Immunodiagnosis of human cysticercosis in cerebrospinal fluid. Antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium

Tapeworm antigens from Taenia crassiceps performed as well as those antigens from Taenia solium in an enzyme-linked immunosorbent assay for the detection of Cysticercus antibodies in 96 cerebrospinal fluid samples from patients with neurocysticercosis and in 96 CSF samples from patients with other varied neurological ailments. Thus, this manageable murine model of experimental cysticercosis solved the problem of antigen supply for clinical and epidemiological applications, and it provided an immediate means of abundant production of antigens for the wide distribution and standardization of immunodiagnostic tests for cysticercosis.     

Determination of herpes simplex virus type-specific antibodies by enzyme-linked immunosorbent assay.

We determined type-specific antibodies to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by an indirect enzyme-linked immunosorbent assay, using as antigens HSV-1 glycoprotein gC-1 and a HSV-2-specific polypeptide purified on affinity columns of monoclonal antibodies. All sera were initially screened for HSV antibodies by the enzyme-linked immunosorbent assay with a pool of Triton X-100-extracted antigens of HSV-1- and HSV-2-infected HEp-2 cells. The titer of HSV antibodies was predicted from a linear regression curve based on the absorbance of the initial 1:50 serum dilution. The sensitivity and specificity of the screening assay and of the assay for type-specific antibodies were established.     

Monoclonal antibody to human renal glomeruli cross-reacts with streptococcal M protein.

Two murine monoclonal antibodies (immunoglobulin M) evoked against human kidney tissue were examined for cross-reactivity with group A streptococci. A glomerulus-specific antibody, PMII.40.H.2, cross-reacted in an enzyme-linked immunosorbent assay with purified pepsin-extracted M proteins from type 6 and 12 streptococci, but not type 1, 3, 5, 19, and 24 M proteins. The cross-reactive monoclonal antibody also opsonized type 6 and 12 streptococci, indicating that it was directed against protective M protein epitopes that were exposed on the surfaces of these organisms. A control antibody, which was tubule specific, did not cross-react with any of the purified M proteins, nor did it opsonize any of the serotypes of streptococci tested. Western immunoblot experiments identified the glomerular cross-reactive antigen as a 43-kilodalton protein. The results demonstrate that M protein of group A streptococci and glomerular antigens of the human kidney possess cross-reactive determinants.     

Evaluation of a serological method for the detection of <Emphasis Type="Italic">Taenia saginata</Emphasis> cysticercosis using serum and meat juice samples

Two peptides, HP6-2 and Ts45S-10, were used as antigens for the detection of antibodies against Taenia saginata cysticercosis in serum and meat juice samples using enzyme-linked immunosorbent assay (ELISA). Positive control samples were obtained from animals experimentally infected (serum) and from animals naturally infected (meat juice). The two peptides and a pooled preparation of both peptides were evaluated, and their cut-off points with both sample categories were calculated. ELISA results from these different peptides were compared. Sensitivity and specificity of HP6-2 using serum were calculated as being 100 and 98%, respectively, showing to be higher than the values for the other antigens used. The average optical density (OD) value for negative samples was 0.646, whereas it was 1.702 for the positive control samples. This peptide was used to examine serum samples from animals with cysts and random field serum samples. For meat juice samples the pooled peptides showed the highest sensitivity and specificity, as they were 100 and 95%, respectively. The average OD values for the negative and the positive reference meat juice samples were 0.379 and 1.291, respectively. The optimal dilution of the meat juice samples for the ELISA was very low, as it was 1:20 using the pooled peptides, compared with 1:800 serum dilution using HP6-2. To the authors’ knowledge, this is the first report of a successful testing for T. saginata cysticercosis using meat juice.     

Characterization of the 8-kilodalton antigens of Taenia solium metacestodes and evaluation of their use in an enzyme-linked immunosorbent assay for serodiagnosis

The Western blot for cysticercosis, which uses lentil lectin purified glycoprotein (LLGP) antigens extracted from the metacestode of Taenia solium, has been the "gold standard" serodiagnostic assay since it was first described in 1989. We report that the diagnostic antigens at 14, 18, and 21 kDa, as well as some larger disulfide-bonded antigens, are actually all members of a very closely related family of proteins, the 8-kDa antigens. The genes for 18 unique, mature proteins have been identified. Nine of these were chemically synthesized and tested in an enzyme-linked immunosorbent assay with a battery of defined serum samples, including 32 cysticercosis-positive serum samples reactive with the 8-kDa antigens of LLGP on Western blotting, 34 serum samples from patients with other parasitic infections, and 15 normal human serum samples. One of the 8-kDa antigens, TsRS1, is 100% sensitive and 100% specific. TsRS1 will be one component of a cocktail of three to four synthetic or recombinant antigens, based on the diagnostic bands of the Western blot, which will be used for the serodiagnosis of cysticercosis.     

