Circulating Antigen Research Articles

Detection of circulating antigen in serum of mice infected with Schistosoma japonicum by immunomagnetic bead ELISA based on IgY

We developed a novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) for detection of circulating antigen (CA) in sera of mice infected with Schistosoma japonicum. The assay involved the use of chicken polyclonal antibodies IgY against soluble egg antigens (SEA) of S. japonicum as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5B labeled horseradish peroxidase (HRP) as a detecting antibody. Two groups of BALB/c mice infected with S. japonicum cercariae were used: lightly infected mice (infected with 10 S. japonicum cercariae) and heavily infected mice (infected with 30 S. japonicum cercariae). The CA was detectable as early as 4 and 5 weeks after infection in the sera of heavily and lightly infected mice, respectively. The CA levels rose rapidly and reached a peak in 8 weeks after infection and then remained a plateau for at least another 6 weeks in both groups. Moreover, the effect of praziquantel on the CA levels was also investigated. The heavily infected mice were treated with praziquantel and the CA levels in sera increased dramatically in the first week post-treatment and then decreased to the control level by 6 weeks after treatment. The novel assay appears to be sensitive for detection of schistosomal antigenemia and valuable to judge the efficacy of chemotherapy in murine schistosomiasis.

Detection of Circulating Antigen with a MAb-Based Sandwich-ELISA and Its Comparison with Specific IgM Detection in Sera of Patients with Hemorrhagic Fever with Renal Syndrome

A monoclonal antibody (1H2) against nucleocapsid protein of Hantavirus was developed. A sandwich enzyme-linked immunosorbent assay (ELISA) with the monoclonal antibody (MAb) was established and evaluated for detecting circulating antigen (CAg) in serum of patients with hemorrhagic fever with renal syndrome (HFRS). Results were compared to that of an immunoglobulin M (IgM)-detecting ELISA. Of 143 patients with HFRS, 106 were positive for CAg of Hantavirus and 128 positive for specific IgM. Among the 15 HFRS patients in whom specific IgM was not detected, 10 were positive for CAg. Of 100 controls including 40 hepatitis B cases, 40 measles cases, and 20 healthy persons all were negative for both CAg and specific IgM. Detection of Hantavirus CAg with a MAb-based sandwich ELISA (MBS-ELISA) established in the present study adds a new diagnostic tool for HFRS, and it increases the diagnostic rate to conventional specific IgM detection, especially for patient in the early stage of HFRS.

Real-Time Quantitative PCR for Determining the Burden ofPlasmodium falciparumParasites during Pregnancy and Infancy

Real-time quantitative PCR (RTQ-PCR) provides a quick, accurate, and reproducible quantification of parasites. However, the value of RTQ-PCR for predicting clinical outcomes of malaria is unknown. Here, we compared RTQ-PCR to microscopy of blood smears, nested PCR (nPCR), and parasite circulating-antigen (CAg) assays for detection of Plasmodium falciparum in pregnant Kenyan women and their infants and related these findings to parity and birth weights in their newborns (n = 554). nPCR was the most sensitive assay for detection of malaria in pregnancy, followed in decreasing order of sensitivity by RTQ-PCR, CAg assays, and blood smears. RTQ-PCR detected a higher frequency of malaria infection (46%) in maternal peripheral blood in primiparous than in multiparous women (35%; P < 0.001), with a >12-fold difference in parasite burden (geometric mean = 25,870 versus 2,143 amplicons/microl blood; P < 0.0001). Similarly, the presence of placental malaria determined by RTQ-PCR was approximately twofold higher in primiparous versus multiparous women (21% versus 13%; P < 0.01). The presence and intensity of malaria infection in pregnant women estimated by RTQ-PCR strongly correlated with low-birth-weight babies, especially in those with high amplicon numbers. RTQ-PCR identified malaria-infected women, missed by blood smear, who were at risk for having underweight offspring. By contrast, malaria detected by nPCR and CAg assay showed a much weaker association with parity or low birth weight. Thus, RTQ-PCR provides an estimate of parasite burden that is more sensitive than blood smear and is predictive of clinical outcomes of malaria infection in pregnant women and newborns.

Clinical and epidemiological features of patients with clonorchiasis.

