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PubMed1983

Repeated application of first-layer antiserum improves immunofluorescence staining: a modification of the indirect immunofluorescence staining procedure.

Repeated application of the first-layer antiserum in the indirect immunofluorescence technique considerably improves the immunostaining. The modified method reveals more antigenic sites and increases the contrast between specific and background stainings, particularly where sparsely distributed antigenic areas are to be investigated. The effect of this novel immunostaining procedure is compared with that of the routine procedure and of other modifications. Possible mechanisms for the improving effect are discussed. A procedure of combined modifications is recommended for exploration of non-abundant antigens or for achieving a high-quality photograph.

Animals↗
PubMed1983

Serotyping of herpes simplex virus isolates: a comparison of BVDU sensitivities, indirect immunofluorescence with monoclonal antibodies, and indirect immunofluorescence with cross-adsorbed rabbit antibodies.

Four methods for typing of herpes simplex virus (HSV) isolates were compared for 43 recent clinical isolates and 3 reference strains of HSV. These isolates were subjected to indirect immunofluorescence using both monoclonal antibodies and cross-adsorbed rabbit antisera. Sensitivity to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was also examined. All isolates which fluoresced with HSV-1 monoclonals were found to be sensitive to BVDU with ID50's ranging from 0.001 to 0.006 micrograms/ml. All isolates labelled as HSV-2 using monoclonal antibodies had ID50's to BVDU ranging from 0.4 to 3.5 micrograms/ml. Results of typing with rabbit cross-adsorbed antisera were less accurate, however. When dilutions were predetermined according to manufacturer's instructions, only 3 of 22 isolates (14%) of HSV-1 were correctly typed. HSV-2 isolates were correctly labelled in 24 of 26 situations (92%). When fluorescence dilution endpoints were compared, however, 21 of 22 (95%) HSV-1 isolates had fluorescence endpoints at a greater dilution with HSV-1 antiserum. Twenty-three of 24 HSV-2 isolates were also correctly typed (96%).

Animals↗
PubMed1968

Relation between Epstein-Barr viral and cell membrane immunofluorescence of Burkitt tumor cells. I. Dependence of cell membrane immunofluorescence on presence of EB virus.

A comparison was made of the immunofluorescence tests for detection of cell membrane and Epstein-Barr virus antigens in cells from Burkitt tumor biopsies or continuous cultures derived therefrom. On the whole, cell membrane fluorescence in established lines appeared to depend not only upon the presence of EBV but to a considerable degree also upon the extent of the persistent viral infection. There was no constant relationship, however, between the results of the two tests and exceptions to the rule were noted. These observations indicate that different antigens are involved in the two tests. Biopsy cells in general and young cultures may reveal strong MIF activity but few, if any, EBV-positive cells. The reverse, the presence of relatively large numbers of EBV antigen-containing cells in the absence of significant MIF reactions, was also noted on occasion in a few established cultures. The possible interpretations of these findings have been discussed.

Antigens↗
PubMed1969

Relation between Epstein-Barr viral and cell membrane immunofluorescence in Burkitt tumor cells. 3. Comparison of blocking of direct membrane immunofluorescence and anti-EBV reactivities of different sera.

Sera from patients with Burkitt's lymphoma (BL), infectious mononucleosis (IM), carcinoma of the postnasal space (Ca PNS), and various controls were investigated for antibodies against the Epstein-Barr virus (EBV) by immunofluorescence on acetone-fixed smears (5) and for antibodies against the distinctive antigenic sites expressed on the surface of viable lymphoblastoid cells within EBV-carrying culture lines (1). The latter were studied by the blocking of direct membrane staining with FITC-conjugated Mutua serum. This serum has been derived from a Burkitt's lymphoma patient in long-term regression after chemotherapy and is free from detectable isoantibodies. It has been used previously as a standard of reference to demonstrate the presence of the membrane antigen(s) on all lines derived from BL biopsies and leukocytes from IM patients. It was found that 102 of 279 (37%) of the sera tested had high anti-EBV titers (>==80) and high membrane-blocking (Bl > 0.5) activity, 124 of 279 (44%) of the sera were low in both tests, 22 of 279 (8%) had low EBV titers (<==80), in spite of a high blocking index, and 31 of 279 (11%) of the sera were low in blocking activity (<0.5), in spite of a high EBV titer. The two tests thus gave concordant results with 81% and discordant with 19% of the sera. The majority of sera from BL patients were high in both tests. IM sera also showed a relationship between the two antibody activities but, in general, both activities were lower than in BL cases. Ca PNS sera seemed to fall into two main groups: (a) high anti-EBV, high blocking or (b) low anti-EBV, low blocking. Control sera, including four isoantisera, showed predominantly low reactivities in both tests.

