistochemically detected in the RA175(-/-) testis. These observations indicate a unique expression pattern of RA175 in the testis and provide clues regarding the mechanism of male infertility in the testis.

NO production in RAW264 cells stimulated with Porphyromonas gingivalis extracellular vesicles

This experiment was carried out in order to prove the inducible nitric oxide synthase (iNOS) expression and the nitric oxide (NO) production in mouse macrophage cells (RAW264) which were stimulated by vesicles released from Porphyromonas gingivalis, and discussed about the role of vesicles in advance periodontal diseases. Production of NO(2) (-) in RAW264 cells was investigated after 0, 1, 3, 6 and 12h of stimulation with P. gingivalis vesicles. NO was analyzed by HPLC-based flow reactor system with Griess reagent. The cells stained by the enzyme-labeled antibody method, after being stimulated with vesicles for 12h. The iNOS proteins, which were expressed in RAW264 cells after 12h of stimulation with vesicles, were detected by western blot. When stimulated with vesicles from W83 and from ATCC33277, the RAW264 cells produced NO, but cell proteins that came in contact with the vesicles were degraded by protease activities in vesicles. When stimulated with vesicles from gingipain-deficient mutant strain KDP136, the RAW264 cells produced NO, but the quality was about 60%, compared with the vesicles from ATCC33277. The results suggest that vesicles are not only just a part of bacterial component, but also are a toxic complex of lipopolysaccharide and protease, and one of the putative virulence factor for periodontal diseases that continue inflammation and cause chronic conditions.

Primary culture of cells derived from human myometrium: both myometrium and cultured cells stained with desmin by the enzyme-labeled antibody method.

To evaluate the characteristics of myometrial cells, we studied primary cultures of cells obtained from uterine muscle. The myometrium was cut into small pieces and suspended in a trypsin solution. The primary cell culture, as a monolayer, could be passaged four times. To confirm that the cultured cells were derived from myometrium, both the myometrium and the cells were stained with desmin by the enzyme-labeled antibody method. These were all positive. As a result, we confirmed that the cultured cells were derived from myometrium.

Protein localization in wax-embedded and frozen sections of bone using immunohistochemistry.

AbstractImmunohistochemistry can provide valuable information regarding protein expression in different cell types at specific stages of differentiation during bone modeling and remodeling. By combining immunohistochemistry with other techniques, it is possible for the researcher to determine protein expression, in relation to mRNA production, enzyme activity and bone remodeling, on the same sample of bone. This chapter covers the localization of protein in human bone by immunohistochemistry using an indirect immunoperoxidase method, and considers both frozen and wax-embedded sections. Methods for immunostaining of plastic embedded tissue can be found in the chapter by Van Leeuwen and Derkx, this volume. Immunohistochemistry is based on incubating high-affinity antibodies on tissue sections to detect patterns of expression for specific antigens within the tissue. This can be achieved using either an immunofluorescence-based technique in which the antibody is conjugated with a fluorochrome, or an enzyme-labeled antibody method. The latter procedure has several advantages; it utilizes the same enzyme complexes for all primary antisera irrespective of its origin in different animal species; stained sections may be permanently mounted; and sections can be viewed using brightfield microscopy. Detection methods have increased the sensitivity of immunohistochemistry. There are now several ways that the signal of antibody-antigen binding can be amplified, thus allowing the use of only small amounts of antibody.KeywordsBone SampleBrightfield MicroscopyBone SectionEthylenediamine Tetraacetic Acid Disodium SaltIndirect Immunoperoxidase MethodThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Analysis of Prognostic Predictors in Idiopathic Membranous Nephropathy

We studied clinical and histologic parameters at the time of renal biopsy of 19 patients with idiopathic membranous nephropathy (IMN) to investigate the predictors for prognosis of IMN. Nineteen patients diagnosed by open renal biopsy between 1988 and 1993 and followed for at least 5 years were divided into two groups according to the latest follow-up renal function. Group I included 16 patients with normal renal function at the last follow-up point. Group II included three patients with end-stage renal failure at the last follow-up point. Antibodies to CD68, CD45RO, α-SMA, collagen IV, and collagen VI were used to investigate glomerular and interstitial changes in biopsy specimens by the indirect enzyme-labeled antibody method. Degree of global glomerulosclerosis, segmental glomerulosclerosis, adhesion to Bowman's capsule, and crescent formation were evaluated by light microscopy (periodic acid–Schiff, periodic acid-metheramine [PAM] staining). The difference between the two groups was analyzed by Mann-Whitney U test. The number of interstitial cells, the number of interstitial CD45RO-positive cells, and increases of interstitial collagen IV and VI were found to be the most important factors for prognosis of IMN. These findings suggest that the extent of tubulointerstitial changes (cellular infiltration and fibrosis) determines the prognosis of renal function in IMN.