Use of Taenia crassiceps cysticercus antigen preparations for detection of antibodies in cerebrospinal fluid samples from patients with neurocysticercosis (Taenia solium).

Antigen extracts obtained from the vesicular fluid of Taenia crassiceps cysticerci and from fractions purified by affinity chromatography with the lectin concanavalin A and the glycoprotein antigen separated by electrophoresis were used for the detection of Taenia solium anticysticercus antibodies. The sensitivity and specificity obtained for all antigens were 100% in enzyme-linked immunosorbent assay with good reproducibility. Using immunoblotting of the three antigens, low-molecular-mass peptides (18 and 14 kDa) were characterized only in cerebrospinal fluid samples from patients with neurocysticercosis. The results confirm that antigen fractions purified from T. crassiceps cisticerci are important sources of specific peptides and proved to be efficient in detecting anti-T. solium antibodies.     

Improved immunodiagnosis of human cysticercosis with scolex protein antigens

Crude antigenic extract (CAE) and a scolex protein antigen (SPA) of Taenia solium cysticerci were used in indirect haemagglutination (IH) and enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies in cysticercosis patients. Complement fixation (CF) was used for comparison with antigens obtained as an alcoholic extract of CAE (ACAE) and SPA (ASPA). For each test, a dilution was chosen which showed no cross-reactivity with sera from patients with other parasitic diseases such as taeniasis, schistosomiasis, ancylostomiasis, ascaridiasis, strongyloidiasis, Chagas' disease and syphilis. For the CF, 14 was the discriminative dilution determined; for IH, 116 and for ELISA, 1256. The CF could detect serum antibodies to cysticerci in 45% of patients when ACAE antigen was used and 73% using ASPA. In the IH, serum antibody was detected in 73% of patients when CAE was used and 86% using SPA. In ELISA 63% of patients were positive when CAE was used and 91% using SPA. The use of SPA improved the serological distinction between infected and uninfected patients in all tests and the ELISA showed a significantly higher capacity to detect infected patients.     

Reactivity of two major allergens isolated from Schistosoma japonicum eggs against IgE and IgG antibodies in human serum

Antigenicity and specificity of two major allergens termed J1 and J2, both of which had been purified from soluble egg antigen (SEA) preparation of Schistosoma japonicum, were examined against serum from schistosomiasis japonica patients. By using enzyme-linked immunosorbent assay, specific IgE or IgG levels to J1 or J2 in the patients' sera correlated well with those to crude SEA, indicating that J1 and J2 are the major antigens in SEA binding human IgE and IgG antibodies. Binding inhibition studies using 125I-J1 and 125I-J2 clearly showed that cross-reactivity was not present between J1 and J2 in terms of their capacity to bind IgG or IgE antibody. When antibody levels of an individual patient to these two purified antigens were compared, a high degree of correlation was observed between them, regardless of the class of antibody. However, specific IgE antibody levels of individual serum to J1 or J2 were not correlated with IgG antibody levels to the respective antigen.     

Detection of circulating excretory secretory antigens in human fascioliasis by sandwich enzyme-linked immunosorbent assay.

We developed a sandwich enzyme-linked immunosorbent assay to detect circulating parasite antigen in humans with fascioliasis. The assay uses antibodies against Fasciola hepatica excretory secretory (ES) antigens. A monoclonal antibody was used to capture circulating ES antigens, and a human polyclonal antibody peroxidase conjugate was used to identify circulating ES antigens. Optimal dilutions of all reagents were determined by block titration. The antigen concentration in sera from patients was estimated by comparing the optical density at 492 nm of test sera with a standard curve. All of the serum samples from 25 patients with parasitological evidence of fascioliasis had a detectable antigen concentration (more than 10 ng/ml). None of the serum samples from 80 patients infected with parasites other than F. hepatica showed a positive reaction, suggesting the absence of cross-reactions in this assay.     

Serotype specificity and immunogenicity of the capsular polymer of Haemophilus pleuropneumoniae serotype 5.

