Immunoglobulin G antibodies directed against protein III block killing of serum-resistant Neisseria gonorrhoeae by immune serum.

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent serum from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by IgG isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, an antigenically conserved outer membrane protein, contained most of the blocking activity in IgG. Antibodies specific for gonococcal porin (protein I), the major outer membrane protein, displayed no blocking function. In separate experiments, immune convalescent DGI serum which did not exhibit bactericidal activity was restored to killing by selective depletion of protein III antibodies by immunoabsorption. These studies indicate that protein III antibodies in normal and immune human serum play a role in serum resistance of N. gonorrhoeae.

Mouse leukemia: therapy with monoclonal antibodies against a thymus differentiation antigen.

Monoclonal antibodies against a thymus cell differentiation antigen (Thy-1.1) were effective in the therapy of a transplanted mouse leukemia. Passive immunization resulted in high titers of cytotoxic antibody in the serum of treated mice and the suppression of metastatic tumor cells. The tumor-suppressive effects of the monoclonal antibodies were amplified by the administration of exogenous complement. This combined antibody and complement therapy resulted in the cure of leukemia in a significant proportion of the treated animals.

Cytotoxic activities of monoclonal antibodies against the envelope proteins of murine leukemia virus.

Monoclonal antibodies against the envelope proteins [gp70 and p15(E)] of murine leukemia virus react with the cell surface of virus-infected cells. The specificity and potency of these antibodies exceed those observed with conventional polyvalent antisera. In cytotoxic assays, certain of the monoclonal anti-gp70 antibodies demonstrate 1000-fold differences in their titer on leukemic and normal thymus cells.

Alteration of lymphoid cells in AKR mice by treatment with monoclonal antibody against Thy-1 antigen.

AKR/J mice treated with high levels of monoclonal anti-Thy-1.1 antibody and C' remained healthy without apparent side effects. Examination of the lymphatic organs of these mice demonstrated a selective depletion of T cells in lymph nodes and spleen. Following cessation of treatment of the levels of anti-Thy-1.1 antibody in the circulation fell and the peripheral lymphatic organs gradually became repopulated with T cells. Depletion occurred in lymph nodes whether or not C' was infused along with the antibody. Although the thymocytes of these mice were coated with anti-Thy-1.1 antibody they were not eliminated by the treatment. The elimination of peripheral but not thymic T cells suggests either a thymic barrier to the penetration of cofactors (possibly antibody-dependent effector cells) or that antibody acts by interfering with the normal traffic of peripheral T cells which normally "home" to the lymph nodes and spleen.

Direct fluorescent monoclonal antibody stain for rapid detection of infant Chlamydia trachomatis infections.

A method of direct fluorescent antibody staining for rapid diagnosis of Chlamydia trachomatis infections in infants is described. This method utilized a fluorescein-conjugated species-specific monoclonal antibody to C trachomatis for detecting chlamydial elementary bodies in smears of the conjunctiva, nasopharynx, oropharynx, anus, and vagina. The sensitivity of direct fluorescent antibody staining was compared with isolation of the organisms in McCoy cells. Thirty-nine infants with purulent conjunctivitis were studied. Diagnosis of C trachomatis conjunctivitis was correctly made by smear in all 16 infants when inflamed eyes were sampled. Positive smears were obtained from 12/14 culture-positive and 4/16 culture-negative nasopharyngeal specimens from infants with chlamydial conjunctivitis. All nasopharyngeal cultures and smears from infants with nonchlamydial conjunctivitis were negative. These results indicate that the direct smear test is a sensitive and specific test for diagnosing C trachomatis infection of the eye and nasopharynx in infants, and this test can be completed within one hour of specimen collection.

Monoclonal antibodies for the diagnosis of sexually transmitted diseases.

Monoclonal antibodies are already being used for the diagnosis of human sexually transmitted diseases. These antibodies can be used to detect a wide range of microorganisms, including bacteria, parasites, and viruses. For both culture and direct tests, monoclonal antibodies showed patterns of specificity and reproducibility that exceeded those available with conventionally prepared antisera. The direct tests for these organisms required less than an hour to perform, representing a major advancement in a diagnosis that previously required 2 to 6 days of culture followed by confirmatory testing. Furthermore, rapid differential diagnosis of infection will now be possible. Because some sexually transmitted diseases may be transmitted simultaneously and share similar clinical manifestations (that is, gonorrhea and chlamydia in cervicitis or urethritis, syphilis or herpes in genital ulcers), it will be possible to differentiate a single from a multiple infection by simultaneous testing of direct samples with the appropriate monoclonal antibody reagents.

Disseminating sexually transmitted infections diagnostics information: the SDI web publication review series.