To study the clinical and epidemiological features of patients with clonorchiasis so as to provide scientific evidences for the diagnosis and prevention of clonorchiasis. Stools from 282 subjects suspected of having clonorchiasis were examined for helminth eggs with modified Kato's thick smear and sedimentation methods, and their sera were tested for HAV-DNA, HBV-DNA, HCV-RNA, HDV-RNA and HEV-RNA with polymerase chain reaction (PCR). Clinical symptoms of patients with clonorchiasis only were analyzed, and their blood samples were tested for circulating antigen (CAg) with Dot-ELISA, eosinophilic granulocyte count, and alanine aminotransferase (ALT). Meanwhile, they were asked to provide data of occupation, eating habit, hygienic habit and knowledge of clonorchiasis. In addition, the ecosystem of the environment in epidemic areas was surveyed. Among the 282 patients, 61 (21.43%) were infected with clonorchis sinensis only, 97 (34.64%) were co-infected with clonorchis sinensis and other pathogens, 92 (32.86%) were infected with hepatitis virus only and 31 (11.07%) neither with clonorchis sinensis nor hepatitis virus. Among the 61 patients with clonorchiasis only, there were 14 (22.95%) subjects with discomfort over hepatic region or epigasfrium, 12 (19.67%) with general malaise or discomfort and inertia in total body, 6 (9.84%) with anorexia, indigestion and nausea, 4 (6.56%) with fever, dizziness and headache (6.56%), and 25 (40.98%) without any symptoms; sixty one (100%) with CAg (+), 98.33% (59/60) with eosinophilic granulocytes increased and 65.00% (39/60) with ALT increased. B-mode ultrasonography revealed 61 cases with dilated and thickened walls of intrahepatic bile duct, and blurred patchy echo acoustic image in liver. Twenty-six cases had stones in the bile duct, 39 cases had slightly enlarged liver with diffuse coarse spots in liver parenchyma. Twenty cases had enlarged gallbladder with thickened coarse wall and image of floating plagues, 9 cases had slightly enlarged spleen. By analysis of epidemiological data, we found that the ecologic environment was favorable for the epidemiology of clonorchiasis. Most patients with clonorchiasis were lack of knowledge about the disease. Their living environment, hygienic habits, eating habits and their occupations were the related factors that caused the prevalence of the disease. The clinical symptoms of clonorchiasis are non-specific, and the main evidences for diagnosis of clonorchiasis should be provided by etiologic examination, B-mode ultrasonography and clinical history. The infection of clonorchis sinensis is related to occupations, bad eating habits and lack of knowledge about prevention of the disease.

Kinetics of circulating antigens in pigs experimentally infected with Taenia solium eggs.

Three groups of four piglets were experimentally infected with different doses (10(3), 10(4) and 10(5)) of Taenia solium eggs whereas a fourth group of two pigs received gravid proglottids. At autopsy 6 months post infection, the two latter pigs were heavily infected with more than 3000 living cysts per kg of muscle. Ten of the 12 other pigs harboured light infections, i.e. between 2 and 107 cysticerci, 42.4% of which were degenerated. The two remaining pigs had no detectable cysts at post mortem examination. Circulating antigens (CA) were detected in the sera of all pigs harbouring living cysticerci using a monoclonal antibody based ELISA. CA were first detected between 2 and 6 weeks post infection and remained present generally throughout the entire observation period even in pigs carrying only five to eight living cysts, although strong fluctuations of the level of CA were observed in some pigs. In animals without living cysts at post mortem CA were only detected for a short period and disappeared presumably when the cysticerci became degenerated. The minimum number of living cysts, which could be detected using this ELISA, was 1.

Age and gender specific patterns of microfilaremia and circulating antigen with specific lymphocyte proliferative responses in Wucherereia bancrofti filariasis.

