检测梅毒螺旋体抗体的纤维膜条方法的建立及其在诊断中的应用

目的：建立以纤维膜为载体的检测梅毒螺旋体抗体的方法,检查病人血清中对梅毒螺旋体多种抗原的抗体,用于梅毒感染的诊断。方法：将基因工程表达及纯化的梅毒螺旋体蛋白tp15、tp17、tp42和tp47分别结合在纤维膜上,用载抗原的纤维膜条检查血清中的抗体,抗体阳性者在相应抗原位置显示出特异条带。结果：梅毒螺旋体感染者血清中存在特异性抗体,在检查的460份临床诊断的患者血清中,对tp15、tp17、tp42和tp47抗原的抗体检出率分别为41.3%、100%、98.7%和51.7%;134份献血员血清抗体阴性。结论：建立的检测梅毒螺旋体感染的方法可同时检查对多种抗原的抗体,以纤维膜条作为诊断条检测血清抗体方法简便,用于临床诊断更特异、更敏感。     

Sera from patients with multiple sclerosis react with human cell T lymphotropic virus-I gag proteins but not env proteins--Western blotting analysis

To study the possible involvement of human T cell lymphotropic virus type I (HTLV-I)-related agent in Japanese multiple sclerosis (MS), we performed a Western blotting analysis, using purified viral antigens, on sera from 46 patients with MS, nine patients with other neurologic diseases, and 11 healthy controls. Of 46 MS patients, 11 (24%) had antibodies reactive with antigens corresponding to the group-specific antigen (gag) proteins (p15, p19, and p24), although the prevalence was lower than that reported in a recent study using an enzyme-linked immunosorbent assay (ELISA). Despite the lower frequency of immunoreactivity, Western blotting technique had merits of identification of multiple antigens and higher specificity for detection of antibodies than ELISA. Those sero-positive patients consisted of four cases with IgG antibodies reactive mainly to the gag p24 and/or p15, four with IgM antibodies mainly to the gag p24 and/or p19, and three with both IgG and IgM antibodies. These immunostaining patterns of MS sera were clearly distinguishable from those of adult T cell leukemia patients who had antibodies to the envelope (env) proteins and its precursors in addition to the gag proteins. The antibody in MS sera was generally of low titer and reactive at a high serum concentration (1/10 dilution). None of the sera from patients with other neurologic diseases and healthy controls had the viral antibodies. These findings indicate that at least one quarter of Japanese MS patients have antibody responses to a hitherto unidentified agent related to HTLV-I, which possibly plays a part, primarily or secondarily, in the pathogenesis of those patients.     

Serum antibodies to whole-cell and recombinant antigens of Borrelia burgdorferi in cottontail rabbits

Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections.     

Pneumocystis carinii: immunoblotting and immunofluorescent analyses of serum antibodies during experimental rat infection and recovery

Serum antibodies to Pneumocystis carinii were measured in rats by the indirect fluorescent antibody and immunoblotting techniques. Serum IgG and IgM antibodies developed with environmental exposure to P. carinii, were low or absent during immunosuppression to induce P. carinii pneumonia, and rose when immunosuppression was withdrawn. The IgG and IgM antibodies formed at the same time, but the titers of each antibody varied in individual rats. Serum IgG antibodies by immunoblotting recognized bands of 45, 50, and 116 kDa as the major reactive moieties of P. carinii. The bands were detected with sera from all rat groups in a temporal pattern which closely paralleled antibody formation by indirect immunofluorescence. The pattern of immunoblotting reactivity varied among individual rats, particularly with immunosuppression. Additional bands were detected with prolonged exposure to P. carinii. Thus, the rat makes both IgG and IgM antibodies to P. carinii, and specific P. carinii antigens identified in this immune response might be targeted for future serologic studies.     

Protective efficacy against group B streptococcal infection in neonatal mice delivered from preimmunized pregnants

Neonatal mice delivered from mothers preimmunized with heated or formalinized whole cell vaccines of type Ia, Ia/c and III/c group B streptococci were infected with each type of bacteria, and then serum antibodies of mothers and neonates who survived the experiments were measured by enzyme-linked immunosorbent assay. The relationship between the protectivity in neonate mice and the antibody titers to the type specific polysaccharide antigens and the protein c antigen of their sera were examined. In the Ia-immunized group which showed high protection against the type Ia infection, anti-Ia IgG antibody titers were low, and anti-protein c IgG antibody was not detected. Type Ia/c and III/c vaccines were highly effective against both type Ia/c and III/c infection, but less effective in type Ia infection. The protein c antigen was identified in both type strains by the double diffusion assay, and the IgG antibodies to the protein c were significantly high in sera of both maternal mice immunized with types Ia/c or III/c organisms and their newborn infants. High titers of the protein c IgG antibody retained 3 to 4 weeks after the last injection of vaccines which corresponded to the period of pregnancy and lactation. Small amounts of IgM antibody to all antigens were detected only in maternal sera. These results suggest that IgG antibodies to the protein c antigen and to the type-specific polysaccharide antigens are equally important protective factors which are transferable from preimmunized mothers to their newborn infants through placenta and/or lactation.     

Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans

Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses.     

Detection and functional characterization of early appearing antibodies in rabbits with experimental syphilis

Following testicular infection of rabbits with Treponema pallidum, different antibodies become detectable initially at the time of healing. Experiments were performed to determine a functional role for these antibodies. Rabbits were sacrificed after 4-8 days. Treponemal numbers steadily increased for 10-12 days. Thereafter, host defenses were sufficiently stimulated to begin clearing the organisms. Antibodies in serum and antibodies localized at the site of infection were quantitated using radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) techniques. Anti-treponemal IgG was detected as early as day 4. Quantities of antibody correspondingly increased with time following infection. Treponema pallidum was harvested 7 and 14 days postinfection and tested for surface antibodies. With increasing days postinfection, more antibody was found on the organisms. Two functional properties of these antibodies were shown. Sera from 24 of 45 rabbits infected for 14 days immobilized T. pallidum in the presence of complement and 14-day sera blocked the attachment of T. pallidum to tissue culture cells. We suggest that antibody-mediated, complement-dependent immobilization of T. pallidum and blockage of attachment are at least partially responsible for healing of testicular lesions.     

An enzyme-linked immunosorbent assay for detection of chronic subclinical Pasteurella pneumotropica infection in mice.

Serum samples from seventy-five, 3- to 12-week-old and 16 retired breeder male Swiss mice from a conventional colony with enzootic chronic subclinical Pasteurella pneumotropica infection were tested by enzyme-linked immunosorbent assay (ELISA) and Western blots for IgG antibodies to whole cell (WC) and lipooligosaccharide (LOS) antigens of P. pneumotropica. In 3- to 12-week-old mice, serum antibody levels to LOS exceeded those to the WC preparation. Western blots of sera from mice in this age group substantiated that a major component of the early IgG antibody response was directed against LOS antigens. Higher antibody levels to both antigen preparations in 3-week-old mice compared to mice 4 and 6 weeks old were interpreted as reflecting a decline in antibodies acquired from the dam. Active immunity indicative of infection was first detected at 8 weeks of age. Serum samples from retired breeder mice (28 weeks of age) also had substantial antibody titers to LOS but, in contrast to sera from mice in the younger age groups, retired breeders had significantly greater IgG reactivity to WC preparations than to LOS antigens. The superior specificity of the LOS antigen compared to the WC preparation in the ELISA was demonstrated by testing serum samples from retired breeder mice against WC and LOS antigens from P. ureae, P. multocida, and P. hemolytica. The reactivity of IgG against LOS antigens from these organisms was negligible, whereas substantial titers were evident to WC antigens. This ELISA, using LOS preparations as antigen, is a useful serologic assay for the detection of subclinical P. pneumotropica infection in mice.     

p185, an immunodominant epitope, is an autoantigen mimotope

An immunodominant peptide (p185(378-394)) derived from the c-erbB2 gene product, was recognized by an anti-DNA antibody, B3, and importantly by two classical DNA-binding proteins, Tgo polymerase and Pa-UDG. These reactivities were inhibited by DNA, confirming that the peptide mimicked DNA. BALB/c mice immunized with p185(378-394) developed significant titers of IgG anti-dsDNA antibodies. Screening of 39 human lupus sera revealed that 5% of these sera possessed reactivity toward p185(378-394). Representative mouse and human sera with anti-p185(378-394) reactivity bound intact p185, and this binding was inhibited by dsDNA. This is the first demonstration of a naturally occurring autoantigen mimotope. The present study identifies a potential antigenic stimulus that might trigger systemic lupus erythematosus in a subset of patients.     

