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At least 73 records · Page 4Linked to original sources
PubMed1993

Skin explant culture: a reliable method for detecting pemphigoid antibodies in pemphigoid sera that are negative by standard immunofluorescence and immunoblotting.

We investigated the presence of bullous pemphigoid antibodies in bullous pemphigoid sera that are negative by standard indirect immunofluorescence. We incubated each of four indirect immunofluorescence-positive bullous pemphigoid sera, seven indirect immunofluorescence-negative bullous pemphigoid sera, one indirect immunofluorescence-negative herpes gestationis serum, three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera, five indirect immunofluorescence-negative epidermolysis bullosa acquisita sera, and two normal human sera with fresh human skin explants in medium 199 at 4 degrees C for 48 h. All bullous pemphigoid sera, herpes gestations serum, and the three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera had IgG that bound the basement membrane zone of skin explants with moderate to marked intensity as demonstrated by immunofluorescence. Normal sera and indirect immunofluorescence-negative epidermolysis bullosa acquisita sera failed to bind the explant basement membrane zone. Immunoblotting of bullous pemphigoid sera showed five of seven indirect immunofluorescence-negative bullous pemphigoid sera to bind high-molecular weight and/or low-molecular weight bullous pemphigoid antigens from epidermal extracts. We conclude that the skin explant culture system is a very sensitive method for the detection of bullous pemphigoid antibodies in all bullous pemphigoid sera.

Adolescent↗
PubMed2006

Value of direct immunofluorescence in predicting remission in pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris is characterized by the presence of autoantibodies to desmogleins. Multiple relapses and remission may occur during the course of the disease. The goal of this study was to determine whether direct immunofluorescence study has any value in detecting immunological remission of pemphigus vulgaris. METHODS: Fifty-seven patients with pemphigus vulgaris who were in clinical remission for at least 3 months, while taking prednisolone 5-7.5 mg/day, were recruited retrospectively for the study. Direct immunofluorescence study had been performed in all patients after a period of at least 3 months in clinical remission. Treatment had been discontinued in all patients with negative results of direct immunofluorescence. RESULTS: Of 57 patients who were in clinical remission, 24 patients (42%) had negative and 33 patients (58%) had positive results of direct immunofluorescence. Eleven patients (46%) with negative results of direct immunofluorescence relapsed within the first year of the follow-up period. Nine patients with negative direct immunofluorescence had a history of more than 6 months of clinical remission before direct immunofluorescence study. Among them, two patients (22%) relapsed. None of four patients with history of more than 12 months of clinical remission before a negative direct immunofluorescence study relapsed. CONCLUSIONS: Negative direct immunofluorescence is an indicator of immunological remission in patients with pemphigus vulgaris after 6-12 months in clinical remission.

Adult↗
PubMed1993

Quantitative and qualitative comparison of DNA amplification by PCR with immunofluorescence staining for diagnosis of Pneumocystis carinii pneumonia.

AIM: To compare the results of DNA amplification by the polymerase chain reaction (PCR) with immunofluorescence staining for detecting Pneumocystis carinii in bronchoalveolar lavage specimens taken from symptomatic HIV seropositive patients with suspected P carinii pneumonia (PCP). METHODS: Bronchoalveolar lavage specimens were obtained from 28 symptomatic HIV seropositive patients. Specimens were examined for P carinii using immunofluorescence, and by DNA amplification with PCR to obtain results on gel electrophoresis (gel) and a more sensitive Southern hybridisation (blot) technique. Specimens positive by immunofluorescence and gel electrophoresis were serially diluted to a 10(-6) concentration and each dilution strength tested for P carinii using PCR to compare quantitatively immunofluorescence with PCR. RESULTS: Of the 28 specimens analysed, 18 were negative for P carinii by both immunofluorescence and PCR, two were positive only by the blot technique of PCR, four were equivocally positive and four unequivocally positive by immunofluorescence. Three of the four equivocally positive patients tested by immunofluorescence were negative for P carinii by PCR, although one was positive by PCR (blot) technique. This patient had clinically confirmed PCP. Of the four unequivocally positive patients tested by immunofluorescence, three were gel and blot positive by PCR and had PCP clinically, but one was negative by both gel and blot techniques, although the patient certainly had PCP on clinical grounds. This patient had received nine days of treatment with high dose co-trimoxazole before bronchoalveolar lavage specimens were obtained. The three specimens positive by gel and blot techniques remained gel positive down to dilutions of between 10(-4) and 10(-6). CONCLUSIONS: PCR results may become negative soon after starting treatment for PCP. Specimens should therefore be taken before, or soon after, starting treatment. PCR seems to be between 10(4) and 10(6) times more sensitive than immunofluorescence.

