Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique

Proteins of Toxoplasma gondii were separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. Immunologically reactive polypeptides were detected by human sera with previously known toxoplasma antibody levels. Heavy chain-specific, peroxidase-conjugated anti-human immunoglobulins were used as the indicator antibodies for the separate identification of IgG and IgM reactive polypeptides. IgG toxoplasma antibodies reacted with several antigens of M_r ≈27 000–67 000, while toxoplasma-specific IgM seemed to detect only a few polypeptides. The M_r of 35 000 for the dominating IgM reactive polypeptide was observed.     

Tumor cell reactivity mediated by IgM antibodies in sera from melanoma patients vaccinated with GM2 ganglioside covalently linked to KLH is increased by IgG antibodies

 Natural IgM antibodies against the melanoma cell-surface ganglioside GM2, and IgM antibodies induced by vaccination with GM2 adherent to bacillus Calmette-Guerin, have been correlated with increased disease-free and overall survival in melanoma patients in previous phase I and II clinical trials. A vaccine containing GM2 covalently attached to keyhole limpet hemocyanin (KLH) plus the immunological adjuvant QS-21 now induces higher-titer, longer-lasting IgM antibodies against GM2 and has recently entered phase III clinical trials. For the first time this new vaccine also induces IgG antibodies against GM2 in the majority of immunized patients. With regard to immunity against bacteria, IgM antibodies have been described to be 1000-fold more effective than IgG antibodies at opsonification, complement-mediated cytotoxicity and protection from bacterial challenge. Though IgG antibodies have the theoretical advantage of being able to mediate antibody-directed cell-mediated cytotoxicity (ADCC), they may inhibit complement mediated IgM effector mechanisms against melanoma cells. Our goal was to confirm the functional characteristics of the anti-GM2 IgM and IgG antibodies induced by vaccination and to determine the impact that IgG antibodies might have on IgM antibody reactivity with GM2-positive tumor cells. Post-immunization sera from seven immunized patients were separated by size-exclusion chromatography into IgM and IgG fractions and a variety of serological assays were performed with the individual fractions and their combinations. Assays identifying specific IgM or IgG reactivity demonstrated partial inhibition by the opposite fraction. However, when the endpoint was complement-mediated lysis or overall antibody binding, which may more faithfully predict in vivo complement-mediated opsonification and lysis, the combinations of IgM and IgG fractions consistently demonstrated higher reactivity than either fraction alone. In addition, ADCC was induced in all seven patients. The results were the same whether the sera were obtained after 2 months or 2 years of immunizations. These findings suggest that IgG antibodies induced by the GM2-KLH plus QS-21 vaccine will not inhibit and should further augment the clinical impact of induced IgM antibodies. Received: 25 April 1996 / Accepted: 21 October 1996     

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis

Abstract This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii . To assess specificity, patient sera were statistically ( χ ², P ≤ 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30–33, 36–37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally isolated strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.     

Radiolabelling of the excretory-secretory and somatic antigens of Anisakis simplex larvae.

Anisakis simplex larvae were cultured in vitro in medium containing 35S-methionine for ten days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptide by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antisera were similarly analysed, using Staphylococcus aureus to absorb immuno-complexes. ES products of Anisakis larvae contained many polypeptides with molecular weights of less than 200 K. 180 KDa and 40 KDa polypeptides in ES products reacted with IgG in Anisakis-infected human sera. Somatic extracts also contained many polypeptides with molecular weights of less than 200 K. One of these polypeptides with a molecular weight of 130 K reacted with IgG in Anisakis-infected human sera. These polypeptides did not react with other nematode-infected human sera.     

The antibody response in Lyme disease.

We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis.     

