Antigen(s) responsible for immunoglobulin G responses specific for the acute stage of Toxoplasma infection in humans.

An agglutination test for immunoglobulin G (IgG) Toxoplasma antibodies with acetone-fixed tachyzoites (AC antigens; AC agglutination test) was positive only with sera from patients during the acute stage of their infection. In contrast, when the test was performed with Formalin-fixed tachyzoites (HS antigens; HS agglutination test), positive results were obtained during both the acute and chronic (latent) stages of the infection. Studies were performed to define the antigen(s) of T. gondii which are detected by IgG antibodies present only during the acute stage of the infection. Sera of mice immunized with AC antigens recognized predominantly 10 antigens of tachyzoites by immunoblot analysis. Sera from individuals with the acute but not chronic infection reacted strongly with these same 10 antigens in immunoblots. Sera of mice immunized with HS antigens recognized more Toxoplasma antigens on immunoblots than did mouse AC antibodies. Absorption of the latter with HS antigens removed detectable reactivity of AC antibodies in immunoblots and in the AC and HS agglutination tests, suggesting that AC antigens are a selected portion of HS antigens. AC antigens were specific for tachyzoites in that AC antibodies reacted with the cell membranes of both forms of the organism in an indirect fluorescent-antibody test. Tachyzoite-specific antigen(s) appear to be useful in differentiating between the acute and chronic stages of Toxoplasma infection through their detection by IgG antibodies.     

Antibody responses to toxoplasma antigens in mice infected with strains of different virulence.

Swiss Webster mice were infected with either the relatively virulent C56 strain or the relatively avirulent C37 strain of Toxoplasma gondii, and the sequence of their antibody response to surface antigens of the parasite was studied. An immunoglobulin M (IgM) agglutinating antibody was the first serologically detectable antibody and was first detected on day 2 in mice infected with the C37 strain and on day 5 in mice infected with the C56 strain. IgG antibodies were first detected on day 8 for both strains. The major component of the IgG antibodies was IgG2:IgG3 antibodies had lower titers, and no IgG1 antibodies were detected. The IgG antibodies were active in direct parasite agglutination and in the complement-dependent cytotoxicity assay of Sabin and Feldman (Science 108:660-663, 1948). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sera from mice infected with T. gondii detected all major radioiodinated surface proteins of toxoplasma tachyzoites. The earliest time point at which these antigens were detected differed for the two strains. Serum from mice infected with C56 strain immunoprecipitated all cell surface antigens by day 10 of infection, whereas serum from mice infected with the C37 strain did not do so until day 15 of infection.     

Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.     

Use of an immunoglobulin G avidity assay based on recombinant antigens for diagnosis of primary Toxoplasma gondii infection during pregnancy

The objective of this work was to develop an antibody-specific immunoglobulin G (IgG) avidity assay to discriminate between acute and latent phases of Toxoplasma gondii infection by using recombinant antigens. One hundred twenty-one serum samples from women who developed IgG antibodies against Toxoplasma during pregnancy were used. The IgG avidities of antibodies directed against epitopes carried by fragments of GRA3, GRA7, MIC3, and SAG1 antigens were measured by performing parallel enzyme immunoassays. The avidity index for Toxoplasma-specific antibodies against a homogeneous mixture of recombinant GRA3, GRA7, MIC3, and SAG1 antigens correlated closely with the IgG avidity of antibodies against lysed whole-cell T. gondii antigen. The avidity assay performed with the recombinant MIC3 antigen highlighted the presence of avidity low-antibodies IgG exclusively in sera collected within 2 months after primary infection. The presence of T. gondii-specific, low-avidity IgG antibodies against recombinant MIC3 antigen can be used to determine the point of infection with T. gondii within a 2-month time frame after infection.     

The production of hybrid cells producing monoclonal antibodies against Toxoplasma gondii

Hybridomas that secrete monoclonal antibody to Toxoplasma gondii have been developed. Two groups of 10 female BALB/c mice each were immunized either over a shorter (71 d) or longer (117 d) period at first with Toxoplasma lysate antigen and afterwards with intact tachyzoites of the RH strain. Higher titres of antibody were obtained with the long-period immunization. The fusion experiments have shown that both schemes of immunization approximately result in the same number (16 and 14% respectively) of hybridoma cell lines producing antigen-specific monoclonal antibodies. Hybridoma cultures secreting antitoxoplasma monoclonal antibodies were screened parallel by indirect immunofluorescence antibody test (IFAT) and enzyme immunoassay (EIA). 16 of the hybridoma cell cultures produced positive results only in the IFAT, 112 reacted only in the EIA and 21 were positive in both tests. The monoclonal antibodies 5B10, 5G6 and 1B2, which are positive in the IFAT form a chemical compound with the major antigens on the surface of RH tachyzoites. The patterns of fluorescence produced by these monoclonal antibodies are in conformity with those produced by using polyclonal sera of Toxoplasma gondii infected hosts (mouse, rabbit, man).     