Adult↗
PubMed1979

Immunofluorescence on avian sarcoma virus-transformed cells: localization of the src gene product.

The localization of the avian sarcoma virus src gene product (termed p60src) was examined by indirect immunofluorescence in cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D (SR-RSV-D). Antiserum to p60src was obtained from rabbits bearing SR-RSV-D-induced tumors, and immunofluorescence was performed on chicken embryo fibroblasts (CEF) transformed with SR-RSV-D, as well as normal rat kidney (NRK) cells transformed by the same virus (termed SR-RK cells). Both acetone and formaldehyde fixation were used for the immunofluorescence tests. The specificity of the anti-tumor serum was first demonstrated in both cell systems by gel electrophoresis of immunoprecipitates prepared from 35S--methionine-labeled cells. Anti-tumor serum precipitated p60src from SR-RSV-D-transformed CEF but not from CEF infected with a transformation-defective mutant of SR-RSV-D. All viral structural proteins and precursors contained in these immunoprecipitates could be eliminated by competition with unlabeled virus. Similar experiments on SR-RK cells indicated that no viral proteins other than p60src were expressed in these cells, and this observation was supported by immunofluorescence tests using antiserum to whole virus. For immunofluorescence localization of p60src, reactions with viral structural proteins were blocked with unlabeled virus. This presaturation step, obligatory for p60src detection in the SR-RSV-D-transformed CEF, was unnecessary when antitumor serum was tested on SR-RK cells, since p60src was the only viral protein detectable in these cells. With acetone-fixed cells, p60src-specific immunofluorescence revealed a characteristic fluorescence pattern which was similar in both cell systems. The principal pattern was diffuse and situated in the cytoplasm. A clear nuclear fluorescence was never observed. Immunofluorescence on formaldehyde-fixed cells also indicated the cytoplasmic location of p60src and revealed a specific subcytoplasmic concentration of the fluorescence. With both fixation methods, an additional fluorescence pattern was seen between cells in contact, and was also found in both SR-RK cells and SR-RSV-D-transformed CEF. Immunofluorescence on viable cells suggested that p60src was not on the surface of these transformed cells. The fluorescence patterns were specific for avian sarcoma virus-transformed cells and were not found in uninfected cells, cells infected with a transformation-defective mutant of SR-RSV-D or cells transformed by an antigenically unrelated murine sarcoma virus. Furthermore, anti-tumor serum did not contain antibodies to proteins of the microtubules or intermediate filaments.

Animals↗
PubMed1982

Evaluation of methods of detecting terminal deoxynucleotidyl transferase in human hematologic malignancies. Comparison of immunofluorescence and enzymatic assays.

Terminal transferase (TdT) activity and antigen have been measured in 267 specimens of human bone marrow and peripheral blood by using a biochemical assay for enzymatic activity and an immunofluorescence test for antigen. Oligo p(dA)50 and dGTP were used as reagents in the biochemical assay and either rabbit anti-calf TdT or rabbit anti-human TdT was used as the primary antibody for immunofluorescence. Because both false-positive and false-negative detection of TdT antigen occurs, the biochemical assay of TdT activity is considered the standard against which immunofluorescence assays must be measured. If specimens of cells contained TdT activity, then the immunofluorescence detected antigen in 91% of cases (rabbit anti-calf TdT) and 95% of cases (rabbit anti-human TdT). When no TdT activity was detected, the immunofluorescence test was positive in 7.8% of cases (rabbit anti-calf TdT) and 5.2% of cases (rabbit anti-human TdT). When air-dried slices were shipped by air mail to a distant location before being stained for immunofluorescence, TdT antigen was detected in only 33% of matched pair cases which contained TdT activity. From this study, the authors conclude that with current methodology, immunofluorescence tests for TdT antigen must be carried out on slides prepared in the testing laboratory and that such tests are reliable in more than 90% of cases. However, because a small percentage of results are false positives and false negatives, the authors suggest that if a patient's clinical response is not consistent with the immunofluorescence TdT result, an enzymatic assay for TdT activity be carried out.