Characterization of immunoglobulin classes of human antibodies to cryptococcus neoformans

Immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) levels were determined by radial immunodiffusion techniques in sera from 11 patients with cryptococcosis. Most specimens showed increased levels of IgM. Studies with fluorescein-labeled monospecific antihuman IgG and IgM, however, indicated that IgG was the immunoglobulin reactive in the indirect fluorescent antibody (IFA) test. In addition, cross-reacting sera from mycotic infections other than cryptococcosis were also shown to contain IFA antibodies of the IgG class. Sera treated with 2-mercaptoethanol continued to react in both the IFA test and the tube agglutination test. No correlation could be established between IgG and IgM concentrations and serological reactivity in the sera evaluated in this study.     

Experimental amebiasis: molecular weight analysis of Entamoeba histolytica antigens recognized by IgG antibodies

The reactivity of sera from experimentally infected animal was studied from 5-60 days postinoculation to determine which of the E. histolytica antigens are recognized frequently to infection. Crude extract of E. histolytica trophozoites was used and sera were examined by immunoelectrotransference assay. It was observed that sera recognized polypeptide with 70 kDa molecular mass after 15 days postinoculation onward and later 14 to 24 polypeptide with molecular weight of 110-22 kDa were recognized. All the amebic antigens (polypeptides) could be recognized by sera till 60 days postinoculated animals. Significance of expression of different amebic polypeptides in terms of pathogenesis needs further investigations.     

Rheumatoid factors from patients with rheumatoid arthritis react with beta 2-microglobulin.

IgM rheumatoid factors (RF) from 18 sera of rheumatoid arthritis (RA) patients isolated from monomeric IgG affinity columns showed strongly positive ELISA reactions with human beta 2-microglobulin (beta 2m), as well as with recombinant beta 2m. When the same RA sera were adsorbed to beta 2m-Sepharose affinity columns, eluted material showed predominant IgM anti-Fc of IgG and anti-beta 2m reactivity. Inhibition reactions with "RF" obtained from IgG affinity columns showed slightly higher reactivity of RF for Fc over beta 2m; however, when RF from the same RA serum had been adsorbed to and eluted from beta 2m affinity columns, beta 2m showed greater inhibition than Fc for RF reacting with either beta 2m or Fc on ELISA plates. Thus two overlapping populations of RF were identified in RA sera showing reactivity with both beta 2m and Fc of IgG. When RF were isolated from IgG columns, affinity was slightly higher for Fc than beta 2m. Conversely, RF eluted from beta 2m Sepharose reacted slightly more with beta 2 m than Fc. Trypsin digests of a polyclonal RA IgM RF showed no beta 2m reactivity in Fc mu 5 fragments. Fab mu RF retained slight anti-Fc IgG but no residual anti-beta 2m activity. Monoclonal human IgM, IgG, or IgA RF either from mixed cryoglobulins or EBV-stimulated RA lymphoid cell lines showed negative or occasional weakly positive anti-beta 2m activity. Overlapping 7-mer peptide ELISA analysis of the entire 99-amino acid sequence of beta 2m showed a major RF-reactive linear hydrophilic sequence at positions 56-60 which included a 3-amino acid exact homology to positions 401, 403, and 404 of the C gamma 3 domain. A peptide encompassing this sequence produced 90% inhibition of RF binding to whole beta 2m. Substitution of neutral glycines for each amino acid throughout the reactive epitope at positions 56-66 indicated that lysine at position 58 aspartic acid at 59, and tryptophane at 60 represented major portions of the RF-reactive epitope. These findings indicate that human RF derived from patients with RA react with other epitopes besides those present on IgG Fc, including epitopes on human beta 2m. For many years serum RF3 found in patients with RA have been regarded as premier examples of autoantibodies to autologous IgG.(ABSTRACT TRUNCATED AT 400 WORDS)     

The level of IgM, IgG and IgA antibodies and total antibodies to the low-molecular, cell-wall protein of Streptococcus group A without type specificity in the serum of patients with a streptococcal infection

