Soluble Antigen Fluorescent Antibody Research Articles

Serodiagnosis of intestinal tuberculosis by enzyme immunoassay and soluble antigen fluorescent antibody tests using a saline extracted antigen

The efficacy of enzyme-linked immunosorbent assay (ELISA) and soluble antigen fluorescent antibody (SAFA) tests in the diagnosis of intestinal tuberculosis was compared using saline extracted surface antigen of M. tuberculosis H 37R a. Of the 24 proved cases of intestinal tuberculosis ELISA detected positivity in 92% compared with 83% detected by the SAFA test. The 2 tests showed a strong correlation with each other and yielded a clear cut difference between patients and controls, although there was some degree of overlap.

Application of the Soluble Antigen Fluorescent Antibody (SAFA) test to the serodiagnosis of rabies

The adaptation and evaluation of the Soluble Antigen Fluorescent Antibody (SAFA) test for the serologic diagnosis of rabies is described. Evaluation of the SAFA test was based on a comparison between serum titers obtained in the SAFA test, the mouse Serum Neutralization (SN) test and in the Indirect Fluorescent Antibody (IFAT) test. Dog, fox raccoon and skunk sera were used for the comparison with mouse SN titers. Human serum was used for the comparison with the IFAT titer. The purity and concentration of the test antigen is a crucial factor in determining the efficiency of the SAFA test for rabies serodiagnosis. Viral antigen obtained by the AlPO 4 gel method for rabies virus purification and concentration was found to be sufficiently purified and concentrated for use in the SAFA test. Conjugate dilution decreased the level of non-specific staining. Although specific activity was also decreased, there was a statistically significant difference ( P ⩽ 0.05; Student's t test) between the rabies positive and the rabies negative serum samples at all conjugate dilutions for all species studied. In three cases (fox, raccoon, skunk) SAFA titers were greater than mouse SN titers. In one case (dog) the SAFA titer was less than the mouse SN titer. The IFAT titer of the human serum sample was greater than the SAFA titer. Comparison of Fluorometer Dial Readings (FDR) of sera obtained in separate protocols demonstrated satisfactory reproducibility of the SAFA test for rabies serodiagnosis.

Toxocaral larva migrans: the use of larval secretory antigens in haemagglutination and soluble antigen fluorescent antibody tests

Toxocara larval excretions and secretions collected from in vitro culture were used as antigen in passive haemagglutination and soluble antigen fluorescent antibody tests for the diagnosis of visceral larva migrans in experimental animals and man. Antibody to toxocaral secretions was detected in rabbits within 13 days of light Toxocara infection (ten larvae per kg) and within four days of heavy infection (10 4 larvae). Antibody was not detected following infection with 10 4 Ascaris suum larvae. In human sera, antibody was detected at low titre in 1 % of 100 healthy adults and in 2 % of 50 children. High titres were observed in one third of 170 patients with suspected visceral larva migrans and in 23 of 27 such patients presenting with an eosinophilia greater than 20 %. In 25 patients with ocular lesions of an undiagnosed nature, four showed significant levels of anti- Toxocara antibody.

An immunofluorescence reaction for Fasciola hepatica using the Defined Antigen Substrate Spheres (DASS) system

The Defined Antigen Substrate Spheres (DASS) system, using Fasciola hepatica antigens, proved to be a promising immunofluorescent antibody test for fascioliasis. The antigen bound beads could be freeze-dried and reconstituted to a spherical form for measurement. Sera of individuals with F. hepatica infections were examined with the Indirect Fluorescent Antibody (IFA) technique on frozen sections of the adult parasite and with the DASS system. Sera of experimentally F. hepatica infected rabbits were examined with the IFA technique, the DASS system, and the Soluble Antigen Fluorescent Antibody (SAFA) technique. A few bovine sera with F. hepatica infection were examined with the DASS system.

Amebic liver abscess in childhood. A case study of a 21-month-old Thai child and a literature review.

A case of a 21-month-old child with hepatic abscess due toEntamoeba histolyticais presented. Serological testing included the soluble antigen fluorescent antibody test and immunoelectrophoresis. A review of the world literature for infants and children with this entity suggests three observations: (1) Wherever the disease is indigenous among adults, it must be considered in the differential diagnosis when children present with dysentery and hepatomegaly. (2) There seems to be an increased prevalence of the childhood condition in southern Africa and Latin America. (3) Children less than 3 years old have hepatic abscess more frequently than older children.

Serodiagnostic test for tuberculosis.

A soluble antigen fluorescent antibody test was used for the serodiagnosis of tuberculosis in both human and nonhuman primates. This test method, when used with various serologically active isolates from mycobacteria, showed promise in detecting and measuring antibody concentration in serum. The test might be superior to the currently used intradermal tuberculin skin test. With further purification of the antigen, the soluble antigen fluorescent antibody test might be useful for identifying the specific stage of the disease and for appraising the efficacy of therapy.