A population from a W. bancrofti- endemic area was evaluated by use of microfilaria(Mf) and adult worm circulating antigen(CAg) enzyme linked immunosorbent assay to demonstrate the relation of these two parameters to age , gender and the clinical status (symptomatic or asymptomatic) of the examined individuals. In addition, antigen-driven T cell proliferation was measured to study the effect of the clinical status and / or infection status on the host responsiveness in lymphatic filariasis. The results showed that the prevalence rates of each of the two markers are clearly age dependent and were significantly higher in males than females. The antigen prevalence rate was consistently higher than the microfilaria prevalence rate in all age groups. Among the asymptomatic and symptomatic patients, the direct relationship between Mf+ and CAg+ remained intact regardless of clinical status. Individuals positive for CAg had significantly lower lymphocyte proliferation responses to parasite antigen than did CAg-negative people. This study re-emphasizes the importance of CAg measurements for accurately assessing the clinical status of filariasis and demonstrates that the presence or absence of active infection, not clinical status is most closely associated with the immune responsiveness in lymphatic filariasis.

Worm burden and host responsiveness in Wuchereria bancrofti infection: use of antigen detection to refine earlier assessments from the South Pacific.

A population from the Wuchereria bancrofti-endemic island of Mauke was reevaluated retrospectively by use of the Og4C3 circulating antigen (CAg) enzyme-linked immunosorbent assay to assess active infection in relation to host responses by age and gender. Use of microfilaremia (Mf) alone misclassified approximately 50% of infected people, although CAg and Mf levels were positively correlated. Levels of CAg peaked between those aged 31-60 years; men aged > 60 years had a significantly higher CAg prevalence (> 90%) than women. Filaria-specific immunoglobulin (Ig) G4 reached maximum levels in both genders at age 51-60 years. By analysis of variance, both age and gender significantly influenced CAg and IgG4, with men having higher levels of both in the total population. Individuals positive for CAg had significantly lower lymphocyte proliferation responses to parasite antigen than did CAg-negative people, regardless of clinical status. This study reemphasizes the importance of CAg measurements for accurately assessing filarial prevalence and clinical status and demonstrates the relationship between active infection and immune responsiveness.

Relevance of circulating antigen detection to follow-up experimental and human cystic hydatid infections.

We analysed specific antibody (Ab) and circulating antigen (CAg) profiles along experimental mouse infection using as control a group of mice immunized with intact but dead parasites. Results from this experiment showed an early major CAg peak followed by a larger Ab peak which partially overlaps with other minor CAg peaks. These results suggest that CAg may be a marker of early mouse infection. In order to study the relevance of these findings in humans we similarly analysed by ELISA 148 sera provided by retrospective post-surgical follow-up of 19 patients. Available records showed that 14 patients developed new cysts one to ten years after surgery while no new disease was observed in the-other five. Some of the former patients showed CAg, as early as two months after surgery while no CAg was observed in the other five patients at any time. In addition, a collection of 38 sera obtained before surgery were similarly tested and five of them showed only CAg, while 18 showed only Ab and 12 sera showed Ab&CAg. These results in humans are consistent with the findings in the mouse experimental model and suggest that CAg may be an early marker of hydatid infection, thus being relevant for post-surgical follow-up.

Detection of a Schistosoma japonicum specific circulating antigen by two-dimensional gel electrophoresis

To detect an adult Schistosoma japonicum circulating antigen, we produced a monoclonal antibody (MAb), termed 1-7B, which recognized a gut-associated excretory–secretory antigen. We characterized the antigen on a two-dimensional gel electrophoretic pattern (2-D pattern) of proteins extracted from S. japonicum adult worms. The antigen formed one vertically elongated spot, with an apparent molecular weight of 60–200 kDa and an isoelectric point of<4. Since the spot was seen on a 2-D pattern of S. japonicum-infected rabbit serum but not on that of normal rabbit serum, it was ascertained that the antigen was a circulating antigen (CA). A polyclonal rabbit antiserum against the spot was prepared and used for immunoblotting analysis with S. japonicum, S. mansoni, Paragonimus miyazakii, Spirometra erinaceieuropaei and Anisakis sp. The antigen was detected only in an extract of S. japonicum. From these results, the antigen is considered to differ from the CAs reported by previous authors, because of its molecular weight, isoelectric point and specificity.

Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein ofMycobacterium tuberculosis H37Ra

With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction(Mtb EST-6) of purifiedMycobacterium tuberculosis H37Ra and affinity purified polyclonal antibodies againstMtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system usingMtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontub~rculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified antiMtb EST antibodies detected circulating antigen in 83% and 61% of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91% respectively) compared to antigen detection (sensitivity of 79% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility ofMtb EST-6 as a diagnostic marker of pulmonary tuberculosis.

