Microphotometric quantitation of the reaction product of several indirect immunoperoxidase methods demonstrating monoclonal antibody binding to antigens immobilized on nitrocellulose

The nitrocellulose model and microphotometry were used to investigate whether in immunoperoxidase cytochemical methods the amount of final reaction product reflects the amount of cell surface antigen. The results obtained with four cytochemical peroxidase methods, i.e., those using diaminobenzidine/H2O2 (DAB/H2O)2, DAB/H2O2/COCl2, DAB/H2O2/imidazole, and silver intensification of the DAB end product, were compared first. The quantitative DAB/H2O2/imidazole method proved to be the most sensitive and was selected for further studies. Cell surface antigens prepared by solubilization of peritoneal macrophages with octyl-beta-D-glucopyranoside were immobilized on nitrocellulose. Monoclonal antibody binding to these cell antigens was detected by peroxidase immunocytochemistry. Comparison of the sensitivity of the indirect immunoperoxidase and the biotin-(strept)avidin immunoperoxidase methods on the basis of the highest detectable dilution of a cell lysate showed that these methods were equally sensitive. A linear relationship between the absorbance of the peroxidase reaction product and the amount of cell lysate immobilized on nitrocellulose was found for all three indirect immunoperoxidase methods. This proves that the amount of final immunocytochemical peroxidase reaction product is proportional to the amount of antigen in cell lysates. However, the relative expression of antigens in intact cells differs from that in cell lysates. Therefore, the present method to solubilize cells and immobilize cell antigens cannot be used to quantitate the antigen content of cells.     

Luminescent detection method for immunodot, Western, and Southern blots

An anti-peroxidase-anti-biotin hybrid hybridoma rat cell line, capable of producing a bispecific monoclonal antibody, has been derived to explore its use in conjunction with a luminol immunodetection system. Luminescence was detected using x-ray film. The method was sufficiently sensitive and effective, but was less sensitive than autoradiographic methods using high-specific-activity 32P-labeled probes. Exposure times, on the other hand, were of the order of seconds rather than days. The direct binding of both peroxidase and biotin by the bispecific monoclonal antibody is simpler but less sensitive than the more conventional indirect method using a commercial peroxidase coupled with anti-rat antibody as a developing antibody.     

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.     

Immunocytochemistry of cerebrospinal fluid

In order to determine how best to study cells in cerebrospinal fluid (CSF) by immunocytochemical techniques, several crucial technical variables and five immunocytochemical methods were examined. Immunocytochemical studies could be performed on either cell suspensions or smears. The method using cell suspensions was more sensitive, producing less background staining, but requiring more cells than that using smears. Among the five methods examined, indirect immunoperoxidase (IP) and indirect immunoalkaline phosphatase (IAP) were comparable in sensitivity. The peroxidase-antiperoxidase (PAP), alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin complex-immunoalkaline phosphatase (ABC-AP) methods were comparable in sensitivity and were more sensitive than either the IP or IAP technique. The peroxidase methods were plagued with problems related to endogenous enzyme activity and the ABC-AP method may exhibit undesirable background staining. Therefore, the IAP method should be used for cell suspensions and the APAAP for cells on smears. In CSF specimens with a small number of cells, immunocytochemical studies should be done on smears by the APAAP method. These conclusions are supported by our experience with CSF specimens from patients with reactive and neoplastic lymphocytoses.     

Identification of Mallory bodies with rhodamine B fluorescence and other stains for keratin

Rhodamine B staining in conjunction with fluorescence microscopy is shown to demonstrate Mallory bodies. Mallory body morphology, localization, and distribution in hepatocytes from griseofulvin-fed mice, human hepatoma, and human alcoholics were similar to those observed in the same tissues after conventional staining methods for Mallory bodies. The presence of these inclusions was further confirmed by specific cytochemical localization with indirect immunoperoxidase labeling, horseradish peroxidase labeling, and electron microscopy. Other tinctorial or histochemical procedures previously used for keratin or prekeratin (modified Mallory stain, Kreyberg method, Pauly method for histidine) also stained Mallory bodies for study with white light microscopy but with decreasing sensitivity respectively. Mallory bodies from mouse and human liver both appear to contain a keratin-like moiety. This entity may be simply, rapidly, and permanently stained with rhodamine B, and selectively and reproducibly demonstrated with fluorescence microscopy.     

The value of epithelial membrane antigen in the diagnosis of ovarian tumors.

The value of epithelial membrane antigen (EMA) in the diagnosis of ovarian tumors was investigated using an indirect immunoperoxidase staining technique on 91 histologic sections (88 tumors and 3 normal tissues) and 39 ascitic fluid smears (28 from patients with epithelial ovarian tumors and 11 from cases of myoma uteri). The rate of positive EMA staining was highest in malignant tumors (89.2%), second highest in tumors of low malignant potential (33.3%) and lowest in benign tumors (25.0%); normal ovarian tissues were negative for EMA. Of the malignant tumors, all 48 serous cystadenocarcinomas (100%) and 18 of 26 mucinous cystadenocarcinomas (69.2%) stained positively for EMA. In serous cystadenocarcinomas, the EMA staining was mainly localized on the luminal membrane of cells in well-differentiated tumors, but appeared on the entire cell surface and cytoplasm of cells in poorly differentiated tumors. The results of EMA staining on ascitic fluid smears were almost the same as the results for the histologic sections. The intensity and the localization of EMA staining were related to the grade of malignancy in these ovarian tumors. In comparison with staining for other antigens (carcinoembryonic antigen, CA-125 and human keratin protein), EMA was found to be one of the most sensitive markers for the diagnosis of ovarian cancer.     