Immunological reactions in chimpanzees experimentally infected with Schistosoma japonicum

Twelve young chimpanzees were infected with a single dose of 50 Schistosoma japonicum cercariae per kilogram of body weight and two others were used as uninfected controls. Serum from 4 of the 12 infected chimpanzees produced passive cutaneous anaphylaxis (PCA) in rhesus mokeys. The PCA antibodies appeared 2 months after exposure to infection and persisted for 1–2 months. Skin tests showed immediate-type hypersensitivity in all infected animals and no delayed hypersensitivity in any of them. The level of IgE immunoglobulin measured by a human gamma E radioimmunoassay increased in the infected chimpanzees. Sera from all infected animals became positive by radioactive antigen microprecipitin (RAMP) assay beginning with the second month and reached a peak 7 months after exposure. Antibody production as measured by the RAMP assay and the soluble antigen fluorescent antibody test followed a different time course of development. Lymphoblastogenesis was demonstrated in all infected animals by incorporation of tritiated thymidine into lymphocyte DNA in response to an antigenic stimulus. DNA synthesis increased progressively in all infected animals reaching a peak 4 months after exposure to infection. Sera from animals treated with an antischistosomal drug gave values which were intermediate between those of the untreated chimpanzees and their uninfected controls. No significant differences in the complement levels were observed between the infected and uninfected chimpanzees.

Trichinella spiralis: Antigen-antibody interaction assayed by radioactive iodinated antigen

A radioactive iodine labeled microprecipitin (RAMP) assay was developed for measuring the binding of antigen by antibody in trichinosis. Fifteen of 18 sera from trichinosis patients were reactive in this assay. No reactions were obtained with 71 sera from healthy persons and with 98 sera from individuals with proven viral, bacterial, mycotic, parasitic, or degenerative diseases. In experimentally infected rats and rabbits, all but one of 75 infected animals gave positive reactions in this assay, whereas all of 104 uninfected controls failed to react. Serum pools from infected and normal rabbits tested at different times with different lots of radioactive antigen gave highly reproducible results. Heating or reduction and alkylation of the immune sera destroyed or markedly reduced the antibody reactions in the RAMP assay. Rabbit immune serum was absorbed with goat antirabbit IgE antiserum. When tested after absorption, the reactivity of the serum in the RAMP assay was significantly reduced and the reactivity in the PCA test was eliminated. However, the titer in the soluble antigen fluorescent antibody test was not reduced. These studies indicate that the RAMP assay can reliably measure antibodies to T. spiralis belonging primarily to the immunoglobulin E class.

Current state of serological tests used to detect blood parasite infections

Various serologic tests used for immunodiagnosis of blood parasite infections are discussed, and the advantages and limitations of each are noted. The procedures include complement fixation, indirect fluorescent antibody, indirect hemagglutination, agglutination, and soluble antigen fluorescent antibody tests. The influence of the quality of antigen on the reliability of the tests is stressed, and the fact that no single procedure is suitable for all applications is emphasized. Problems peculiar to preparation of antigens from intraerythrocytic parasites are noted. In addition, an adaptation of the quantitatively standardized complement fixation procedure for critical, objective evaluation of antigens is described.

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.

A Radioactive Antigen Microprecipitin (RAMP) Assay for Schistosomiasis

An assay for measuring antigen binding by antibody in schistosomiasis was developed using a fraction of Schistosoma mansoni antigen labeled with radioactive iodine. This assay which is not de- signed for the routine serological diagnosis of schistosomiasis was designated radioactive antigen micro- precipitin (RAMP). It is highly reproducible, quantitative, and may serve as an indicator of the immune response in schistosomiasis. Serum samples from patients with proven schistosomiasis and other diseases were assayed for their ability to precipitate 125I-labeled S. mansoni antigen. Eighty-one per cent of 104 sera from schistosomiasis patients were reactive in the RAMP assay; no reactions were obtained with 90 sera from healthy persons. A few nonspecific reactions were obtained in other disease states. The RAMP assay and the passive cutaneous anaphylaxis (PCA) tests were compared using 85 serum specimens from infected schistosomiasis patients reactive in the soluble antigen fluorescent antibody (SAFA) technique. The RAMP assay and the PCA test were both reactive with 48 of these specimens. Three specimens in which reaginic antibodies had been detected failed to react in the RAMP assay and 17 sera which reacted in the RAMP assay gave negative results in the PCA test. Fractions of pooled human anti-S. mansoni sera were tested in the RAMP assay and the results were compared with those obtained with the same frac- tions in the SAFA and PCA tests. The first fraction from DEAE-A-25 contained the major amounts of IgG and IgA immunoglobulins and all of the detectable SAFA reaction, while fraction 5 contained RAMP and PCA activity but no detectable SAFA antibodies. Heating or reduction and alkylation of the immune serum destroyed or markedly reduced the antibody reactions in the RAMP assay and PCA test, but did not affect the reactivity of the SAFA technique. When rabbit S. mansoni antiserum was absorbed with goat antirabbit IgE serum, the reactivity of the serum was greatly reduced in the RAMP assay, elimi- nated in the PCA test, and was unchanged in the SAFA technique. The RAMP assay and PCA test cor- related closely in demonstrating the time-course development of antibodies in animals infected with schis- tosomiasis, but the antibodies detected by the SAFA technique followed an entirely different time course. These studies indicate that the RAMP assay can reliably measure antibodies in all immunoglobulin classes and particularly in IgE, and this assay is a means of demonstrating primary binding of antigen by antibody in schistosomiasis.