Serotyping of Haemophilus pleuropneumoniae and serologic assays for detection of serotype-specific antibody are problematic due to the potential cross-reactivity of the crude antigens used for raising immune serum or for serology. The capsular polymer (CP) of H. pleuropneumoniae serotype 5 was investigated for serotype-specific activity with antiserum to whole cells or with antiserum made monospecific to CP by adsorption with a capsule-deficient mutant. When antiserum to whole cells or monospecific antiserum to CP was tested against purified CP from serotypes 1 to 7 by immunodiffusion or enzyme-linked immunosorbent assay, only capsules of serotype 5 were reactive. In addition, only encapsulated serotype 5 cells reacted with serum monospecific to CP in an indirect immunofluorescent-antibody assay. Serotype-specific antibody was completely inhibited in each assay by preincubation of purified CP with the serum. Antiserum to whole cells of H. pleuropneumoniae serotype 5 contained antibodies to proteins and lipopolysaccharide; these antibodies cross-reacted with antigens of heterologous serotypes by dot-blot enzyme-linked immunosorbent assay and immunoblotting. The antigenic activity of CP was stable after heating for at least 30 min at 100 degrees C. High titers of antibody to CP were present in the sera of rabbits immunized intravenously with whole log-phase cells or in the convalescent sera of pigs experimentally infected with H. pleuropneumoniae serotype 5. However, the purified CP was poorly immunogenic in rabbits and swine. Our results indicate that the capsule is the serotype-specific antigen of H. pleuropneumoniae and that a monospecific antiserum to capsule or purified capsule should be used for serotyping or serologic assays, respectively.     

Use of Taenia crassiceps Cysticercus Antigen Preparations for Detection of Antibodies in Cerebrospinal Fluid Samples from Patients with Neurocysticercosis (Taenia solium)

Antigen extracts obtained from the vesicular fluid of Taenia crassiceps cysticerci and from fractions purified by affinity chromatography with the lectin concanavalin A and the glycoprotein antigen separated by electrophoresis were used for the detection of Taenia solium anticysticercus antibodies. The sensitivity and specificity obtained for all antigens were 100% in enzyme-linked immunosorbent assay with good reproducibility. Using immunoblotting of the three antigens, low-molecular-mass peptides (18 and 14 kDa) were characterized only in cerebrospinal fluid samples from patients with neurocysticercosis. The results confirm that antigen fractions purified from T. crassiceps cisticerci are important sources of specific peptides and proved to be efficient in detecting anti-T. solium antibodies.     

Enzyme-linked immunosorbent assay for quantifying antigens of Dermatophagoides farinae and D. pteronyssinus (Acari: Pyroglyphidae) in house dust samples

Double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody as capture and detector antibodies was developed for quantifying antigens of Dermatophagoides farinae Hughes and D. pteronyssinus (Trouessart) mites contained in house dust samples. This monoclonal antibody was directed against mite antigens that were also reactive to immunoglobulin E antibody in all of 10 serum samples obtained from patients allergic to mites. Histological study using fluorescent antibody revealed that the monoclonal antibody was bound to the major part of the D. farinae mite body section including fecal matter and cuticles. The detection limit of the assay system was 0.17 microgram of soluble antigens of both mite species and the antigen amount corresponding to 0.5 mites per microplate well, whether in live or dead mites. This system did not react to antigens of Tyrophagus putrescentiae (Schrank) and Cheyletus malaccensis Oudemans. Slight inhibition of less than or equal to 21.3% was observed with nonspecific substances contained in house dust, such as wool, cotton, human dander, human hair, soil, and biscuit, but no direct ELISA reactions were obtained with any of these materials. In 49 house dust samples, ELISA was significantly correlated with the conventional microscopic observation method.     

A convenient method for epitope competition analysis of two monoclonal antibodies for their antigen binding

Choosing optimum pair of capturing antibody and detecting antibody when developing monoclonal antibody (MAb)-based, sandwich enzyme-linked immunosorbent assays is a time-consuming process requiring the coupling of individual antibodies to an enzyme like horseradish peroxidase or alkaline phosphatase. The MAbs required for the two-site sandwich ELISA should bind to distinct epitopes of the antigen, and their binding should not be mutually exclusive. To determine if two monoclonal antibodies would occupy distinct sites of their antigen in binding, and enzyme-linked immunosorbent assay was devised, which is easy-to-use and does not require any coupling of monoclonal antibodies to enzymes. Microplate wells are coated with rabbit polyclonal antibodies raised against the same antigen of MAbs. After blocking, a limited amount of the antigen is added for incubation with the rabbit antibodies. Mouse monoclonal antibody 1 (Mab 1) is added to saturation. A serial dilution of MAb 2 (for analysis) or MAb 1 (for control) is added subsequently. An enzyme-labeled, goat anti-mouse secondary antibody and its substrates are added for color development. Thus, the epitope competition of two MAbs for their antigen binding is easily determined by the measurement and comparison of color development between the two MAb additions.     