OBJECTIVES: The World Health Organization Sexually Transmitted Diseases Diagnostics Initiative (SDI) website publication review seeks to provide health care providers in all geographic and economic settings with timely, critical, and concise information concerning new developments in laboratory and field diagnosis of sexually transmitted infections (STI). METHODS: Since 2003, the website (www.who.int/std_diagnostics/literature_reviews) has disseminated information in the form of annotated abstracts and commentaries on articles covering studies of STI laboratory-based and rapid assays that are commercially available or under development. Articles identified through searches of PubMed, specific journals, and by referrals from Editorial Board members are selected for inclusion if they meet pre-specified criteria. The objectives, methods, results, and conclusions for each article are summarised and board members are invited to prepare commentaries addressing study design and applicability of findings to end users. RESULTS: Currently, 91 STI diagnostics experts from 17 countries on six continents serve on the Editorial Board. Twelve quarterly issues have been posted that include summaries of 214 original and 17 review articles published from January 2002 through March 2005, with expert commentaries on 153 articles. Interest in the site has increased every year. In 2005, over 36 700 unique visitors from more than 100 countries viewed over 75,000 pages of information. CONCLUSIONS: The SDI Publication Review series has the potential to contribute to SDI's goal of improving care for patients with STI by increasing knowledge and awareness of STI diagnostics. Given the proliferation of internet-based STI testing services, this website may be broadened to meet the needs of a wider range of users.

An AIDS test that travels well.

PIP: A new dipstick test will enable health workers in remote Third World communities to screen blood for human immunodeficiency virus (HIV) and monitor the spread of HIV infection. The test, developed by the Program for Appropriate Technology in Health (PATH), works without electricity, instrumentation, or cold chain and is locally manufacturable. Results are available in 20 minutes, and the cost is 25 cents per sample. Antibodies to both HIV-1 and HIV-2 are detectable. In the test, a plastic dipstick shaped like a comb with 8 test strips is dipped into blood samples, rinsed, and soaked in a reagent solution. The appearance of a red dot on a dipstick tooth indicates a positive finding. Field trials in Uganda, Kenya, Brazil, China, Indonesia, India, and Thailand indicate the dipstick is as reliable as screening tests already on the market; the false-positive rate was under 2%. India and Indonesia are already manufacturing the new test, and Kenya, Uganda, Cameroon, China, and Thailand have expressed interest. The low cost of the dipstick may enable its use in seroprevalence surveys in previously unstudied regions of the Third World. Such a survey was just conducted among a randomly selected group of pregnant women from across Haiti. Blood samples from simple finger pricks collected on filter paper yielded highly reliable results.^ieng

Gonococcal protein I-specific opsonic IgG in normal human serum.

Pooled normal human serum (NHS), as well as 10 individual NHS samples, markedly inhibited the reaction between monoclonal antibodies and their cognate epitopes on protein I of serum-sensitive, serum-resistant, and disseminated gonococcal infection-associated strains of Neisseria gonorrhoeae, as determined by ELISA inhibition. IgG was the immunoglobulin class responsible for the inhibition. Only the Fab fragment of IgG was inhibitory, making it likely that the IgG reacted specifically with protein I. After absorption with purified protein I, NHS did not inhibit the binding of a protein III-specific monoclonal antibody, thus excluding the possibility that protein III-specific antibodies in NHS masked epitopes on protein I. In addition, lipopolysaccharide-specific IgG in NHS did not appear to contribute to the inhibition of monoclonal antibody binding to protein I. The IgG from NHS was opsonic; opsonization was prevented by coating gonococci with the Fab fragment of protein I-specific monoclonal antibodies.

Analysis and enrichment of murine natural killer cells with the fluorescence-activated cell sorter.

The antiserum anti-NK 1.1 defines an alloantigen specific for natural killer (NK) cells on normal C57BL/6 spleen cells (SC). With complement this antiserum lysed an insignificant percentage of SC, yet could deplete SC suspensions of NK effector cells. The antiserum was also used in this study to indirectly fluorescein label NK cells. The anti-NK 1.1 serum labeled 10 to 15% of nonadherent, nylon column passed SC. The labeled cells were analyzed on the fluorescence-activated cell sorter (FACS), by using flow fluorometry, and were found to be small to medium-sized cells. SC were sorted on the FACS into labeled (NK-1+) and unlabeled (NK-1-) populations, and assayed for NK activity on YAC-1 cells. When compared with control SC, labeled NK-1+ cells were enriched 4- to 13-fold in lytic activity, whereas unlabeled NK-1- cells had little if any NK effector function. Thirty to 60% of the labeled SC adhered to YAC-1 tumor cells in a visual target binding cell assay. The percentage of lymphocytes in the sorted NK-1+ population that bound YAC-1 cells was over 3-fold greater than in unsorted control SC preparations. NK-1- sorted cells did not bind YAC-1 targets. In a preliminary experiment NK-1+ sorted SC inhibited outgrowth of YAC in A/J mice, whereas NK-1- sorted cells did not.