Antigenic complexity of Treponema pallidum: antigenicity and surface localization of major polypeptides

The protein structure of Treponema pallidum was characterized by two-dimensional electrophoresis (2DE), consisting of isoelectric focusing (IEF, pH 5 to 7) in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Up to 85 major polypeptide species could be detected in the organisms in 2DE gels by Coomassie Blue staining. The antigenicity of the individual polypeptides was determined by transferring the 2DE pattern to nitrocellulose paper and utilizing a sensitive immunoperoxidase procedure to demonstrate the reactivity of immunoglobulins in sera obtained from rabbits infected intratesticularly at least 6 mo previously. The infected rabbit serum reacted with virtually every major polypeptide detectable by protein staining techniques, indicating that infected rabbits produce antibodies against nearly all major T. pallidum proteins at the time when the animals exhibit systemic resistance to reinfection. Surface radioiodination of freshly purified T. pallidum by an Iodogen procedure yielded preferential labeling of a major polypeptide with an apparent m.w. of 39,000. The results of this study indicate that the antigenic complexity of T. pallidum is much greater than described previously. The 39-kd polypeptide appears to be a major surface constituent of T. pallidum and as such may play an important role in the induction of immunity to syphilis.     

Characterization of the serum requirement for macrophage-mediated killing of Treponema pallidum ssp. pallidum: Relationship to the development of opsonizing antibodies

Abstract Serum pools were collected from rabbits bled at various times after intra-testicular infection with Treponema pallidum ssp. pallidum . These were tested for their ability to opsonize T. pallidum and promote killing of the organisms by macrophages. Compared to normal sera, significant opsonization was first seen on day 10 of infection as measured by both ingestion ( P < 0.001) and macrophage-mediated killing ( P = 0.006); significant levels of functional antibodies persisted through 300 days of infection. Although opsonic activity peaked early in infection, antibodies that promoted optimal macrophage-mediated killing developed much later, suggesting that these two functions may represent activities of antibodies with differing specificities or affinities. The initial development of antibodies that augment both phagocytosis and killing corresponds with the in vivo clearance of treponemes from the primary site of infection. These observations support the hypothesis that macrophages are the major effector mechanism for elimination of T. pallidum during early syphilis infection.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

Immune immobilization of Treponema pallidum: antibody and complement interactions revisited

The Treponema pallidum immobilization test was designed for serodiagnosis of syphilis and is dependent upon specific antibody and a heat labile component of normal serum. Investigators have shown the component to be dependent upon divalent cations and it is presumed to be complement. Experiments were performed to reevaluate the interactions of antibody and complement and the mechanism of immobilization. The loss of treponemal motility was correlated to the loss of complement activity in the reaction mixture. When motility of treponemes incubated with immune serum IgG and complement had dropped to 50% (3.4 h), 72% of the available complement had been consumed. At the same time, treponemes incubated with normal serum IgG and complement were 82% motile and only 51% of the complement had been consumed. C6 deficient rabbit serum and C4 deficient guinea pig serum were used in conjunction with immune serum IgG to determine which components of the complement cascade were necessary for immobilization. Treponemes were not immobilized by either sera. Results suggest that the heat labile factor in normal sera is complement, that both early and late components of the complement cascade are necessary, and that the reaction proceeds via the classical complement pathway. Although T. pallidum is susceptible to the actions of antibody and complement, the organisms must interact with these components for at least 2 h before immobilization will result.     

Characterization of the antigenic determinants and host components in immune complexes from patients with secondary syphilis

Sera from patients with secondary syphilis were evaluated for abnormal levels of circulating immune complexes (IC), immunoglobulins (Ig), and complement components. Clq-solid-phase assays (Clq-SPA) that made use of monoclonal and polyclonal antibodies directed against IgG subclasses indicated that human IC were composed primarily of IgG3 and IgG1; these findings appeared consistent with subclass profile responses of electrophoretically transferred blots (Western blots) of Treponema pallidum reacted with syphilitic sera. Complexes were isolated from reactive sera by polyethylene glycol precipitation followed by either anti-Clq column chromatography or protein A-Sepharose chromatography. Although qualitative and quantitative differences were noted, all purified materials contained a treponemal polypeptide antigen with a m.w. of approximately 87,000. Subsequent analysis of this polypeptide, which was also present in purified IC from rabbits with experimental syphilis, suggests that it may represent the fibronectin receptor of the organism. The 76,000 and 66,000 materials, earlier identified in purified rabbit IC, appeared to represent C-terminal degradation products of fibronectin presumably of host origin, rather than treponemal antigens. Although fibronectin binds avidly to Clq and could represent a co-precipitable contaminant throughout the isolation procedure, anti-fibronectin antibodies in the sera of patients detectable by radioimmunoassay and the present of antibodies to 76,000 and 66,000 dalton fibronectin fragments in the globulin fractions of disassociated complexes argues against such a conclusion.     