Recent developments in serologic methods for the diagnosis of coccidioidomycosis. 3. A soluble antigen fluorescent antibody test.

A rapid serologic test using a fluorescein isothiocyanate-conjugated (FITC) anti-IgG serum has been evaluated. Human serum reacted with antigen (coccidioidin)-coated millipore disks provided the matrix for testing. Values were obtained from fluorometer readings. Results of testing more than 100 sera have indicated a close correlation with complement-fixation and agar gel precipitin inhibition (AGP1) titers. Sera tested were positive by either slide latex agglutination (SLA) or immunodiffusion (ID) tests, or both. The soluble antigen fluorescent antibody (SAFA) test appears to be worthy of consideration for use in laboratories not equipped to perform complement-fixation testing. As with the AGPI test, the SAFA procedure can be used with single serum specimens, with consequent earlier reporting of results. Preliminary studies suggest that varying the final FITC reagent might allow evaluation of the contribution of IgM and IgA to positive serology.

Trichinella spiralis and Schistosoma mansoni: Specificity of in vitro, leukocyte-mediated histamine release from rabbit platelets

The specificity of the allergic release of histamine from rabbits infected with Trichinella spiralis or Schistosoma mansoni was examined using five different helminth antigens. Significant release could be demonstrated with homologous antigen in all 20 infected rabbits. Although several cross reactions in the infected animals were observed with non- Trichinella and Schistosoma antigens, the frequency of the cross reactions was considerably less than that observed in complement fixation or soluble antigen fluorescent antibody tests.

Echinococcus granulosus and E. multilocularis: Soluble antigen fluorescent antibody test

A soluble antigen fluorescent antibody test for the laboratory diagnosis of human echinococcosis is described. A satisfactory degree of sensitivity, specificity, and reproducibility was obtained in this test when an antigen extracted from scolices and fractionated by Chromatographic methods was employed. Conversely, fractions of hydatid fluid obtained in the same manner did not permit a desirable degree of specificity, since a large percentage of sera from individuals with parasitic, bacterial, and mycotic infections reacted in the test. Antigen extracted from Echinococcus granulosus scolices permitted the detection of E. multilocularis infections as well. This technique may be suited as a screening procedure in investigating the seroepidemiology of hydatid disease and for the mass screening of domestic animals in highly endemic areas.

Epidemiologic and serologic investigations of filariasis in indigenous populations and American soldiers in South Vietnam.

Abstract Studies were accomplished in residents of South Vietnam to determine the geographic extent, prevalence, and periodicity of filariasis. Examination of concentrated, peripheral-blood samples revealed endemic foci of infection with Wuchereria bancrofti in Montagnard tribespeople residing in areas north and west of Saigon. No infections were demonstrated in residents of Saigon, in a central highland village, or in a Mekong River delta district. Prevalences in endemic areas ranged from 5 to 22%. In 12 persons with microfilaremia, quantitative studies on blood specimens collected at regular intervals over a 24-hour span demonstrated a nocturnal periodicity. Some specimens were obtained from native residents and U.S. servicemen for determination of filarial antibodies with the soluble-antigen fluorescent-antibody (SAFA) test. In native residents of endemic areas, the rates of seropositivity were 84% in those with microfilaremia, and 51% in those without demonstrable microfilaremia (total equals 55% seropositive). Rates of seropositivity in U. S. servicemen were 11% in endemic areas compared with 3% in apparently non-endemic areas. Moreover, when U. S. servicemen in an endemic area were characterized according to type of duty, the rate of positive reactions in field troops was three times higher than that of base-camp personnel. One U. S. field soldier, who was first seen with inguinal adenopathy and peripheral hypereosinophilia, was found to have circulating microfilariae of W. bancrofti and a positive SAFA test. The high rates of seropositivity in native residents and U. S. servicemen in endemic areas probably reflect immunologic responses to the Filarioidea that infect man or other animals, or to both.

A soluble antigen fluorescent antibody test for the serodiagnosis of Mycobacterium tuberculosis infection.

