Fluorescein-labeled Antiserum Research Articles

Use of Immunofluorescence to Identify Clostridium botulinum Types A, B, and E

T HE DE'TECTION of Clostridium, botulinum in food or cultures usually requires the demonstration of toxin by mouse inoculation and the identification of toxic type by mouse protection tests using the specific antitoxins. Since cultural and animal tests are very time consuming, there is a need for specific, more rapid methods to detect C. botulinuim in food. The fluorescent antibody (FA) technique has been useful in the rapid identification of pathogens (1). If a specific FA method could be developed for detecting C. botulinum organisms, it would greatly facilitate examination of food and cultures. A search of the relevant literature showed interesting and varied findings. Kalitina (2), using a fluorescein-labeled antiserum prepared against a formalinized culture of C. botulinutm type A, found that all types of C. botulinum organisms were stained but none of the other species tested were stained. Bulatova and Kabanova (3) found that only types A and B were stained by fluorescein-labeled antiserum prepared against type A organisms, but 3 of 17 strains of C. sporogenes weire sta,ined. Using FA techniques, Walker and Batty (4) found it possible to differentiate among three important groups: proteolytic types A, B, and F; nonproteolytic types C and D; and nonproteolytic type E. No cross-reactions with C. sporogenes were observed by Walker and Batty. All three groups of investigators agreed that the PA technique holds great promise. This study was undertaken to obtain data on the application of a direct FA technique for the detection and identification of types A, B, and E C. botulinum organisms in food and in cultures.

Cellular origin of hyaluronateprotein in human synovial membrane.

Bright fluorescent staining, which indicates the presence of hyaluronateprotein, was observed in the lining cells of the synovial membrane following application of rabbit antiserum to hyaluronateprotein and a fluorescein-labeled antiserum to rabbit gamma-globulin. Staining was shown to be specific and due to antigenic determinants on or closely associated with the protein moiety of hyaluronateprotein.

Specificity of immunofluorescent staining for study of Aspergillus flavus in soil.

Fluorescein-labeled antiserum prepared with Aspergillus flavus strain CS was tested for specificity by staining fungi grown in soil in the vicinity of buried slides. All 14 strains of A. flavus fluoresced as intensely or nearly as intensely as the antigen control. Among 21 isolates of species of Aspergillus other than A. flavus, 17 reacted with moderate to low fluorescence at intensities readily distinguishable from that of A. flavus. The fluorescence of the remaining four cultures, and particularly A. sydowi, was indistinguishable from that of A. flavus. Fungi other than aspergilli were generally nonreactive. Interfering cross-reactions were encountered for one strain of Spicaria and one strain of Stemphylium; three isolates could not be evaluated because of interfering autofluorescence. An additional 22 isolates were either wholly negative or had a low order of fluorescence. Agglutination tests between each of the fungi and A. flavus CS serum revealed close agreement between agglutination titer and fluorescent-staining reaction. Unknown fungi freshly isolated from soil were checked for reaction to the A. flavus labeled antiserum; only one isolate gave a pronounced staining reaction, and that one proved to be a strain of A. flavus. In a simplified ecological model, the fluorescent-antibody technique was used to follow the development of A. flavus in mixed culture in soil with five other soil fungi.

Specificity of Immunofluorescent Staining for Study of Aspergillus flavus in Soil

Fluorescein-labeled antiserum prepared with Aspergillus flavus strain CS was tested for specificity by staining fungi grown in soil in the vicinity of buried slides. All 14 strains of A. flavus fluoresced as intensely or nearly as intensely as the antigen control. Among 21 isolates of species of Aspergillus other than A. flavus, 17 reacted with moderate to low fluorescence at intensities readily distinguishable from that of A. flavus. The fluorescence of the remaining four cultures, and particularly A. sydowi, was indistinguishable from that of A. flavus. Fungi other than aspergilli were generally nonreactive. Interfering cross-reactions were encountered for one strain of Spicaria and one strain of Stemphylium; three isolates could not be evaluated because of interfering autofluorescence. An additional 22 isolates were either wholly negative or had a low order of fluorescence. Agglutination tests between each of the fungi and A. flavus CS serum revealed close agreement between agglutination titer and fluorescent-staining reaction. Unknown fungi freshly isolated from soil were checked for reaction to the A. flavus labeled antiserum; only one isolate gave a pronounced staining reaction, and that one proved to be a strain of A. flavus. In a simplified ecological model, the fluorescent-antibody technique was used to follow the development of A. flavus in mixed culture in soil with five other soil fungi.

DETECTION OF A PLASMODIUM BERGHEI-ANTIBODY COMPLEX FORMED IN VIVO.

SummaryDirect staining of Plasmodium berghei with fluorescein-labeled antiserum from infected rodents indicated that these animals are capable of producing an antibody against the parasite. A naturally occurring P. berghei-antibody complex was detected by exposing parasitized erythrocytes from infected mice to fluoresceinlabeled anti-mouse globulin.