<I>Actinobacillus pleuropneumoniae</I>による子豚の後躯麻痺

Two piglets aged 2 months in an integrally-operated swinery presented posterior trunk palsy and astasia. Autopsy revealed an abscess 3cm in diameter between the thoracic vertebral column and pleura, reaching the vertebral canal and compressing the medulla, which was curved with hemorrhage and neuronal degeneration and necrosis. Actinobacillus pleuropneumoniae (Ap) serotype 2 was isolated from the abscess. Neutrophil and lymphocyte infiltration was seen in some alveoli in the lungs. Ap serotype 2-specific antigen was detected inthe abscess and pulmonary lesions by enzyme-labeled antibody method. Antibody against Ap serotype 2 was detectable in not only the two diseased cases but also all other piglets reared in the same swinery.

Lysozyme localization in human gastric and duodenal epithelium. An immunocytochemical study.

The distribution of lysozyme in normal gastric and duodenal mucosa was studied by light- and electron-microscopic immunocytochemical techniques (direct enzyme-labeled antibody method). In the duodenal mucosa, lysozyme was found in the Paneth cells and the epithelial cells of Brunner's glands. Electron-microscopically, lysozyme was found in rough endoplasmic reticulum and perinuclear spaces, which were assumed to be protein-synthesizing organelles, and also in the secretory granules of Paneth cells. Additionally, lysozyme was detected in the stomach in mucinous granules and in some parts of the rough endoplasmic reticulum within the epithelial cells of the pyloric glands, the mucous neck cells of the fundic glands, and in several surface epithelial cells of the plyoric and fundic regions. This suggests that some quantity of lysozyme in gastrointestinal secretion originates from the gastric and duodenal glands, and that it acts as a defense mechanism in the gastrointestinal tract.

LIVER METASTASIS AND BONE METASTASIS IN THREE CASES OF RECTAL CARCINOID

In Japan, rectal carcinoid has so far been regarded as a disease of comparatively low incidence and as benign in most cases. Up to 1982, however, 25 cases of metastatic carcinoid had been reported.During the past 10 years, we have experienced 7 cases of rectal carcinoid, in 3 of which liver and bone metastasis wea present.Neuron-specific enolase stain was positive in the 6 cases of rectal carcinoid examined by the enzyme-labeled antibody method. We discuss these 7 cases with additional discussion of the rectal carcinoid-producing mother cell.

Characteristic Differences in Immunohistochemical Localization of New Placental Proteins (PP1, PP19, PP21) in the Human Placenta

The immunohistochemical localization in the human placenta of new placental proteins PP1, PP19, and PP21 was clarified using modified indirect enzyme-labeled antibody method and compared with that of pregnancy-specific beta 1-glycoprotein (SP1). The major results are as follows: positive staining for PP1 was seen at the nucleus and cytoplasm of villous cytotrophoblasts, the X cells at the basal plate, and of chorionic trophoblasts, while the decidua cells and amnion were not stained. PP19 was characteristically seen in the nucleus and cytoplasm of syncytiotrophoblasts. X cells in basal plate, chorionic trophoblasts, and maternal leukocytes. The villous cytotrophoblasts, decidua cells, and amnion were not stained. PP21 localization was found at the microvilli and basal membrane of syncytiotrophoblasts and at the cytotrophoblast plasma membrane of the chorionic villus in early gestation. In late gestation, increased staining was seen at the syncytiotrophoblast microvilli and the villous basement membrane, and moderate staining at plasma membrane of the amniotic epithelium and chorionic trophoblasts. SP1 was found only at the syncytiotrophoblast cytoplasm of chorionic villi. Studies using these four placental proteins simultaneously may therefore provide a new key learning about unknown metabolic functions of trophoblasts.

Detection of the cholestatic factor in the liver tissue of patients with acute intrahepatic cholestasis.

A novel lymphokine, which we have designated as cholestatic factor (CF), was produced from peripheral blood lymphocytes of patients with drug-induced allergic intrahepatic cholestasis by stimulation with a causative drug in the presence of the liver soluble fraction containing liver-specific lipoprotein (LSP). Marked reductions in bile flow and bile acid excretion were induced in rats by injecting CF through a mesenteric vein. In order to confirm the presence of CF in the liver tissue of patients, we attempted to detect this lymphokine by using the enzyme-labelled antibody method. As a result, CF was found in the liver tissue of eleven out of thirty-eight patients with acute intrahepatic cholestasis including one with hepatitis A type, one with hepatitis B type, two with hepatitis non-A non-B type, five with drug-induced allergic hepatitis, one with alcoholic hepatitis and one with lupoid hepatitis. In contrast, CF was undetectable in the liver tissue of patients without intrahepatic cholestasis. These results may additionally support our assumption that CF plays an important role in the induction of intrahepatic cholestasis in various liver diseases.