Sensitivity of immunofluorescence with monoclonal antibodies for detection of Chlamydia trachomatis inclusions in cell culture.

Monoclonal antibodies which recognize the species-specific major outer membrane protein antigen of Chlamydia trachomatis were used for immunofluorescence staining of chlamydial inclusions in cell culture. A total of 115 clinical specimens were inoculated onto replicate HeLa 229 cell monolayers and assayed for chlamydial inclusions by immunofluorescence staining and Giemsa staining. Of the isolates, 38 were detected by immunofluorescence staining on passage 1 and 1 was detected on passage 2; 23 isolates on passage 1 and 13 isolates on passage 2 were detected by Giemsa staining. Immunofluorescence staining was significantly more sensitive than Giemsa staining for detecting chlamydial inclusions, particularly from specimens containing low titers of Chlamydia.

Antigenicity of Neisseria gonorrhoeae outer membrane protein(s) III detected by immunoprecipitation and Western blot transfer with a monoclonal antibody.

Some properties of gonococcal outer membrane III were studied by using a monoclonal antibody (2E6) in Western blot transfer and immunoprecipitation reactions. By Western blot, the 2E6 monoclonal had a single antigenic target: outer membrane protein III. All three gonococcal strains examined exhibited identical reactivities for their protein(s) III with this monoclonal. When 125I-labeled gonococci were incubated with monoclonal antibody 2E6, lysed in Zwittergent, and antibody-antigen complexes were immunoprecipitated with protein A-Sepharose, both outer membrane proteins I and III were present in the immunoprecipitates regardless of the gonococcal strain used. Exposure of gonococci to Iodogen and 2-mercaptoethanol influences the electrophoretic migration characteristics of protein(s) III in polyacrylamide gel electrophoresis with sodium dodecyl sulfate; the antigenicity of protein(s) III was unaltered when assessed by Western blot transfer after reaction with Iodogen or 2-mercaptoethanol. These data demonstrate that outer membrane protein III is a surface-exposed antigenic moiety common to diverse strains of gonococci.

Antigenic differences in the surface mannoproteins of Candida albicans as revealed by monoclonal antibodies.

Monoclonal antibodies to Candida albicans were prepared with blastoconidia bearing germ tubes used as the immunogen. Four antibodies reacted by immunofluorescence with surfaces of C. albicans as well as Candida stellatoidea, Candida tropicalis, and several strains of C. albicans, but not with Torulopsis glabrata. One antibody reacted with Saccharomyces cerevisiae. In addition, the monoclonal antibodies precipitated material of approximately 200 kilodaltons when tested against metabolically labeled blastoconidia digests. The monoclonal antibodies exhibited heterogeneous staining of C. albicans surfaces, as shown by immunofluorescence. None of the monoclonal antibodies were specific to germ tubes. More importantly, however, two of the monoclonal antibodies reacted with the mannoprotein precipitin arc of C. albicans that was produced by reference rabbit polyclonal antisera by crossed immunoelectrophoresis, thus linking the heterogeneity seen by immunofluorescence to the heterogeneity in mannoproteins. Finally, three of the monoclonal antibodies reacted with a glycan fraction of cell digests, indicating their reactivity with the carbohydrate portion of the mannoprotein.

[Classification of Gonococcus: auxanologic and serologic aspects using monoclonal antibodies]. / Typisierung des Gonokokkus: Auxotypie und Serotypie mit monoklonalen Antikörpern.

500 N. gonorrhoeae strains from the area of Lübeck/West Germany, collected in 1976-84, were auxologically and serologically classified by means of monoclonal antibodies from the Genetic Systems Corporation, Seattle, Washington (USA), against gonococcal outer membrane protein I. During the first years, auxological classification showed high proportions of AHU-strains (40%), which amounted to 20% over the total observation period. PPNG and DGI strains have characteristic type patterns. Our synopsis of serovars corresponds with the results obtained in a recent world-wide study. Combined auxological and serological classification shows two N. gonorrhoeae pools, 18% each, which cannot be further distinguished: AHU/A1 and prototrophic/B3. The advantages of the system have been proved in answering plain epidemiological questions. For further discrimination, an additional independent classification method might be useful.

Hemorrhagic proctitis due to lymphogranuloma venereum serogroup L2. Diagnosis by fluorescent monoclonal antibody.