A soluble antigen fluorescent antibody (SAFA) test for the serodiagnosis of tuberculosis has been described. In these preliminary studies, three antigens derived from Mycobacterium tuberculosis were evaluated in SAFA tests on 42 human sera collected at various intervals during therapy. The C protein fraction proved to be the most specific, sensitive antigen and, in contrast to intradermal tests, antibodies detected in the SAFA test appeared to indicate current or recent active tuberculosis. Although individuals with advanced or far advanced disease demonstrated considerable evidence of energy, it was believed that this would not pose a serious problem in detection of early infection. The majority of patients demonstrated a significant rise in antibody titers following initiation of therapy and maximal levels were reached within 60 to 90 days. The findings indicate that the SAFA test has excellent potential for detection of active tuberculosis and for appraising the efficacy of therapy.

A soluble antigen fluorescent antibody (SAFA) test for the immunodiagnosis of trichinosis in man and experimental animals

The use of the soluble antigen fluorescent antibody (SAFA) test for the laboratory diagnosis of trichinosis is described. Twenty-six of 28 specimens from humans with proven trichinosis and 2 of 90 specimens from healthy controls reacted in this test. Except for schistosomiasis, reactions with sera from individuals infected with other parasitic agents rarely occurred. Elution of dried blood specimens obtained on absorbent paper could be tested satisfactorily by this technic. Results with mice, rats, and rabbits experimentally infected with single and multiple doses of Trichinella spiralis larvae showed that SAFA antibodies could be detected 2 weeks after inoculation and reached a peak 4–5 weeks later. A high antibody titer was sustained until the end of the experiment, approximately 8 months after infection.

Mass diagnostic test using Plasmodium falciparum and chimpanzee erythrocyte lysate

A soluble antigen fluorescent antibody test was developed for the serologic diagnosis of human malaria by using Plasmodium falciparum-parasitized erythrocyte lysates from experimentally infected chimpanzees as antigen. A stable antigen fractionated by sequential elution with chromatography on DEAE Sephadex A-25 was reactive at high dilutions. Therefore, approximately 50,000 tests could be performed with the amount of antigen normally collected from one infected chimpanzee. The technic was evaluated with 581 human serum specimens from selected individuals. Reactions were observed in 269 of 270 specimens from malaria patients whereas 129 of 132 specimens from healthy controls gave negative results. Occasional cross-reactions were observed with sera from individuals with infections other than malaria. Most of these reactions were obtained with sera from individuals with leprosy, leishmaniasis, American trypanosomiasis or hookworm in areas which are endemic for malaria. The high degree of sensitivity, specificity and reproducibility of the test, the objectivity of mechanical reading, the availability of large amounts of P. faldparum antigen and the possibility of processing many specimens semiautomatically in a relatively short time suggest that this technic may be well suited as a screening procedure in investigating the sero-epidemiology of malaria and for the mass screening of potential blood donors.

Soluble antigen fluorescent antibody test for amebiasis

The use of the soluble antigen fluorescent antibody test for the laboratory diagnosis of human amebiasis is described. Although, in general, higher titers were observed in sera from patients who had amebic liver abscesses, a high degree of sensitivity was observed with this test also when sera from patients with amebic dysentery and asymptomatic individuals were used. A relatively high degree of reproducibility of results was observed. While a number of nonspecific staining reactions occurred with sera from patients with visceral leishmaniasis, the specificity with other sera suggests that this procedure may be useful in the serological diagnosis of human amebiasis.

Relative sensitivity and specificity of soluble antigens (metabolic and somatic) and whole cercariae in fluorescent antibody tests for schistosomiasis in humans and rabbits

The diagnostic value of six fluorescent antibody procedures employing different antigens has been investigated with 391 human serum specimens. The sensitivity of the various procedures was determined on 123 specimens from individuals with proven infection with Schistosoma mansoni and/or S. haematobium. The specificity of the procedure was evaluated on 144 serum specimens from individuals with infections other than schistosomiasis and with collagen diseases, and on 124 specimens from healthy controls. The highest degree of sensitivity was obtained when fixed whole cercariae were used as the source of antigen or when somatic products of delipidized cercariae were used in the soluble antigen fluorescent antibody test. The highest degree of specificity was obtained when fixed intact cercariae were used as the source of antigen or when cercarial somatic and adult metabolic products were used in the soluble antigen fluorescent antibody test. Adult somatic and metabolic antigens conferred to the test the lowest degree of sensitivity. Cercarial metabolic antigen resulted in the lowest degree of specificity. The serologic response of rabbits to single graded doses of cercariae was studied quantitatively. With all antigens, the titers increased rapidily and reached a peak between 6 and 13 weeks after infection. When intact fixed cercariae were used as antigen, the titer was directly related to the size of infection.