Isotypic analysis of humoral immune responses in rhesus monkeys to an adult microsomal antigen of Schistosoma mansoni: an indicator of successful treatment.

A study was undertaken to examine the potential role of immunodiagnostic methods in determining successful chemotherapy in schistosomiasis. Fifteen rhesus monkeys were infected with 1,500 Schistosoma mansoni (Puerto Rico strain) cercariae, and 10 of the monkeys were then treated with a curative dose of praziquantel 13 weeks after infection. Five monkeys remained untreated. One monkey was not successfully cured, as confirmed by the presence of both male and female worms at the time of perfusion. Serum samples were longitudinally collected and specific Ig isotypes were quantified with an adult microsomal antigen of S. mansoni using the FAST-ELISA. Specific isotypes were detected with monoclonal antibodies specific for each human Ig isotype, followed by a peroxidase-conjugated anti-mouse Ig. Longitudinally, all monkeys showed similar isotype patterns. Isotypes increased for the first nine weeks following infection, and then began to decrease. Ten to 14 days following treatment, all isotypes increased. The Ig isotype responses of all monkeys followed classic patterns of isotype expression. A ratio of pretreatment (week 13) IgG1 absorbance values to post-treatment IgG1 absorbance values was generated for each monkey. All successfully treated monkeys, determined to be worm-free by perfusion, had IgG1 ratios at week 53 greater than 2.4 (range 2.4-181). The untreated monkeys and the single monkey that was a treatment failure had IgG1 ratios less than 2.1 (range 0.09-2.05) for the same time period.

Serodiagnosis of parasitic diseases.

In this review on serodiagnosis of parasitic diseases, antibody detection, antigen detection, use of monoclonal antibodies in parasitic serodiagnosis, molecular biological technology, and skin tests are discussed. The focus at the Centers for Disease Control on developing improved antigens, a truly quantitative FAST-enzyme-linked immunosorbent assay, and the very specific immunoblot assays for antibody detection is highlighted. The last two assays are suitable for field studies. Identification of patient response in terms of immunoglobulin class or immunoglobulin G subclass isotypes or both is discussed. Immunoglobulin isotypes may asist in defining the stage of some diseases. In other instances, use of a particular anti-isotype conjugate may increase the specificity of the assay. Monoclonal antibodies have played important roles in antigen purification and identification, in competitive antibody assays with increased sensitivity and specificity, and in assays for antigen detection in serum, body fluids, or excreta. Molecular biological technology has allowed significant advances in the production of defined parasitic serodiagnostic antigens.

A specific diagnostic antigen of Echinococcus granulosus with an apparent molecular weight of 8 kDA.

An echinococcus antigen with an apparent molecular weight of 8 kDa was identified as diagnostically important. An immunoblot assay using this antigen was 91% sensitive for surgically confirmed Echinococcus granulosus hydatid disease of the liver. Specificity was 100% for echinococcosis. Marked cross-reactivity was observed with serum specimens from patients with E. multilocularis and E. vogeli infections. The 8 kDa component was not related to the widely recognized echinococcus antigen 5.

Diagnosis of paragonimiasis by immunoblot.

A sensitive and specific immunoblot assay was used to rapidly and accurately diagnose paragonimiasis. The immunoreactivity of a complex Paragonimus westermani Chaffee antigen was evaluated by SDS-PAGE and Western blot analysis. Initial probing with pooled human serum from proven Paragonimus infections revealed many bands, including a significant antibody response to an approximately 8,000 molecular weight (8 kDa) protein. Forty-three of 45 proven paragonimiasis serum specimens had antibodies to this diagnostic band. Of 29 normal serum specimens and 210 serum specimens from patients with other parasitic and nonparasitic infections, only 1 serum, from a schistosomiasis haematobium patient, reacted positively. These results indicate that our immunoblot for paragonimiasis, which uses a comparatively crude antigen, is highly sensitive (96%) and specific (99%).

The present status of serodiagnosis and seroepidemiology of schistosomiasis.