Comparison of naturally acquired antibody responses against the C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 under low transmission and unstable malaria conditions in Sri Lanka

We report here, for the first time, a comparison of naturally acquired antibody responses to the 42 and 19 kDa C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 assayed by ELISA using p42 and p19 baculovirus-derived recombinant proteins, respectively. Test populations comprised patients with microscopy confirmed acute P. vivax infections from two regions endemic for vivax malaria where low transmission and unstable malaria conditions prevail, and a non-endemic urban area, in Sri Lanka. The antibody prevalence to the two proteins, both at the individual and population levels, tend to respond more to p42 than to p19 in all test areas, where >14% of individuals preferentially recognized p42, compared with <2% for p19. In patients with no previous exposure to malaria, 21% preferentially recognized p42, whereas none exclusively recognized p19. A significantly lower prevalence of anti-p19 IgM, but not anti-p42 IgM, was observed among residents from endemic areas compared with their non-endemic counterparts. Individuals from both endemic areas produced significantly less anti-p19 IgM compared with anti-p42 IgM. IgG1 was the predominant IgG isotype for both antigens in all individuals. With increasing exposure to malaria in both endemic areas, anti-p19 antibody responses were dominated by the functionally important IgG1 and IgG3 isotypes, with a concurrent reduction in IgM that was lacking in the non-endemic residents. This antibody switch was also reflected for PvAMA-1 as we previously reported with the identical battery of sera. In contrast, the antibody switch for p42 was restricted to endemic residents with more extensive exposure. These results suggest that an IgM-dominated antibody response against the p42 polymorphic region in endemic residents may interfere with the development of an IgG-dominated "protective" isotype shift to p19, that may complicate vaccine development.     

A Hospital-Based Serological Survey of Cryptosporidiosis in the Republic of Korea

The seroprevalence of cryptosporidiosis was examined using patients'' sera collected from hospitals located in 4 different areas of the Republic of Korea. ELISA was used to measure antibody titers against Cryptosporidium parvum antigens from a total of 2,394 serum samples, which were collected randomly from patients in local hospitals; 1) Chungbuk National University Hospital, 2) Konkuk University Hospital, 3) local hospitals in Chuncheon, Gangwon-do (province), 4) Jeonnam National University Hospital, from 2002 through 2003. Of the 2,394 samples assayed, 34%, 26%, and 56% were positive for C. parvum-specific IgG, IgM, and IgA antibodies, respectively. Positive IgG titers were most common in sera from Jeonnam National University Hospital, Gwangju, Jeollanam-do, and positive IgM titers were most common in sera from Chungbuk National University Hospital, Cheongju, Chuncheongbuk-do. The seropositivity was positively correlated with age for both the IgG and IgA antibodies but was negatively correlated with age for the IgM antibodies. Western blotting revealed that 92%, 83%, and 77% of sera positive for IgG, IgM, and IgA ELISA reacted with 27-kDa antigens, respectively. These results suggested that infection with Cryptosporidium in hospital patients occurs more commonly than previously reported in the Republic of Korea.     

False-Positive Anti-Toxoplasma Fluorescent-Antibody Tests in Patients with Antinuclear Antibodies

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.     

Antibodies against Toxoplasma gondii in the Pacific bottlenose dolphin (Tursiops aduncus) from the Solomon Islands

Serum samples from 58 Pacific bottlenose dolphins (Tursiops aduncus) from the Solomon Islands were tested for the IgG antibody to Toxoplasma gondii by the latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. The ELISA cut-off value was taken as OD > or = 0.276, and the final dilution ratio, recognized as positive, was represented by the end titer. In 25 of 58 samples, no antibody activity was detected by LAT and ELISA. In 8 of 58 samples, anti-T. gondii IgG antibodies were detected by both LAT and ELISA, with titers of greater than 1 : 64 and 1 : 160, respectively. By immunoblotting, the 8 serum samples producing higher titers showed specific antibody IgG binding to several antigens on the T. gondii lane, but not on the Neospora caninum lane. No specific bands were noted on the lanes for either parasite in the 25 serum samples for which no antibody activity was detected. The specific binding of IgG antibodies to T. gondii antigens observed for serum samples producing higher titers suggests that Pacific bottlenose dolphins from the Solomon islands are exposed to T. gondii.     

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.