A comparative study on specific antibodies and circulating antigen (CAA) in serum and parasitological findings for diagnosis of schistosomiasis mansoni in an endemic area in Tanzania

A baseline study to evaluate the prevalence of Schistosoma mansoni infection as well as the diagnostic efficacy of serodiagnostic tests was performed in Kabaganga village, Kome island, Lake Victoria, Tanzania. A total of 1108 individuals were examined parasitologically and clinically. Egg excretion was demonstrated by one-sample Kato-Katz test. Specific IgG1 and IgG4 antibodies against S. mansoni adult worm (SAWA) and egg (SEA) antigens as well as circulating anodic antigen (CAA) were determined in serum samples from 250 of these subjects. As a control population 41 individuals from a non-endemic area were examined parasitologically, clinically and serologically. In the parasitologically examined Kabaganga population 45% were excreting eggs. The pattern of egg excretion was typical for an endemic area with a peak in the age group 10–14 years. Sixty-five percent of the serologically tested villagers were positive in the CAA test. A total of 80% were positive in either of the two tests, indicating an active infection. In 67–95% of these individuals the levels of isotype specific antibodies were increased. The prevalence of CAA positivity corresponded fairly well with that of Kato-Katz results in the age groups 10–29 years, but in the younger age groups a considerably greater number of individuals were positive in the CAA test than in the Kato-Katz test. The results obtained indicate that virtually all of the Kabaganga villagers, regardless of age, had an ongoing, active infection or had previously been infected with S. mansoni. This population, therefore, may be useful for evaluation of the diagnostic efficacy of various antibody tests. The highest degree of discrimination between the endemic and the non-endemic village populations was noted for anti-egg IgG4 antibodies. It is concluded that the combined determination of parasite eggs in faeces and CAA in serum provides high sensitivity as regards active infection. Increased levels of isotype-specific antibodies, particularly of the IgG4 subclass, is a sensitive indicator of past or present infection, and the prevalence of individuals with such increased levels may be a simple and reliable indicator of the frequency of schistosomiasis in a community.

Detection of Circulating Antigens of Toxoplasma gondii in Human Infection

Seventy-nine serum specimens from pregnant women and 29 from immunocompromised patients (12 from graft recipients and 17 from patients with acquired immunodeficiency syndrome) were classified into three groups according to their serologic status to Toxoplasma gondii as determined by immunofluorescence and an enzyme-linked immunosorbent assay (ELISA): no antibodies (group 1), acute acquired infection (group 2), and reactivation (group 3). These samples were tested for the presence of circulating antigens (CAg) of T. gondii by capture ELISA and immunoblotting. The presence of CAg was detected by at least one of the two techniques in six of 31 subjects in group 1, 51 of 68 subjects in group 2, and seven of nine subjects in group 3. Of a total of 108 serum specimens, 28 were found to be T. gondii-positive by capture ELISA, 57 by immunoblotting, and 21 by both techniques. Among the nine polypeptides detected by immunoblotting, 38 recognized p14, 17 recognized p8, and 16 recognized p8, and 16 recognized p30. These results demonstrate that the detection of CAg can aid in the diagnosis of infection by T. gondii in humans, especially in immunocompromised patients whose serologic response can be impaired.

Use of specific antibody and circulating antigen serum levels in the hydatid immunodiagnosis of asymptomatic population

Levels of specific antibodies (Ab) and circulating antigens (CAg) were tested by ELISA in sera from 115 surgically confirmed hydatid patients, 41 individuals exhibiting other parasitic and unrelated diseases and 69 healthy subjects. Addition of CAg data to Ab detection in this sera collection increased sensitivity from 85% (only Ab) to 89% (Ab + CAg). Combination of ultrasonography with Ab and CAg serology for diagnosis of asymptomatic population in endemic areas was analyzed. One field survey (163 persons) involved both blood extraction and ultrasonography to all the population. Three people exhibited cyst images and all of them were Ab positive, while 6 Ab and 1 CAg positive individuals exhibited no cyst image. Another survey (1620 persons) involved a selection of 85 subjects for serology according to ultrasound data and record of family hydatid history. Twelve per cent exhibited no hydatid image being serologically positive and 14% were serologically negative but exhibited cyst image. Ultrasonography and serology (Ab and CAg) should be used in combination to maximize the diagnostic yield in asymptomatic population.

