Monoclonal antibodies identify new Toxoplasma gondii soluble antigens

In order to characterize Toxoplasma gondii antigens, we have produced a panel of monoclonal antibodies specific for the parasite. A total of 22 hybridomas were derived from the spleen cells of mice immunized either with a 100,000 g supernatant of a sonicate from the RH strain (called F3), or chronically infected with the Wiktor or the 76K strain. Except for one hybridoma producing an IgM, all the hybridomas derived from mice immunized with F3 produced IgG1 antibodies while those obtained from chronically infected mice produced antibodies belonging to the IgG2b, IgG2a and IgM subclasses. Western-blot analysis showed that the panel of monoclonal antibodies defines at least 7 distinct antigens or antigen families. An antigen of apparent Mw 25 kD present exclusively in the 100,000 g supernatant of the T. gondii sonicate was recognized by the majority of monoclonal antibodies derived from mice immunized with the F3 fraction. Two other antigens of apparent Mw 27 kD and 29 kD present in the soluble and insoluble fractions of the sonicate were also identified. Monoclonal antibodies against the previously described 21 kD and 31 kD surface antigens and belonging to the IgG2a but also to the IgG1 subclasses were able to mediate lysis of the parasite in the presence of human non immune serum. The 22 monoclonal antibodies did not identify antigenic differences between the two independently isolated RH and Wiktor strains.     

Monoclonal antibodies against Neisseria gonorrhoeae: production of antibodies directed against a strain-specific cell surface antigen.

Hybridomas secreting monoclonal antibodies against an apparent strain-specific cell surface antigen of Neisseria gonorrhoeae were produced. Spleen cells from BALB/c mice immunized with whole gonococci were fused with mouse myeloma cell line Sp2/0, and hybrid cells were selected in culture. One hybridoma that secreted antibodies reactive with the immunizing strain was cloned by limiting dilution to obtain cell lines secreting monoclonal antibodies. These antibodies reacted with purified outer membranes from the immunizing strain as well as with whole gonococci. Binding of antibodies to whole gonococci was highly strain specific, with most gonococcal strains showing less than 1% of the binding with the immunizing strain. Antibodies did not bind to the other Neisseria species tested. Binding of monoclonal antibodies to whole gonococci of the immunizing strain was not dependent on state of piliation. The extent of antibody binding did vary in different colonial variants of the immunizing strain. Antibody bound to cells from colonies that were transparent or of intermediate opacity, but did not bind to cells from deeply opaque colony variants.     

Monoclonal antibodies directed against AFAP-110 recognize species-specific and conserved epitopes.

The actin filament-associated protein, AFAP-110, is a Src SH2/SH3 binding partner that can modulate changes in actin filament structure. AFAP-110 contains a carboxy terminal motif that facilitates actin filament interactions, as well as amino terminal protein binding motifs, including an SH3 binding motif, two SH2 binding motifs, and two Pleckstrin homology domains. Two monoclonal antibodies (MAbs) were developed that recognized epitopes in either the amino terminus (MAb 4C3) or the carboxy terminus (anti-AFAP-110) of AFAP-110. Site-directed mutations that change key proline residues to alanine in the SH3 binding motif and an adjacent proline-rich motif abrogated MAb 4C3 binding. These same mutations have been shown to prevent SH3 interactions between AFAP-110 and Src527F. These data indicate that MAb 4C3 recognizes an epitope that is part of the SH3 binding motif. Interestingly, MAb 4C3 is not efficiently reactive with mammalian homologs of AFAP-110. Sequence analysis of a putative cDNA clone that encodes the amino terminus of the human AFAP-110 isoform predicted a one amino acid difference within this epitope, indicating a mechanism for species-specific binding by MAb 4C3. A second, MAb anti-AFAP-110, recognizes AFAP-110 across species and binds to an epitope within the carboxy terminus. This epitope includes the 5th heptad repeat of the carboxy terminal, leucine zipper motif (amino acids 592-598)--a motif that facilitates self-associations and may regulate the function of AFAP-110. These MAbs will be useful for analyzing the effects of AFAP-110 upon cell morphology and actin filament integrity. In addition, the avian-specific MAb 4C3 may be useful for studying the effects of avian AFAP-110 constructs expressed in mammalian cells, by providing an internal epitope tag.     

Standardized quantitative enzyme-linked immunoassay for antibodies to Toxoplasma gondii.