Definitive diagnosis of lymphogranuloma venereum is impeded by difficulty in culturing the causative agent and by serologic cross-reactivity between Chlamydia trachomatis L1, L2, and L3, which can cause the disease, and the many other serotypes of C. trachomatis, which do not. In a 23-year-old man with massive rectal bleeding, an exudative rectal ulcer, and inguinal lymphadenopathy, serologic findings were compatible with a recent lymphogranuloma venereum infection, but stains and cultures of lymph-node aspirates were negative, and biopsy specimens of the rectum and lymph nodes showed only nonspecific inflammatory changes. A diagnosis of lymphogranuloma venereum was made when intracellular organisms and inclusion bodies were demonstrated in rectal submucosal tissue by fluorescein-tagged monoclonal antibodies directed against both chlamydial group antigens and L2 serotype antigen. This technique was of particular value in this patient because it specifically identified an unusual cause of severe gastrointestinal bleeding.

Presence of T cell-associated surface antigens on murine NK cells.

The expression of T cell-associated surface antigens on natural killer (NK) spleen cells of C57BL/6 mice was evaluated by cytotoxic depletion experiments with alloantisera prepared against the Thy 1, Ly 1, Ly 2, Ly 5, Ly 6, and NK 1 antigens. The NK activity of these nonimmunized spleen cells for YAC-1 leukemia cells was dramatically reduced by antisera to the Ly 5 and NK 1 antigens. Variable results were obtained with anti-Ly 6 sera--certain pools of this antiserum decreased the NK activity, whereas other pools showed only negligible effects. The NK activity of the same cell suspensions was not affected by antisera to the Thy 1, Ly 1, and Ly 2 antigens. In parallel tests the T cell-associated cell surface antigens of alloimmune T killer cells were similarly evaluated by cytotoxic depletion experiments. In this case, the activity of these cells was consistently diminished by antisera to the Thy 1, Ly 2, Ly 5, and Ly 6 antigens, but not by antisera to the Ly 1 and NK 1 antigens. On this basis it was concluded that the NK cells expressed a restricted subset of T cell-associated alloantigens and therefore may have been derived from the T cell lineage of lymphocytes.

Monoclonal antibodies to Chlamydia trachomatis: antibody specificities and antigen characterization.

Nineteen independent hybrid cell lines that produce monoclonal antibodies to Chlamydia trachomatis surface antigens were prepared by the fusion of mouse myeloma cells with lymphocytes of mice that were immunized with C. trachomatis immunotypes B, C, and L2. Seven serologically distinct reaction patterns were detected by microimmunofluorescence (micro-IF) of elementary body (EB) preparations when culture fluids were tested against a panel of 18 chlamydial serotyping reference strains. These reaction patterns demonstrated genus-, species-, subspecies-, and type-specific distributions. Additionally, these antibodies were tested in parallel against reticulate body (RB) preparations of several chlamydial strains. Monoclonal antibodies that reacted with genus-specific antigens reacted preferentially with RB, whereas antibodies that reacted to species-, subspecies-, or type-specific antigens reacted equivalently to both RB and EB. Physiochemical characterization of antigens recognized by the different monoclonal antibodies was assessed by heat treatment, pronase digestion, periodate oxidation, and immuno-blot techniques. The genus-specific antigen was a heat-stable, pronase-resistant, and relatively periodate-sensitive component of less than 10,000 m.w. The species-, subspecies-, and type-specific antigens were heat stable, pronase sensitive, and periodate resistant. The antibodies that detected species- and subspecies-specific antigens predominantly reacted in immuno-blots with the 40,000 m.w. major outer membrane protein. These monoclonal antibodies now provide a new approach for the precise serologic classification and detection of different C. trachomatis strains.

Inhibition of Trichomonas vaginalis motility by monoclonal antibodies is associated with reduced adherence to HeLa cell monolayers.

Adherence of trichomonads to host epithelial cells appears to be a critical step in the pathogenesis of trichomoniasis. We evaluated the effect of a panel of 10 monoclonal antibodies on attachment of [35S]methionine-radiolabeled Trichomonas vaginalis strains to HeLa cell monolayers. Of 10 monoclonal antibodies, 3 totally eliminated motility of PHS2J strain trichomonads and reduced their adherence to 48 to 60% of control values (P less than 0.001). However, none of the monoclonal antibodies affected motility or adherence of STD13 strain trichomonads. Although the antibodies all reacted with PHS2J trichomonads by immunofluorescence, there was no correlation between inhibition of adherence and findings on either immunofluorescence or radioimmunoprecipitation. Direct microscopic observations showed that incubation with the monoclonal antibodies did not cause cytolysis of T. vaginalis. In quantitative cultures there was no difference in the number of colonies produced by parasites that had been incubated with antibodies that inhibited or had no effect on adherence. We conclude that our monoclonal antibodies reduced adherence not by cytotoxic effects or by competing for specific sites mediating adherence of the protozoa, but by inhibiting motility of T. vaginalis.