Antigens↗
PubMed1969

Relation between Epstein-Barr viral and cell membrane immunofluorescence in Burkitt tumor cells. IV. Differentiation between antibodies responsible for membrane and viral immunofluorescence.

Previous reports (1, 2) have established that the expression of certain distinctive membrane antigen(s) on the surface of Burkitt's lymphoma (BL) and infectious mononucleosis (IM) cells is dependent on the presence of Epstein-Barr virus (EBV) in the cell line. The investigations reported here provide evidence that antibodies directed against EBV antigens, as revealed by the immunofluorescence test on acetone-fixed smears (8), and the membrane reactive antibodies, although often present in the same serum, are nevertheless distinctly different. Absorption of Mutua serum, the standard reference serum for demonstrating membrane antigen(s) on BL and IM cells, with BL cells completely removed anti-membrane activity without significantly affecting the anti-EBV antibody titer. Furthermore, sera were found which contained one type of antibody but not the other. Sera with high anti-membrane but low anti-EBV activity were found among relatives of BL patients. These sera reacted with the membranes of EBV-carrying BL and IM cells in essentially the same way, i.e., against the same spectrum of target cells, as the EBV-positive Mutua serum. They were unable to block the membrane reactivity of FITC-conjugated Mutua serum, however. In some cases they showed weak but incomplete blocking. One such EBV-negative, membrane-positive BL relative serum (Robert) was conjugated with FITC and used for direct staining of BL and IM cells. Again, this conjugate reacted against the same target cell spectrum as a Mutua conjugate, but its reactivity was completely blocked by a number of Burkitt patients' sera, although unconjugated Robert serum did not block the Mutua-conjugate. A number of other membrane-positive BL relative sera also failed to block Mutua, but completely blocked the Robert conjugate. A number of Swedish and African control sera and an isoantiserum gave no blocking against Robert conjugate. It therefore appears that the Mutua conjugate contains at least two antibody specificities against the EBV-determined membrane antigens. One, but not the other, is shared with the antibody specificity present in Robert's serum and a number of other sera from relatives of BL patients.

Antigens↗
PubMed1978

[Immunofluorescence with the PI-3 virus--study of various working conditions for the detection of viral infection using immunofluorescence on various types of cell cultures].

We used the fluorescence method for the investigation of the sensitivity of several kinds of cell cultures to the infection with the parainfluenza virus 3 (PI-3). Cultures from calf kidneys were the most sensitive while we did not determine any differences between primary cultures and cultures of the first and second subpassages and/or freshly cultivated or incubated cultures over seven days at a temperature of 37 degrees C. Equal values of infection titres like on the cultures of calf kidneys were determined by immunofluorescence also on the kidney cells of lambs though the presence of the infection was not accompanied by cytopathic changes. Infection of pig kidney cells appeared only after the inoculation of 10(3) TKID50 and higher doses of the virus, the infection having a very slow course of development without detectable cytopathic changes. Fluorescent findings were identical in different tissues. Antibodies present in the culture medium stopped the spreading of the infection by neutralizing the virus released from the cells, however, not the primary infection. The increase in the content of antibodies in the medium led - by inhibiting the intercellular virus - to the slowing down of the growth of primary fluorescent lesions.

