Optimization of binding capacity and specificity of protein G on various solid matrices for immunoglobulins.

Streptococcal protein G is a more versatile and efficient alternative to staphylococcal protein A in purifying immunoglobin G (IgG) isotypes from various animal species. Optimizations are most dramatic with goat IgG, which binds protein G 55 times better than protein A. Using GammaBind G (a recombinant form of protein G (Genex Corp.)), we optimized binding capacity and specificity for IgG. Protein G was covalently coupled to three different matrices (CNBr-Sepharose, Tresyl-Sepharose, and Affigel-10) and compared with protein A-CNBr-Sepharose. Equal volumes of human, mouse, and goat serum samples were equilibrated into Hepes/NaOH buffers with various ionic strengths (i.e., concentrations of NaCl) and pH values and allowed to bind to affinity columns of proteins G and A. Bound ligands were eluted with 8.0 M urea, 0.05-M Tris/HCl, pH 8.00. Bound fractions were assayed for protein concentration and analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis. The optimal conditions for binding IgG to protein G are 1.0 M NaCl and pH 8.0 for human, mouse, and goat.

Prevalence and comparison of serologic assays, necropsy, and tongue examination for the diagnosis of porcine cysticercosis in Peru.

Swine cysticercosis, a severe zoonotic disease which is part of the Taenia solium life cycle, causes major economic losses in pig husbandry. Throughout South America, farmers diagnose cysticercosis by examining the tongues of their pigs for cysticercus nodules. Farmers do not bring pigs believed to be infected to the slaughterhouse for fear of confiscation. Therefore, reliable statistics on porcine cysticercosis can only be acquired at the household level. We examined the utility of the tongue test as a diagnostic tool for porcine cysticercosis. The results of the tongue test was compared with 2 serologic methods for the detection of cysticercosis, the enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunoelectrotransfer blot assay (EITB), and with necropsy results. We examined 11 animals from an endemic area (Huancayo) and 42 animals from an area free of cysticercosis (Lima). The tongue test has a sensitivity of 70% and a specificity of 100%, the EITB a sensitivity and specificity of 100%, and the ELISA a sensitivity of 79% and a specificity of 75%. Thus, the tongue examination, being a test essentially without cost and having fair sensitivity and high specificity, can be useful in epidemiological surveys. Prevalence for porcine cysticercosis in Huancayo is 23.4% by tongue examination, 31.2% by necropsy, 37.7% by ELISA, and 51.9% by EITB.

Immunotherapy for porcine cysticercosis: implications for prevention of human disease. Cysticercosis Working Group in Peru.

Taenia solium cysticercosis is an important cause of human disease in many developing countries. Porcine cysticercosis is a vital link in the transmission of this disease and impairs meat production. A treatment for porcine cysticercosis may be an effective way of preventing human disease that would also benefit pig farmers, facilitating control programs in disease-endemic regions. Previous research suggests that reinfection with cysticercosis or immunotherapy with cysticercal antigens may cause degeneration of cysticerci, potentially curing porcine cysticercosis. Therefore, a blinded, randomized, controlled study to assess the efficacy and safety of immunotherapy in 28 naturally parasitized pigs was performed. Four groups of pigs with similar weights were inoculated twice with membrane-enriched cysticercal antigens (MA), saline, aqueous-soluble crude cysticercal antigens (AA) in adjuvant (Freund's complete then incomplete), or adjuvant alone. Immunotherapy was well tolerated but had no consistent effect on the macroscopic appearance of cysticerci or eosinophil count. Histopathologic findings were variable, with both severe and minimal inflammatory reactions seen in adjacent cysticerci in all pigs. Nine (64%) of 14 pigs given immunotherapy developed new antibody bands on electroimmunotransfer blot compared with one (7%) of 14 control pigs (P < 0.01). Treatment with AA in adjuvant caused a significant increase in the proportion of cysticerci that failed to evaginate and were, therefore, not viable for infecting humans (34% for pigs given AA in adjuvant compared with 10% for adjuvant alone; P < 0.04). Although immunotherapy caused a statistically significant decrease in the viability of cysticerci, this immunologic reaction was not great enough to prevent human disease.

