Indirect Immunofluorescence Antibody Staining Research Articles

The population of a high-virulence strain of Salmonella enterica serovar Enteritidis in subcutaneously infected partridge: a quantitative time-course study using real-time PCR

This research was undertaken to determine the population of a high-virulence strain of Salmonella enterica serovar Enteritidis in partridge by a fluorescent quencher PCR assay and to correlate these findings with the results obtained from the immunohistochemical localization and histopathological examinations of selected Salmonella enterica serovar Enteritidis-infected tissues. To make the results meaningful, a side-by-side bacteriology method (indirect immuno-fluorescent antibody staining) was performed too. The results of indirect immuno-fluorescent antibody staining and immunohistochemical localization were similar to the fluorescent quencher PCR assay. The time course of the appearance of bacterial antigens and tissue lesions in various tissues was coincident with the levels of the bacterial DNA loads at the infection sites. This suggests that Salmonella enterica serovar Enteritidis loads in internal organs are closely correlated with the progression of the infection.

Immune Cell Phenotyping Using Flow Cytometry

AbstractImmunophenotyping of lymphoid tissues, particularly spleen and thymus, has been used in the field of immunotoxicology for many years as part of a tiered screening approach for detecting immunotoxicity following chemical exposure. This unit describes basic procedures for direct and indirect immunofluorescent antibody staining of surface proteins on lymphoid cells from tissues or blood. Protocols for resolution of dead cells and for fixation of cells are also included. In addition, information on antibody selection and titration, fluorochrome choices, isotype controls, compensation, standardization of flow cytometric analysis, data analysis, and statistical issues is presented.

T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus spike protein elicit a specific T-cell immune response in patients who recover from SARS.

The immunogenicity of HLA-A2-restricted T-cell epitopes in the S protein of the Severe acute respiratory syndrome coronavirus (SARS-CoV) and of human coronavirus strain 229e (HCoV-229e) was analyzed for the elicitation of a T-cell immune response in donors who had fully recovered from SARS-CoV infection. We employed online database analysis to compare the differences in the amino acid sequences of the homologous T epitopes of HCoV-229e and SARS-CoV. The identified T-cell epitope peptides were synthesized, and their binding affinities for HLA-A2 were validated and compared in the T2 cell system. The immunogenicity of all these peptides was assessed by using T cells obtained from donors who had fully recovered from SARS-CoV infection and from healthy donors with no history of SARS-CoV infection. HLA-A2 typing by indirect immunofluorescent antibody staining showed that 51.6% of SARS-CoV-infected patients were HLA-A2 positive. Online database analysis and the T2 cell binding test disclosed that the number of HLA-A2-restricted immunogenic epitopes of the S protein of SARS-CoV was decreased or even lost in comparison with the homologous sequences of the S protein of HCoV-229e. Among the peptides used in the study, the affinity of peptides from HCoV-229e (H77 and H881) and peptides from SARS-CoV (S978 and S1203) for binding to HLA-A2 was higher than that of other sequences. The gamma interferon (IFN-gamma) release Elispot assay revealed that only SARS-CoV-specific peptides S1203 and S978 induced a high frequency of IFN-gamma-secreting T-cell response in HLA-A2(+) donors who had fully recovered from SARS-CoV infection; such a T-cell epitope-specific response was not observed in HLA-A2(+) healthy donors or in HLA-A2(-) donors who had been infected with SARS-CoV after full recovery. Thus, T-cell epitopes S1203 and S978 are immunogenic and elicit an overt specific T-cell response in HLA-A2(+) SARS-CoV-infected patients.

Infection of plant material derived from Solanum acaule with Clavibacter michiganensis ssp. sepedonicus: temperature as a determining factor in immunity of S. acaule to bacterial ring rot

The development of potato (Solanum tuberosum) cultivars immune (completely free of infective agents) to bacterial ring rot disease, caused by Clavibacter michiganensis ssp. sepedonicus, would be a significant step toward eradication of the disease. Earlier results suggested that the wild potato species Solanum acaule, acc. PI472655 (genotype 7‐8), was immune to C. michiganensis ssp. sepedonicus. The goal of the present study was to investigate the possibility of transferring this desirable trait to cultivated potato through interspecific somatic hybridization. Eight different somatic hybrids between S. acaule and S. tuberosum, with three different genome ratios, were tested for susceptibility to infection in the glasshouse using two C. michiganensis ssp. sepedonicus strains. All the somatic hybrids expressed symptoms of ring rot and were susceptible to infection as determined by IFAS (indirect immunofluorescent antibody staining) tests. The genome compositions of the hybrids influenced bacterial titres. Most of the hybrids with a higher proportion of the S. acaule genome contained lower numbers of C. michiganensis ssp. sepedonicus cells than hybrids with lower proportions of the S. acaule genome. In growth chamber tests, temperature was found to be a determining factor in the expression of immunity in the tetraploid S. acaule 7‐8 line. At 21°C, S. acaule 7‐8 was immune to infection by C. michiganensis ssp. sepedonicus, but at 15°C, plants supported large populations of the pathogen. However, none of the S. acaule plants expressed disease symptoms. Thus, S. acaule exhibits temperature‐dependent immunity to infection by C. michiganensis ssp. sepedonicus and should be considered tolerant rather than immune.

