Fluorescent Antibody Procedure Research Articles

Separation of Treponema pallidum from tissue substances by continuous-flow zonal centrifugation.

The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 x 10(9) (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum. Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure.

Reactivity of treponemal and nontreponemal tests with cerebrospinal fluid from syphilitic chimpanzees.

Samples of cerebrospinal fluid (CSF) and serum collected over a period of approximately four years from four chimpanzees infected with Treponema pallidum were studied. The VDRL slide test, the Treponema pallidum immobilization-200 (TPI-200) test, and several fluorescent-antibody procedures were used in the examination of the spinal fluid. Sera were examined with the VDRL slide and fluorescent treponemal antibody-absorption (FTA-ABS) tests. None of the animals showed reactivity in the CSF before inoculation. The immunofluorescence techniques usually detected reactivity in the CSF before the VDRL slide test. The TPI-200 test was considerably less reactive with CSF than either the immunofluorescence procedures or the VDRL slide test. Reactivity of the immunofluorescence and VDRL slide tests with the CSF was delayed and weaker than reactivity of these tests with serum. Results suggest that evaluation of the FTA technique with CSF of humans might lead to use of an FTA test in diagnosis of syphilis of the central nervous system of humans.

Separation of Treponema pallidum from Tissue Substances by Continuous-Flow Zonal Centrifugation

The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 x 10(9) (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum. Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure.

Efficacy of a Fluorescent-Antibody Procedure for Identifying Bacillus cereus in Foods

One hundred and seventeen strains of Bacillus were examined by the fluorescent-antibody technique by using the globulin fraction of serum prepared against spores of B. cereus T. All but one strain of the 59 B. cereus tested fluoresced at the exosporium surface. Fluorescent staining of B. anthracis, B. thuringiensis, and B. mycoides was also observed. Absorption of the globulin fraction with B. anthracis and B. mycoides resulted in the elimination of staining of these organisms. Absorption with B. thuringiensis ATCC 10792 removed antibodies reacting with 6 of the strains of B. thuringiensis tested. Absorption with B. thuringiensis var. galleriae removed antibodies against B. cereus to such a degree that the globulin fraction was unusable.

Advances in methodology for immunodiagnosis of parasitic diseases

During recent years, significant progress has been made in the development and improvement of procedures for the immunodiagnosis of parasitic diseases. The purpose of this review is to briefly discuss the inherent advantages and limitations of various immunodiagnostic methods currently in general use, to delineate areas in which further research is needed, and to call attention to new technics that may prove to be valuable additions to the present armamentarium of procedures for the immunodiagnosis of parasitic infections. It is well established that the specificity and sensitivity of an immunodiagnostic procedure depend on the quality of the antigen employed. The introduction of better methods for isolating the serologically active antigen components from crude parasite extracts has significantly improved the pathognomonic value of many immunodiagnostic tests. Since the chemical nature of the major specific antigen(s) differs among the various parasites, no single method is universally suitable for antigen fraetionation. Studies illustrating the variety of physicochemical fractionation procedures that have been used successfully for the isolation of specific parasite antigens are noted. In addition, attention is called to the potential advantages of employing exoantigens (i.e., excretions and secretions of living parasites) in immunodiagnostic tests. Procedures most frequently employed for immunodiagnosis of parasitic diseases are discussed in some detail and include: complement fixation; indirect hemagglutination; indirect immunofluorescence procedures using whole organisms as antigens; flocculation tests employing lecithin-cholesterol crystals, bentonite or latex particles as the antigen matrix; card tests using antigen-sensitized lecithin-cholesterol crystals with added charcoal; agglutination reactions; precipitin tests; and hypersensitivity reactions. The advantages and limitations of these procedures are noted and areas in which their use could be extended are suggested. Certain less commonly employed procedures that have shown considerable promise for serodiagnosis or for basic studies on the immunology of parasitic diseases also are discussed. These include immunoelectrophoresis, the recently developed soluble antigen fluorescent antibody procedure, in vitro histamine release by in vivo-sensitized leukocytes, passive cutaneous anaphylaxis, conglutinating complement absorption procedures, and serological adhesion (immune adherence) reactions. Problems associated with the wide variations of methodology employed in various diagnostic laboratories are noted and the need for improved standardization of antigens and technics is emphasized.