Discrepancies between cerebral computed tomography and western blot in the diagnosis of neurocysticercosis. The Cysticercosis Working Group in Peru (Clinical Studies Coordination Board).

Serum samples from sequential patients who underwent cerebral computed axial tomography (CT) scan in a Peruvian radiologic clinic were tested by the highly sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB) test to detect antibodies to Taenia solium. The results of the EITB test were compared with those obtained by CT scan for the diagnosis of neurocysticercosis. Of the 383 patients sampled, 32 (8%) were seropositive. The results of CT and EITB were frequently discrepant. When compared with the EITB assay, the CT scan was 44% sensitive and 95% specific. The sensitivity of CT increased to 63% if less specific images (single calcifications, granulomas, or hydrocephalus) were included. The CT scan for diagnosis of cysticercosis can best be used in conjunction with a reliable serologic test such as the EITB.

Treatment of porcine cysticercosis with albendazole.

In a randomized, controlled study, the efficacy and safety of two different schemes of albendazole therapy for treatment of porcine cysticercosis were tested. Seventeen naturally infected pigs were divided into three groups and treated per os with albendazole (50 mg/kg single dose), albendazole (30 mg/kg every day for three days), or given no treatment, respectively. Serologic responses were monitored with the enzyme-linked electroimmunotransfer blot assay. Pigs were humanely killed 12 weeks after treatment, necropsied, and the number of parasites was recorded. Scolex evagination was used to assess viability of the cysts. Both albendazole-treated groups had significant side effects (anorexia, lethargy). Only a single viable cyst was recovered from the brain of one animal after therapy in the multiple-dose group, and the single-dose therapy left 11% of the cysts viable. In contrast, more than 90% of muscle cysts were found to be viable in the untreated group. Although albendazole therapy for three days was found to be highly effective, side effects and the need for multiple doses would still prevent its widespread use.

Use of sentinel pigs to monitor environmental Taenia solium contamination. The Cysticercosis Working Group in Peru (CWG).

We tested a novel approach to assay Taenia solium prevalence using the enzyme-linked immunoelectrotransfer blot assay in sentinel piglets to determine environmental contamination with T. solium eggs in a disease-endemic zone in Peru. Twelve sentinel piglets from an area where the disease is not present were tested at two months of age, moved to an area where the disease is endemic, and retested at the of age nine months. Sentinel piglets native from this T. solium-endemic area were also tested concurrently at two and nine months of age. Of the non-native pigs, 33% (4 of 12) acquired new infection. Of the 28 native pigs tested, 64% (18 of 28) acquired the infection. In a subset of the native piglets from seronegative sows, 44% (4 of 9) were infected at five months of age. Serodiagnosis of sentinel piglets is a practical method to detect T. solium eggs in the environment. Furthermore, it permits indirect assessment of human risk, which may be useful for monitoring the efficacy of intervention programs.

Immunodiagnosis of human cysticercosis (Taenia solium): a field comparison of an antibody-enzyme-linked immunosorbent assay (ELISA), an antigen-ELISA, and an enzyme-linked immunoelectrotransfer blot (EITB) assay in Peru. The Cysticercosis Working Group in Peru (CWG).

We compared results of an enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunoelectrotransfer blot (EITB) assay for the diagnosis of cysticercosis in sera and cerebrospinal fluid (CSF). Sera from 34 patients with confirmed cysticercosis were tested by both ELISA and EITB assays. Cerebrospinal fluid from some of these patients was also tested by ELISA for the presence of antibody (AB-ELISA) (n = 21) and antigen (AG-ELISA) (n = 15). Specificity in sera was examined by testing 51 serum samples from Bangladesh, where cysticercosis is not endemic. Cross-reactivity was evaluated in sera from patients with Echinococcus granulosus (hydatid) and Hymenolepis nana infections. Sensitivity in detecting cysticercosis in sera was 94% by EITB and 65% by AB-ELISA (P less than 0.01). Sensitivities in the CSF tested by EITB, AB-ELISA, and AG-ELISA were 86%, 62%, and 67%, respectively. The specificity of the EITB was 100%, while that of AB-ELISA was 63% (P less than 0.01). Cross-reactions occurred in the AB-ELISA with 11% and 20% of sera from hydatid and H. nana patients, respectively. Our results demonstrate that the EITB is the best assay available for the diagnosis of cysticercosis in both sera and CSF.