The levels of IgM, IgG and IgA antibodies, as well as total antibodies, to group A streptococcal low-molecular cell-wall protein without type specificity were studied in the sera of patients with primary erysipelas, rheumatism in the active and inactive phases, seronegative rheumatoid arthritis, as well as in the sera of healthy donors. The average level of antibodies to low-molecular protein in the sera of all groups of patients was significantly higher than the sera of healthy donors. The analysis of the distribution of antibodies in accordance with their isotypes revealed the specific features of response, characteristic of each group of patients. For rheumatism patients, the positive correlation between response to low-molecular protein and response to group-specific polysaccharide A was established. This correlation was most pronounced in patients with rheumatism in the inactive phase.     

Comparison between autoantibodies arising during Trypanosoma cruzi infection in mice and natural autoantibodies

The autoantibodies induced in (C57BL/6 x BALB/c)F1 mice during Trypanosoma cruzi (CL strain) infection were analyzed and compared with natural autoantibodies present in healthy mice. Mice were killed at intervals after infection and their sera were tested by enzyme immunoassay against a panel of self- and non-self-Ag: actin, myoglobin, myosin, tubulin, DNA, and TNP-OVA. The level of IgM and IgG autoantibodies against all Ag started to increase from day 15 until 6 wk after the parasite infection. The high level of all autoantibodies persisted 3 mo postinfection, and 1 yr later, half of the mice still had elevated levels of IgM and IgG autoantibodies, particularly antitubulin IgG antibodies. IgM and IgG were isolated from pools of normal and infected mouse sera and their binding capacity to all Ag was compared. The titers of infected mouse sera were increased and the slopes of both IgM and IgG binding curves of autoantibodies to actin, myosin, and tubulin were greater than those of control mouse sera, indicating higher affinities. The average dissociation constant of the IgG2a autoantibody to mouse tubulin was 5 times lower than that of natural antitubulin IgG2a antibodies. Furthermore, absorption of the IgG from infected mouse sera onto a tubulin immunoadsorbent removed half the reactivity with tubulin and also with myosin, actin and parasite extracts. The eluted antibodies bound the same Ag. When IgG were further analyzed by Western blot on proteolytic fragments of tubulin, we found that antibodies from both groups bound to the same broad spectrum of polypeptide bands. However, additional fragments were recognized by antibodies from infected mice. All these results indicate that the autoantibodies naturally present in mice are significantly affected after infection with T. cruzi, in quantity as well as in specificity and affinity.     

A highly conserved 72,000 dalton centromeric antigen reactive with autoantibodies from patients with progressive systemic sclerosis

An autoantibody reactive with a 72,000 dalton centromeric antigen was detected by immunoblotting with the use of a nuclear enriched HeLa cell preparation in 42 of 77 patients with progressive systemic sclerosis (PSS). Reactivity with the 72,000 dalton polypeptide was associated with anti-centromere autoantibodies (ACA) detected by immunofluorescence (IF), and the antigen was highly conserved, being present in both human cells and Leishmania tropica. Thirty-five (83%) of the 42 sera reactive with the 72,000 dalton polypeptide also reacted with a 19,500 dalton polypeptide, and antibodies eluted from both the 72,000 dalton and the 19,500 dalton polypeptides reacted with the centromere when retested by IF on intact HEp2 cells, demonstrating that both polypeptides are antigenic components of the centromere. Only one of the 42 sera had precipitating antibodies to the Scl-70 antigen detected by counterimmunoelectrophoresis, indicating that the 72,000 dalton polypeptide was not related to the previously described Scl-70 antigen. The other 35 of the 77 sera tested were negative for ACA, although all had ANA, with the main patterns of IF being fine speckling of the nucleus (18 sera) and homogeneous or speckled staining of the nucleolus (17 sera). Anti-Scl-70 antibodies were detected in 17 of these 35 patients, 15 (88%) of whom reacted with an 89,000 dalton polypeptide, one with a 140,000 dalton polypeptide, and one with a 74,000 dalton polypeptide. Ten of the 15 sera reacting with the 89,000 dalton polypeptide also reacted with a 74,000 dalton polypeptide, and 2-D gel analysis suggested a relationship between the two molecules. Clinically defined types of scleroderma tended to associate with antibodies to particular molecular antigenic specificities. Thirty-seven (88%) of the 42 patients reactive with the 72,000 dalton polypeptide had sclerodactyly and features of the CREST syndrome, whereas patients reactive with the 89,000 dalton polypeptide and with Scl-70 tended to have more extensive cutaneous and visceral involvement.     