The literature of the past 4-5 yr on serodiagnosis and seroepidemiology of schistosomiasis is reviewed. A variety of assays with different antigens are being used for serodiagnosis. Several purified antigens appear to be sensitive and specific, but have little if any capability of indicating duration of infection, parasite burden, or effect of chemotherapy. The results of long-term posttherapy field studies indicate that serology has a role in monitoring control programs. Standardized serologic assays and the need for International Standard Reference Sera are emphasized. A standardized enzyme-linked immunosorbent assay based on the Falcon Assay Screening Test system (FAST-ELISA), and involving a standard reference serum pool, is suitable for both serodiagnosis and field studies. Measurement of circulating antigens as a parameter of active infection is considered to have increased potential, compared with antibody measurement, in management of clinical disease and in control programs. Recombinant DNA technology may be useful for producing standard antigens for use in assays measuring antibody or circulating antigen. Time-resolved immunofluorescence involving europium-labeled conjugates may provide the increased assay sensitivity needed for measurement of circulating antigen.

Serodiagnosis of Schistosoma mansoni with microsomal adult worm antigen in an enzyme-linked immunosorbent assay using a standard curve developed with a reference serum pool.

A standardized microtest plate enzyme-linked immunosorbent assay was developed using the microsomal fraction of adult worms of Schistosoma mansoni (MAMA) as antigen. The standard reference serum pool was prepared from acutely and chronically infected rhesus monkeys and was shown to be appropriate as a standard for measuring the levels of reactivity of the unknowns. The standard serum pool was arbitrarily designated as having 100 activity units per microliter. The levels of reactivity of the unknowns were expressed as activity units per microliter. Serum specimens were obtained from 190 patients infected with S. mansoni in the Caribbean, South America, and Africa. Serum was obtained from small numbers of patients infected with S. haematobium, S. japonicum, or S. mekongi. Controls were 136 patients with other helminthic infections, 142 patients with protozoal or other diseases with liver involvement, and 81 healthy serum donors. The J index (or predictability) of the assay was calculated to determine the significant level of reactivity. The assay has a predictability of 95% for both patients with S. mansoni infections and those with other infections. The sensitivity of the assay for S. mansoni infections was 96%, and the specificity (in terms of cross-reactions with infections with other parasite genera or with other liver diseases) was 99%. The heterologous Schistosoma species showed a markedly lower level of reactivity, with an overall sensitivity of 55%. This is in accord with the species-specificity previously recognized in MAMA, and emphasizes the need for standard reference pools of human sera prepared from patients infected with single species of each of the Schistosoma. Use of these pools in assays with antigens of the respective schistosome species would allow optimum serologic evaluation.

Standardization of serological assays for parasitic diseases with schistosomiasis and toxocariasis as prototypes.

A standardized microtest plate enzyme-linked immunosorbent assay (ELISA) was developed using the microsomal fraction of adult worms of Schistosoma mansoni (MAMA) as antigen. A standard reference serum pool was prepared and arbitrarily designated as having 100 activity units per microliter. The precision of the test, the suitability as a standard of the reference serum pool, and the J index as a method of determining the significant level of reactivity were evaluated. The predictability of the assay for patients with S. mansoni infections and for non-schistosomiasis patients was 95%. Preliminary studies with the truly quantitative microtest plate kinetic ELISA indicate that this assay is suitable for standardization for schistosomiasis and toxoplasmosis. The need for WHO International Reference Sera with designated International Units and for well-defined batteries of sera for evaluating the specificity and sensitivity of assays was emphasized.

Quantitative capacities of glutaraldehyde and sodium m-periodate coupled peroxidase-anti-human IgG conjugates in enzyme-linked immunoassays.

Covalently linked peroxidase-anti-human IgG conjugates were prepared by either glutaraldehyde or NaIO4 coupling techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows that the glutaraldehyde coupled conjugate is composed of generally lower molecular weight components than the NaIO4 coupled product. The NaIO4 conjugate, when used to quantitate human immunoglobulin (Ig) in enzyme-linked immunoassays, appears to be highly sensitive in that small amounts of Ig elicited relatively high reactivities. The quantitative range of this type of conjugates, where reactivities are linearly proportional to the amount of human Ig present, is, however, extremely narrow (0.01-0.10 micrograms/ml of human IgG). Conversely, the glutaraldehyde coupled type conjugate is capable of sustaining a much wider range of linearity (0.01-0.6 micrograms/ml), but with a more gradual rise of reactivity which corresponds well to the amount of human Ig present. Conjugates prepared with glutaraldehyde are thus more useful in quantitative assays where wide quantitative ranges are desirable. NaIO4 conjugates on the other hand, are more suited to qualitative assays where sensitivity is more important.

