Use Of Polyclonal Antiserum Research Articles

Monoclonal antibody and its use in the diagnosis of livestock diseases

Since the discovery of hybridoma cells by Kohler and Milstein, the uses of monoclonal antibody (mAb), the protein produced by such cells are in vogue. Such antibodies with single isotype have higher specificity, and the serological tests employed in show higher reproducibility compared to those with use of polyclonal antisera. There are several procedures of mAb production which vary considerably but the principle remains the same which states that antigens introduced into animals generally result in the stimulation of lymphocytes, some of which produce antibody of only one type, although the isotype may change. The developments in the field of cell culture and transfection technology have lead to the production of improved qualities of mAbs. In general, monoclonal antibodies are important reagents used in biomedical research, such as, in the field of diagnostics and therapeutics as well as targeted drug delivery systems. They have got importance not only for infectious diseases caused by microbes and parasites, but also for cancer, metabolic and hormonal disorders, in the diagnosis of lymphoid and myeloid malignancies and tissue typing, enzyme linked immunosorbent assay (ELISA) (especially blocking ELISA), radio immunoassay (RIA), serotyping of pathogens and their immunological intervention with passive antibody, anti-idiotype inhibition or magic bullet therapy with cytotoxic agents coupled with antimouse specific antibody. The application of mAbs in diagnosis of various livestock diseases is an important area of concern as these diseases are a major and increasingly important factor reducing livestock productivity in various parts of the world. In this context, the application of mAbs for diagnosis of important bacterial diseases viz., Anthrax, Brucellosis, Paratuberculosis, Leptospirosis, Listeriosis, Clostridial infections and Mycoplasmosis (CBPP), fungal diseases viz., Zygomycosis, Cryptococcosis, Histoplasmosis and Paracoccidiodomycosis, viral diseases viz., Foot-and-mouth disease (FMD), Infectious bovine rhinotracheitis/Infectious pustular vulvovaginitis (IBR/IPV), Rota viral diarrhoea, Blue tongue, Rabies, Classical swine fever and re-emerging viral diseases like Hendra and Nipah viral infections and parasitic diseases viz., dirofilariosis, and Trichinellosis and haemportozoan diseases (including Trypanosomiasis, Leishmaniasis, Anaplasmosis, infections caused by Plasmodium spp. as well as tick borne diseases) have been discussed thoroughly along with the specifications of the diagnostic assays for each disease for the convenience of the diagnosticians, researchers, scientists and students to employ such assays, both in field and laboratories to strengthen the disease control programme.

Epitope selection to male specific antigens for sex selection in swine

Immunological approaches to gender selection have been contemplated since the discovery of the family of male-specific H-Y antigens found only on the surface of male cells. H-Y antigens are able to elicit an immune reaction when cells or tissues from a male donor are grafted to a female recipient. We describe here the development and testing of an inexpensive approach using polyclonal antibodies against four specific H-Y outer membrane proteins male enhanced antigen 1 (MEA 1), male enhanced antigen 2 (MEA 2), sex determining region Y (SRY) and testis determining factor (TDF). Epitopes based on hydrophilic primary sequences of the proteins were synthesized, N-terminal biotin-labeled, linked to streptavidin and mixed with a Ribi adjuvant prior to immunization in rabbits. The antiserum was tested to determine affinity to swine spermatozoa using anti-motility, flow cytometry and motility and sedimentation chambers. Fluorescent microscopy and fluorescent in situ hybridization (FISH) was used to identify the percentage of motile spermatozoa that contained the Y chromosome. We found that the polyclonal antibodies had high affinity to the spermatozoa leading to a cessation of motility. Furthermore, the majority of these non-motile spermatozoa contained the Y chromosome. We conclude that the use of polyclonal antiserum against synthetic H-Y peptide antigens may be an inexpensive and simple means to inhibit the motility of swine spermatozoa bearing the Y chromosome.

Anti-Infective Strategies of the Future: Is there Room for Species-Specific Antibacterial Agents?