Human ovarian surface epithelium in primary culture

Summary The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17β-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17β-HSD activity, and presence of keratin. Supported by a grant and a research associateship to N. A. by the National Cancer Institute of Canada.     

Human ovarian surface epithelium in primary culture

The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17 beta-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17 beta-HSD activity, and presence of keratin.     

GDF15基因在卵巢上皮性癌组织中的表达及其临床意义

目的:探讨生长分化因子GDF15(Growth Differentiation Factor 15)基因在卵巢上皮性癌组织中的表达及其与铂类耐药的相关性。方法:应用免疫组化、western blot、RT-PCR等方法对80例原发性卵巢癌组织和卵巢癌顺铂敏感/耐药株A2780和CP70、SKOV3和SKOV3/DDP中生长分化因子GDF15表达水平进行测定。结果:生长分化因子GDF15的表达强度与卵巢癌铂类耐药性显著相关。在卵巢癌顺铂耐药株CP70、SKOV3/DDP中GDF15表达水平较顺铂敏感株A2780、SKOV3明显增高。结论:GDF15表达水平与卵巢癌发生发展及铂类耐药相关,对于卵巢癌患者早期筛选、预测预后具有一定的临床指导价值。     

A Simultaneous Visualization of the Antioxidant Enzymes Glutathione Peroxidase and Catalase on Polyacrylamide Gels

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (I) running samples on a 7. 5% polyacryla-mide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25-2.0 mM). (3) soaking the gel in GSH plus H_zO_z or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride I% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase and catalase was 0.25rnM; I.5mM glutathione was the best concentration for visualization of glutathione peroxidase alone. The method is sensitive enough to detect catalase and glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase. glutathione peroxidase is heat resistant (68°C. 1min), but sensitive to 10mM sodium iodoacetate.     

Efficiency and sensitivity of indirect immunoperoxidase methods

The peroxidase-antiperoxidase (PAP) complex method has repeatedly been claimed to be more sensitive and antibody efficient than the indirect peroxidase labeled antibody method. However, most studies comparing these methods used tissue sections as the test material. However, test systems with known amounts of antigen will allow more reliable comparison of these methods and quantitative evaluation of method sensitivity. We therefore compared the antibody efficiency and sensitivity of these methods for the detection of human chorionic gonadotropin in an enzyme linked immunosorbent assay (ELISA), an antigen spot test (AST) and tissue sections of choriocarcinoma. In the PAP technique rabbit PAP and goat anti-rabbit antibody were applied. The same antibody was peroxidase-labeled with the periodate technique and used in the labeled antibody method. In the ELISA the PAP method resulted in slightly higher antibody efficiency than the labeled antibody method. At low primary antibody dilutions the intensity of the reaction decreased with the PAP method but remained high with the labeled antibody method, in the ELISA as well as on tissue sections. In the AST the labeled antibody method and the PAP method appeared to be equally sensitive.     

Sensitive fluorometric assays for glutathione peroxidase and reductase

A microfluorometric adaptation of the method of D. E. Paglia and W. N. Valentine has been made which can assay glutathione peroxidase activity in less than 100 μg of tissue. As in the original method, the oxidized glutathione produced in the reaction is coupled to the oxidation of NADPH by glutathione reductase. No inhibition by NADPH was found. A similar method can be used to measure glutathione reductase. These methods have been used to assay glutathione peroxidase and reductase in rat brain and in a neuronal and a glial cell line using samples containing 15 μg of protein. The assays are sensitive enough to allow multiple determinations of the enzymes in brain regions, organotypic tissue cultures, and microwell cell cultures.     

A Simultaneous Visualization of the Antioxidant Enzymes Glutathione Peroxidase and Catalase on Polyacrylamide Gels

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (I) running samples on a 7. 5% polyacryla-mide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25–2.0 mM). (3) soaking the gel in GSH plus H_zO_z or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride I% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase and catalase was 0.25rnM; I.5mM glutathione was the best concentration for visualization of glutathione peroxidase alone. The method is sensitive enough to detect catalase and glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase. glutathione peroxidase is heat resistant (68°C. 1min), but sensitive to 10mM sodium iodoacetate.     