A carrier state of mumps virus in human conjunctiva cells. I. General characteristics.

Mumps virus produced a carrier state in human conjunctiva cells that was maintained for more than 100 subcultures over a period of 3 years. Antiserum in the medium was not required. The virus had little apparent effect on the cells which grew at a rate similar to uninfected control cells. Mumps virus was regularly found in the culture medium at levels about 0.9 log higher than the cell-associated virus. When first tested after 30 subcultures, the virus was found to have lost its cytopathogenicity for cells ordinarily susceptible to mumps virus, but was identifiable as mumps virus by neutralization with specific antiserum. Use of fluorescein-labeled antiserum revealed that 80 to 95 per cent of cells in the carrier cultures contained mumps virus antigen. The antigen was concentrated in a few sharply circumscribed, discrete masses in the cell cytoplasm rather than in many granules throughout the cytoplasm as is characteristic of cell infection by cytopathogenic mumps virus. The carrier cultures were resistant to the destructive effect of a cytopathogenic line of mumps virus, but showed little resistance to the cytopathogenic effect of vesicular stomatitis, Sendai, or Newcastle disease viruses.

Transfer of nephrotoxic glomerulonephritis in rats by means of white blood cells.

TRANSFER of experimental glomerulonephritis on originally healthy rats has been accomplished in this and other laboratories by means of parabiosis1,2. Transfer was observed in random-bred as well as in inbred rats, either in short-term experiments or after intervals up to 71 days between institution of nephrotoxic serum glomerulonephritis in the primarily nephritic animal, and parabiotic conjunction with the originally healthy partner3–5. Fluorescein-labelled antisera were used to exclude the involvement of the primarily injected heterologous (rabbit) antibodies in the transfer phenomenon6. From these experiments it was derived that a secondary nephritis-producing factor, appearing as a consequence of the nephrotoxic serum glomerulonephritis in the circulation of the primarily nephritic rat, was transferred to the primarily healthy animal capable of attacking the corresponding organs of the latter.

ANTIBODIES TO ESTRONE-PROTEIN CONJUGATES: III. TISSUE LOCALIZATION OF ESTROGENS

Evidence was obtained that the mature rat ovary contained cellular component(s) which cross-reacted with fluorescein-labelled antiserum to estrone-2-carbamido-HSA.

Staining toxoplasma gondii with fluorescein-labelled antibody. II. A new serologic test for antibodies to Toxoplasma based upon inhibition of specific staining.

A new serologic test for antibodies to Toxoplasma is described, which is based upon inhibition of specific staining with fluorescent antibody. In performing the test, a mixture of the test serum and known fluorescein-labelled antiserum is added to a dried smear of toxoplasms for 1 hour at 37 degrees C. The smear is then rinsed and examined with a fluorescence microscope. Reduction in the brightness of fluorescence, as compared to that of a negative control slide, indicates the presence of antibody in the test serum. A comparison of the results of this test with those of the methylene blue dye test showed a strong parallelism between the two sets of results. On the other hand, the complement-fixation test for toxoplasmosis did not yield nearly as many positives as the inhibition test. The specificity of the new test was studied by comparing it with dye test results and clinical histories in human patients, and by testing a group of animals immunized with a variety of non-Toxoplasma antigens. No evidence of cross-reactions was obtained in the latter series. Some advantages and disadvantages of the inhibition test are discussed.

Studies on the Metabolism of Fibrinogen in Two Patients with Congenital Afibrinogenemia

Abstract 1. The rate of disappearance from the plasma of intravenously administered fibrinogen has been studied immunochemically in two children with congenital afibrinogenemia. 2. About half of the administered fibrinogen disappeared from the circulation in the first forty-eight hours, thus emphasizing the importance of the extravascular pool of plasma protein. 3. After the first two days, the fibrinogen followed a logarithmic decay curve, with a half life of four days; this is so close to the half life of 5.6 days estimated from radioisotope turnover studies that it indicates that the fundamental defect in these patients is a failure to synthesize adequate quantities of fibrinogen. 4. Traces of fibrinogen were detected immunochemically in the plasma of each patient; in one of them, the small amount found could not readily be attributed to prior infusion of blood or blood products. 5. The connective tissue in biopsy specimens of skin and muscle from one patient, when examined with fluorescein-labelled antisera, was found to be specifically deficient in fibrinogen. This deficit was quickly rectified by intravenous administration of fibrinogen.

Distribution of mumps virus in tissue cultures as determined by fluorescein-labeled antiserum.

SummaryFluorescein-labeled antibody has been successfully employed in the localization of mumps virus in cultures of various types of chick embryonic tissues infected in vitro. The distribution of the virus was found to be restricted to the cytoplasm of the cells which came in direct contact with the inoculum irrespective of the kind of tissue used in the culture.