抗人红细胞膜抗原非凝集型单克隆抗体的研制及特性鉴定

目的:制备抗人红细胞膜抗原的非凝集型单克隆抗体(mAb)并鉴定其特性。方法:应用杂交瘤技术,以人O型红细胞膜抗原免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,用聚凝胺试管法筛选识别红细胞表面共同抗原的抗体,玻片凝集实验剔除凝集型抗体(完全抗体),再将分泌非凝集型mAb(不完全抗体)的杂交瘤细胞株用有限稀释法克隆化3次。对杂交瘤细胞的稳定性和mAb的特性进行鉴定。结果:获得1株可稳定分泌mAb的杂交瘤细胞2E8。mAb2E8为IgG1类,可特异性地识别红细胞膜上的H抗原,没有种属交叉血凝反应。杂交瘤细胞的培养上清与人的A、B、AB和O型红细胞均能产生强凝集,凝集效价为1∶1024,腹水mAb的凝集效价达到1∶64×106。mAb的亲和力用凝集试验检测,出现血凝的时间为7s,3min以内凝块>1mm。结论:成功地制备了针对红细胞膜H抗原的非凝集性mAb,此mAb的凝集效价、相对亲和力及特异性均较良好,为构建双特异性抗体奠定了基础。     

Ultrastructural localization of an intracellular Toxoplasma protein that induces protection in mice.

We report the ultrastructural localization of Toxoplasma antigens recognized by monoclonal antibody F3G3, which protects mice against lethal challenge. By using colloidal immunogold labeling, F3G3 failed to react with the surface of intact Toxoplasma cells, confirming previous observations that it recognizes an intracellular antigen. Immunoperoxidase labeling with F3G3 was obtained only when Toxoplasma cells were previously exposed to Triton X-100. A specific immunoperoxidase reaction was located beneath the surface membrane in the region of the pellicle and within the elaborate network of vesicles which are extruded from the surface of Toxoplasma cells during entry into host cells. Similar results were obtained with mouse polyclonal sera and a second monoclonal antibody, 6-86-1E11, both of which were produced against the F3G3 affinity-purified protein. The location of the epitope recognized by F3G3 was confirmed by purifying T. gondii-derived surface membrane vesicles, separating these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. Monoclonal antibody 6-86-1E11 and the analogous polyclonal sera reacted with the antigenically related proteins of 58 and 28 kilodaltons that were found both in the intact organisms and in the purified surface membrane vesicles. These results indicate that the epitope recognized by F3G3 is located beneath the Toxoplasma cell surface membrane and is contained within plasma membrane-derived vesicles.     

Detection of specific immunoglobulin E in patients with toxoplasmosis.

An immunocapture assay was developed to detect Toxoplasma gondii-specific immunoglobulin E (IgE) in sera from adults with acute acquired infection or reactivation and from babies with congenital toxoplasmosis. The components of this assay were monoclonal antibody to human IgE, samples from patients, and T. gondii tachyzoites treated with Formalin. When T. gondii-specific IgE antibodies were present, visually detectable agglutination occurred. Sera, umbilical cord blood, fetal blood, cerebrospinal fluid, and amniotic fluid were tested by this method. Specific IgE antibodies were detected in sera from 25 (86%) of 29 adults who developed specific IgG antibody during pregnancy or had specific IgA and IgM antibodies. Specific IgE was present early during infection, at the time that IgM antibodies were present, and slightly preceding the presence of specific IgA antibodies. In 23 patients tested serially, IgE antibodies never persisted for longer than 4 months. No nonspecific anti-T. gondii IgE was detected in sera from uninfected individuals. Maternal IgE antibodies did not cross the placenta. In sera of patients with congenital toxoplasmosis, specific IgE antibodies were found at birth, during the first year of life, and during immunologic recrudescence following discontinuation of pyrimethamine-sulfonamide therapy. The IgE immunocapture assay is simple to perform. It is especially useful for determining when T. gondii was acquired by recently infected pregnant women.     