Detection of trophoblastic beta 1-glycoprotein in tumor tissue and blood in ovarian neoplasms

Using immunochemical analysis methods (the reaction of precipitation in agar, immunoenzymatic method, immunofluorescence), trophoblastic beta 1-glycoprotein (TBG) concentration in tumour tissue and in the blood serum of patients with ovarian cancer was studied. By rabbits immunization with glycoprotein fraction of ovarian adenocarcinoma, dissolved in 0.6 M sulfosalicylic acid, the authors obtained antibodies to TBG. Immunoenzymatic method showed, that TBG level is raised during ovarian cancer (more than 3 micrograms/l): in 18% of tumour extracts, in 12.5% of blood sera samples and in 41.6% of cases in ascites fluid. Utilizing indirect immunofluorescence method morphological structures of trophoblastic type were identified in paraffin sections of ovarian adenocarcinoma. The authors suppose, that such structures may be responsible for TBG biosynthesis in ovarian tumours.     

Azo dying of α-keratin material improves microbial keratinase screening and standardization

Microbial conversion through enzymatic reactions has received a lot of attention as a cost-effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo-dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well-known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure-based assays (3-fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.     

蝶蛹金小蜂卵黄蛋白单克隆抗体的制备及其应用方法的建立

为深入研究寄生蜂卵黄发生及其内分泌调控,特采用杂交瘤细胞技术,制备4株能稳定分泌抗蝶蛹金小蜂Pteromalus puparum卵黄蛋白（vitellin, Vt）的单克隆抗体（mAb）,即PpVt mAb1,PpVt mAb2,PpVt mAb3和PpVt mAb4。这4株单克隆抗体的重链和轻链的亚类均分别为IgG1和κ类型,不仅特异性识别Vt,而且识别雌蜂血淋巴中卵黄原蛋白（vitellogenin,Vg）,但与雄蜂体液无反应。通过比较4种不同的ELISA方法,确定了微量检测蝶蛹金小蜂体内Vg/Vt的最适ELISA法,即双夹心ELISA法。该方法可用于单头雌蜂体内Vg/Vt的检测,其检测灵敏度为20 ng/mL。用Western 免疫印迹的方法证实了该蜂Vg的合成始于刚羽化的成虫,并在羽化后12～36 h内含量达到高峰。     

Colour development of immunogold-labelled antibodies for light microscopy

Summary We describe an improvement of the immunogold technique, which is based on the colour development of silver-intensified immunogold signals. This method (referred to as the coloured-SIG technique) was found to be as sensitive as the silver-intensified immunogold method and more sensitive (in two of the three tested systems) than immunoenzymatic procedures, such as the peroxidase/antiperoxidase technique or the avidin-biotin system. The coloured SIG-method results in either a magneta-red or a cyan-blue final reaction product. Therefore, this new improvement of the immunogold technique should be useful in (1) double-staining methods, (2) immunoblot methods and (3) conventional immunostaining methods.Supported by the Robert-Bosch-Foundation, Stuttgart     

Resolution of optical isomers by chiral high-performance liquid chromatography: separation of dihydrodiols and tetrahydrodiols of benzo[a]pyrene and benz[a]anthracene

A competitive enzyme-linked immunoassay (CELIA) for human serum angiotensin-1-converting enzyme (ACE) was developed. The sensitivity was amplified by using a secondary antibody and an avidin biotin-conjugated horseradish peroxidase complex as the enzyme-labeled reagent. This configuration was compared to three other configurations for an indirect CELIA, and was found to be the most sensitive. A sensitivity of 39 ng/ml ACE was achieved with intraassay and interassay coefficients of variation of 6.3 and 8%, respectively. CELIA will detect ACE in human serum without interference from either pharmacological or endogenous ACE inhibitors. In normal human volunteers, ACE values obtained using CELIA correlated well with values obtained by enzymatic assay.     

Comparative study of procedures for histological detection of lectin binding by use of Griffonia simplicifolia agglutinin I and gastrointestinal mucosa of the rat

The histological localisation of alpha-D-galactopyranosyl residues in glycoconjugates of rat stomach and duodenal mucosae was studied by use of Griffonia simplicifolia agglutinin I, i.e. the isolectin mixture (A + B) and the isolectin B4 (B4). Cryostat sections which were either unfixed or acetone fixed and paraffin sections from both ethanol-acetic acid and formaldehyde fixed tissue blocks were compared. Cellular details were better preserved in paraffin than in cryostat sections. Reactivity of cells binding GS I was less sensitive after formaldehyde than after ethanol-acetic acid fixation inasmuch as higher concentrations of lectins were needed. This drawback could be overcome by trypsinisation of the sections. The binding pattern of GS I (A + B) corresponded with that of GS I (B4) in either cryostat or paraffin sections. GS I was detected in the cytoplasm of parietal cells and in Brunner's gland cells. In duodenal crypts and villi, lectin was bound to supranuclear regions in the cytoplasm of columnar and goblet cells. The staining efficiency of fluorescein (FITC), horseradish peroxidase (HRP) and colloidal gold particle (CGP) labels in both direct and indirect lectin stainings was compared. Under all experimental conditions, indirect methods required lower concentrations of lectins than direct ones; indirect procedures increased sensitivity about 5-10 fold. CGP labels were always of highest sensitivity when gold particles were further developed by a silver precipitation method. HRP was not as efficient in lectin localisation as CGP, but cytochemical staining was more convenient in routine work. Direct FITC labellings proved to be of lowest sensitivity.     

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)