Prevalence of antibodies to Francisella tularensis in forest workers from different regions of Poland

In the present study we evaluate the prevalence of antibodies to F. tularensis in 480 serum samples obtained from healthy forest workers from different regions of Poland. The investigations were performed using the tube agglutination test and ELISA. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. In none serum samples we detected antibodies to F. tularensis by tube agglutination test. Of the 480 tested sera IgA antibodies were detected by ELISA in 4.6%, antibodies IgG in 3.8% and antibodies IgM in 2.70% serum samples. The results of our study showed that antibodies to F. tularensis were slightly, but not statistically significant, more often diagnosed in healthy forest workers than healthy blood donors.     

Microagglutination Procedures for Febrile Agglutination Tests

Febrile agglutination tests were done by using as antigens Brucella abortus, Salmonella group D, Proteus OX19, and Pasteurella tularensis. Comparison of results from 23 sera showed that the microtechnique, rapid slide, and test tube methods gave similar titers, although those from the microtechnique were generally higher. The sensitivity of the microtechnique depended upon the concentration of antigen, and, to obtain reproducible results, the optimal concentration of antigens had to be determined by preliminary titrations against specific, positive control antisera. Readability of reactions in the microtechnique was enhanced by adding the dye Safranin O to diluent for antigen and by use of V-type, rather than U-type, microtiter plates. Tests were also done to determine the effects of dye and salt concentrations, pH, and temperature of incubation upon the titer of agglutinations by the microtechnique. Our results indicated that the microtechnique could be used for agglutination tests involving febrile antigens. The procedure is less time-consuming than the tube method and requires less antigen and serum than the latter method or the rapid slide method.     

Value and Interpretation of Serological Tests for the Diagnosis of Cryptococcosis

MAXIMAL SEROLOGICAL DIAGNOSIS OF CRYPTOCOCCOSIS MAY BE ACCOMPLISHED THROUGH THE CONCURRENT USE OF THREE TESTS: the latex agglutination (LA) test for cryptococcal antigen, and the indirect fluorescent antibody (IFA) and tube agglutination (TA) tests for Cryptococcus neoformans antibodies. These tests were applied to 141 serum and cerebral spinal fluid specimens from 66 culturally proven cases of cryptococcosis and to 42 sera from normal subjects and from patients with other systemic mycotic diseases. The LA test was sensitive and completely specific; of the sera from proven cases, 55% were positive. With the TA test, 37% of the specimens were positive and the test was highly specific. With the IFA test, 38% of the specimens were positive and the test appears to be the least specific of the three. Cross-reactions were most evident with blastomycosis and histoplasmosis case sera. When the three tests were used concurrently, 87% of the cryptococcosis case specimens were positive and permitted a presumptive diagnosis of C. neoformans infections in 61 (92%) of the 66 patients whose specimens were examined.     

Yersinia Yop-Specific IgA antibodies in Hungarian blood donors

Sera of 112 healthy Hungarian blood donors were tested for the presence of Yersinia enterocolitica and Y. pseudotuberculosis-specific agglutinins by tube agglutination, and for that of yersinia outer membrane protein (Yop)-specific IgA antibodies by ELISA. The positive results of this latter assay were confirmed by immunoblot. Only one sample gave a positive agglutination reaction with Y. enterocolitica antigen (group 03) and four exhibited an equivocal reaction with Y. pseudotuberculosis antigens (groups II and IV). Contrary to the low incidence of agglutinins, 15.1% of the samples showed a positive Yop-specific IgA reaction, while further 5.3% samples fell into the equivocal range by ELISA (17 and 6 specimens, respectively). Eleven of these samples (9.8% of all specimens tested) were also positive by immunoblot for the presence of Yop-specific IgA antibodies. These data suggest a higher incidence of yersinia infections than the 1.0-1.4 per 10(5) population predicted on the basis of stool culture results.     

Microtiter Plate Agglutination Test for Salmonella Antibodies

Similar results were obtained when testing human sera for Salmonella antibodies by the tube agglutination test and by the Microtiter plate agglutination test. The plate test was easier to perform and saved time, space, antigen, and serum.     