A quantitative and standardized enzyme-linked immunosorbent assay is described which uses lyophilized antigen-coated disks for the detection of human antibodies to Toxoplasma gondii. It does not require serial dilution of the test specimen, and the objective absorbance readings are converted into international units per milliliter traceable to the World Health Organization's reference standard preparation of toxoplasma antibodies. It was shown to be highly specific, reproducible, and sensitive. The reagents were stable, without biohazard, and ready for use. Studies of various parameters in the assay indicated that the test conditions were relatively flexible. The enzyme-linked immunosorbent assay results correlated satisfactorily with the methylene blue dye test, the indirect immunofluorescence test, and the passive hemagglutination test.     

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).     

Monoclonal antibodies selectively directed against the cell wall surface of Mycobacterium tuberculosis.

In the last few years several monoclonal antibodies against Mycobacterium tuberculosis have been described, but their use as diagnostic tools has been limited. In this study we describe eight monoclonal antibodies against M. tuberculosis for diagnostic purposes. The monoclonal antibodies were selected after enzyme-linked immunosorbent assay screening with whole bacterial suspensions of mycobacteria and other bacterial species. Four monoclonal antibodies (BS100, BS101, BS102, and BS104) reacted with a whole bacterial suspension of M. tuberculosis but not with the other mycobacteria. When tested with a cytoplasmic fraction of mycobacteria the same monoclonal antibodies showed a broad cross-reactivity. Therefore the monoclonal antibodies showed not specific but selective binding to M. tuberculosis. The molecular size of the recognized antigens ranged from 12 to 71 kilodaltons as determined by the immunoblotting technique. The ability to differentiate M. tuberculosis from mycobacteria other than tuberculosis was investigated by enzyme-linked immunosorbent assay with 131 freshly cultured strains of M. tuberculosis from patients and 36 strains of mycobacteria other than tuberculosis. The monoclonal antibody BS104 could clearly distinguish between M. tuberculosis and other mycobacterial species.     

Monoclonal antibodies directed against human airway secretions. Localization and characterization of antigens.

Cellular mechanisms of normal airway mucus secretion and their alterations in chronic obstructive lung disease are poorly understood. To aid in their study, the authors have produced a panel of monoclonal antibodies directed against various constituents of human airway secretions. Two fusions yielded 401 hybridoma-containing cultures. Supernatants from 150 of these cultures stained human tracheal secretory cells by immunofluorescence. Twenty-nine hybridomas were selected for expansion because they selectively stained a single cell type or displayed another interesting distribution. Antigens were further characterized by their localization in glycol methacrylate sections of human trachea, sensitivity to periodate oxidations, selective affinity for fraction peaks obtained by Sepharose 4B chromatography, and reactivity with molecules of various sizes, as estimated by SDS-PAGE. These antibodies will be useful for 1) quantitative detection of antigens in sputum or lavage samples by immunoassay and 2) purification and biochemical characterization of molecular constituents of airway secretions in health and disease.     

Monoclonal antibodies against rat immunoglobulin kappa chains

Monoclonal antibodies directed against rat kappa light chains have been generated by immunizing SJL/J mice with soluble rat immunoglobulin, followed by fusion of immune spleen cells with the P3-X63-Ag8 myeloma cell line. Monoclonal antibodies from three of these hybridoma cell lines, MAR 18.5, 80.2, and 103.6, have been extensively characterized. MAR 18.5, 80.2, and 103.6 antibodies are of the gamma 2a kappa isotype, and bind strongly to protein A, allowing easy purification. Monoclonal antibody from clone 18.5 binds equally well to Ig of both RI-1a and RI-1b allotypes, whereas 80.2 and 103.6 antibodies selectively bind to RI-1b. These monoclonal antibodies can be FITC conjugated for use as a second antibody in indirect immunofluorescence assays, or radiolabeled for use in radio immunoassays requiring a specific antirat kappa antibody. The antiallotype specific monoclonal antibodies also may be of use in the study of rat immunoglobulin genetics.     

Monoclonal antibodies against rat leukocyte surface antigens

Summary: Monoclonal antibodies have proven to be powerful tools for studying the properties of leukocyte surface antigens and the cells that express them. In the past decades many monoclonal antibodies (mAb) for identifying the different rat leukocyte surface antigens have been described. A list of mAb is provided in Table 1 below. The rat leukocyte surface antigens are divided into different sections, including rat CD antigens (a), rat leukocyte surface antigens without CD designation (b), rat major histocompatibility complex (MHC) antigens (c), rat T-cell receptors (d) and rat immunoglobulins (e). The molecular and functional characteristics of rat leukocyte surface antigens are discussed in more detail in some of the other chapters of this issue (e.g. Van den Berg et al., p. 45). A more extensive overview of the properties of leukocyte surface antigens is provided by Barclay et al. (1).     