Application of the enzyme-linked immunoelectrotransfer blot to filter paper blood spots to estimate seroprevalence of cysticercosis in Bolivia.

An enzyme-linked immunoelectrotransfer blot (EITB) assay was used to study the prevalence of cysticercosis in rural Bolivia. Dried blood spots on filter paper from fingersticks were used as assay samples. Before the serosurvey, experiments were performed to show that samples eluted from dried whole blood on filter paper exhibited no decrease in sensitivity when compared with the more traditional serum samples used in the EITB. Fingerstick blood dried on filter paper is a convenient, economical way of transporting and storing field samples for epidemiologic surveys of cysticercosis in developing countries. This report shows the utility of this sample collection method in underdeveloped countries where refrigeration is not possible and where venipuncture is a problem. Blood was obtained from randomly selected residents in three rural regions of Bolivia: Chuquisaca (n = 1,859), Cochabamba (n = 1,516), and Tarija (n = 1,010). The estimated seroprevalence on 10% of the sample collected for the three regions were 9%, 4.5%, and 2%, respectively.

Geographic clustering and seroprevalence of schistosomiasis in Puerto Rico (1995).

A systematic, island-wide survey for schistosomiasis in Puerto Rico has not been conducted for more than 40 years. In 1974, a thorough survey of Boqueron de Las Piedras, a small community, showed a prevalence of 40%. No additional information on prevalence in Puerto Rico has been obtained during the ensuing 21 years. Concern for the public health of residents and visitors prompted the formation of the Bilharzia Commission in 1994 and the systematic serosurvey reported herein. Two thousand nine hundred fifty-five plasma samples from healthy donors were obtained randomly from the Red Cross in March and April 1995. Sex, resident municipalities, and age of the donors were recorded. The donors were from all but three of 79 municipalities in Puerto Rico. No sample was available from the three out island municipalities of Mona, Vieques, and Culebra. Male donors (n = 2,027) outnumbered females (n = 928) by more than 2:1, ages ranged from nine to 76 years with most (85.3%) between 19 and 51 years of age. All samples were tested with the Falcon assay screening test:enzyme-linked immunosorbent assay (FAST:ELISA) with microsomal antigens of Schistosoma mansoni. All FAST:ELISA+ samples were confirmed by enzyme-linked immunoelectrotransfer blot (EITB). Our data showed that 15.4% were FAST:ELISA+, and 10.6% were confirmed by EITB; 13.5% of the males and 4.1% of the females were EITB+. If we exclude those municipalities with fewer than five samples, the prevalence of EITB+ ranged from 0% to 38.5%, with the highest seroprevalence rates (21.1-38.5%) concentrated in 17 municipalities, which accounted for 48% of all seropositive samples. These 17 municipalities, however, contain only 18% of the total population of Puerto Rico. Two areas of high seroprevalence rates center around Jayuya (38.5%) and Naguabo (36.4%). The previously surveyed area of Boqueron is located in Las Piedras (35.3%), adjacent to Naguabo. In addition, we found 10% (21) of our total 215 donors less than 25 years of age to be EITB+ and all but two are residents of the high prevalence districts. These data strongly support the contention that schistosomiasis has been transmitted in a focal fashion during the past approximately 20 years.

Seroprevalence of cysticercosis in an Orthodox Jewish community.

Neurocysticercosis cases were identified in 1991 in an Orthodox Jewish community. Transmission was linked to tapeworm-infected immigrant housekeepers from countries where Taenia solium is endemic. To evaluate the extent of and risks for locally acquired cysticercosis, a seroprevalence survey was conducted in 9% of the households in this community. Cysticercosis antibodies were detected in 23 (1.3%) of 1,789 persons from 612 families. All 23 seropositive persons were asymptomatic, and no intracerebral lesions were found for the 21 seropositive persons who underwent brain imaging. Seropositivity was associated with female sex (relative risk [RR] = 2.45, P = 0.049), hiring a domestic worker for child care duties (RR = 3.79, P = 0.05), and with employees from Central America (RR = 2.70, P = 0.0001). Exposure to T. solium in this community is unexpectedly high. Widespread employment of domestic workers from endemic regions and high employee turnover contributes to exposure risk.