Determination of tularemia antibodies by an immunoenzyme method on a solid-phase carrier (ELISA)

ELISA "sandwich" techniques have been developed and the optimum assay conditions for detecting specific antibodies in human serum samples have been determined. The possibility of using these techniques for the determination of the level of antibodies to tularemia antigens in the sera of persons immunized with live tularemia vaccine has been shown. Statistically significant differences in the level of antibodies to tularemia antigen in the sera of immunized and nonimmunized persons have been established. The comparative study of five serological methods - ELISA, the agglutination test, the passive hemagglutination test, the immunofluorescence test and the defined antigen substrate sera ( DASS ) techniques - has revealed the advantage of ELISA, whose sensitivity has proved to be considerably higher than that of all other methods used in our work.     

Detection of antibodies in serum and cerebrospinal fluid to Toxoplasma gondii by indirect latex agglutination test and enzyme-linked immunosorbent assay.

Sensitivity of anti-Toxoplasma antibody (IgG) test by enzyme-linked immunosorbent assay (ELISA) was evaluated in comparison with indirect latex agglutination (ILA) using 2,016 paired human samples of serum and cerebrospinal fluid (CSF). The samples were collected from neurologic patients in Korea with mass lesions in central nervous system (CNS) as revealed by imaging diagnosis (CT/MRI). When the sera were screened for anti-Toxoplasma antibody by ILA, 76 cases(3.8%) were positive (1:32 or higher titers). In the paired samples of CSF, no positive reactions were observed. When ELISA was performed using PBS extract of Percoll purified tachyzoites as antigen, cut-off absorbance was determined as 0.40 for serum and 0.27 for CSF tests. The antibody positive rates by ELISA were 7.0% in serum and 5.6% in CSF. Of them, 40 cases (2.0%) showed positive reactions in both serum and CSF. The antibody positive rates were higher in groups older than 40 years. The rates were higher in male (4.7% by ILA, 8.3% by ELISA) than in female (2.2% by ILA, 5.0% by ELISA). The rates in CSF showed no such sex difference. ELISA showed twice higher positive rates when serum was tested, and was sensitive enough to detect specific antibodies in CSF. Etiologic relations between positive antibody tests and CNS lesions remained unknown.     

Pasteurella multocida toxin type D serological assay as an alternative to the toxin neutralisation lethality test in mice.

The objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Pasteurella multocida toxin type D, that correlated to a mouse lethality test. Currently, the mouse lethality test is one of several tests used world-wide to evaluate serological responses in animals immunised with vaccines containing toxoids. The mouse lethality test involves injecting mice with a mixture of toxin and test serum sample (from animals that have been vaccinated with a toxoid), and then determining antibody titre of the test serum from the number of mice that survive. Thus, the titre calculated is based on the neutralising activity of the test serum. The mouse lethality test requires large numbers of animals and causes severe distress to the animals. Organisations world-wide are working towards alternatives to animals in the development and control of biological products for human and veterinary use. Additionally, the mouse lethality test is labour-intensive, costly and lacks robustness and may be difficult to reproduce between different technicians. We have developed a double sandwich ELISA to measure anti- P. multocida toxoid type D antibodies in swine serum. Sera from swine immunised with vaccines containing type D toxoid showed good correlation to the mouse lethality assay (Spearman analysis=0.94 and Pearson analysis=0.84). When compared to the mouse lethality test, titres obtained using the ELISA format had higher correlation with protective immunity (i.e., lower turbinate atrophy) following challenge with virulent P. multocida. The ELISA assay is more robust, reproducible and costs less than the mouse lethality assay; and it complements efforts to reduce the use of animals in testing.     

Attempts to demonstrate specific antibody responses in the vitreous body of the eye

Postmortem serum and vitreous humor specimens obtained from 31 autopsied human bodies were assayed for specific antibody responses to adenoviruses, RS virus and Mycoplasma pneumoniae using the complement-fixation (CF) test and the ELISA procedure (in 23 of the bodies examined). The antibody responses as measured by the CF test were negative in all vitreous body samples tested, with the ELISA five specimens gave a positive reaction at a titre 1 : 40 and one at 1 : 80. These positive antibody titres turned out to invariably coincide with the high-titre antibody levels in the serum. Implicitly, at high-titre levels in the serum these antibodies tend to penetrate in the vitreous body of the eye.     

Brucella abortus in coyotes. I. A serologic and bacteriologic survey in eastern Texas.

Prevalence of Brucella abortus serum antibodies in coyotes from east central Texas was determined by the buffered Brucella antigen (card test), rivanol, standard agglutination tube, and cold complement fixation tube tests. Eighteen percent (9 of 51) of the coyotes were positive serologically. B. abortus biotype 1 was isolated from various tissues from 7 of 43 coyotes by bacteriologic culture. Congenital transmission was found.     