Antigens↗
PubMed1997

Immunoperoxidase and immunofluorescent staining of Rickettsia rickettsii in skin biopsies. A comparative study.

OBJECTIVE: To compare immunofluorescent and immunoperoxidase staining of Rickettsia rickettsii in skin biopsies of patients suspected of having Rocky Mountain spotted fever (RMSF). DESIGN: Immunofluorescent staining results for R rickettsii from skin biopsies of patients suspected of having RMSF were obtained by computer and chart review. Immunoperoxidase staining for R rickettsii was performed on formalin-fixed, paraffin-embedded skin biopsies from the same patient population. PATIENTS: Twenty-six patients who were clinically suspected of having RMSF were included in this study. Skin biopsies of these patients were examined for evidence of RMSF by immunofluorescence and routine histology. MAIN OUTCOME MEASURES: The sensitivity and specificity of both immunofluorescent and immunoperoxidase staining techniques were calculated. The chi 2 method was used to assess significance. RESULTS: Both tests were highly significant for the detection of R rickettsii (P < .01). The sensitivity and specificity of the immunofluorescent and immunoperoxidase staining techniques for the identification of RMSF were identical. No significant difference between these tests was identified (P > .05). CONCLUSION: The sensitivity and specificity of immunofluorescent and immunoperoxidase staining of R rickettsii in routinely processed, paraffin-embedded skin biopsies of patients suspected of having RMSF are identical. Although not as rapid as the immunofluorescent technique, immunoperoxidase staining of R rickettsii has advantages over the immunofluorescent technique; these include easier antigen localization and concomitant viewing of the corresponding histopathology.

Biopsy↗
PubMed2006

Detection of human metapneumovirus in respiratory secretions by reverse-transcriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells.

Over two winters in Newcastle upon Tyne, respiratory secretions, negative by immunofluorescence staining for other respiratory viruses, were tested for the presence of human metapneumovirus (HMPV) by RT-PCR. In the second winter, specimens were also tested by immunofluorescence staining with an anti-HMPV polyclonal rabbit antiserum and immunofluorescence positive specimens were inoculated into a line of human bronchiolar cells, 16HBE140. Overall, 55 of 549 (10%) specimens tested were positive for HMPV by RT-PCR. Of 162 specimens tested by both RT/PCR and immunofluorescence staining, 23 were positive by both techniques. Of five specimens positive by RT-PCR alone, only one was confirmed with a second set of primers. Of three specimens positive by immunofluorescence alone, only one was confirmed by virus culture. All four previously recognized sub-genotypes of the virus were identified by both RT-PCR and immunofluorescence staining. Sub-genotype A1 was prevalent in the first winter and B1 prevalent in the second. HMPV replication and virus isolation rates were higher in 16HBE140 cells than in monkey kidney cells and did not require exogenous trypsin. Low passage isolates of both sub-genotypes A2 and B1 replicated slowly reaching peak titers only 12 days after inoculation. In summary, single round RT/PCR and immunofluorescence staining with a polyclonal rabbit antiserum proved of equal sensitivity in the diagnosis of HMPV infection in respiratory secretions both detecting 96% of confirmed positive specimens. 16HBE40 cells provided a significant improvement on monkey kidney cells for the isolation and propagation of the virus.

Animals↗
PubMed1975

Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-neurohypophypopseal system.