Fast dipstick dye immunoassay for detection of immunoglobulin G (IgG) and IgM antibodies of human toxoplasmosis

A dipstick dye immunoassay (DDIA) was developed to detect immunoglobulin G (IgG) or IgM antibodies of toxoplasmosis infection in humans. The assays employ a blue colloidal dye particles (D-1) conjugated to sheep anti-human IgG and rabbit anti-human IgM as the visualizing agents and a soluble antigen of tachyzoites of Toxoplasma gondii strain RH (TSA) as the detective antigen. The mixture of dye-labeled anti-human antibody-special human antibody was captured by TSA onto a nitrocellulose membrane dipstick by means of immunochromatography. The assays are rapid (the whole test can be completed within 15 min), simple, and cheap, and they don't require any equipment. They are sensitive and specific for the detection of anti-Toxoplasma IgG or IgM antibodies and generally agree closely with the results from the enzyme-linked immunosorbent assay. The assays are especially suitable for field applications.     

Differential expression of Epstein-Barr virus membrane antigens defined with monoclonal antibodies

Four different mouse hybrid cell lines producing IgG2b monoclonal antibodies against Epstein-Barr virus (EBV) membrane antigens (MA) were analyzed. The antigens defined by these antibodies, MA-2, MA-4, MA-5, and MA-7, were expressed only on EBV-producing cells. The antigens were induced on P3HR-1 cells by treatment with tumor-promoting agent (TPA) plus n-butyrate, and this induction was inhibited by treatment with phosphonoacetic acid (PAA) or acyclovir. Most MA monoclonal antibodies neutralized the infectivity of EBV in vitro in the presence of complement. The monoclonal antibody MA-4 precipitated two polypeptides with mol wt of 340K and 240K, while other monoclonal antibodies MA-2, MA-5, and MA-7, did only 340K peptide. The frequency of positive cells in MA-induced cells varied for each monoclonal antibody tested. It was also found that MA-4 (anti-340K and 240K) antibody reacted on both chemically induced cells and EBV-superinfected cells, but others did only on chemically induced cells. It was suggested that MA had a different pattern of expression between chemically induced cells and EBV-superinfected cells.     

Major surface protein of Toxoplasma gondii (p30) contains an immunodominant region with repetitive epitopes

Four monoclonal antibodies (mAb) against surface antigens of tachyzoites of Toxoplasma gondii were produced. Immunoprecipitation of extracts of 125I-labeled tachyzoites identified the same polypeptide with apparent molecular weight of 30 000 (p30). A competition binding assay indicated that a single region of p30 was recognized by all 4 of the mAb. Furthermore, we found that single mAb inhibited 25-50% of the specific binding of antibodies of patients with toxoplasmosis to the antigenic extract of tachyzoites. It appears, therefore, that p30 is the most immunogenic constituent of tachyzoites, and that a single region of this molecule contains most of the immunogenic activity. Finally, a two-site/one-antibody immunoradiometric assay with the same mAb indicated that the p30 molecule is multivalent with respect to the expression of a single epitope.     

Monoclonal antibodies against the nonhemagglutinating fimbrial antigen 1C (pseudotype 1) of Escherichia coli.

Hybridoma-derived monoclonal antibodies were produced with fimbrial preparations from Escherichia coli 20025 (04:K12:H-) with fimbrial (F) antigens 1C, 13, one related to 12, and one preliminarily termed y and from E. coli 2980 (018ac:K5:H-) with F antigens 1C and 8. Two clones of subclonal hybrid cells were studied which produced monoclonal antibodies (mc-20025-F2b, immunoglobulin G2b [IgG2b]; mc-2980-F2, IgG1) that were reactive with E. coli 20025, 2980, and a number of additional strains which exhibited the F1C antigen. Results of enzyme-linked immunosorbent assay and Western blot analysis indicated that the antibodies had F1C specificity, and competitive enzyme-linked immunosorbent assay with 125I-labeled antibodies showed that they recognized different epitopes on the fimbrial subunit. Neither of the antibodies agglutinated F1C-fimbriated E. coli but bound to the bacteria. There was no binding to E. coli without F1C fimbriae.     

Mouse monoclonal antibody to a latent epitope of leucocyte receptors for leukotriene B4.