Western blot antibody determination in sera from patients diagnosed with Anisakis sensitization with different antigenic fractions of Anisakis simplex purified by affinity chromatography

Using Western blot techniques, the specificities of crude and purified (PAK and PAS) Anisakis simplex antigens were compared against 24 sera from patients diagnosed with Anisakis sensitization. All patients recognized a 60 kDa protein against the A. simplex crude extract, while 37.5% and 12.5% reacted with proteins of 40 and 25 kDa, respectively, when IgG was tested. In the case of IgE determination, 41.6% of sera were negative, while 12.5% and 20.8% appeared to cross-react against Toxocara canis and Ascaris suum, respectively. When the PAK antigen (A. simplex antigen purified by means of a column of IgG anti-A. simplex) was tested, immune recognition towards the 60, 40 and 25 kDa proteins increased in 83.3%, 16.7% and 4.2%, respectively, when the Ig antibodies were tested. In the case of the PAS antigen (PAK antigen purified by means of a column of IgG anti-A. suum), the reaction against the 40 and 25 kDa proteins increased to 45.8% and 25%, respectively, when Ig antibodies were used. Finally, when the EAS antigen (eluted from the anti-A. suum column after PAK purification) was tested, 83.3% of the assayed sera reacted against the 14 kDa protein, when the Ig antibodies, IgG and IgM immunoglobulins were measured. With the IgE determination, the reactions were observed in 41.7% of patients with proteins between 60 and 35 kDa against the PAS antigen. With the EAS antigen, reactive bands of 184, 84 and 14 kDa appeared. In conclusion, in the purification process of the A. simplex larval crude extract, the proteins implicated in cross-reactions with Ascaris and Toxocara were eliminated, with an important concentration of proteins responsible for the induction of specific responses.     

Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies.

Monoclonal antibodies to varicella-zoster virus were used to study viral glycoproteins by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on the viral glycoproteins immunoprecipitated, the five monoclonal antibodies fell into three groups. Two antibodies, 4B7 and 8G9 (group 1), immunoprecipitated a single glycoprotein of molecular weight (MW) 118,000 (118K glycoprotein) and had high neutralizing activity in the absence of complement. One antibody, 3C7 (group 2), which lacked neutralizing activity, immunoprecipitated two glycoproteins of MWs 120,000 and 118,000 and a glycoprotein giving a diffuse band in the region of 64,000 to 65,000. Pulse-chase experiments and experiments with monensin as an inhibitor of glycosylation suggested that the 120K polypeptide was derived by glycosylation of the 118K polypeptide and that a 43K antigen was processed into the 64 to 65K glycoprotein. Two antibodies, 3G8 and 4E6 (group 3), both had neutralizing activity only in the presence of complement, and both immunoprecipitated at least five polypeptides, with MWs ranging from 50,000 to 90,000. Antibody 3G8 was isotype immunoglobulin G2b (IgG2b), and its immunoprecipitating activity was stronger than that of 4E6, which was isotype IgG1. Pulse-chase experiments with antibody 3G8 showed that lower-MW glycopeptides chased into three polypeptides of MWs 90,000, 80,000, and 60,000 by 24 h. Immunoprecipitation experiments with antibody 3G8 on infected cells treated with glycosylation inhibitors 2-deoxyglucose, monensin, and tunicamycin, suggested that a prominent, early-appearing 70K polypeptide may have been processed into the glycoproteins of higher MWs and that the 60K polypeptide may have been derived by glycosylation of polypeptides of lower MWs.     