Serum-free culturing of adult Schistosoma mansoni in dialysis bags for the production of excretory/secretory antigens.

Antigenic excretory/secretory (E/S) products from Schistosoma mansoni are potentially important in the development of diagnostic assays used to detect circulating antigens in schistosomiasis. The E/S products to be used as antigen(s) for this development must, by necessity, be free of exogenous proteins. The ability to extend serum-free in vitro culture of adult worms is, therefore, essential. Adult worms were perfused from mice, washed in serum-free RPMI-1640 with antibiotics, and placed in sterile dialysis bags, molecular weight cut-off 10 kDa, at a concentration of 100 worms in 1 ml of serum-free, supplemented RPMI-1640. Each bag was then placed in a flask of supplemented RPMI-1640 with 10% fetal calf serum in a humidified incubator at 37 C, 7% CO2. At days 1, 3, 5, 8, and 12, worms were collected; E/S culture medium was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Worm survival rates were 85% after 1 day in culture, dropping gradually to 65% on day 8, and then to 38% on day 12. Silver stain for total protein and immunoblot exposed to positive human infection serum showed E/S culture media from days 3 and 5 having the least complex banding pattern. The quantitative specific activity of E/S, as measured by antigen-limiting Falcon assay screening test system-enzyme-linked immunosorbent assays against human infection serum, indicates E/S antigenicity closely follows the attrition of worms and, therefore, may be directed against the release of somatic antigens by dead worms. Culturing S. mansoni in dialysis tubing is useful in deriving E/S products.

Rapid, low-technology field- and laboratory-applicable enzyme-linked immunosorbent assays for immunodiagnosis of Schistosoma mansoni.

Simple and rapid polystyrene- and nitrocellulose-based enzyme-linked immunosorbent assays were developed for detecting antibodies against adult Schistosoma mansoni microsomal antigens. The polystyrene test uses the Nunc Immuno Stick System. A single dilution of the antibody source being tested, the conjugate, and the substrate (3,3',5,5'-tetramethylbenzidine) are placed in tubes. Dried, antigen-coated polystyrene sticks are then exposed to the reagents by immersion. Once the sticks are sensitized, an entire assay can be completed in 8 min. Positive reactions result in a rich blue color in the substrate tube and can be distinguished with the naked eye. In the nitrocellulose-based test, a nitrocellulose sheet with antigen drawn in a line by pen is cut to produce identical strips. The ligand-binding steps and washings are performed in the troughs of incubation trays. The exposure times required for a single dilution of the antibody source being tested, the conjugate, and the substrate (3,3'-diaminobenzidine) are 5 min, 5 min, and 7.5 min, respectively. Once sensitized strips are available, an entire assay can be run in 50 min. Both techniques can assay serum or whole blood. The characteristics of polystyrene- and nitrocellulose-based techniques allow them to be used successfully in field studies and in minimally equipped laboratories.

Optimization of the covalent conjugating procedure (NaIO4) of horseradish peroxidase to antibodies for use in enzyme-linked immunosorbent assay.

The procedure for covalent conjugation of horseradish peroxidase (POD) to goat anti-human IgG (GAHG) molecules was systematically optimized in terms of reactant molar ratio, time of reaction, pH, and temperature. The optimum conjugation procedure was defined by the conditions that produced an enzyme-labeled Ab with the highest specific activity in immunosorbent assays for normal human IgG (NHIgG). The best conditions are: Sodium meta-periodate (NaIO4) to Ab molar ratio during oxidation is 40:1; time of oxidation is 5 min at 37 degrees C; oxidation reaction is conducted at pH 5.0; the molar ratio of POD:GAHG is 6:1; the conjugation time is 24 h at 4 degrees C; and the optimal conjugation pH is 10.0. A conjugate constructed under these conditions is capable of generating 1.8 and 12.6 times more specific signals (delta A650nm/min) than the best and worst commercial conjugates, respectively. This conjugate is also able to detect NHIgG at a concentration of 2.25 x 10(-13) M, a sensitivity 25 times that achieved by most comparable commercial products in identical assays.

