ELISA for detection of IgM and IgG antibodies to sandfly fever sicilian virus

• •An enzyme-linked immunosorbent assay (ELISA) was developed to detect specific human immunoglobulin G and M antibodies to sandfly fever Sicilian (SFS) virus. Acute and early convalescent serum pairs with ⩾ 7 days between the 2 specimens were available from 20 patients and all showed significant optical density (OD) increase and significant titre rise (⩾ 4-fold) by IgG ELISA. However, negative or borderline-positive sera were found as late as 11 days after onset of symptoms when tested by IgG ELISA.
• •Specific IgM antibodies were detected during the first week of symptoms, and maximum OD values were obtained during the first 4 weeks after onset of disease. The IgM OD values declined over the following 3–9 months. All sera collected later than 14 months post-onset were negative by IgM ELISA.
• •The combination of early antibody response and the need to test only one serum specimen gives IgM ELISA an advantage over IgG ELISA in patient diagnosis.
• •The IgG ELISA was also evaluated as a seroepidemiological tool and compared to a plaque reduction neutralization test (PRNT) using sera from a normal Cypriot population. Of 183 sera tested, 34 (19%) were positive in plaque reduction neutralization tests (PRNT) and 113 (62%) by IgG ELISA. A number of PRNT-negative sera were strongly positive by IgG ELISA and also by indirect immunofluorescence test, which may suggest the presence of a virus related to SFS in Cyprus which has not yet been isolated.

     

Infectivity-enhancing antibodies to Ebola virus glycoprotein

Ebola virus causes severe hemorrhagic fever in primates, resulting in mortality rates of up to 100%, yet there are no satisfactory biologic explanations for this extreme virulence. Here we show that antisera produced by DNA immunization with a plasmid encoding the surface glycoprotein (GP) of the Zaire strain of Ebola virus enhances the infectivity of vesicular stomatitis virus pseudotyped with the GP. Substantially weaker enhancement was observed with antiserum to the GP of the Reston strain, which is much less pathogenic in humans than the Ebola Zaire and Sudan viruses. The enhancing activity was abolished by heat but was increased in the presence of complement system inhibitors, suggesting that heat-labile factors other than the complement system are required for this effect. We also generated an anti-Zaire GP monoclonal antibody that enhanced viral infectivity and another that neutralized it, indicating the presence of distinct epitopes for these properties. Our findings suggest that antibody-dependent enhancement of infectivity may account for the extreme virulence of the virus. They also raise issues about the development of Ebola virus vaccines and the use of passive prophylaxis or therapy with Ebola virus GP antibodies.     

Detection of IgG and IgM antibodies with ELISA technique in human trichomoniasis

The direct wet mount examination of vaginal secretion, widely applied for the diagnosis of Trichomonas vaginalis infection in woman patients, is rapid and economical, however, the sensitivity of this technique is not so high. In this study enzyme-linked immunosorbent assay (ELISA) was employed for the detection of serum anti-T. vaginalis IgG and IgM antibodies from 30 vaginal trichomoniasis patients and 30 non-infected healthy persons. The results were as follows: 1. Serum ELISA-IgG value was 0.37 +/- 0.134 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.21 +/- 0.054 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgG antibody were 70.0% and 96.7%, respectively. 2. Serum ELISA-IgM value was 0.33 +/- 0.177 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.11 +/- 0.051 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgM antibody were 70.0% and 96.7%, respectively. 3. The ELISA-IgG values showed a significant correlation with ELISA-IgM values (r = 0.77, p less than 0.005). With above results, it is assumed that ELISA is a reliable method for the diagnosis of T. vaginalis infection and simultaneous measurement of serum IgG and IgM with this technique is recommended.     

Characterization of IgM of Indian major carps and their cross-reactivity with anti-fish IgM antibodies

Indian major carps (IMC), rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were immunized with bovine serum albumin and the serum immunoglobulin M (IgM) was purified by affinity chromatography. The heavy and light chain of IgM of all the three species of IMC were about 88 and 26 kDa, respectively. Anti-fish IgM antibody against all the three species were raised in mice and the reaction of anti-fish IgM antibodies with IgM of all the three species of IMC were studied by Western blot. The anti-fish IgM antibodies reacted strongly with the heavy chain of the same species against which it was raised while the reactions with the heavy chain of other species were milder indicating some degree of epitope sharing among the heavy chains of IgM of IMCs. However, there was no cross-reaction with the light chain of any of the IgM.     

A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus

Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of ³H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.     

Binding of human IgM from a rheumatoid factor to IgG of 12 animal species

The binding of IgM from a rheumatoid factor (RF-IgM) to IgG from 12 animal species was analyzed by an ELISA system. The RF-IgM bound various animal IgG with dissimilar affinities. The binding of RF-IgM to animal IgG was inhibited by addition of protein A, which binds some animal IgG by recognizing the junctional site on CH2-CH3 domains in the Fc region. As previously reported, no significant correlation was observed between the binding of RF-IgM to IgG and the content of galactose-free oligosaccharides, which is increased in IgG of rheumatoid arthritis patients or autoimmune mice. We suggest that the crucial epitope of IgG for RF-IgM binding is not the oligosaccharide structure generated specifically in IgG of autoimmune diseases but that RF-IgM may recognize a certain protein conformation of a region in IgG near the binding site of protein A.     