The laminin-nidogen complex is a ligand for a specific splice isoform of the transmembrane protein tyrosine phosphatase LAR.

Leukocyte antigen-related protein (LAR) is a prototype for a family of transmembrane protein tyrosine phosphatases whose extracellular domain is composed of three Ig and several fibronectin type III (FnIII) domains. Complex alternative splicing of the LAR-FnIII domains 4-8 has been observed. The extracellular matrix laminin-nidogen complex was identified as a ligand for the LAR-FnIII domain 5 (Fn5) using a series of GST-LAR-FnIII domain fusion proteins and testing them in in vitro ligand-binding assays. LAR- laminin-nidogen binding was regulated by alternative splicing of a small exon within the LAR-Fn5 so that inclusion of this exon sequence resulted in disruption of the laminin-nidogen-binding activity. Long cellular processes were observed when HeLa cells were plated on laminin-nidogen, but not when plated on a fibronectin surface. Indirect immunofluorescent antibody staining revealed high expression of LAR in a punctate pattern, throughout the length of these cellular processes observed on laminin-nidogen. Antibody-induced cross-linking of LAR inhibited formation of these cellular processes, and inhibition was correlated with changes in cellular actin cytoskeletal structure. Thus, LAR-laminin-nidogen binding may play a role in regulating cell signaling induced by laminin-nidogen, resulting in cell morphological changes.

Plant-to-plant transmission of the bacterial ring rot pathogenClavibacter michiganensis subsp.sepedonicus

Plant-to-plant transmission of the bacterial ring rot (BRR) pathogen,Clavibacter michiganensis subsp.sepedonicus (Spieck. et Kott.) Skapt. et Burkh. of potato (Solanum tuberosum L.), was studied in field trials over a three year period. Healthy and infected seed tubers were planted 35 cm apart. In one treatment, a subsurface barrier was placed between the healthy and infected seed tubers separating the root systems of neighbouring plants. In this treatment, none of 216 plants grown from healthy seed tubers was infected at harvest, as determined by indirect immunofluorescence antibody staining (IFAS) with monoclonal antibodies. In the other treatment, no subsurface barrier was used. In this treatment, two of 368 plants (0.5%) grown from healthy seed tubers were infected at harvest. It is concluded that plant-to-plant transmission may occur but at very low frequency, and is unlikely to play a significant role compared with the potential of transmission by shared potato handling equipment.

Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts

The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel‐end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 106–7.4 × 107 colony‐forming units per ml. Following concentration by high‐speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi‐selective media, enzyme‐linked immunosorbent assay (ELISA), indirect immunofluorescent‐antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre‐enrichment of samples in semi‐selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi‐selective medium and tomato bioassay could detect fewer than 104 cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.

Replication of human herpesvirus type 6 (strain AJ) in JJHan cells grown in protein-free medium

The human T-cell line JJHan, commonly used as a substrate for human herpesvirus type 6 (HHV-6) replication, was established in a protein-free 1:1 mixture of Ham's F12 and Iscove's modified Dulbecco's medium. After 250 passages (4 years) in protein-free medium, the cells, designated JJHan-PF, were cocultured with JJHan cells chronically infected with strain AJ of HHV-6 conventionally grown in serum-supplemented medium. The production of viral antigen was monitored by indirect immunofluorescent antibody staining. The infectivity of the virus in JJHan-PF cells was similar to that in JJHan cultures as shown by HHV-6 antigen detection using a convalescent phase serum. The results show that JJHan-PF cells provide a sensitive substrate for HHV-6 replication without the interference of serum or other proteins usually added to the culture medium.