Immunoglobulin subclasses of antibodies to human T-cell leukemia/lymphoma virus I-associated antigens in acquired immune deficiency syndrome and lymphadenopathy syndrome

Immunoglobulin G (IgG) and IgM antibodies to human T-cell leukemia/lymphoma virus-I (HTLV-I)-associated membrane antigens (HTLV-I-MA) were assayed by indirect cytospin immunofluorescence, and IgG and IgM antibodies to purified HTLV-I were assayed by enzyme-linked immunosorbent assay in sera from 119 immunologically well-characterized promiscuous male homosexuals in The Netherlands, of whom 9 suffered from acquired immune deficiency syndrome (AIDS), 18 suffered from lymphadenopathy syndrome (LAS), and 5 suffered from gay bowel syndrome. Antibodies to HTLV-I-MA were present in four of nine AIDS patients, including one patient with antibodies to purified HTLV-I. Antibodies to HTLV-I-MA were present in 6 of 18 LAS patients, including 3 patients with antibodies to purified HTLV-I. Of five patients with gay bowel syndrome, one had IgG and IgM antibodies to HTLV-I-MA. Of the four HTLV-I seropositive AIDS patients, two had IgG and IgM antibodies to HTLV-I or HTLV-I-MA, one had only IgG antibodies, and one had only IgM antibodies. Of the six HTLV-I seropositive LAS patients, four had IgG and IgM antibodies to HTLV-I or HTLV-I-MA, and two had only IgM antibodies. In the sera from 27 healthy homosexuals with and 60 without T-cell subset imbalances, no antibodies to HTLV-I or HTLV-I-MA were detected.     

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

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

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

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

Evaluation of immunoglobin isotype specific to Leishmania in tegumentary American leishmaniasis

Leishmania-specific immunoglobulin subclass response was evaluated in 133 patients infected with Leishmania braziliensis. The indirect immunofluorescent antibody test (IFAT) was employed with amastigotes of L. mexicana amazonensis as antigen. Among the 133 sera obtained at consultation for diagnosis of active lesions, IgM was detected in 54 following absorption with Staphylococcus aureus Cowan strain I, and in 5 sera prior to absorption. IgM reactive with Leishmania antigen was only found in sera from patients whose lesions had evolved over the past two months or less. Leishmania-specific IgG was detected in all sera prior to absorption. Sera obtained at the time of recurrence or after complete healing of lesions presented only specific IgG. The combined use of the Montenegro skin test and specific IgM increased the sensitivity of immunodiagnostic methods in patients with lesions of less than 2 months duration. Normal control volunteers were negative for specific IgM and unreactive to Montenegro skin testing. Among 16 patients with non-leishmanial lesions, 3 with sporotrichosis showed IgG reactive with Leishmania; none, including 4 with lesions of less than two months duration, showed specific IgM. We conclude that in patients infected with L. braziliensis the presence of specific IgG and IgM is associated with the time of lesion evolution and the primary or recurrent nature of the lesions. In addition, the combined use of IgM titer and Montenegro reactivity is of potential utility in the diagnosis of early lesions.     

Detection of IgG binding to Schistosoma mansoni recombinant protein RP26 is a sensitive and specific method for acute schistosomiasis diagnosis