Broad-spectrum antibiotics, directed against conserved bacterial targets, are the mainstay of antibacterial therapy. Increasing resistance, however, demands new strategies. Over time a number of therapeutic concepts have evolved, starting out with the use of polyclonal antisera, which were rapidly replaced by the easier to use antibiotics. Other concepts, such as immunotherapy, radioimmunotherapy, anti-virulence agents, phage therapy and others are under evaluation and often limited in application. In the discovery process of new antibiotics in the pharmaceutical industry quite a number of new agents have emerged, which exhibit a surprisingly high degree of species-specificity. None of them has been considered for development so far. Some examples from the literature which show selectivity for Helicobacter pylori, Pseudomonas aeruginosa, Haemophilus influenzae, Staphylococcus aureus, anaerobes, and others, will be discussed here. It is postulated that there is a room for such agents in future antibacterial therapy, e.g. in difficult to treat infections caused by nonfermenters such as multiresistant P. aerugoinosa, Acinetobacter, Enterobacteriaceae, and S.aureus, including MRSA. Their application would include monotherapy as well as combination therapy with other antibiotics, anti-virulence agents or immunotherapy and these possibilities would greatly expand the current anti-infective armamentarium.

Sarcocystis Falcatula—Associated Encephalitis in a Free-Ranging Great Horned Owl (Bubo Virginianus)

A great horned owl (Bubo virginianus) was admitted to a rehabilitation clinic with severe neurologic signs that were unresponsive to supportive care. The animal was euthanatized because of a poor prognosis. Marked granulomatous encephalitis with focal brainstem malacia was detected microscopically. The brainstem was the most severely affected brain location and the only place in which schizonts and merozoites, morphologically compatible with Sarcocystis spp., were detected. Immunohistochemistry with the use of polyclonal antisera indicated the presence of Sarcocystis falcatula. The species identification of the protozoa as S. falcatula was confirmed by polymerase chain reaction. To the author's knowledge, this is the first report of spontaneous S. falcatula-associated encephalitis in a great horned owl.

Antibody binding to hazelnut (Corylus avellana) proteins: the effects of extraction procedure and hazelnut source

The quality of an applied protein extract is important in both serological and in vivo diagnosis of allergy, and for allergen detection methods. In the present study the effects of the extraction procedure and hazelnut source on antibody binding to hazelnut (Corylus avellana) proteins were investigated. An overnight extraction procedure in tris–glycine buffer at 45°C was compared to a quick, vigorous extraction in a tris buffer at room temperature (RT). Antibody binding was studied by the use of polyclonal antiserum, a monoclonal antibody against the pollen-unrelated allergen Cor a 9, and human IgE from hazelnut allergic individuals. The extraction procedure, as well as the storage time and degree of processing of hazelnuts affected the protein constitution of the extract, as visualised by SDS–PAGE. Fresh and roasted (10 min at 170°C) hazelnuts generally contained a higher amount of intact hazelnut proteins than elder (>6 months) raw hazelnuts stored at RT. The time of storage of raw hazelnuts was inversely related to the amounts of Cor a 9 and IgE-binding proteins. In general, a rapid extraction procedure increased the efficacy of protein extraction and antibody-binding capacity to the proteins compared to an overnight extraction protocol. The present study indicates that for the production of hazelnut protein extracts, the use of fresh hazelnuts is important. A quick, vigorous extraction in a tris buffer might contribute positively, at least for extraction of Cor a 9.