Accumulation of immunoreactive opsin on plasma membranes in degenerating rod cells of rd/rd mutant mice.

Immunoreactive opsin was detectable in the apical portion of normally developing photoreceptor cells on postnatal day 3 by the indirect enzyme-labeled antibody method. Immunoreactivity increased and had extended from the central retina to the periphery by the advanced stages of development. In the rd mutant retinas, accumulated opsin was present in the apical portion and in the outer nuclear layer on postnatal day 8. Immunoreactive opsin mainly was present in the outer nuclear layer by day 14, even being detectable on day 28. No immunoreactivity was present in the remaining cones. Electron microscopic immunocytochemistry confirmed the association of immunoreactive opsin with the persistent rod cell plasma membrane. Molecular weight of immunoreactive opsin in 14-day-old rd mutant mouse retina, as estimated by gel filtration chromatography, was large and did not seem to be degraded. These findings indicate that accumulated rhodopsin continues to function in the plasma membrane because an electroretinogram could be made after day 14 for the rd mutant mouse retina.

Immunocytochemical and enzymecytochemical studies on the intracellular transport mechanism of secretory immunoglobulin A and lactoferrin in human salivary glands.

The intracellular transport mechanism of secretory immunoglobulin A (sIgA) has been immunocytochemically defined in human submandibular glands. To examine the properties of the intracytoplasmic vesicles which contain IgA, the enzyme labeled antibody method for SC and IgA and Novikoff's method for acid phosphatase (ACPase) activity were employed on the same sections. The intracytoplasmic vesicles containing IgA and SC in the serous acinar cells were free of ACPase activity and were thus distinguishable from lysosomes. Neither IgA nor SC was localized in the secretory granules. Lactoferrin was localized in the secretory granules and glandular lumen of the serous acinar cells, but not in the cytoplasmic vesicles, which were also free of ACPase activity. These findings suggests that the transport of sIgA was performed by intracytoplasmic vesicles and that lactoferrin is discharged from secretory granules into the lumen and finally makes a "rendezvous" with sIgA in the lumen of acinar cells.

Morphological and immunocytochemical study of rat pancreatic beta cell changes induced by cyclizine.

Wistar rats were treated daily with cyclizine (50-75 mg kg-1) and autopsied every week until the eighth week of the treatment. Although no evident change could be detected by either serum analysis or by light microscopy from the first week to the fourth week, electron microscopy revealed that beta cells showed depletion of secretory granules and cystic dilation of the rough endoplasmic reticulum from the first week. A significantly higher level of plasma glucose and a lower level of plasma insulin than those of untreated rats were recognized from the fifth week. A diabetic pattern was also observed in the glucose tolerance test which was conducted at the seventh week. From the sixth week, large cytoplasmic vacuoles were observed light microscopically in islet cells. Electron microscopic examination revealed that the vacuoles originated from the rough endoplasmic reticulum in beta cells, while no prominent change was found on the Golgi apparatus. In addition to the above findings, immunocytochemical study with anti-insulin serum demonstrated that the staining intensity of the beta cell cytoplasm became weaker (as judged by light microscopy) from the fifth week, but large cytoplasmic vacuoles were stained positively under both the light microscope (PAP method) and the electron microscope (enzyme-labelled antibody method). These findings suggested that the depletion of secretory granules and accumulation of insulin or its precursor in the rough endoplasmic reticulum occurred in beta cells. The diabetogenic effect of cyclizine on rats is discussed with reference to the immunocytochemical findings.

Combined application of monoclonal antibody to human la antigens and avidinblotin-peroxidase staining method in immunoelectron microscopy

Immunological detection of cellular and tissue antigens have been based on the widely used original enzyme labeled antibody method or the unlabeled antibody, peroxidase-anti-peroxidase (PAP) method. Recently, an improved immunoenzymatic technique for light microscopy using the avidin-biotinperoxidase complex (ABC) system was developed for cell and tissue staining, and found to be superior to the PAP technique in terms of sensitivity and specificity. The application of the ABC method in immunoelectron microscopy (IEM), however, has not been extensively evaluated. This study demonstrates that the ABC method is suitable for localizing specific cellular antigen at the ultrastructural level with monoclonal antibody (MAb), the production of which by somatic cell hybridization is now a well established procedure.

Immunocytochemical localization of T antigen in cells of BK virus-induced hamster brain tumor.