We recently described the first recombinant Schistosoma mansoni protein RP26, which was capable of acute infection diagnosis. The aim of the present work was to further characterize the RP26 diagnostic properties in immunoblot and enzyme-linked immunosorbent (ELISA) assays. Testing sera from uninfected donors and sera from patients with acute or chronic Schistosoma infection by Western blot immunoassay revealed 100% specificity and 100% sensitivity for acute infection identification. Sera from uninfected, acute, and chronic schistosomiasis were also probed for IgG, IgG4, IgA, and IgM reactivity to RP26 plus soluble egg antigens (SEA) in ELISA. The mean IgG reactivity to RP26 by sera from acute schistosomiasis patients was significantly higher than the chronic ones. The IgG4, IgA, and IgM reactivities to RP26 were low and similar in both infected groups. The mean IgA and IgM reactivities to SEA were significantly higher in the group of acute compared to chronic group, whereas mean IgG4 reactivity was higher in chronic group. To estimate the specificity of Schistosoma infection diagnosis sera from patients infected with other different parasites were tested to detect IgG reactivity to RP26 and IgA and IgM reactivity to SEA. For IgA against SEA detection, 72% of sera were positive and 48% of sera were positive for IgM detection. Based on these results we can suggest that detection of sera IgG binding to RP26 is a sensitive and specific method for acute schistosomiasis diagnosis. Therefore, RP26 is a candidate for immunodiagnostic kit development.     

Identification of adenovirus 2 early region 4 polypeptides by in vitro translation and tryptic peptide map analysis.

The mRNA species encoded by early region 4 (E4) (map position [mp] 91.5 to 99.3) of adenovirus 2 were isolated from the polysomes of infected KB cells and were purified by hybridization to the cloned HindIII-F fragment (mp 89.5 to 97.3) or to EcoRI-C fragment (mp 89.7 to 100). The mRNA's were translated in vitro using [35S]methionine as a labeled precursor in rabbit reticulocyte lysates treated with micrococcal nuclease as well as in wheat germ lysates. Five major (35,000-molecular-weight [35K], 23K, 22K, 21K, 18K) polypeptides were observed when the reticulocyte lysate was used. The 23K, 22K, 21K, and 18K polypeptides were also observed with the wheat germ lysate, as well as a very prominent 11K polypeptide; the 35K polypeptide was not observed. Assignment of these polypeptides to E4 was further established by hybrid arrested translation. Two-dimensional gel electrophoresis of a wheat germ translate resolved five polypeptides ranging from 18K to 23K, the major 11K polypeptide, and polypeptides of 10K and 9K. The in vitro 23K to 18K and 11K polypeptides migrated to approximately the same positions on two-dimensional gels as did seven 26K to 21K polypeptides and an 11K polypeptide synthesized in vivo (Brackmann et al., J. Biol. Chem, 255:6772--6779, 1980). Two-dimensional tryptic peptide maps demonstrated that the 35K, 23K, 22K, 21K, and 18K polypeptides are related. The peptide map of 11K is different from those of the above polypeptides, although 11K may share one tryptic methionine polypeptide with them. These results indicate that E4 encodes a major 11K polypeptide, as well as major 35K, 23K, 22K, 21K, and 18K polypeptides.     

Haemorrhagic fever with renal syndrome: evaluation of ELISA for detection of Puumala-virus-specific IgG and IgM

IgM and IgG ELISA to Puumala virus were evaluated using sera from patients with haemorrhagic fever with renal syndrome (HFRS) from different geographical regions: Sweden, Denmark, Norway, Belgium and the European USSR.IgM ELISA proved useful in the diagnosis of HFRS in patients from all the regions mentioned above. Specific IgM could be detected as early as day 1 post onset of disease, and patients remained IgM-positive for several months. Specific IgG ELISA antibodies were also frequently detected in acute sera, and acute-convalescent serum pairs often failed to show a significant titre rise or increase in optical density (OD) values. This limits the use of IgG ELISA in patient diagnosis. Sera collected 2 years after infection revealed higher IgG ELISA OD readings than convalescent sera, and very high values were still detectable 10 to 20 years postinfection. IgG ELISA is therefore useful for the testing of immunity and in seroepidemiological studies.Acute and convalescent sera from HFRS patients in Korea and the Asian USSR showed no or only very weak reactivity in the Puumala virus IgG and IgM ELISA. These results are consistent with the “one-way” crossing described earlier.