Serodiagnosis and seroepidemiology of human unilocular hydatidosis in Jordan

A total of 2182 serum samples from 38 patients with surgically confirmed unilocular hydatidosis, 19 clinically assessed patients, 15 patients with parasitic infections other than hydatidosis, 104 hospital outpatients, and 2006 normal Jordanians were serodiagnosed for the presence of IgG antibodies against hydatid fluid, circulating immune complexes (CIC), and/or hydatid circulating antigen (CA). Anti-hydatid IgG antibodies were detected in the sera of 77.4% of patients with hydatid disease and persist for very long periods postsurgery. As many as 54.1% of patients with hydatidosis had positive levels of CIC, and 16.1% had circulating antigen in their sera. The search for circulating antigen and CIC decreased the number of false-negative hydatid cases from seven to three, and the combined sensitivity of the assays thus increased from 77.4% to 90.3%. Using the immunoblot technique, 16- and < 14.4-kDa Echinococcus granulosus-specific bands were detected in sera from 54.1% and 61.5% of patients with hydatid disease who were tested before and after surgery, respectively. The seropositivity rate for anti-hydatid IgG antibodies was 2.4% for the general Jordanian population and 5.8% for hospital outpatients.

Assessment of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B for detection of human hydatid circulating antigens.

Four monoclonal antibodies (MAb) to Echinococcus granulosus Antigen 5 (Ag5) and Antigen B (AgB) were assessed in an enzyme-linked immunosorbent assay (ELISA) for detection of circulating antigens (CAg) in sera of human patients with E. granulosus infection. Around 5.5-8% of 200 sera from 42 surgically proven hydatid patients contained detectable CAg by individual MAb. The combined detection rate for CAg, using four MAb, was 19% (38/200). Although hydatid CAg was detected by MAb in at least one serum sample from 21 of 42 patients, some patients remained negative in the assay regardless of the time when serum samples were taken (pre- or post-operatively), or of the continuing presence of hydatid cysts, their location or fertility. In addition, it was observed that the binding capacity of MAb for sheep hydatid cyst fluid antigen (SHCF) was somewhat reduced in the presence of normal human serum. The CAg detection assay would only be useful for assessment of hydatid infection status in patients with detectable CAg in serum samples.

The susceptibility of adult schistosomes to immune attrition.

Mouse infection models are described that demonstrate reduction of egg production in Schistosoma haematobium infections and both worm loss and reduced fecundity in S. bovis infections. Neither phenomenon could be shown in S. mansoni infected mice. The immunological basis for these anti-adult responses was inferred by comparison with infections in T-cell deprived mice and by serum transfer of the ability to reduce a S. bovis worm burden into immunocompromised hosts. Vaccination with irradiation attenuated parasites was also shown to have consequences for the adults of a challenge infection of S. haematobium and S. bovis specifically. Prior vaccination resulted in an abrogation of the anti-fecundity and adult worm elimination that occurred in non-vaccinated similarly infected mice. These models are being used to define the targets and mechanisms involved in anti-adult attrition. A serological assay, quantitation of a circulating antigen (CAA) has been assessed for its ability to measure worm burdens of different species of schistosome in mice. This assay will be used to question whether anti-adult immunity contributes to the pattern of infection with S. mansoni and S. haematobium in man.

Kinetic of circulating antigens by capture ELISA and immunoblotting in murine toxoplasmosis

Capture ELISA and immunoblotting techniques were applied for the detection of Toxoplasma gondii (T.g) circulating antigens (CAG) in serum specimens of OF-1 mice infected with virulent RH, or cystogenic PGD strains. The capture ELISA assay allowed to detect CAG from the first day of infection to the death of animals with the two T.g strains, although CAG rates were higher when the RH strain was used. Immunoblotting confirmed these findings. Moreover, immunoblotting allowed to identify 7 CAG (MW: 22 kDa to 110 kDa) in serum specimens of mice infected with the RH strain and only 3 (MW: 75 kDa to 110 kDa) in serum specimens of mice infected with the PGD strain. These results are discussed in the light of the evolutive ways of experimental infection by T.g.