Rapid Detection of Salmonella Microcolonies by Fluorescent Antibody

A microcolony fluorescent-antibody (FA) procedure for detecting salmonellae was compared to the usual direct FA procedure on 304 environmental, food, and feed samples. The microcolony FA test detected all of the specimens found positive by culture, whereas the direct FA missed 3.1% of them. Both FA tests revealed stained organisms in some of the culturally negative specimens. The microcolony FA test has several advantages over the direct FA test: ease of examining the smears, elimination of the fluorescent background material, and increased sensitivity.

One-day fluorescent-antibody procedure for detecting salmonellae in frozen and dried foods.

The indirect fluorescent-antibody technique was used to examine 422 food samples for the presence of salmonellae. A cultural phase involving a 16-hr preenrichment in buffered nutrient broth-milk medium followed by a 4- to 5-hr subculture into fresh medium of the same composition was evaluated. This procedure yielded a sufficient population of salmonellae so that no false-negative results were obtained. Of the 31 false-positives obtained, 12 samples yielded positive cultural results upon extensive subculture of the original enrichment broths. Yeast cells and both vegetative and spore forms of bacilli were observed to fluoresce when stained with anti-Salmonella serum. Efforts to ascertain the cause of these cross-reactions and several alternate explanations are discussed.

Fluorescent-Antibody Marker for Vaccine-Induced Rubella Antibodies

Tests with LLC-MK2 and BHK-21 cells showed that the fluorescent-antibody (FA) procedure can be used to differentiate antibodies resulting from natural infection and antibodies stimulated by rubella vaccine virus. Negative or weak FA reactions in LLC-MK2 cells and strong reactions in BHK-21 cells indicated antibody stimulated by the vaccine virus; strong reactions in both cell systems reflected naturally acquired immunity.

Immunoglobulin Class of Natural Human Antibodies Reactive with Treponema pallidum

To aid the development of serological tests for incubating syphilis, a characterization was made of the background of natural anti-Treponema pallidum antibodies against which the initial immune response to syphilis takes place. Sera from presumed normal persons were studied with monospecific antisera in an indirect fluorescent-antibody procedure. Of 36 sera tested at a 1:5 dilution, all showed IgG reactivity with T. pallidum (Nichols strain), 58% showed IgM reactivity, and 20% showed IgA reactivity. The titer of IgG reactivity was considerably higher than that of the other two immunoglobulins. Heating the sera for 1 hr at 65 C abolished IgA and IgM anti-T. pallidum reactivity, but one-third of the sera retained IgG reactivity. Human Cohn fractions II and III(1) from three commercial sources contained mostly IgG antibodies reactive with T. pallidum, but IgM and IgA antibodies were also present. IgG reactivity was found in a pool of presumably normal maternal sera from 20 mothers and in a corresponding pool of umbilical cord serum from their infants. IgM and IgA reactivities were present only in the maternal serum pool.

One-Day Fluorescent-Antibody Procedure for Detecting Salmonellae in Frozen and Dried Foods 1

The indirect fluorescent-antibody technique was used to examine 422 food samples for the presence of salmonellae. A cultural phase involving a 16-hr preenrichment in buffered nutrient broth-milk medium followed by a 4- to 5-hr subculture into fresh medium of the same composition was evaluated. This procedure yielded a sufficient population of salmonellae so that no false-negative results were obtained. Of the 31 false-positives obtained, 12 samples yielded positive cultural results upon extensive subculture of the original enrichment broths. Yeast cells and both vegetative and spore forms of bacilli were observed to fluoresce when stained with anti-Salmonella serum. Efforts to ascertain the cause of these cross-reactions and several alternate explanations are discussed.

Fluorescent antibody test for neonatal congenital syphilis: A progress report

Sera from newborn infants with reactive VDRL and FTA-ABS tests were examined in an indirect IgM fluorescent antibody procedure. This experimental test was reactive in all 18 cases of neonatal syphilis and nonreactive in all clinically normal infants studied. Although the γM-FTA-ABS test appeared to be at least as specific as the VDRL and FTA-ABS tests, further study is necessary in order to definitely establish its sensitivity. The test appears to be specific for neonatal congenital syphilis and sometimes reverts to normal after adequate treatment.