Rodents infected with Schistosoma mansoni produce cytolytic IgG and IgM antibodies to the Lewis x antigen.

Schistosoma mansoni is a blood fluke that produces glycoconjugates containing the Lewis x antigen (Le(x)) Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->R. However, Le(x) antigen is also normally expressed in many tissues of adult rodents. We now report that mice and hamsters chronically infected with S.mansoni generate high titers of both IgM and IgG antibodies reactive with Le(x) and that no reactivity is present in sera from uninfected animals. Anti-Le(x) antibodies were detected by ELISA using the Le(x)-containing neoglycoprotein lacto-N-fucopentaoseIII-BSA. The IgG in infected animals consists of IgG1, IgG2a, and IgG2b subclasses and binds to Protein A-Sepharose. The sera of infected animals reacts only with Le(x) antigen and has no reactivity toward either Le(a) or sialyl Le(x). The IgM response to Le(x) is detectable at week 2, whereas the IgG response is detectable at weeks 5-6 following infection of mice. The sera of infected mice and hamsters can mediate the complement-dependent cytolysis (CDC) of cells expressing surface Le(x). This cytolytic activity is exclusively effected by the anti-Le(x) antibodies, since their removal from sera by adsorption depletes the sera of CDC activity. Thus, the abundant expression of the Le(x) antigens by the parasite elicits cytolytic antibodies reactive with a host antigen.

Schistosoma mansoni infection in humans and primates induces cytolytic antibodies to surface Le(x) determinants on myeloid cells.

The Lewis x antigen (Le(x); Gal beta 1-4[Fuc alpha 1-3]GlcNac beta1-R), which is present on the surfaces of human cells, is also synthesized by the human helminthic parasite Schistosoma mansoni. We now report that IgM and IgG antibodies to Le(x) antigens are present in the sera of humans and rhesus monkeys infected with S. mansoni, whereas these antibodies are completely absent in uninfected individuals. The sera from infected humans and monkeys mediate specific complement-dependent cytolysis of human promyelocytic leukemic HL-60 cells, which bear surface Le(x) antigen. Furthermore, the major activity in sera from infected individuals toward HL-60 cells is due to anti-Le(x) reactivity.

Guatemalan human onchocerciasis. II. Evidence for IgG3 involvement in acquired immunity to Onchocerca volvulus and identification of possible immune-associated antigens.

Ag-specific isotypic differences in immune response to Onchocerca volvulus Ag were assessed for 778 long term residents of endemic Guatemalan areas by quantitative ELISA with 5-min incubation steps and immunoblot. The study population was separated into five groups based on clinical status: N+F+, N+F-, N-F+, N-F-H+, and N-F-H-, where N = O. volvulus adults (nodule), F = microfiladermia, and H = history of O. volvulus infection. A subset of 44 individuals with high exposure to onchocerciasis from the N-F-H- group were critically evaluated and designated as "putatively immune." IgG1 reactivity to O. volvulus Ag was elevated in the majority of infected persons, but not in putatively immune individuals. Specific IgG3 levels, however, were equally elevated in all groups. The majority of N+F- persons also had elevated IgG1 levels, but they were lower than those found in F+ persons. IgG3 reactivities to a group of antigens at 20 kDa (GP20) were seen in many uninfected persons and some N+F- persons. In contrast, most F+ persons, react to this Ag with IgG1 and not IgG3. A mangabey inoculated with the infectious larval stage of O. volvulus (L3), but showed no signs of infection, began to recognize GP20 at 2 wk postinoculation. Early recognition of GP20 was possibly elicited by the larval stage. Purified nodule Ag from N+F+ individuals contained GP20, however, identical nodule Ag prepared from N+F- individuals did not. These data suggest that GP20 Ag may be common to both uterine microfilaria and the infectious larval stages. The fact that GP20 is predominantly recognized by IgG3 in putatively immune persons and some N+F- persons suggests that this increased IgG3 activity may be important in acquired immunity to onchocerciasis.

Diagnosis of cysticercosis in endemic regions. The Cysticercosis Working Group in Peru.