Development of a blocking enzyme-linked immunosorbent assay for the detection of avian polyomavirus-specific antibodies

Avian polyomavirus, described originally as budgerigar fledgling disease virus, has been associated with devastating contagious disease outbreaks in budgerigar aviaries. At present, this virus affects a wide range of psittacine and non-psittacine birds worldwide, and the serum neutralisation test is used for the serodiagnosis of avian polyomavirus infections. A blocking enzyme-linked immunosorbent assay was developed for the screening of large numbers of sera collected from various avian species. The assay employs a monoclonal antibody directed against the major structural protein VP1 as a blocking antibody in a sandwich blocking procedure. Either purified avian polyomavirus particles or avian polyomavirus VP1 expressed in recombinant baculovirus-infected Sf9 cells were used as antigen. The specificity of the blocking enzyme-linked immunosorbent assay was evaluated by testing sera directed against mammalian polyomaviruses. Using sera obtained from chicken infected experimentally with avian polyomavirus and a collection of psittacine field-origin sera, a good correlation was observed between the results of the blocking enzyme-linked immunosorbent assay and the serum neutralisation test. However, the blocking enzyme-linked immunosorbent assay is more rapid and more economic. Both, avian polyomavirus particles and VP1 produced by recombinant DNA technology proved to be suitable antigens.     

Cysticercosis immunodiagnosis using 18- and 14-kilodalton proteins from Taenia crassiceps cysticercus antigens obtained by immunoaffinity chromatography

Monoclonal antibodies (MAb) against Taenia crassiceps and Taenia solium cysticerci were produced and showed cross-reactivity with a 14-kDa protein from T. solium and with 18- and 14-kDa proteins from T. crassiceps. These MAbs and antibodies from cerebrospinal fluid (CSF) as well as serum samples from patients with neurocysticercosis (NC) reacted with 18- and 14-kDa T. crassiceps proteins purified by immunoaffinity chromatography using a Sepharose column coupled with MAbs (anti-excretory/secretory or anti-vesicular fluid antigens). Immunoaffinity-purified 18- and 14-kDa proteins were used in the design of a diagnostic enzyme-linked immunosorbent assay (ELISA) to detect antibodies in 23 CSF and 20 serum samples from patients with NC, showing 100% sensitivity. The test specificity was determined using 42 noninflammatory CSF samples and 70 inflammatory CSF samples from patients with other neurological disorders (OND), showing 100% and 99.1% (confidence interval, 97.3% to 100%) specificity, respectively. A false-positive CSF sample result in the OND group was from a human immunodeficiency virus-positive patient with meningoencephalitis. By using serum samples from 194 healthy individuals, the specificity was 100%. Analysis of an additional 16 serum samples from individuals with other parasitic diseases (13 with intestinal parasitosis and 3 with schistosomiasis) showed negative results. Three (10%) serum samples from patients with hydatidosis were positive in our ELISA and in ELISA with T. solium cysticerci antigens. Two of them were also positive by immunoblotting. The use of 18- and 14-kDa T. crassiceps immunoaffinity-purified proteins for detection of anti-cysticercus antibodies in CSF and/or serum samples using an ELISA system showed a good performance and high specificity for serum samples, dispensing with the use of confirmatory tests, such as immunoblotting, for checking specificity.     

Serological diagnosis of human cysticercosis by use of recombinant antigens from Taenia solium cysticerci.

A Taenia solium metacestode cDNA expression library in the lambda ZAPII vector was screened with pooled sera from patients with neurocysticercosis. Sixty primary clones were identified and shown to belong to two classes. The clones NC-3 and NC-9 did not reveal any significant homologies to sequences deposited in the databases and were further characterized. Both recombinant antigens were expressed as glutathione S-transferase fusion proteins and applied for serological diagnosis of human cysticercosis. An enzyme-linked immunosorbent assay was established and evaluated with 27 serum samples of La Réunion and Madagascar patients with cysticercosis. Diagnosis in these patients was established with radiological and serological procedures. For antigen NC-3 a sensitivity of 96.3% and a specificity of 91.5% for the serodiagnosis were achieved. In contrast, the sensitivity of antigen NC-9 was only 33.3%.     