SALMONELLAE AND THE FLUORESCENT-ANTIBODY TECHNIQUE: A CURRENT EVALUATION

The increased microbiological surveillance of foods by government regulatory agencies and industry has shown that salmonellae are an important cause of food-borne disease. The significance of salmonellae in food-borne disease has made it mandatory for regulatory agencies as well as industry to develop a rapid, reliable, and reproducible method for the recovery of salmonellae in feeds and foods.Standard cultural procedures have become increasingly sensitive but not without a proportional increase in the time necessary for salmonellae detection and confirmation. This test may require from 5 to 7 days elapsed time with all of the implied disadvantages.One of the most promising areas of activity is the rapid microbiological-serological technique employing an improved fluorescent-antibody procedure. While much has been done by the medical technologist, the application of this technique is relatively new in food microbiology. The published data show the method to be highly promising and possibly even more sensitive than the standard cultural procedure.This technique is not without its inherent problems. Like all methods under investigation, there are differences in procedure, validity, and interpretation of the data. These are cited, with a description and critique of the work done, in order to give the reader an opportunity to evaluate the state of the art.

Rabies in wild animals trapped for pelts.

The brains of 1,634 wild animals trapped for pelts were examined for rabies by the fluorescent antibody procedure. Twenty-one of 268 striped skunks and 2 of 52 spotted skunks examined were positive for rabies. Thirteen hundred twenty three animals including 7 badgers, 65 coyotes, 365 foxes, 1 weasel, 377 mink, 52 muskrats, 208 opossums, 219 racoons and 1 wolf were negative.

Fluorescent-antibody technique in detection of salmonellae in animal feed and feed ingredients.

Comparative studies of fluorescent antibody procedure and a cultural method for the detection of Salmonella were made on 1,013 feed and feed-ingredient samples. The agreement between the two methods was 92.1%. There were more false positives (5.7%) than false negatives (2.2%). Of the 22 false negatives, 15 (68%) were obtained on meat meal. Of the total number of samples, 37% were meat meal. An additional study of 73 samples of meat meal indicated that correlation between methods was better than correlation between samples.

Determination and analysis of Actinomyces israelii serotypes by fluorescent-antibody procedures.

This study was undertaken to determine the antigenic relationships between serotypes of Actinomyces israelii with fluorescent-antibody (FA) procedures. In addition, the antigenic relationships between A. israelii and other members of the genus Actinomyces were studied by the same methods. Seven FA conjugates were used to determine the serological characteristics of 28 isolates believed to represent A. israelii, serotypes 1 and 2. The results showed that the lower dilutions of serotype 1 conjugates stained serotype 2 antigens; however, serotype 2 conjugates did not stain serotype 1 antigens. Serotype 1 conjugate could be made specific by adsorption. A. israelii serotype 1 conjugate cross-reacted also with A. naeslundii, but this cross-reaction could be eliminated by adsorption or dilution. Serotype 2 conjugate appeared to be specific for A. israelii serotype 2. Adsorption studies revealed antigenic variants among the various A. israelii serotype 1 and 2 isolates. However, all isolates could be identified by direct FA staining with appropriate conjugates. One isolate previously identified as A. israelii was shown, on the basis of FA studies, to be an A. naeslundii. A polyvalent diagnostic reagent was prepared which was specific for A. israelii serotypes 1 and 2. This reagent should find application in diagnostic and reference laboratories.

Accelerated Immunofluorescence Procedure for the Detection of Salmonella in Foods and Animal By-Products

An accelerated, direct immunofluorescent-antibody procedure was developed for the detection of Salmonella in food products. This method includes pre-enrichment and selective enrichment but eliminates many of the washing and smear treatments present in existing methods. Commercially available fluorescein-conjugated somatic antiserum was used in comparing this method with conventional culture, biochemical, and serological procedures. The 894 samples tested represented 39 different products. The fluorescent-antibody procedure detected Salmonella in 216 test samples as compared to 205 positives recovered by using the standard culture procedures. In no instance did the fluorescent-antibody procedure fail to detect a Salmonella positive which had been detected by the standard procedure. With a three-tube, most-probable-number procedure, the fluorescent-antibody method was able to detect Salmonella at a level of 0.036 organism per g. In addition to being a more rapid method for the detection of Salmonella, it has proven to be comparable to conventional culture procedures.