Quantifying aphid predation rates of generalist predators in the field

The community of predators within agroecosystems has the potential to restrict aphid populations, especially early in the season before exponential increases in density and prior to the arrival of specialist natural enemies. Although direct observations of predation, laboratory feeding trials and manipulative field studies have been used to estimate levels of biological control exerted by different species (or potentially negative interactions between them), it is often difficult to extrapolate results to naturally occurring interactions in the field.Over 100 investigations have utilized gut-content analysis to estimate aphid predation rates by predators. Throughout the last century, gut dissection, which enables the visual identification of aphid body parts, has been used in over 50% of studies although accurate identification and quantification of predation is difficult. Other techniques have included radio-labelling of prey, dissection of faecal samples, electrophoresis, stable isotope analysis and use of polyclonal antisera. In recent studies of invertebrate predation, monoclonal antibodies have been the most frequently applied technique but advances in molecular biology have enabled the detection of species-specific DNA sequences. The use of these applications to quantify predation by aphidophagous predators will be reviewed, with emphasis on potential sources of error and difficulties of quantitative interpretation. Despite the considerable focus currently directed towards molecular approaches, antibody-based techniques are likely to remain an important tool for studying predation rates of pests in the field, especially when antibodies have already been developed. However, the study of multiple predation events within complex generalist predator food webs is only likely through the detection of species-specific DNA sequences using molecular techniques.

Photoregulation of Protochlorophyllide Oxidoreductase and Rubisco Large Subunit Accumulation in Phytochrome A-Deficient Transgenic Tobacco Plants

Some morphogenetic responses, induced by far red (FR) light in tobacco plants (Nicotiana tabacum L.), were studied. The inhibitory effect of FR irradiation on chlorophyll synthesis in transgenic plants with reduced phytochrome A content was almost absent. Phytochrome A-mediated repression of the por gene was demonstrated with the use of polyclonal antiserum against protochlorophyllide oxidoreductase. Continuous FR light induced the accumulation of Rubisco large subunits in wild-type but not in transgenic tobacco plants. Our data confirm the suggestion that phytochrome A mediates photoregulation of the synthesis of these proteins.

Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei.

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200-kDa antigen of B. pseudomallei cross-reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide-positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain.

Isolation and growth of a cytopathic agent from multiple sclerosis brain tissue.

Although many studies support a role for viruses in multiple sclerosis (MS) etiopathology, no specific agent has been consistently associated with significant numbers of MS patients without concomitant detection in non-MS controls. Previous studies have shown the presence of viral-like structures in MS plaques, although the specificity of these structures for MS has been questioned. The present study describes the use of polyclonal antisera against feline and human brain-derived cytopathic agents and immunoaffinity chromatography to purify and partially characterize possible virus-like structures from MS brain tissue. Chromatography eluates from 4 MS brains contained pleomorphic particles up to 350 nm in diameter and tubular/filamentous-like structures approximately 10-18 nm in thickness. Inoculation of primary rat glial cell cultures with chromatography eluates from MS brain tissue resulted in a reproducible pattern of cytopathic effects in the form of multinucleation in cells identified immunocytochemically as oligodendrocytes. Antisera raised against the feline and MS-derived cytopathic agents were used to successfully immunolabel infected oligodendrocyte-like cells and syncytia and to detect a 66,000 M(r) protein on Western blots of inoculated cultures or concentrated MS brain eluates. Similar structures, cytopathic effects (CPE) and protein expression were not observed in eluates from 5 control brains or in cultures inoculated with control brain eluates. These studies demonstrate that cytopathic, virus-like structures can be isolated from MS brain tissue using antisera raised against a cytopathic agent rescued from demyelinating brain lesions in cats. The identity of this agent and its possible role in MS aetio-pathology remains unknown.

Sorbin in the porcine gastrointestinal tract and pancreas: an immunocytochemical analysis.

Sorbin is a 153-amino acid peptide that was initially discovered in the porcine duodenum. We have reported previously that this peptide regulates intestinal electrolyte transport and have described accumulation sites in the rat digestive tract. In the present study, we investigated the anatomical distribution and the site(s) of sorbin production in the porcine digestive tract using immunocytochemistry. The use of polyclonal antisera, which by cross-reaction studies were shown to be specific for different regions of the molecule, revealed a diversified distribution. Sorbin predominated in endocrine cells preferentially localized in the pyloric glands, duodenal crypts of Lieberkühn, and pancreatic islets; in the gastrointestinal tract, sorbin coexisted with Met-enkephalin or with substance P in a small fraction of serotonin-storing [enterochromaffin (ED)] cells, i.e. EC2 cells and EC1 cells, respectively; in the pancreas, sorbin coexisted with insulin in the beta-cells, also considered as serotonin-storing cells in the pig, and with EC cells in the exocrine pancreas. An enteric neuronal system containing sorbin was also reported. Our results demonstrate that sorbin is a component of the serotonin-storing cell type in the porcine gastrointestinal tract and pancreas, and suggest potential directions to investigate the functions of this new regulatory peptide.