Human blood polymorphonuclear (PMN) leucocytes and human leucocytes of the HL-60 line, which were induced to differentiate by 1,25-dihydroxyvitamin D3, express stereospecific receptors for the potent chemotactic mediator, leukotriene B4 (LTB4), that is derived by 5-lipoxygenation from arachidonic acid. Monoclonal antibodies to LTB4 receptors (LTB4-R) were generated by immunizing BALB/c mice with partially purified PMN leucocyte membrane proteins, and fusing their splenocytes with P3X63Ag8 mouse myeloma cells. Hybridoma supernatants were screened initially by binding to PMN leucocyte LTB4-R protein, which had been affinity cross-linked with aminopropylamide (APA)-LTB4 and immobilized in plastic wells through attachment of the linked APA-LTB4 to adherent Fab of monoclonal anti-LTB4. Of the three clones producing antibodies which bound to LTB4-R, 0.5 mg/ml of one IgG3k antibody, termed E2, precipitated over 90% of the [3H]LTB4-binding activity of solubilized PMN leucocyte membrane proteins. E2 also bound to a radiolabelled protein of 70,000-80,000 MW from 125I-labelled PMN leucocyte membranes [35S]-labelled HL-60 cell membranes, and PMN leucocyte membranes affinity-labelled with [3H]APA-LTB4, that was identical in size to the LTB4-R precipitated by the rabbit IgG anti-idiotypic antibodies. E2 did not bind to intact PMN leucocytes or modify the binding of [3H]LTB4 by PMN leucocytes. The binding of E2 to LTB4-R in purified membranes of PMN leucocytes was less than one-fourth of that observed for the anti-idiotypic antibodies, but increased substantially after solubilization of the LTB4-R. The E2 monoclonal antibody thus recognizes a partially latent substituent of LTB4-R, which does not contribute to combining site function.     

Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.     

Monoclonal autoantibodies from insulin-dependent diabetic patients: autoantibodies against beta-cell surface or cytoplasmic antigens.

To investigate the target antigen(s) recognized during the autoimmune process in insulin-dependent diabetes mellitus (IDDM), we produced human monoclonal antibodies by Epstein-Barr virus transformation of peripheral blood lymphocytes from a large number (n = 50) of newly diagnosed IDDM patients. Screening by indirect immunofluorescence assay, using the RINm5F rat insulinoma cell line and eight other human or rat tumour cell lines, was performed to identify monoclonal antibodies that reacted with either membrane or cytoplasmic antigens. Eighteen IgM monoclonal antibodies reacting with cytoplasmic antigens of RIN cells were obtained; 14 of them also showed a staining of the cytoplasm of various non-beta-cell lines, while four displayed a binding restricted to beta-cells among the panel tested. However, among three monoclonal antibodies reacting with the membrane of RIN cells, one (HMD-1) produced an IgG antibody with a binding restricted to the membrane of beta-cells (RIN, HIT, and normal rat islet cells). The membrane antigens of HMD-1 were identified in Western blotting as proteins with molecular weights of 64 and 70 kD. This antibody had no apparent cytotoxic effect on RIN cells. These data suggest that, apart from 'natural autoantibodies,' it is feasible to obtain human monoclonal antibodies from IDDM patients that bind specifically to the beta-cell cytoplasm or to the beta-cell membrane.     

Monoclonal rat antibodies directed against Toxoplasma gondii suitable for studying tachyzoite-bradyzoite interconversion in vivo.

We previously reported the in vitro analysis of stage differentiation of Toxoplasma gondii in murine bone marrow-derived macrophages. The purpose of this study was to generate monoclonal rat antibodies that might be suitable for investigating tachyzoite-bradyzoite interconversion in vivo with the murine model. Immunization of Fischer rats with cysts of T. gondii NTE resulted in the generation of seven monoclonal antibodies of the immunoglobulin G2a, G2b, or M isotype, which were further characterized by the immunoblot technique, immunofluorescence assay, immunohistology, and immunoelectron microscopy. Immunoblots demonstrated specific reactivity of five monoclonal antibodies with proteins with molecular masses of 40, 52, 55, 60, 64, 65, and 115 kDa. One antibody (CC2) appeared to recognize a differently expressed antigen depending on the parasite stage, reacting with a 40-kDa molecule in tachyzoites and a 115-kDa antigen in bradyzoites and oocysts. Several other monoclonal antibodies were shown to be stage specific and to react in immunofluorescence assays or in immunoblots with either tachyzoites or bradyzoites. Kinetics of stage conversion in vitro could be monitored by immunofluorescence with two of these monoclonal antibodies. Preliminary immunohistological investigations of tissue sections from infected mice demonstrated the possible usefulness of these monoclonal antibodies for future in vivo studies on stage differentiation of T. gondii in the murine system.     

