Immunoperoxidase Staining Of Cells Research Articles

Comparative evaluation of novel African swine fever virus (ASF) antibody detection techniques derived from specific ASF viral genotypes with the OIE internationally prescribed serological tests

The presence of antibodies against African swine fever (ASF), a complex fatal notifiable OIE disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. The early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness make antibody detection techniques essential in control programmes. Recent reports appear to indicate that the serological tests recommended by the OIE for ASF monitoring are much less effective in East and Southern Africa where viral genetic and antigenic diversity is the greatest. We report herein an extensive analysis including more than 1000 field and experimental infection sera, in which the OIE recommended tests are compared with antigen-specific ELISAs and immuno-peroxidase staining of cells (IPT). The antibody detection results generated using new antigen-specific tests, developed in this study, which are based on production of antigen fractions generated by infection and virus purification from COS-1 cells, showed strong concordance with the OIE tests. We therefore conclude that the lack of success is not attributable to antigenic polymorphism and may be related to the specific characteristics of the local breeds African pigs.

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10−8 M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.

Limits of Detection of Bluetongue Virus with Different Assay Systems

The sensitivity of different assay systems for detecting low concentrations of bluetongue virus (BTV) were compared. These assays included blind passage on baby hamster kidney (BHK-21) cells and on cattle pulmonary artery endothelial (CPAE) cells, immunoperoxidase staining of cells on multiwell slides, and cDNA/RNA hybridization of BTV infected cells. Nine serial 10-fold dilutions of a cell culture-adapted BTV serotype 11 were tested (each dilution was treated as a separate sample) in all assays. Visual inspection for cytopathic effects (CPE) during 3 passages in BHK-21 cells detected samples that contained greater than or equal to 3 plaque forming units (PFU)/ml of BTV. Evidence of CPE during 3 passages in CPAE cells detected samples that contained greater than or equal to 0.3 PFU/ml of BTV. A limit of detection (greater than or equal to 0.3 PFU/ml) was obtained faster by immunoperoxidase staining of BTV-inoculated CPAE cells on multiwell slides and incubated for 3 days. The cDNA/RNA hybridizations of CPAE and BHK-21 cells incubated for 2 or 3 days, respectively, with BTV dilution samples detected samples that contained greater than or equal to 30 PFU/ml. Of the assay systems examined, immunoperoxidase staining of CPAE cells on multiwell slides inoculated with cell culture-adapted BTV was the most sensitive and fastest assay for definitive virus identification.

Reovirus serotype 1 intestinal infection: a novel replicative cycle with ileal disease.

After oral inoculation, reovirus serotype 1 strain Lang was shown to specifically infect the epithelial cells of the ileum, while sparing the epithelial cells in the duodenum, jejunum, and colon. The initial site of replication was localized in cells of the crypts of Lieberkühn adjacent to Peyer's patches. Virus was subsequently found by immunoperoxidase staining in cells migrating up the crypt-villus complex throughout the ileum. The severity of the pathological changes in the ileum was proportional to the concentration of the viral inoculum. This site-specific infection of the ileum by reovirus may provide a model for diseases that are restricted to specific sites in the intestine.

Enzyme-linked immunoassay to monitor the purification of, and to screen for monoclonal antibodies against, C3b receptor.

An enzyme-linked immunosorbent assay both to screen for monoclonal anti-bodies to the C3b receptor and to monitor its purification was developed. The test requires only purified C3, converted to either the hemolytically no longer active iC3 or C3b. An NP-40 lysate of tonsil cells can be used as a source of CR1 in this test which works best under hypotonic conditions facilitating the interaction of iC3/C3b and CR1. Two monoclonal antibodies to CR1 were produced using this test for the screening of hybridomas. The identity of the molecule recognized by these antibodies with CR1 is demonstrated by immunoprecipitation and Western blot studies, as well as immunofluorescence and immunoperoxidase staining of cells and tissues known to contain CR1. Fractions from lentil-lectin and DEAE-Sephadex columns containing CR1 can be identified using this test.