Comparative sensitivity of different serological tests for seromonitoring and surveillance of Edwardsiella tarda infection of Indian major carps

Different serological tests viz. indirect ELISA, indirect blocking ELISA, competitive ELISA and serum agglutination tests were evaluated to detect antibodies against Edwardsiella tarda in naturally infected fish sera for seromonitoring and epizootiological studies. Approximately 66.6, 62.5, 57.6 and 16.6% of the field sera samples were found to be positive by indirect ELISA, competitive ELISA, indirect blocking ELISA and serum agglutination test, respectively. The percentage of serum samples positive for E. tarda antibodies in serum agglutination, competitive ELISA and indirect blocking ELISA, when compared with indirect ELISA, were 33.3, 83.6 and 66.6%, respectively, but its use was restricted due to the requirement of several conjugates against different fish species and the difficulty in assaying large numbers of serum samples from different fish species in a limited time to enable seromonitoring of the disease prevalence. No significant difference (P<0.05) in the mean optical density value was found in indirect and competitive ELISA. Although the competitive ELISA was slightly less sensitive than the indirect ELISA, it could accommodate a large number of serum samples with one anti-rabbit conjugate, and the need for different fish conjugates as required in indirect ELISA was eliminated. As in medical and veterinary practices, these tests can now be used in aquaculture practices for seromonitoring and study of pre-exposure of Indian major carps to pathogens in enzootic areas.     

Revision of the antigenic structure of genus Listeria

O-antigenic structure of genus Listeria was studied, using antisera (obtained from rabbits) against different O-antigens of reference strains of each serovar. The titres of sera were determined by agglutination using antigens of the same reference strains as well. Some differences from the actual scheme were found: serum antifactor-IX gave a lower titre than expected against antigens 4ab and 6b, while the titre observed against antigen 4b was higher than the expected in this case. Serum antifactor-VIII presented a higher titre than could be expected against antigen 6b. The strains of serovars 4d and 4e used in this experience were impossible to distinguish, and could have been classified in the same serovar. We could not obtain serum antifactor-XI from serovar 6b after several trials. From these differences we propose some modifications of the current antigenic scheme of genus Listeria.     

Estudio comparativo de las reacciones serologicas cuantitativas con un antigeno metabolico de Aspergillus fumigatus

Summary The following quantitative serologic reactions: agar-gel immunodiffusion, complement-fixation, opposite electrophoresis and latex particle agglutination tests have been performed in 38 sera from mycologically proved pulmonary aspergillosis cases. A metabolic antigen from a strain ofAspergillus fumigatus according toAjello et al technic modified by us, has been employed. Sera from 120 subjects suffering from non-mycotic lung conditions, as well as 10 sera from histoplasmosis cases, 10 sera from S. A. blastomycosis and 2 sera from patients with lung aspergillosis produced byA. niger, gave negative results with the above mentioned seroligic reactions.One hundred per cent of positive results were obtained with the complement-fixation test (titre ranging from 1/20 to 1/1280), agar-gel immunodiffusion test (titre up to 1/64) and the opposite immunoelectrophoresis (titre ranging from 1/2 to 1/256). Twenty five per cent negative and 4 non-specific results were registered with the latex particle agglutination test.A correlation of the number of serum precipition bands obtained by the electrophoresis technic with the titre of the quantitative serologic reactions, as well as a correlation of the titre of the circulating antibodies with the severity of the clinical form of aspergillosis seems to be present.Electrophoretic motility of the specific antibody performed in 10 sera showed results like the IgM in 1 instance and an intermediate position between IgA and IgG in 9 samples.     

Modified Agglutination Test for Pasteurella tularensis

Several modifications in technique were incorporated into the standard agglutination test for Pasteurella tularensis. Reciprocal shaking of all tubes in a Kahn shaker was introduced to increase the rate of agglutination and quantity of agglutinated cell mass, making it possible to report preliminary results within 4 hr. Increased incubation time at a higher temperature was used to favor the rate of agglutination. A serum control for each serum tested was necessary to detect false positive tests. Finally, a verification procedure with 5% NaCl used as the diluent was instituted to prevent these false positive reactions.     