The use of polyclonal antisera and blocking of antibodies in the identification of marine dinoflagellates: species-specific and clone-specific antisera against Gymnodinium and Alexandrium

Separate polyclonal antibodies were developed against cell surface antigens of the 4 dinoflagellate species: (a) Gymnodinium catenatum; (b) a morphologically similar non-toxic dinoflagellate Gyrodinium sp.; (c) Alexandrium minutum; and (d) morphologically similar A. lusitanicum. Blocking of antibodies to obtain clone-specificity were performed. Positive immunofluorescence reactions were visualized with epifluorescence microscopy as well as spectrofluorometry using FITC indirect immunofluorescence assay. Specificity and cross reactivity were tested with cells from laboratory cultures as well as cells from natural samples. Polyclonal antisera were species-specific (for G. catenatum and Gyrodinium sp.) and genus-specific (for Alexandrium). After blocking the antisera were clone-specific. An excellent labelling was obtained with fresh cells and from those preserved in 4% buffered formaldehyde. The blocking of the antibodies shows great promise as a technique to obtain clone-specific antisera.

Clonal diversity among strains of Escherichia coli incriminated in turkey colisepticemia

The extent of genetic differentiation among 80 Escherichia coli isolates collected from turkeys with acute colisepticemia was assessed based on allelic variation at 20 enzyme-encoding loci detected by multilocus enzyme electrophoresis. Isolates were polymorphic at 117 loci and were classified into 32 multilocus genotypes, delineating clones, that differed on average at 36% of the loci. In the total sample, 29 (36%) of the isolates belonged to one of two closely related clones, differing only in a single electromorph, and 11 of these isolates were serogroup O78. Most isolates fell into one of 4 genetically distinct clusters of strains. Three of these clusters represent E. coli clone complexes that have been previously identified in avian diseases and a fourth cluster which is specific to coliseptecimia in turkeys. Most (73%) isolates produced aerobactin, whereas none produced hemolysins. Assays for detecting K1 capsules. including the use of polyclonal antisera, monoclonal antibodies, and K1-specific bacteriophages, gave variable resutls, but showed that overall 18% of the strains from colisepticemia were K1 encapsulated with most of the K1 + isolates found in one clone cluster. The results show that many cases of colisepticemia in turkey flocks are caused by a small number of pathogenic clones representing at least three distinct clone complexes.

Induction of various cytochromes CYP2B, CYP2C and CYP3A by phenobarbitone in non-human primates.