Detection of Equine Antibodies to Babesia caballi by Recombinant B. caballi Rhoptry-Associated Protein 1 in a Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.     

Characterization of Mycobacterium tuberculosis antigen 5 epitopes by using a panel of 19 monoclonal antibodies.

Antigen 5 is a 35,000-dalton protein which has been purified from culture filtrates of Mycobacterium tuberculosis and shown by immunoprecipitation to be restricted in distribution to M. tuberculosis and M. bovis among 14 mycobacterial species studied. We raised 19 antigen 5-reactive monoclonal antibodies and used them to characterize epitopes of this antigen by enzyme-linked immunosorbent assay and immunoabsorbent affinity chromatography. Fifteen monoclonal antibodies, all immunoglobulin M (IgM), cross-reacted in two major patterns with culture filtrates from five species of mycobacteria and with purified mycobacterial arabinomannan and arabinogalactan. All 15 monoclonal antibodies also reacted with M. tuberculosis antigen 6. Immunoabsorbent affinity columns prepared with these antibodies yielded principally arabinomannan and arabinogalactan. Four monoclonal antibodies, three IgG2a and one IgM, reacted by enzyme-linked immunosorbent assay with antigens 5 and 6 exclusively and not with mycobacterial culture filtrates or polysaccharides. All four monoclonal antibodies yielded antigen 5 and small amounts of antigen 6 when used for immunoabsorbent affinity chromatography. We conclude from these studies that antigen 5 has two nonspecific epitopes, possibly carbohydrate in nature, and one apparent species-specific epitope which is shared with antigen 6.     

Enzyme-linked immunosorbent assays using novel Japanese encephalitis virus antigen improve the accuracy of clinical diagnosis of flavivirus infections

The cross-reactive antibodies induced by flavivirus infections confound serodiagnosis and pathogenesis, especially in secondary infections caused by antigenically closely related yet distinct flaviviruses. The envelope (E) glycoprotein fusion peptide contains immunodominant cross-reactive determinants. Using a recombinant Japanese encephalitis virus (JEV) premembrane and E expression plasmid producing JEV virus-like particles (VLPs), dramatic reductions in cross-reactivity were produced by the G106K-L107D (KD) double-mutant VLP against a panel of flavivirus murine monoclonal antibodies. Human serum panels from patients with recent flavivirus infections were analyzed to compare the accuracy of JEV wild-type (WT) and KD VLPs as serodiagnostic antigens in enzyme-linked immunosorbent assays. Statistical analysis demonstrated significant differences in assay performances for accurate determination of current JEV infections between WT and KD antigens by detecting immunoglobulin M antibodies at a serum dilution of 1:4,000 (likelihood ratios = 2.74 [WT] and 22 [KD]). The application and continued development of cross-reactivity-reduced antigens should improve both flavivirus infection serodiagnosis and estimates of disease burden.     

Humoral antibody response against Bacteroides gingivalis-specific antigen recognized by monoclonal antibody in adult periodontal patients.

An antigen recognized by monoclonal antibody specific for Bacteroides gingivalis was purified in the presence of 0.5% (wt/vol) beta-octyl-glucoside by immunoadsorbent column chromatography. The purified antigen was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining, and the pattern of SDS-PAGE agreed with that of immunoblotting. The molecule exhibited an apparent molecular weight of about 62,000 by SDS-PAGE. The antigen was sensitive to trypsin, Staphylococcus aureus V8 protease, DNase I and II, and heating, but insensitive to RNase and neuraminidase. By the enzyme-linked immunosorbent assay method, the purified antigen was not cross-reactive with rat polyclonal antibodies to each of several black-pigmented Bacteroides species, Fusobacterium nucleatum, Eikenella corrodens, and Actinobacillus actinomycetemcomitans. These results indicate that the purified antigen is specific for B. gingivalis. Humoral antibody titers in adult periodontal patients against the specific antigen were measured by the enzyme-linked immunosorbent assay. Serum immunoglobulin G antibody titers against the specific antigen in adult periodontal patients correlated significantly with the severity of periodontal disease. However, such significant correlation was not observed with serum immunoglobulin M antibody titers.