Identification and localization of proteins in gregarines that are immunologically related to smooth muscle α-actinin

Summary The immunological cross-reactivity of proteins from gregarine cell extracts with polyclonal and monoclonal antibodies against smooth muscle α-actinin was demonstrated by immunoblotting in one- and two dimensional gel electrophoresis and by immunofluorescence microscopy. In immunoblotting, two proteins of Gregarina blaberae with Mr = 90,000 and Mr = 66,000 and with isoelectric points of 5.5 and 5.9, respectively, showed cross-reactivity with anti-α-actinin antibodies. Electrophoretic analysis showed that the two proteins were associated with ghosts and a cytoplasmic extract of trophozoite and sexual stages. The Mr = 90,000 and the Mr = 66,000 proteins were extracted from G. blaberae ghosts by 6 M urea in high ionic strength buffer. Indirect immunofluorescence antibody staining showed these polypeptides to be localized in the cell cortex, within longitudinal lines underlying the folds of G. blaberae (Polycystidae) and Lecudina pellucida (Monocystidae), and with lines separating the large folds of Selenidium pendula (Selenidiidae). The presence of two distinct forms (Mr = 90,000 and Mr = 66,000) of α-actinin is discussed with regard to the motility of gregarines.

Detection of giardia and cryptosporidium in marine waters

Raw sewage disposal in marine waters is a common practice in many countries. This practice raises health risk concerns of possible transmission of Giardia and Cryptosporidium. Both of these protozoa have been shown to be transmitted by recreational swimming. To date no studies have determined the efficiency of their detection and concentration in marine waters. This study evaluated the efficiency of their detection in tap water and from marine waters in Hawaii with two different filter types. This study compared a polypropylene fiber cartridge filter, DPPPY (1.0 μm nominal porosity) (Cuno, Meriden, CT) which is typically used for parasite detection and the Filterite negatively charged filter (0.45μm) (Filtemp Sales, Inc., Phoenix, AZ). The latter would allow for both viruses and parasites to be concentrated simultaneously. The organisms were removed from the filter by passing the eluent through the filters in the opposite direction of collection and detected by indirect immunofluorescence antibody staining specific for Giardia and Cryptosporidium. Processing was simpler and faster with the Filterite filter and the overall efficiency for both Giardia and Cryptosporidium detection was greater. These methods are currently being used for the detection of the oocysts and cysts at bathing beaches in Hawaii impacted by marine sewage discharge.

Detection of Giardia and Cryptosporidium in marine waters

Raw sewage disposal in marine waters is a common practice in many countries. This practice raises health risk concerns of possible transmission of Giardia and Cryptosporidium. Both of these protozoa have been shown to be transmitted by recreational swimming. To date no studies have determined the efficiency of their detection and concentration in marine waters. This study evaluated the efficiency of their detection in tap water and from marine waters in Hawaii with two different filter types. This study compared a polypropylene fiber cartridge filter, DPPPY (1.0 µm nominal porosity) (Cuno, Meriden, CT) which is typically used for parasite detection and the Filterite negatively charged filter (0.45µm) (Filtemp Sales, Inc., Phoenix, AZ). The latter would allow for both viruses and parasites to be concentrated simultaneously. The organisms were removed from the filter by passing the eluent through the filters in the opposite direction of collection and detected by indirect immunofluorescence antibody staining specific for Giardia and Cryptosporidium. Processing was simpler and faster with the Filterite filter and the overall efficiency for both Giardia and Cryptosporidium detection was greater. These methods are currently being used for the detection of the oocysts and cysts at bathing beaches in Hawaii impacted by marine sewage discharge.

Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.

Potato Flea Beetle (Coleoptera: Chrysomelidae) Evaluated as a Possible Vector of Ring Rot Bacterium in Potatoes

Commercial tolerances for the incidence of bacterial ring rot caused by Clavibacter michiganense subsp. sepedonicum (Spieck. & Kotth.), Davis, Gillaspie, Vidaver &Harris in potatoes, Solanum tuberosum L., are zero, requiring strict regulations to reduce field spread of the disease. Few insects have been shown to be directly associated with the dissemination of ring rot bacteria. Concentrations of bacteria within potato leaves as potential inoculum and the ability of the potato flea beetle, Epitrix cucumeris (Harris), to acquire and disseminate the ring rot bacterium were investigated. There was considerable variation in the concentrations of ring rot bacteria within potato leaves. The highest numbers of bacteria were found in the midveins. Fewest bacteria were present in the mesophyll of the laminae, but the basal sections of laminae contained more bacteria than did the midrib sections from leaflet tips. Potato flea beetles acquired the bacteria in 24 h or less. Beetles exposed to infected plant tissue were confirmed positive for the pathogen using indirect immunofluorescence antibody staining (IFAS). In addition, scanning electron photomicrographs of infected flea beetles showed their mouthparts to be contaminated with bacteria that were the same size and morphology as the ring rot pathogen. Positive IFAS reactions of potato test plants that had been fed upon by potato flea beetles previously exposed to ring rot-infected plants represented support for transmission of the bacteria by the beetles. Mode of transmission appears to be through feeding activity because IFAS analyses of regurgitants and fecal material from contaminated beetles were negative.