The localization of both the large T and small t tumor (T) antigens in cultured cells (Vn 12 cells) of hamster brain tumors induced with BK virus (BKV), a new human papovavirus, was studied by an enzyme labelled antibody method at both the light and electron microscopic levels. Under the light microscope, BKV T antigen was observed in the nucleus, except for the nucleoli, of cells in interphase, and under the electron microscope it was observed in the nucleus except for the nucleoli and nuclear membrane. BKV T antigen appears to be closely associated with nuclear chromatin as previously reported for simian virus 40 tumor antigen (SV40 T antigen). The intracellular localization of BKV T antigen was the same as that of SV40 T antigen. In metaphase, BKV T antigen seems to be distributed diffusely throughout the cytoplasm except for the chromosomes. In telophase, BKV T antigen transfers from the cytoplasm to the nucleus. The migration of BKV T antigen during the cell cycle is thought to be related to the function of T antigen.

Immunocytochemical approach to virus-induced brain tumors - from the aspect of T (tumor) antigen.

Recently isolated human papovaviruses such as JC and BK viruses are not only antigenically related to SV40, but also neuro-oncogenic in experimental animals. Cells transformed in vitro or brain tumors induced by these viruses are usually free of infectious virus. However, the causual relationship between the tumor or transformed cells and the viruses can be immunocytochemically demonstrated by the presence of virus-specific non-virion antigens such as T (tumor) antigen. In the present study, the authors successfully applied a sensitive enzyme-labelled antibody method using Fab' fragment to localizing T antigens in SV40 or BK virus-induced brain tumors and infected cells. T antigens of SV40 and BK virus are immunocytochemically indistinguishable. Positive staining for T antigen is seen as a granular pattern in the tumor cell nuclei, but not the nucleoli, in the interphase and it appears as a loose network of electron-dense reaction precipitates associated with nuclear chromatin, whereas the chromosomes in mitosis do not seem to be tagged with immunoperoxidase reaction products for T antigens. On the other hand, T antigen is first observed in the cytoplasm of SV40-infected cells as early as 3 hours postinfection with subsequent transport to the nuclei within 24 hours. The intensity of staining of T antigen is markedly reduced because of maturation of virions in the infected cell nuclei 24 hours after infection. The authors believe that extensive studies of papovavirus T antigens are necessary to elucidate papovavirus oncology and may provide more information regarding possible viral etiology of human tumors of the central nervous system.

Newly developed techniques of enzyme-labelled antibody method

newly developed techniques of enzyme-labelled antibody method

Light and electron microscopical combined staining by immunohistochemical and enzyme histochemical methods using rat prolactin-secreting pituitary tumours.

Combined immunohistochemical staining (IHCS) and enzyme histochemical staining (EHCS) methods for light microscopy (LM) and electron microscopy (EM) are reported, using oestrogen-induced rat pituitary tumours. For LM, combined staining for alkaline phosphatase and acid phosphatase by EHCS, using the azo dye method, and for prolactin and ACTH by IHCS, using the enzyme-labelled antibody method, gave the best results on 1 microgram glycol methacrylate sections. For EM, combined staining by EHCS on 30 microgram tissue sections followed by IHCS for prolactin on ultrathin Epon sections (enzyme-labelled antibody method) provided acceptable results. By these combined staining methods, the neoplastic prolactin cells were shown to have close affinity to rich alkaline phosphatase-positive capillaries and to possess an alkaline phosphatase-positive cell membrane. Furthermore, they revealed acid phosphatase-positive lysosomal and secretory granules. These combined staining methods may be valuable in studies on the actual functional status of cells.

Clinical applications of the enzyme labeled antibody method. Immunoperoxidase methods in diagnostic histopathology.

Histological criteria for the definition of disease entities have largely been established with light microscopy of conventionally stained and routinely processed tissue sections. More or less specific histochemical staining procedures and more recently enzyme-histochemical and quantitative histo- and cytochemical techniques in some cases provided additional criteria. In the last decade, however, the introduction of immunofluorescence and more recently the different immunoperoxidase methods have significantly influenced the scope of contemporary histopathology. Especially, the possibility to use immunoperoxidase methods on routinely processed tissue specimens has offered new dimensions in diagnostic pathology. These methods proved of particular importance for: 1) The development of new criteria for diagnosis, prognosis and treatment (e.g. immunological classification of lymphoma; plasmacell typing in intestinal inflammatory conditions; human chorionic gonadotropin and alpha-fetoprotein in germ cell tumors of the testis). 2) The possibility of etiological diagnosis (e.g. the recognition of hepatitis B viral antigens in liver biopsy specimens; histological typing of causative micro-organisms in inflammatory conditions). 3) The recognition of disease entities that were hitherto unrecognized (e.g. hyperplasia of parafolicular C-cells in the thyroid in multiple endocrine neoplasia syndromes; gastric G-cell hyperplasia as a variant of Zollinger-Ellisons syndrome). 4) Functional analysis of tissue components (e.g. hormone content of pituitary and pancreatic adenomas; cytoplasmic differentiation produces in "undifferentiated" tumors). It can be expected that immunoenzymehistochemistry will soon play a major role in routine diagnostic histopathology.