Taenia solium cysticercosis is a frequent cause of neurological disease in developing countries. Specific diagnosis of cysticercosis is difficult. We obtained serum and/or CSF samples from 204 consecutive patients admitted to a neurological ward in Lima, Peru, and looked for antibodies specific for T solium with the enzyme-linked immunoelectrotransfer blot (EITB) assay. 21 (12%) of 173 serum samples from these patients were EITB-positive. In contrast, only 2 (1.5%) of 135 patients attending a public endoscopy clinic and 1 (1%) of 88 patients attending a private endoscopy clinic were seropositive. 1 (1%) of 98 pregnant women living in a Lima shanty town was EITB-positive. 15 (58%) of 26 neurology patients diagnosed clinically as having cysticercosis were seronegative. Routine screening by EITB of all patients with neurological symptoms from areas of endemic cysticercosis would avoid misdiagnosis of this common and treatable disease.

Clinical evaluation of the cysticercosis enzyme-linked immunoelectrotransfer blot in patients with neurocysticercosis.

During the 3 years that the enzyme-linked immunoelectrotransfer blot (EITB) assay for the diagnosis of human cysticercosis has been in use at the Centers for Disease Control, 50 patients with both pathologically confirmed neurocysticercosis and computed tomographic (CT) or magnetic resonance imaging (MRI) scan results were identified. Of 32 patients with two or more lesions, 94% had detectable antibodies by EITB compared with 28% of 18 patients with single lesions. Patients with only calcified cysts (single or multiple) were less likely to have EITB-positive results than were those with noncalcified, enhancing lesions. Antibody was detectable more frequently in serum than in cerebrospinal fluid, regardless of the number or apparent condition of the cysts. These findings confirm that the EITB assay for cysticercosis antibodies is highly sensitive in patients with multiple, enhancing intracranial lesions but is less sensitive in patients with single lesions and in those with calcified lesions.

Cysticercosis as a major cause of epilepsy in Peru. The Cysticercosis Working Group in Peru (CWG)

In countries where cysticercosis is endemic, the proportion of epilepsy due to cysticercosis is not well documented. To investigate the association between cysticercosis and epilepsy, we used the enzyme-linked immunoelectrotransfer blot (EITB) assay to detect serum antibodies to Taenia solium in 498 consecutive outpatients at a neurology clinic in Lima, Peru. Every patient was classified as epileptic (n = 189) or non-epileptic (n = 309) after neurological, and where possible electroencephalographic, examination. A substantially higher proportion of epileptic than non-epileptic patients was seropositive in the EITB (22 [12%] vs 8 [3%], p < 0.001). 19% of epileptic patients born outside Lima, 20% of those with late-onset epilepsy, and 29% of patients with both these characteristics were seropositive. Thus, in Peru, cysticercosis is an important aetiological factor for epilepsy.

Guatemalan human onchocerciasis. I. Systematic analysis of patient populations, nodular antigens, and specific isotypic reactions.

The population from five Guatemalan plantations in areas endemic for onchocerciasis was surveyed, and 1032 individuals were recruited to participate in our study. From physical examination, past clinical history (5 to 8 yr), laboratory evidence and sample availability, a group of 778 long term residents with confirmed disease status were selected for detailed examination. We were able to identify 268 long term residents of endemic areas who had never been infected, 44 of these are from hyper- and mesoendemic areas. The 44 uninfected individuals from the hyper- and mesoendemic areas, because of their considerable exposure to this disease, were classified as "putatively immune." Intact nodules containing adult worms of Onchocerca volvulus were homogenized in the presence of protease inhibitors and fractionated into particulate and aqueous isotonic soluble antigens. Systematic analysis of these Ag fractions showed considerable amounts of Ig, presumably associated with Ag in the form of immune complexes. Individual specific antibody reactions from all 778 patients to nodule Ag were examined. Reactions to O. volvulus antigens by antibodies from patients with confirmed parasitic infections were almost exclusively restricted to IgG1 and IgG4 isotypes. Antigenic activity appeared to be primarily associated with low molecular mass (14 to 29 kDa) components. Some competitive blocking of antibody activities of other isotypes by IgG1 was observed, most notable was that of IgG3 and IgA. IgG4 and IgM activities were not significantly blocked.