Development and characterization of monoclonal antibodies to Ebola virus glycoprotein

Balb/С mice were immunized with recombinant Ebola virus glycoprotein. Following the selection, screening, and cloning of murine hybridomas, we obtained five genetically stable clones of monoclonal antibodies GPE118 (IgG), GPE274 (IgM), GPE325 (IgM), GPE463 (IgM), and GPE534 (IgG). These antibodies were isolated and purified from the ascitic fluid of Balb/С mice using Protein G affinity chromatography (for IgG) and euglobulin precipitation (for IgM). To select at least three candidate antibodies for testing in biological assays as components of an antibody cocktail for the prophylaxis and treatment of hemorrhagic fever, we carried out an immunochemical analysis of the epitope specificity of the isolated antibodies. Based on the data of immunoblotting and sandwich ELISA, it became evident that the epitope recognized by GPE 534 differs from the epitopes recognized by the monoclonal antibodies GPE 118 and GPE 325. The last two antibodies also have different epitope specificity: it follows from the immunoblotting data and from the data on the binding of these antibodies with the intact and oxidized (partly deglycosylated) recombinant glycoprotein. For the biological activity studies and the development of recombinant counterparts, we selected three candidate high-affinity monoclonal antibodies GPE 534, GPE 118, and GPE 325.     

Recombinant full-size human antibody to Ebola virus

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids, pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 × 10⁷ ± 1.5 × 10⁷ M^?1. Like the parent single-chain antibody 4D1, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.     

Development of ELISAs for quantification of HMFG1-specific human anti-mouse IgG and IgM antibodies

The aim of this study was to develop and validate ELISAs for quantification of HAMA-IgM and HAMA-IgG in serum of patients with ovarian cancer who enrolled in a large international randomized phase III trial of intraperitoneal Yttrium-90-labeled HMFG1 murine monoclonal antibody therapy. The capture antibody of these 2 assays was the murine antibody HMFG1, while mouse anti-human IgM-HRP or mouse anti-human IgG(Fc)-HRP served as tracer antibodies. A pool of HAMA-positive serum samples was used to prepare a series of assay standards and another pool served as reference preparation. The analytical sensitivity of the HAMA-IgM assay was 2.5 arbitrary units per mL (AU/mL) and 4.7 AU/mL for the HAMA-IgG ELISA. Diluted serum samples showed good parallelism with the HAMA-IgM and HAMA-IgG standard dose-response curves. Within-assay coefficient of variation was 7.5% for HAMA-IgM and 6.5% for HAMA-IgG. Between-assay variation was 14.2% for HAMA-IgM and 15.3% for HAMA-IgG. The developed HAMA-IgM and HAMA-IgG ELISAs show satisfactory reliability criteria (sensitivity, parallelism and precision) and are suitable for monitoring of HAMA-IgM and HAMA-IgG responses in ovarian cancer patients. These ELISAs will be used to monitor the development of HAMAs in patients who received radioimmunotherapy with murine HMFG1.     

Chimeric fab fragments of antibodies to recombinant Ebola virus glycoprotein

Previously, we have determined the nucleotide and amino acid sequences of the variable domains of three mouse monoclonal antibodies specific to the individual epitopes of the Ebola virus glycoprotein: GPE118 (IgG), GPE325 (IgM) and GPE534 (IgG) [1]. In the present paper, chimeric Fab fragments of Fab118, Fab325, and Fab534 antibodies were obtained based on the variable domains of murine antibodies by attaching CH1 and CL constant regions of human kappa-IgG1 to them. The recombinant chimeric Fab fragments were synthesized in the heterologous expression system Escherichia coli, isolated and purified using metal chelate affinity chromatography. The immunochemical properties of the obtained Fab fragments were studied by immunoblotting techniques as well as indirect and competitive ELISA using recombinant Ebola virus proteins: EBOV rGPdTM (recombinant glycoprotein of Ebola hemorrhagic fever virus without the transmembrane domain), NP (nucleoprotein) and VP40 (structural protein). The identity of recombinant chimeric Fab fragments, as well as their specificity to the recombinant glycoprotein of Ebola hemorrhagic fever virus (EBOV GP) was proved. The results of indirect ELISA evidence the absence of immunological cross-reactivity to NP and VP40 proteins of Ebola virus. The dissociation constants of the antigen-antibody complex K _d equal to 5.0, 1.0 and 1.0 nM for Fab118, Fab325 and Fab534, respectively, were determined; they indicate high affinity of the obtained experimental samples to EBOV GP. The epitope specificity of Fab fragments was studied using a panel of commercial neutralizing antibodies. It was found that all studied antibodies to EBOV GP are targeted to different epitopes, while the epitopes of the recombinant chimeric Fab fragments and original murine monoclonal antibodies (mAbs) coincide. All the obtained and studied mAbs to EBOV GP are specific to epitopes that coincide or overlap the epitopes of three commercial neutralizing mAbs to Ebola virus: epitopes Fab118 and Fab325 overlap the epitope of the known commercial mAb h13F6; Fab325 epitope also overlaps mAb c6D8 epitope; Fab534 epitope is located near mAb KZ52 conformational epitope, in the formation of which amino acid residues of GP1 and GP2 domains of EBOV GP are involved.     