Demonstration of species-specific and cross-reactive components of the adult microsomal antigens from Schistosoma mansoni and S. japonicum (MAMA and JAMA).

Analysis of human serum reactivities to the Schistosoma mansoni adult microsomal antigens (MAMA) showed that S. japonicum and S. haematobium infection sera, as a rule, did not react as well to MAMA as did the homologous S. mansoni infection sera. The degree of species specificity, although not absolute, was quite pronounced. Purification of the corresponding microsomal antigens from S. japonicum adults (JAMA) and subsequent assays with both homologous and heterologous infection sera show a distinct and reciprocating species specificity between S. mansoni and S. japonicum microsomal antigens. The specificities of these antigens were quantitated by k-ELISA. Qualitative analysis of active antigenic components for both JAMA and MAMA involved assay by the "Western blot" or enzyme-linked immunoelectrotransfer blot (EITB). The EITB patterns of both antigens, after resolution by SDS-PAGE, show species-specific reactive bands at the 16,000 to 35,000 m.w. region. S. japonicum-specific antigens are located at the 18,000 to 35,000 m.w. region whereas S. mansoni-specific antigens were associated with m.w. components of 16,000 to 29,000. High m.w. antigen components (greater than 40,000) of both JAMA and MAMA are recognized by both heterologous and homologous infection sera and are thus not species specific. The demonstration of the clear separation of species-specific antigen bands of JAMA and MAMA by physical size offers a unique opportunity to isolate and characterize the species specificities of antibody-antigen reactions in these parasitic infections.

Evaluation of serologic tests for Pneumocystis carinii antibody and antigenemia in patients with acquired immunodeficiency syndrome.

Sera from 68 patients with acquired immunodeficiency syndrome and 135 controls were used to evaluate the indirect immunofluorescence and enzyme-linked immunosorbent assays for detection of antibodies to Pneumocystis carinii and a counterimmunoelectrophoresis assay for detection of circulating Pneumocystis antigen. None of these assays was helpful in the diagnosis of P. carinii pneumonia. An improved assay for antigenemia is needed to differentiate between clinical and subclinical infection.

Schistosoma mansoni adult microsomal antigens, a serologic reagent. I. Systematic fractionation, quantitation, and characterization of antigenic components.

A systematic and quantitative search was conducted to identify and isolate a serologically pertinent antigen with high specific activity and low cross-reactivity from adult worms of Schistosoma mansoni. Adult worms of S. mansoni quickly thawed from liquid N2 temperatures were disrupted by controlled homogenization in isotonic buffered sucrose. Differential centrifugation of the homogenate yielded three particulate and one soluble fractions: the 480 x G pellet (nuclear), the 7650 x G pellet (mitochondrial), the 360,000 x G pellet (microsomal), and the 360,000 x G supernatant (cytosol). Quantitative analysis indicated a major concentration of specific antigenic activities in the microsomal fraction. Further purifications of the urea-solubilized, n-butanol-treated microsomal particles by gel filtration and ionic-exchange chromatography resulted in a microsomal antigen (MAMA) possessing high specific activity and low cross-reactivity. The final purification post-ionic exchange chromatograph showed a 30-fold increase of specific antigen activity over that of the cytosol fraction. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunotransfer blot by the "Western blot" technique (EITB) indicated specific antigenic activities in association with several different m.w. bands (heterogeneous m.w. by extrapolation = 4.3 to 11.9 x 10(6), 2.0 x 10(5), and 2.0 x 10(4) daltons) of the MAMA fraction. When compared with other reported serologic antigens, MAMA showed substantially higher specific activity and lower cross-reactivity.