Comparison of six commercial enzyme linked immunosorbent assays for detecting IgM antibodies against Toxoplasma gondii.

To evaluate the usefulness of different commercial enzyme linked immunosorbent assays (ELISAs) for the detection of IgM antibodies against Toxoplasma gondii the results of six of these assays for a panel of 81 sera were compared. The following tests were selected: Toxoplasma gondii IgM ELISA (Clark Laboratories), Toxoplasma IgM EIA (Labsystems), Toxo-M EIA (Abbott), Toxonostika M (Organon), Toxo M Enzyme Immunoassay (Hybritech) and Platelia Toxo IgM (Diagnostics Pasteur). An antibody capture ELISA developed at our laboratory was used as the reference test. An IgM immunoblotting assay was also performed. Four (Toxoplasma IgM EIA, Tox-M EIA, Toxonostika M, and Platelia Toxo IgM) of the commercial IgM ELISAs gave a high sensitivity and a high specificity. Toxo-M EIA, Toxonostika M, Toxoplasma IgM EIA and the Toxo M Enzyme Immunoassay were too insensitive, and the Toxoplasma gondii IgM ELISA was both insensitive and unspecific. No remarkable differences were observed between the results of indirect or antibody capture ELISAs, and between the results of ELISAs performed with polyclonal or monoclonal antibodies.     

Monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus

Neutralizing monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus (LACV) were prepared. Two antibodies immunoprecipitated the 120 kDa virus attachment protein for vertebrate cells, G1, while five immunoprecipitated the 35 kDa G2 protein, whose function is currently unknown. Two monoclonal antibodies were obtained that specifically precipitated both G1 and G2 from [35S]cysteine labeled LACV infected cell lysates. The G2 specific monoclonal antibodies had high neutralizing titers when assayed in mosquito cells but limited ability to neutralize virus in mammalian cells. The G1/G2 specific antibodies neutralized virus infectivity in both vertebrate and invertebrate cells at high titers. These results suggest that G2 is involved in the interaction of virus with mosquito cells and that G1 and G2 may share a common structural epitope relevant to their role as attachment proteins in vertebrate and mosquito cells. Monoclonal antibodies directed against G2 or G1/G2 have not previously been reported and should be useful tools for characterizing the biological functions of these molecules in the divergent micro-environments of vertebrate and invertebrate hosts.     

Latex agglutination test for detection of antibodies to Toxoplasma gondii.

A resurgence of interest in Toxoplasma gondii has occurred because this coccidian parasite causes lethal infections in immunologically compromised hosts and is responsible for at least 3,000 congenitally infected infants in the United States annually. Thus, rapid, specific, and inexpensive serologic tests are required for routine screening of patients, especially pregnant women. We have developed a latex agglutination test for antibodies to T. gondii which utilizes covalently coupled T. gondii antigens. When compared with an indirect immunofluorescence assay, the latex test had a sensitivity of 94% and specificity of 100%. Compared with an enzyme-linked immunosorbent assay, the latex test had 86% sensitivity and 100% specificity. When testing samples which exhibited nonspecific polar staining by the immunofluorescence assay, the enzyme-linked immunosorbent assay had a 50% false-positive rate, whereas the latex agglutination test yielded no false-positive results. Thus, the latex agglutination test provided an efficacious method for routine serological screening for antibodies to T. gondii.     

Rapid immunochromatographic test using recombinant SAG2 for detection of antibodies against Toxoplasma gondii in cats

An immunochromatographic test using recombinant truncated surface antigen 2 for detection of antibodies against Toxoplasma gondii was developed. Evaluation of detection of the antibody in mice and cats suggests that this test is rapid, simple, accurate, relatively inexpensive, and suitable for use under field conditions.     

Enzyme-linked immunosorbent assay for antibodies to Toxoplasma gondii polysaccharides in human toxoplasmosis.