Male and female patas (Erythrocebus patas) and cynomolgus (Macaca fascicularis) monkeys were treated with phenobarbitone (PB) and examined for the induction of various cytochrome P450 (P450)-mediated drug metabolizing enzymes. Hydroxylation of testosterone at the 6 beta, 2 beta, and 15 beta positions, metabolites normally associated with CYP3A P450s increased 2- to 5-fold in PB-treated animals. Induction of this P450 family was confirmed by the use of polyclonal antisera directed against the human CYP3A enzymes which inhibited both induced and constitutive 6 beta- and 15 beta-hydroxylase activities in both species of monkeys. The enzymatic activities testosterone 16 beta-hydroxylation, pentoxyresorufin O-dealkylation [PROD], and benzyloxyresorufin O-dealkylation [BZROD] typically associated with the rodent CYP2B subfamily in rodents were also examined. Testosterone 16 beta-hydroxylation activity was highly induced up to 15-fold in both species of monkeys to maximal levels similar to those induced in PB-treated male rats. BZROD was similarly induced up to 10-fold in both species of monkeys, but the maximal levels of BZROD achieved were substantially lower than those obtained in PB-treated rats. Finally, PROD yielded an idiosyncratic response showing substantial induction (> 30-fold) in certain patas monkeys (4 out of 8) but minimal (< 5-fold) induction in the other patas monkeys (4 out of 8) or any of the cynomolgus monkeys (0 out of 8). Immunodetection of various cytochromes using polyclonal antisera directed against rat cytochromes confirmed the induction of CYP3A proteins as well as the induction of protein(s) immunologically cross-reactive with rat CYP2B P450s. BZROD, PROD, or testosterone 16 beta-hydroxylase activities, were not inhibited by antibody to CYP3A P450s. However, high concentrations of polyclonal antiserum directed against rat CYP2B inhibited all three activities in PB-induced patas monkeys. In contrast, this antiserum failed to inhibit the hydroxylation of testosterone at the 6 beta or 2 beta positions. Induction of the CYP2C subfamily was observed by immunochemical detection. Interestingly, induction of this subfamily appeared to be more pronounced in cynomolgus than in patas monkeys. Finally, we failed to observe significant sex-dependent differences in P450-mediated enzymatic activities in either control or induced monkeys. These results confirm the induction of a similar spectrum of P450 proteins by PB in non-human primates to that which has previously been observed in rodents.

Identification and isolation of the Leishmania transferrin receptor.

In a previous report, we have presented several lines of evidence, derived from widely different methodologies, suggesting that Leishmania has specific receptors for transferrin with a Kd similar to the mammalian transferrin receptor. This paper describes the identification, purification, and biochemical characterization of Leishmania transferrin receptor. The Leishmania transferrin receptor, detected on intact parasites by immunoperoxidase staining, was first identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis, using 125I-transferrin, as a 70-kDa protein. It has been isolated initially from Leishmania infantum promastigotes using affinity chromatography on a transferrin-Sepharose column and, subsequently, from Leishmania major promastigotes. The use of polyclonal antisera to the purified 70-kDa Leishmania transferrin receptor and to the purified rat transferrin receptor showed that the two receptors are antigenically distinct. The 70-kDa Leishmania transferrin receptor was subsequently characterized as an integral membrane glycoprotein. The monomeric state of the Leishmania transferrin receptor was demonstrated by gel filtration of purified receptor complexed with 125I-transferrin. Thus, the Leishmania transferrin receptor, unlike the mammalian receptor, is not a disulfide-linked dimer but a single 70-kDa polypeptide.

Isolation, immunolocalization, and sperm-association of three proteins of 18, 25, and 29 kilodaltons secreted by the mouse epididymis.

Three murine epididymal secretory proteins have been characterized by their site of synthesis, sperm association, and tissue localization by use of polyclonal antisera and immunochemistry. Mouse epididymal protein 7 (MEP 7) was localized initially within the supranuclear regions of some principal epithelial cells in the proximal corpus while other cells remained unstained. In the mid-proximal corpus, all principal cells and stereocilia were stained, and luminal staining increased from corpus to cauda. Some clear cells in the distal corpus and cauda also showed immunoperoxidase staining. Sequential extraction of caudal spermatozoa indicated that MEP 7 was predominantly loosely associated with spermatozoa and that only a small amount of MEP 7 required detergent to extract it from spermatozoa. Examination of other rodent caudal fluids revealed a related protein in rat caudal fluid of 32 kDa, and amino acid sequence analysis of MEP 7 showed a 68% sequence similarity with rat proteins AEG and D/E. MEP 9 immunolocalized within the cytoplasm of all principal cells of the distal caput. In a transition zone between the distal caput and the corpus, some principal cells were stained while others were not. Distal to the corpus, the principal cell staining gradually decreased. In the distal caput and proximal corpus, large heavily stained droplets associated with spermatozoa were seen in the lumen. The staining intensity of these droplets also decreased from corpus to cauda. The clear cells of the distal corpus and cauda did not stain with the antibody to MEP 9. Sequential extraction of caudal spermatozoa showed that some MEP 9 was extractable under low-salt conditions, whereas extraction with 0.1% Triton X-100 was required to remove all MEP 9, indicating it was firmly associated with spermatozoa. The antibody to MEP 9 cross-reacted with a 25-kDa protein present in rat caudal fluid. MEP 10 was localized within the cytoplasm of the principal cells, the stereocilia, and the lumen of the epididymis at the junction of the distal caput and corpus. In the distal corpus, a large number of clear cells were stained, but very few of these cells stained in the cauda. MEP 10 dissociated completely from caudal spermatozoa under low-salt conditions, indicating that it was not firmly bound to spermatozoa. The antiserum to MEP 10 cross-reacted with proteins present in rat and guinea pig caudal fluid. The related rat protein migrated at approximately 20 kDa. Amino acid sequence analysis of MEP 10 revealed an 86% sequence similarity with rat proteins B and C.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of Polyclonal Antisera and Monoclonal Antibodies to Examine Serological Relationships Among Three Filamentous Viruses of Sweetpotato