Prevalence of Entamoeba histolytica in patients with schistosomal colonic polyposis.

Two hundred and fifty-seven Egyptian patients were classified into three groups: patients with schistosomal colonic polyposis, those with simple schistosomiasis without polyposis, and a non-schistosomal group. A diagnosis of schistosomiasis was made by clinical history and examination plus three fresh stool examinations or a rectal biopsy. The presence of schistosomal colonic polyps was established by sigmoidoscopy and biopsy of polyps. Stool examinations were made on all individuals, using the merthiolate-iodine-formaldehyde technique to detect Entamoeba histolytica. We found the prevalence of amebiasis in the group with schistosomal colonic polyposis (37%) to be significantly higher than that in the non-schistosomal group (11%) and in the schistosomal group without polyposis (15%). The difference in prevalence of amebiasis between the simple schistosomal and non-schistosomal groups was not significant.

Schistosoma mansoni adult microsomal antigens, a serologic reagent. II. Specificity of antibody responses to the S. mansoni microsomal antigen (MAMA).

The purified Schistosoma mansoni adult microsomal antigen, MAMA, was used in the quantitative single-tube kinetic dependent enzyme-linked immunosorbent assay (k-ELISA) to measure antibody levels of various human patient sera. The 511 serum specimens tested were from patients with both homologous and heterologous infections. Sera from U.S., Egyptian, Brazilian, and Puerto Rican patients infected with S. mansoni reacted strongly with MAMA. Chinese patients infected with S. japonicum, and Nigerians or Egyptians infected with S. haematobium produced much lower responses to this antigen than those infected with S. mansoni. Sera from patients with echinococcosis, filariasis, paragonimiasis, clonorchiasis, trichinosis, amebiasis, and hepatitis and from healthy uninfected control individuals generally contained no detectable antibodies against this antigen. The S. mansoni adult microsomal antigen, MAMA, therefore, appears to be a highly potent and specific reagent for the serodiagnosis of S. mansoni infections.

Fractionation of Pneumocystis carinii antigens used in an enzyme-linked immunosorbent assay for antibodies and in the production of antiserum for detecting Pneumocystis carinii antigenemia.

Cyst-rich suspensions of Pneumocystis carinii were obtained by differential and gradient centrifugation from heavily infected rat lungs. After preparation of an aqueous-soluble extract of the cyst-rich material, the insoluble residue was extracted with 8 M urea. Small amounts of infected human lung tissue and uninfected rat and human lung were processed similarly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both human and rat infected lung extracts contained a large protein (greater than 200,000 daltons). This component was not present in extracts of uninfected lung. In addition, an HCl-soluble extract was prepared from the cyst-rich suspension from infected rat lung. The urea-extracted antigen was most reactive in an enzyme-linked immunosorbent assay. Rabbit antiserum against the HCl-soluble antigen detected circulating antigen in patients' sera in a counterimmunoelectrophoresis assay.

Detection of specific antibody by enzyme-linked immunosorbent assay and antigenemia by counterimmunoelectrophoresis in humans infected with Pneumocystis carinii.

A urea-soluble extract of cyst-rich material from rat lung heavily infected with Pneumocystis carinii was evaluated in an enzyme-linked immunosorption assay for antibody in 461 human sera. The highest level of reactivity occurred in sera submitted for serodiagnosis from proved or highly suspect cases. However, the range of reactivities in these groups, many of whom were on immunosuppressive therapy, was very wide. A more restricted lower range of reactivity was observed in both hospital-family contacts and healthy Serum Bank donors. Because of the overlap in levels of reactivity between the pneumocystosis and control groups, no concise cutoff value to separate infected from noninfected individuals could be made. Specificity of the reactions was shown by absorption of patients' and control sera with uninfected and P. carinii-infected human and rat lung tissue. The data support the concept that P. carinii is highly prevalent as a latent agent in the general population and is provoked to cause clinically manifest disease in the compromised host. Detection of circulating antigen appeared to be specific and possibly a useful adjunct to diagnosis, as 10 of the 14 proved or highly suspect patients with antigenemia did not have measurable antibody to P. carinii.