Recombinant full-size human antibody to Ebola virus

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids. pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 x 10(7) +/- 1.5 x 10(7) M(-1). Like the parent single-chain antibody 4DI, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.     

Study of interaction between IgG and IgM antibodies against rubella virus by the immunofluorescence method.

In immunoglobulin fractions or after elimination of IgG by absorption the immunofluorescence test for rubella IgM antibodies is more sensitive than in whole serum. Blocking of IgM activity by IgG antibodies was eliminated when the time of incubation of the serum with virus antigen was prolonged. After prolonged incubation higher titres of rubella antibodies were also obtained in the IgM immunoglobulin fractions. Protein A in Staphylococcus aureus suspension effectively absorbs antibodies of IgG class. The IgM antibody titres in absorbed sera of patients infected with rubella were in some cases 2 to 4 times higher than in unabsorbed sera.     

Class IgG,IgM and IgA antibodies againstStaphylococcus aureus antigens in human serum and saliva

Using the ELISA method antibodies against the sonicate, teichoic acid (TA) and exoproducts ofStaphylococcus aureus were determined in sera and saliva of healthy individuals. Main serum antibodies against all the antigens used were shown to be class IgG antibodies. However, antigens of the sonicate stimulated significantly even the systemic IgA response. In the saliva class IgA antibodies predominated, but IgG antibody levels against TA and exoproducts approached the level of IgA antibodies. Levels of IgM antibodies against all antigens tested were low in both the serum and saliva which corresponds with the anamnestic type of response. On the basis of these results one may assume that not only IgG, but also IgA antibodies are important in the systemic immunity against staphylococcal infection and in the immunity of mucous membranes; besides IgA, even class IgG antibodies play an important role.     

Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency

Human IgG4 antibodies directed against phospholipase A, the P1 antigen from Dermatophago?des pteronyssinus extracts, and cat albumin were found unable to cross-link antigen. Previously, it was demonstrated that IgG4 antibodies, in contrast to IgG1 antibodies, did not cross-link Sepharose-bound antigen and antigen added in solution. To eliminate the possibility that this phenomenon was caused by preferential binding of both IgG4 Fab fragments to the solid-phase-bound antigen, cross-linking of antigen was studied in a fluid-phase system. In this test, incapability of IgG4 antibodies to bridge two antigens was also found. As a result of such a phenomenon, it is expected that immune complexes formed by IgG4 antibodies will be considerably smaller than complexes formed by IgG1. This was confirmed by analysis of the molecular size profiles of IgG1- and IgG4-containing immune complexes in sucrose-density gradients. Moreover, IgG1 was able to precipitate antigen in a radioimmunoprecipitation test, whereas precipitation was not demonstrable by the same amount of IgG4 antibodies. Even 3% polyethylene glycol 8,000 did not precipitate the small IgG4-containing immune complexes efficiently. The antibodies studied were of a high-affinity type, and there was no significant difference in association constants between IgG1 and IgG4 antibodies. Therefore, we were not able to confirm observations reported in the literature that the IgG4 subclass is associated with a low-affinity antibody response; probably, the affinity of the IgG4 antibodies was underestimated by other investigators because of the polyethylene glycol precipitation technique used to separate antibody-bound and free antigen. Our findings stress the point that IgG4 antibodies take a special place in the immune response upon chronic exposure to antigen.     

Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies

Western Blot analysis revealed that both IgM and IgG antibodies present in the sera of humans infected with Toxoplasma gondii recognize three major antigens with apparent m.w. of 32,000, 22,000, and 6000, respectively. In addition, IgG antibodies recognized at least 17 other antigenic components. After subcellular fractionation, enrichment of the three major antigens recognized by IgM and IgG antibodies by the membrane fraction was observed. Solubilization of membrane-enriched preparations with a mixture of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that reacted with IgM or IgG antibodies. Treatment of Toxoplasma lysate preparations and various fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. Lipase treatment had no effect on the number or nature of antigens recognized by IgM antibody. Treatment with pronase and trypsin eliminated the 32,000 and 22,000 m.w. antigenic components detected by IgM antibodies, whereas such treatment had no effect on the 6000 m.w. component. Periodic acid-Schiff staining of polyacrylamide gels of Toxoplasma sonicates revealed the presence of three components corresponding to m.w. of 62,000, 45,000, and 6000, respectively. At least 15 components, including the 6000 m.w. component, directly bound concanavalin A.