Fluorescent-Antibody Technique in Detection of Salmonellae in Animal Feed and Feed Ingredients

Comparative studies of fluorescent antibody procedure and a cultural method for the detection of Salmonella were made on 1,013 feed and feed-ingredient samples. The agreement between the two methods was 92.1%. There were more false positives (5.7%) than false negatives (2.2%). Of the 22 false negatives, 15 (68%) were obtained on meat meal. Of the total number of samples, 37% were meat meal. An additional study of 73 samples of meat meal indicated that correlation between methods was better than correlation between samples.

Physiological and serological comparisons among strains of Mycoplasma granularum and Mycoplasma laidlawii.

Mycoplasma granularum strains grew on a medium devoid of animal serum or of serum fractions containing sterols; all strains possessed properties, including carotenoid biosynthesis, similar to those described for M. laidlawii. Some common antigenic components were noted among M. granularum and M. laidlawii strains by indirect fluorescent-antibody tests. The growth of M. granularum strains was slightly inhibited by antiserum to M. laidlawii PG-8, and the electrophoretic patterns of cell proteins of the M. granularum strains showed a close resemblance to that of M. laidlawii. However, direct fluorescent-antibody procedures performed on colonies grown on a serum-free medium clearly distinguished M. granularum from M. laidlawii. The occurrence of nonsterol-requiring mycoplasmas, in addition to M. laidlawii, raises questions as to the taxonomy of M. granularum and of the saprophytic mycoplasmas in general.

Lymphoid Leukosis Virus: A Survey of Commercial Breeding Flocks for Genetic Resistance and Incidence of Embryo Infection

Viruses in the avian leukosis-sarcoma group are known to cause a number of neoplasms, including visceral lymphomatosis (lymphoid leukosis), erythroblastosis, myeloblastosis, myelocytomatosis, nephroblastomas, endotheliomas, sarcomas, and possibly osteopetrosis. Infection by any one of the viruses in this group causes the formation of a group-specific antigen detectable by the COFAL test, a complement-fixation procedure (17). Properties reliant upon the viral coat antigens, however, have permitted the identification of at least 3 distinct subgroups (A, B, and C), two of which (A and B) are known to exist in commercial chicken populations (4,14,15). These properties are: 1) antigenicity, as measured by virus neutralization or fluorescent antibody procedures; 2) ability to induce resistance to a given strain of Rous sarcoma virus (RSV) in chick embryo fibroblasts; and 3) infectivity for various genetically defined cell populations. Several of the virus types, e.g. lymphoid leukosis virus (LLV), avian myeloblastosis virus (AMV), and RSV, have strains representing more than one subgroup (14,15). Rubin, who first noted the resistance phenomenon in 2 above, introduced the acronym RIF, for resistance-inducing factor, for an isolate of LLV (12). The term "RIF virus" has since been loosely applied to other similar isolates, and RIF activity has referred to the resistance phenomenon regardless of the virus type.

Spirochete from the rabbit tick, Haemaphysalis leporispalustris (Packard). I. Isolation and preliminary characterization.

An unidentified spirochete, referred to as the 277F agent, was isolated from Haemaphysalis leporispalustris ticks from two cottontail rabbits by inoculation of the tick suspension into embryonated chicken eggs. Because of its minute width (0.1 mu), the organism was difficult to see when stained by the Giemsa method, but was readily demonstrated by silver impregnation or fluorescent-antibody procedures. In dark-field microscopy, the spirochetes appeared uniformly and rather tightly coiled, and exhibited typical corkscrewlike motility. After yolk-sac inoculation, the agent was highly lethal for chick embryos and was recovered in large quantity from several embryonic tissues and fluids. It could also be maintained in nonfertile eggs and in an enriched liquid medium. This previously undescribed spirochete could pass through Berkefeld N but not Seitz EK filters. It was relatively resistant to heat and to penicillin or sulfadiazine, but was markedly inhibited by streptomycin, broad-spectrum antibiotics, and homologous neutralizing antiserum. Of several species of animals tested for susceptibility to this spirochete, only the snowshoe hare gave evidence of infection.