The present paper deals with the development of an immunofluorescence procedure that allows specific localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system(hnx) of the rat. Antibodies against arginine vasopressin (AVP), lysine-vasopressin (LVP) and oxytocin were raised by injecting these hormones that were covalently bound to thyroglobulin into rabbits. The vasopressin-immunized rabbits showed periods of diabetes insipidus, while histoloty of the "hns revealed an intact neurosecretory system with signs of increased endogenous hormone synthesis in the supraoptic nucleus and increased release in the neuro-hypophysis of some rabbits. The daily water intake of the oxytocin-immunized rabbits was similar to that of control rabbits. The development of antibodies against vasopressin as measured in the immunofluorescence procedure showed a course that was quite different from the curve of the titer as determined by radioimmunoassay (RIA). Also the specificity of the antibodies used in the immunofluorescence procedure was found to be quite different from their specificity in a RIA system. Potency and specificity of the antibodies have to be studied therefore within the immunofluorescence procedure itself. Using freshly frozen acetone-postfixed hypotalami or pituitaries, no sharp localization of immunofluorescence could be obtained in the HNS. Therefore prefixation was performed. Both, the type and the duration of prefixation revealed quite different results regarding the immunofluorescence in the neurosecretory cell boides in the hypotalamus and of their endings in the neurohypophysis. The best immunofluorescence results were obtained using 6 hours glyoxal-prefixation for the hypothalamus and 24 hours formalin-prefixation for the pituitary. The cross-reaction of the antibodies for oxytocin or vasopressin was tested on synthetic hormones that were bound to CNBR-activated agarose beads and mounted on glass sides. All anti-plasmas showed cross-reaction on beads containing the heterologou- antigen. The plasmas were purified by incubation with beads containing the heterologous hormone until the cross-reacting component had been removed. Using purified antibodies, the distribution of oxytocin and vasopressin cells within the HNS was investigated. More oxytocin containing cells were localized in the rostral part and more vasopressin in the caudal part of both, the supraoptic (SON) and paraventricular nucleus (PVN). Comparable percentages of oxytocin and vasopressin containing cells were found in the SON and PVN. The absolute amount of oxytocin containing cells was 2.5 times more in the SON than in the PVN, which seems to contradict the "classical" view that the PVN predominantly or entirely synthetizes oxytocin. In addition, fluorescence was found using antobodies against vasopressin in the suprachiasmatic nucleus in Wistar rats and heterozygous Brattleboro rats, but not in this nucleus of homozygous Brattleboros.

Animals↗
PubMed1983

Diagnosis of autoimmune skin disease in the dog: correlation between histopathologic, direct immunofluorescent and clinical findings.

Histopathologic, immunopathologic and clinical evaluation of two hundred thirty dogs resulted in a diagnosis of canine autoimmune skin disease (AISD) in eighty four of the suspected cases. Pemphigus foliaceus was the most common diagnosis, followed by bullous pemphigoid and pemphigus vulgaris. The highest percentage of cases were mixed breeds, followed by shelties, collies, German shepherds and poodles. There was no apparent age or sex predilection. Eighty seven percent of the eighty four cases of AISD were diagnosed on the basis of supportive clinical criteria in addition to compatible histopathologic and/or direct immunofluorescent findings. Thirteen percent of the cases with a final clinical diagnosis of AISD lacked supportive immunohistopathologic criteria on biopsy and the diagnosis was based upon clinical appearance, exclusion of other dermatoses, and response to immunosuppressive therapy. This latter group included four cases of SLE diagnosed using a combination of clinical criteria and a positive test for antinuclear antibody. Thirty three percent of the AISD cases were diagnosed on the basis of compatible findings on both histopathology and direct immunofluorescence. A histopathologic diagnosis of AISD was made in sixty nine percent of the cases. Positive direct immunofluorescence was found in fifty two percent of the cases. Thirty five percent of the AISD cases were diagnosed on histopathologic criteria without supportive immunofluorescent findings, while nineteen percent had positive direct immunofluorescence with nonspecific histopathologic changes. The number of definitive positive results was greater for direct immunofluorescence (38%) than for histopathology (25%). There was an equal incidence (21%) of false positive results among non-AISD cases with both tests. Indirect immunofluorescence appears to have little value in the diagnosis of canine AISD. The recommended diagnostic approach to canine AISD involves the use of both histopathologic and immunopathologic evaluations.

Animals↗