Insect transmission of the bacterial ring rot pathogen

Three species of potato-infesting insects were evaluated under laboratory and field conditions for their capability to transmitCorynebacterium sepedonicum (Spieck. & Kotth.) Skapt. & Burkh., the bacterial ring rot (BRR) pathogen of potatoes. Laboratory and field studies confirmed the vector capability of the Colorado potato beetle (CPB),Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) and the green peach aphid (GPA),Myzus persicae (Sulzer) (Homoptera: Aphididae). Both adults and larvae of the CPB proved to be vectors of the pathogen. Only adult GPA were evaluated. Bacterial transmission was most likely mechanical: analyses of the CPB mouthparts stained by the indirect immunofluorescence antibody staining (IFAS) procedure confirmed the presence of the bacterium after the beetles were exposed to infected plant tissue. Eggs, haemolymph, feces, regurgitations, and macerates of the digestive tract failed to show the presence of the bacterium. The aster leafhopper,Macrosteles fascifrons (St∮al) (Homoptera: Cicadellidae) failed to acquire and transmitCorynebacterium sepedonicum.

Contrasts in antigen expression in the erythrocytic and exoerythrocytic stages of rodent malaria

The time and site of expression of five antigens, recognized by monoclonal antibodies raised against blood-stage parasites, were studied in the exoerythrocytic stage of Plasmodium berghei using indirect immunofluorescent antibody staining. Two monoclonal antibodies (W 3.5, I 2.6), which stain the cytoplasm of infected erythrocytes, did not stain the cytoplasm of the infected liver cell but stained the parasite itself suggesting a difference in the antigenic architecture of the erythrocytic and exoerythrocytic parasites. Another antibody (17.6.1) revealed a further difference in the antigenic composition of the blood and liver-stage parasites being expressed almost exclusively in the former. Two others (C139 and 17.3.9) showed broadly similar patterns of expression in these two stages of the malarial life-cycle.

ウイルス分離にて診断した非定型ムンプスの２症例

Although mumps virus (MV) infection that induces bilaterally inflammatory reaction such as swelling and tenderness in parotid salivary glands is easily diagnosed, virological and serological investigations are needed for a final diagnosis of an atypical case that suggests MV infection accompanying solitary swelling of certain major salivary glands. In general, it can be serologically diagnosed as MV infection if the serum level of antibody raised against MV at the convalescence of the disease is increased by 4 times in comparison to that at the acute phase. It is considered a major drawback of this diawgnostic method that the final decision is not obtained until recovery from the disease. In the present communication, we describe two atypical cases of MV infection that was diagnosed by MV isolation from the saliva of patients in the initial phase of the disease.Two adult patients, complaining of an unilateral swelling of parotid salivary gland, were examined in our clinic. Since we suspected virus infection, the saliva from these patients was inoculated into green monkey kidnkey Vero cells. Several days later, cytopathic change that seemed to be induced by virus infection, appeared in the inoculated monolayers. Examination of the cells with the indirect immunofluorescent antibody staining technique and transmission electron microscopy revealed that MV replicated in the inoculated Vero cells. Thus, these cases could be diagnosed as MV infection early, at the first visit to our clinic.

Germline-specific Antigens Identified by Monoclonal Antibodies in the Nematode Caenorhabditis elegans1 : (Caenorhabditis elegans/monoclonal antibody/cell-specific antigen/germinal plasm/asymmetry).

Of 27 monoclonal antibodies identified to react, by indirect immunofluorescent antibody staining, with specific cells and tissues of the nematode Caenorhabditis elegans, we report here three monoclonal antibodies pertaining to the gonadal tissues. One antibody defines an antigen that is distributed over the entire embryo at earlier development and later becomes unique to the gonad, including mature oocytes. The antigens recognized by the other two are distributed asymmetrically in the posterior region of the fertilized egg's cytoplasm destined to become the germline precursor cell. Each antigen is successively segregated only to the germline precursor cells of the developing embryo and, postembryonically, is uniquely localized around the germline cell nuclei of the larvae and adults.