Fast Dipstick Dye Immunoassay for Detection of Immunoglobulin G (IgG) and IgM Antibodies of Human Toxoplasmosis

A dipstick dye immunoassay (DDIA) was developed to detect immunoglobulin G (IgG) or IgM antibodies of toxoplasmosis infection in humans. The assays employ a blue colloidal dye particles (D-1) conjugated to sheep anti-human IgG and rabbit anti-human IgM as the visualizing agents and a soluble antigen of tachyzoites of Toxoplasma gondii strain RH (TSA) as the detective antigen. The mixture of dye-labeled anti-human antibody-special human antibody was captured by TSA onto a nitrocellulose membrane dipstick by means of immunochromatography. The assays are rapid (the whole test can be completed within 15 min), simple, and cheap, and they don't require any equipment. They are sensitive and specific for the detection of anti-Toxoplasma IgG or IgM antibodies and generally agree closely with the results from the enzyme-linked immunosorbent assay. The assays are especially suitable for field applications.     

Induction of human gamma interferon by structurally defined polypeptide fragments of group A streptococcal M protein.

Antigens of Toxoplasma gondii eluted from polyacrylamide gels after electrophoresis under reducing conditions were examined for their capacity to react with antibodies from infected humans, to induce antibody formation, and to protect mice against T. gondii. Antigens with approximate molecular weights of 35,000 and 14,000 strongly reacted in an enzyme-linked immunosorbent assay of human immunoglobulin M (IgM) and IgG antibodies to T. gondii. Mice injected with the eluted antigen preparations formed antibodies that reacted differently in four serological tests for Toxoplasma antibodies. All mice formed antibodies that reacted in the IgG and IgM enzyme-linked immunosorbent assay. Antibodies reacting in the conventional indirect immunofluorescent antibody test were detected in all mice except those injected with low-molecular-weight (14,000 or less) antigens. Sabin-Feldman dye test antibodies were not detected in any of the mice. Antibodies reacting in the latex agglutination test were detected mainly in mice injected with antigens with an approximate molecular weight of 66,000. Challenge of the injected mice with a lethal inoculum of T. gondii revealed that the highest survival rate was in animals that received antigens with approximate molecular weights of 35,000 and 14,000.     

Use of monoclonal antibodies to detect antigens of Toxoplasma gondii in serum and other body fluids.

Monoclonal antibodies to Toxoplasma gondii were used in an enzyme-linked immunosorbent assay to detect antigens of the parasite in toxoplasma lysate, in peritoneal fluid of mice, and in sera from humans acutely infected with T. gondii. Four of the six monoclonal antibodies were able to detect antigens of toxoplasma in these specimens. Control sera from individuals not infected with T. gondii and from individuals chronically infected with the parasite were negative in the enzyme-linked immunosorbent assay. Sera from individuals not infected with T. gondii but with positive titers from rheumatoid factor were also negative; 2 or 10 sera from individuals not infected with T. gondii but with positive titers for antinuclear antibodies reacted with the monoclonal antibodies. When the results of the enzyme-linked immunosorbent assay with monoclonal antibodies and with the F(ab)2 fraction of an immunoglobulin G from a rabbit infected with T. gondii were compared, it was noted that the F(ab)2 was more active in detecting parasite antigens than were the monoclonal antibodies. Thus, although monoclonal antibodies can be used to detect antigens of T. gondii in sera and other body fluids, polyvalent antibody (such as the F(ab)2 fraction) appears to be more satisfactory for this purpose.     

Characterization of two monoclonal antibodies against teguments of adult and schistosomula of Schistosoma japonicum.

Two monoclonal antibodies (McAbs) were produced from BALB/c mice hyperimmunized with tegumental extract of Schistosoma japonicum (Chinese strain). The two McAbs were characterized with regard to antibody isotype, antigen binding specificity and parasite stage specificity. One McAb, 8G9-5, was identified as IgM, whereas the other McAb, 9E7, was determined to be IgG2a. Immunoblotting assay indicated that McAb 8G9-5 binds strongly to the band of tegumental antigens of Mr 64 kDa but also binds weakly to other bands at 116, 105, 97, 54, 50, 47 and 45 kDa, whereas 9E7 McAb reacts specifically at Mr 54 kDa. Anatomical localization of the antigens in the adult worm by indirect immunofluorescence assay indicated that the target epitopes of McAb 8G9-5 are in the intra-tegumental structure, whereas the McAb 9E7 epitope is on the surface membrane. The two McAbs also react at similar sites within the teguments of schistosomula and lung worms.