A polysaccharide fraction from Toxoplasma gondii was adsorbed to polystyrene plates, and the enzyme-linked immunosorbent assay was performed (poly-ELISA) with peroxidase-labeled anti-immunoglobulin G and anti-immunoglobulin M antibodies. A comparison was made with a T. gondii total protein extract ELISA (protein ELISA) in serum samples presenting different toxoplasmosis serological patterns, as indicated by a battery of tests for toxoplasmosis. Very low titers and negative results were seen for immunoglobulin G poly-ELISA both for serum samples corresponding to ancient or transitional-period infections (serological patterns II and III) and for samples of recent or acute toxoplasmosis (pattern I). On the contrary, immunoglobulin M poly-ELISA furnished high titer results for pattern I sera, and a very close agreement of titers was seen between immunoglobulin M protein ELISA and immunoglobulin M poly-ELISA. When the polysaccharide fraction was added to pattern I sera, a complete blocking of immunoglobulin M antibody reactivity resulted only for poly-ELISA. In the same way, the total protein extract could completely block only reactivity for protein ELISA. In both cases, a limited decrease in titers was observed for respective heterologous assays.     

Monoclonal antibodies directed against human fibroblast interferon: characterization and functional studies

Spleen cells from BALB/c mice, immunized with SDS-acrylamide gel purified human fibroblast interferon (HuIFN-beta) were fused with mouse myeloma cells. Culture fluids from the resulting hybrid cells were screened for HuIFN-beta specificity by the following tests: a solid-phase RIA using partially purified HuIFN-beta, a protein-transfer RIA using electrophoretically resolved and immobilized HuIFN-beta, and a bioassay which tests residual HuIFN-beta activity in the supernatant following immunoprecipitation. Six HuIFN-beta-specific hybridomas were identified; two were capable of partially neutralizing HuIFN-beta activity. All six antibodies bind to the beta 1-IFN polypeptide synthesized in E. coli cells containing a cloned beta 1-IFN DNA sequence. All six monoclonal antibodies were found to be IgG3/kappa.     

Evaluation of a rapid screening immunoassay for antibodies to Toxoplasma gondii.

A total of 349 human serum samples were examined for anti-Toxoplasma antibodies by the Murex Single Use Diagnostic System (SUDS) qualitative screening test, indirect hemagglutination assay (IHA), and indirect immunofluorescence assay (IFA). Concordant results with SUDS and IHA were obtained for 91.9% of serum samples; 8.9% were SUDS+ and IHA-; none were SUDS- and IHA+. Comparison of the SUDS assay with IFA showed a concordance of 95.3%, with a sensitivity of 97.5% and a specificity of 94.7%. Moreover, the positive and negative predictive values were 84.9 and 99.2%, respectively, when results of the SUDS assay and IFA were compared. The SUDS assay is a rapid, simple test requiring no instrumentation and can be performed on 50 microliters of serum, features which make this an excellent screening test for detecting anti-Toxoplasma antibodies, particularly in outpatient settings.     

Cellular immune reactions directed against Toxoplasma gondii with special emphasis on the central nervous system

Toxoplasma gondii is an obligate intracellular parasite which, after primary infection of humans, is maintained in a dormant state by the host cellular immune system. In the event of an acquired immunosuppression, those parasites surviving as dormant cysts in the host may undergo a change in status, proliferate and cause a life-threatening toxoplasmic encephalitis. Over the last decade much knowlege has accumulated concerning the immune response against T. gondii. This review focuses attention particularly on the anti-parasitic effector mechanisms and the cellular immune reactions in the central nervous system during the course of reactivated toxoplasmic encephalitis. Received: 20 August 1996     

Monoclonal antibodies against rat mast cells differentiate between subtypes

Four monoclonal mouse anti rat mast cell antibodies were selected which detect an antigenic determinant occuring on connective tissue mast cells of the rat. A strong antigen density was found on peritoneal mast cells whereas pleural and mesenteric mast cells exhibit considerably smaller amounts of the antigen. It does not occur on lung mast cells and basophils, thus permitting a mast cell subtype differentiation according to the expression of a surface antigen. The monoclonal antibodies do not react with IgE or IgE Fc-receptor determinants and do not interfere with the histamine secretion from peritoneal mast cells.     

Monoclonal antibodies directed against human myoglobin: characterization and application in a bideterminant radioimmunoassay

Monoclonal hybridoma cell lines secreting antibodies directed against human myoglobin were selected. Two of these cell lines were grown in mouse ascitic fluid resulting in the production of large quantities of antibody. Antimyoglobin antibodies isolated from the ascitic fluids were employed in the development of the sensitive solid-phase, bideterminant radioimmunoassay for human myoglobin that uniquely recognizes two different epitopes on the same molecule.