The opsonizing activity of the sera of hamadryas baboons immunized with a preparation of the outer membranes of Francisella tularensis (based on data from the luminol-dependent luminescence method)]

The opsonizing properties of sera obtained from hamadryas baboons immunized with the preparation of F. tularensis outer membranes (OM) were studied with the use of luminol-dependent chemiluminescence (CL) of whole blood. The immunization of monkeys with the OM preparation was shown to lead to the formation of functionally active antibodies possessing opsonizing properties with respect to virulent F. tularensis. Immune sera obtained from the animals immunized with live vaccine and from those immunized with OM preparation had no essential differences in their opsonizing properties. The level of IgG antibodies in immune sera correlated with the CL parameters of whole blood in the presence of F. tularensis opsonized with these sera. Increased CL of phagocytes observed after addition of bacteria and immune sera under test to whole blood taken from a nonimmune donor made it possible to evaluate the functional activity of antibodies, thus permitting its use as a test for the evaluation of the effectiveness of new vaccine preparations.     

Monoclonal antibodies against hepatitis B virus surface antigen: preparation and characterization

Hybridomas secreting HBsAg antibodies were obtained by fusing murine myeloma cell line P3-X63-Ag8 to spleen cells of BALB/c mice sensitized with HBsAg. The surface antigen used for immunization of mice was prepared by purification from pooled human plasma specimens. Resulting monoclonal antibodies were detected by the SPRIA method. Clones producing highest anti-HBs titres were used to prepare mouse ascitic fluids. Monoclonal antibodies in ascitic fluid reached a titre of 10(6) to 10(7) at a protein concentration of 1 mg per ml. Two of the prepared monoclonal antibodies, HBS-01 and HBS-02, both belonging to IgG1 subclass of immunoglobulins, were selected for further study in order to assess their potential useability in the commercial ELISA kit. The pI values for HBS-01 ranged from 6.60 to 6.85, for HBS-02 from 5.6 to 6.1. In solid phase ELISA test the use of HBS-01 antibody improved accuracy of the assay by increasing its detection sensitivity for HBsAg subtypes adw and ayw in the reference serum; this sensitivity was evidently much better than that seen with the commercially available rabbit polyclonal anti-HBsAg antibody. The monoclonal antibody HBS-01 is specific to the determinant "a", which makes it suitable for use in ELISA test aimed at HBsAg detection. The antibody HBS-02 showed a markedly better reaction with HBsAg subtype adw than subtype ayw and can thus be used with advantage for their discrimination.     

Antigenic characteristics of various strains of Campylobacter jejuni. Preliminary note: technic of extraction of soluble thermostable antigens

Immune serum from rabbit has been obtained against Campylobacter jejuni ATCC no 29428 from the same strain an antigen of the outer Envelope was prepared by EDTA extraction. The specificity of antibodies against the outer antigen has been observed by direct agglutination, C.F., passive haemoagglutination and houcherlony test in agar. In eight other strains of Campylobacter isolated from patients, cross reactions with the ATCC no 29428 strain have been stored, by direct agglutination: only one strain (H) cross-reacted with the immune serum in use.     

Comparative study of cell-immunoenzymatic methods for the estimation of IgG and IgM anti-brucella antibodies in the diagnosis of human brucellosis

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11 % positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM anti-brucella antibodies.     

Lipopolysaccharide O-antigen antibody-based detection of the fish pathogen Flavobacterium psychrophilum

Several yellow-pigmented species within the family Flavobacteriaceae are commonly associated with diseases in fish and are difficult to speciate due to their fastidious, slow-growing nature and cross-reactive antigens. Here we report the development of specific, antibody-diagnostic tests for Flavobacterium psychrophilum, the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. A unique antigen from F. psychrophilum, the lipopolysaccharide (LPS) O-polysaccharide (O-PS), formed the basis for the antibody test. LPS O-PS was purified and conjugated to keyhole limpet haemocyanin and bovine serum albumin for the generation of rabbit immune sera and the development of antibody-based diagnostic tests. Rabbit polyclonal anti-O-PS serum was highly specific for F. psychrophilum, without the need for prior cross-absorption with related bacteria and was the basis of an effective ELISA diagnostic test. Antibodies were purified from rabbit anti-O-PS serum and adsorbed onto coloured latex beads for the development of a specific, bead agglutination assay for F. psychrophilum.     

Comparative study of cell-immunoenzymatic methods for the estimation of IgG and IgM anti-Brucella antibodies in the diagnosis of human brucellosis

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11% positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM antibrucella antibodies.