Toxoplasma gondii antigens: Analysis by SDS-PAGE and immunoblotting; Relation with circulating antigens

Cytoplasmic antigenic extracts (CAE) and membranar antigenic extracts (MAE) from Toxoplasma gondii (T.g) purified trophozoites RH strain, were obtained after hypotonic lysis and sonication. Analysis in Polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie blue staining revealed 21 polypeptides (PP) in CAE and 8 in MAE, whereas the immunoblotting technique detected 28 PP in CAE and 23 in MAE. Immunoblotting is then more sensitive than Coomassie blue staining. This finding is particularly marked for MAE and could be explained by the high antigenicity of some PP that have a lower concentration than the threshold sensitivity of SDS-PAGE. In contrast, some PP in sufficient amount to be detected by SDS-PAGE did not show any antigenic activity since not found by immunoblotting. The molecular weight (MW) ranged from 8 to 130 kDa for cytoplasmic PP and from 4 to 110 kDa for membranar PP. Circulating antigens (CAG) from serum specimens of mice infected with the same strain of T.g were analysed by immunoblotting in comparison with CAE and MAE profiles. From the 7 bands obtained with CAG, 4 were shown to correspond to CAE PP, 2 to common CAE and MAE PP and only 1 to MAE PP (MW: 30 kDa).

Schistosoma mansoni: Detection of circulating antigens in murine schistosomiasis by antigen-capture sandwich ELISA using a monoclonal antibody

A monoclonal antibody (MAb) 5H11/B1 that reacts with a repeating epitope on an excretory-secretory (E + S) antigen of adult worms of Schistosoma mansoni was used in the detection of circulating antigen (CA) in sera from S. mansoni-infected mice using an antigen-capture sandwich ELISA. Trichloroacetic acid (TCA) pretreatment of sera from mice infected for 8 or 16 weeks precipitated immune complexes and/or dissociated CA and allowed its detection. Sera obtained 8 weeks after infection contained high levels of CA. Upon treatment with praziquantel (100 mg/kg body wt), this level was significantly less within 1 week. A strong correlation was found between the worm count determined by perfusion and the level of antigenemia detected by the 5H11/B1 assay in light and heavy infection ( r = 0.80). Based on the results of both TCA pretreatment and sodium periodate treatment, the 5H11/B1 sandwich ELISA assay detects a repeating carbohydrate epitope on an E + S antigen. This system appears to be a sensitive assay for the detection of schistosomal antigenemia in murine schistosomiasis. Studies on the detection of antigenemia in human schistosomiasis using this assay are in progress.

Immunoblot analysis of the circulating antigens occurring in serum of rats infected with Angiostrongylus cantonensis.

Following the infection of rats with Angiostrongylus cantonensis, the occurrence and molecular features of circulating antigens (CAs) were analyzed, with special reference to their origin in worms, by SDS-PAGE combined with an immunoblotting technique. Antisera against the CAs were obtained by immunizing rats with sera from rats 35, 91, and 150 days postinfection. The anti-sera, referred to as anti-CA(35), anti-CA(91), and anti-CA(150), respectively, formed one or two precipitin lines when tested in Ouchterlony plates against extracts of digestive organs (DE) and of reproductive organs (RE) from adult female worms. When the anti-CA(35) was used as a blotting antibody under nonreducing conditions, a set of clearly spaced narrow bands with molecular weights (mol. wt.) in the range of 90,000-180,000 daltons developed only in the case of the DE. Besides the antigen(s), additional bands with mol.wt. of 115,000 and 185,000 daltons were revealed in the case of the RE when two other antisera were used. Immunoblot analysis of the immunoprecipitates, derived from anti-CA(150) and sera of infected rats, revealed the occurrence of two types of protein as the major CAs: one had a mol.wt. in the range of 140,000-180,000 daltons and was found in the serum 14 days postinfection, and the other, with a mol.wt. of 185,000 daltons, was found in the serum 35-150 days postinfection. Immunohistochemical studies localized the CAs predominantly in the cytoplasm of both uterine eggs and maturing oocytes.