Animals↗
PubMedDate not supplied

Comparison of three techniques for the detection of antibodies to double-stranded DNA: immunofluorescence on Trypanosoma gambiense, immunofluorescence on Crithidia luciliae and radioimmunoassay using the Farr technique.

Three techniques for the detection of antibodies against double-stranded DNA were compared: two immunofluorescent (IF) techniques using either Trypanosoma gambiense or Crithidia luciliae as antigen and a radioimmunoassay (RIA) based on the Farr technique. The IF on T. gambiense or C. luciliae gives very similar results but the reaction on C. luciliae is easier to read and to interpret. The use of C. luciliae permits the swift elimination of the two main causes of errors: antibodies against the nucleus of Trypanosomidae and antibodies reacting with the base of the flagella. The RIA appears as a very sensitive test since six sera gave a positive reaction with the RIA and a negative reaction with the IF on C. luciliae. However, the RIA gives in nine cases false positive reactions and in four cases false negative results. Those last four sera contained specific antibodies with low avidity for double-stranded DNA.

Animals↗
PubMed1982

Immunofluorescence and Treponema infection: a method using immunofluorescence to study rabbit testicular tissue infected with T pallidum and T pertenue.

We compared immunofluorescent staining of rabbit testicular tissue infected with Treponema pallidum or T pertenue, and fixed in Bouin's fixative, 95% cold ethyl alcohol with 1% glacial acetic acid, or routine 10% buffered formalin solution. The fixative of choice clearly was Bouin's. Although we studied only rabbit tissue, we assume that these fixatives will work well in human biopsy or autopsy material when identification of pathogenic Treponema is needed.

Animals↗
PubMed1979

Antigenic activity in adult Dipetalonema viteae in the indirect immunofluorescent test against sera from filariasis patients--the immunofluorescent histological search for "pure" antigen.

Using IFAT, it has been shown that isolated egg-shells and uterine fluid of Dipetalonema viteae are the most potent antigens in heterologous systems using human sera from patients infected with Wuchereria bancrofti, Onchocerca volvulus and Loa loa, as well as in homologous systems using sera from animals infected with D. viteae. It is suggested that these antigens are unlikely to be highly species-specific, and that anatomical isolation of antigens is a necessary prerequisite to immunochemical analysis aimed at the preparation of a "pure" antigen.

Animals↗
PubMed1981

Studies in cutaneous immunofluorescence. II. Histological and direct immunofluorescence observations in clinically normal skin of patients with rheumatoid arthritis.

Twenty patients with rheumatoid arthritis (RA) had a biopsy taken from clinically normal skin. These were examined for histological and immunological abnormalities and were compared to those of 8 patients with osteoarthritis (OA). No 'lupus band; linear deposition of immunoglobulin or complement at the dermo-epidermal interface was seen in any patient in either group. Perivascular deposits were seen in 5 out of 20 (25%) patients with RA. These were IgM in all 5 cases with additional C3 in 2 cases (10%) and additional fibrin in one case (5%). No immunoprotein deposits were seen in specimens from any OA patient. 4 of the 5 patients with perivascular immunoprotein deposits had circulating ANAs present and dilutions of 1/256 or higher but normal DNA binding. A sparse perivascular, predominantly lymphocytic infiltrate was seen in 13 out of 20 (65%) patients with RA and 3 of 8 (35%) patients with OA.

Arthritis, Rheumatoid↗
PubMed1975

Studies on defined immunofluorescence in clinical immunopathology. II. Relationship of F/P ratios pf conjugates and staining properties in indirect immunofluorescence.

The effect of conjugate F/P ratios on titers of patient antibodies was evaluated with two model systems in IIF chessboard titrations, notably, antinuclear antibodies and the intercellular antibodies of pemphigus. The plateau titers of both antibodies and F/P conjugate ratios were analyzed and proved to have a high degree of linear relationship statistically. Single titrations of other tissue antibodies, including skin basement membrane antibodies of bullous pemphigoid, MTA, GPA, SMA, and ThA with the same group of conjugates yielded similar information. A quantitative relationship between the two parameters was established.

Animals↗