Sweetpotato is susceptible to infection by several filamentous viruses of which only the aphid-transmitted sweetpotato feathery mottle virus (SPFMV) has been well-characterized. Sweetpotato mild mottle (SPMMV; transmitted by whiteflies) and sweetpotato latent (SPLV; for which no vector has been identified) viruses, although well-recognized, have not been definitively classified. The serological relationships among these viruses were re-examined with SPFMV-, SPLV- and SPMMV-specific polyclonal antisera and potyvirus cross-reactive monoclonal antibodies (MAbs) (...)

Immunocytochemical localization in the central nervous system of four different insect species of molecules immunoreactive against peptides present in the caudodorsal cells of Lymnaea stagnalis

The use of polyclonal antisera directed against three peptides (ovulation hormone (CDCH), α, and β caudodorsal cell peptide (α- and β-CDCP)) produced by the caudodorsal cells of the snail Lymnaea stagnalis resulted in positive immunoreaction in Sarcophaga bullata (Diptera), Leptinotarsa decemlineata (Coleoptera), Locusta migratoria, and Periplaneta americana (Orthoptera). In three species, colocalization was detected using the antisera against CDCH and α-CDCP.

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.

Immunohistochemical Expression of Keratin Proteins in Urinary Bladder Carcinoma

Transitional carcinomas of the urinary bladder were examined immunohistochemically for keratin proteins with the use of polyclonal antiserum (TK, 41-65 kDa) and 3 monoclonal antibodies (KL 1, 55-57 kDa; PKK 1, nos. 19, 18, 8; and K 8.12, nos. 16, 13). Umbrella cells gave particularly strong staining for TK, KL 1 and PKK 1, whereas they were negative for K 8.12. Basal- and intermediate-layer cells in urothelial epithelium were moderately positive for all keratins. Brunn's nests cells showed comparatively slight or moderate keratin staining, and K 8.12 staining of Brunn's nests was higher than in urothelial epithelial cells. Transitional carcinoma (grades I and II) indicated uniform keratin distribution, and staining was strong with TK, while that of KL 1, PKK 1 and K 8.12 varied, and grade III tumors showed the lowest intensity of staining. K 8.12 staining in papillary transitional carcinomas was strongly positive in basal located tumor cells, as compared with apical tumor cells. Squamous cell carcinoma was varying positive to keratin reactions dependent on the degree of keratinization. Heterogenity of keratin distribution in papillary transitional carcinomas was given between basal tumor cells and well differentiated tumor cells including umbrella-like cells.

The production and methods of use of polyclonal antisera to the pathogenic organisms Aeromonas salmonicida, Yersinia ruckeri and Renibacterium salmoninarum

The production and methods of use of polyclonal antisera to the pathogenic organisms <i>Aeromonas salmonicida, Yersinia ruckeri</i> and <i>Renibacterium salmoninarum</i>