Comparison of antibody responses in Atlantic cod (Gadus morhua L.) to Aeromonas salmonicida and Vibrio anguillarum

The immune system of Atlantic cod (Gadus morhua L.) differ from other bony fish species in that no or only very low increases in antibody levels are detected post-immunization with Vibrio salmonicida or V. anguillarum. Here, we report the results from the first study on comparison of antibody responses in cod to Aeromonas salmonicida and V. anguillarum.

A. salmonicida appear to induce a stronger antibody response in cod compared to V. anguillarum, and more individuals immunized with A. salmonicida revealed a response compared to those immunized with V. anguillarum. The antibody responses to both bacterial species were mainly toward LPS, and the results indicate that cod antibodies are able to differentiate between LPS antigens from typical and atypical A. salmonicida strains.     

Monoclonal antibodies against Vipera lebetina venom nerve growth factor cross-react with other snake venom nerve growth factors

Nerve growth factor (NGF) was isolated from the venom of Vipera lebetina and was purified to homogeneity as judged by SDS gel electrophoresis. The biologically active NGF was used to immunize BALB/c mouse, and the spleen cells from immunized mouse were fused with mouse PAI myeloma cells. Forty-seven hybrid cell lines, secreting monoclonal antobodies to V. lebetina NGF, were isolated and nine of them purified from ascitic fluids. The isolated antibodies define two partially overlapping epitopes of the V. lebetina NGF which are not involved in the biological activity of the molecule. Both epitopes are also present on the β-NGF from the mouse salivary gland and on the NGFs from the following snake venoms: V. lebetina, V. ursini, V. berus berus, Echis carinatus, Bungarus caeruleus, Agkistrodon halys, Naja naja oxiana. Naja naja atra and Naja naja, but not on the bovine seminal plasma NGF. The mol. wts of the NGFs in these snake venoms were determined by Western immunoblot with monoclonal antibodies. The mol. wts of the NGFs from V. ursini (37,000), E. carinatus (36,000, 44,000) and A. halys (29,000) were determined for the first time.     

抗巨细胞病毒早晚期抗原单克隆抗体制备及在病毒培养物鉴定中的初步应用

目的 制备抗CMV早晚期抗原单克隆抗体并在病毒培养物鉴定中初步应用.方法 CMV感染MRC-5细胞24h、48h和72h后甲醛灭活的可溶性抗原免疫BALB/c小鼠,小鼠脾细胞与骨髓瘤细胞NS-1进行融合.酶免法筛查阳性杂交瘤并有限性稀释法进行克隆.免疫荧光及印迹对筛选后的克隆进行鉴定,最终获得的杂交瘤制备腹水并使用Protein G进行纯化.纯化后单抗应用于CAP室间质评标本以及临床标本CMV培养物的鉴定.结果 3只小鼠脾细胞与骨髓瘤细胞融合后初步筛查出约110株阳性克隆.剔除与其它抗原有交叉反应、抗体效价低、非全覆盖早晚期抗原的克隆最终获得抗CMV单抗杂交瘤1株,即23B5-1.免疫荧光显示23B5-1株单抗与CMV感染后3h-120h的MRC-5细胞均反应,即覆盖即刻、早期和晚期抗原.免疫印迹试验显示23B5-1株单抗与CMV抗原25KD-50KD之间的5个蛋白条带结合.23B5-1株单抗免疫球蛋白亚型为IgG1.Protein G纯化腹水后的单抗效价≥1∶12800.纯化单抗染色鉴定6份室间质评及10份临床标本CMV培养物全部符合.结论 初步应用显示制备的抗CMV早晚期抗原单克隆抗体性能良好.     

Serological analysis of dermatophyte isolates with monoclonal antibodies produced against Microsporum canis.

Hybridoma cells produced by fusing myeloma cells with spleen cells from mice immunized with a soluble antigen of Microsporum canis yielded 30 antibody-producing clones. Six of these clones, propagated as ascites tumors in mice, showed two different types of monoclonal antibodies. The type 1 monoclonal antibody reacted with 17 heterologous and 10 homologous dermatophyte antigens. Type 2 monoclonal antibodies were unable to precipitate three antigens from different isolates of M. canis, thus suggesting the occurrence of different serotypes within the species.     

Enzyme-linked immunosorbent assay for quantifying antigens of Dermatophagoides farinae and D. pteronyssinus (Acari: Pyroglyphidae) in house dust samples

Double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody as capture and detector antibodies was developed for quantifying antigens of Dermatophagoides farinae Hughes and D. pteronyssinus (Trouessart) mites contained in house dust samples. This monoclonal antibody was directed against mite antigens that were also reactive to immunoglobulin E antibody in all of 10 serum samples obtained from patients allergic to mites. Histological study using fluorescent antibody revealed that the monoclonal antibody was bound to the major part of the D. farinae mite body section including fecal matter and cuticles. The detection limit of the assay system was 0.17 microgram of soluble antigens of both mite species and the antigen amount corresponding to 0.5 mites per microplate well, whether in live or dead mites. This system did not react to antigens of Tyrophagus putrescentiae (Schrank) and Cheyletus malaccensis Oudemans. Slight inhibition of less than or equal to 21.3% was observed with nonspecific substances contained in house dust, such as wool, cotton, human dander, human hair, soil, and biscuit, but no direct ELISA reactions were obtained with any of these materials. In 49 house dust samples, ELISA was significantly correlated with the conventional microscopic observation method.     

Acquisition and dissemination mechanisms of CTXΦ in Vibrio cholerae: New paradigm for dif residents

Vibrio cholerae (V. cholerae) genome is equipped with a number of integrative mobile genetic element (IMGE) like prophages, plasmids, transposons or genomic islands, which provides fitness factors that help the pathogen to survive in changing environmental conditions. Metagenomic analyses of clinical and environmental V. cholerae isolates revealed that dimer resolution sites (dif) harbor several structurally and functionally distinct IMGEs. All IMGEs present in the dif region exploit chromosomally encoded tyrosine recombinases, XerC and XerD, for integration. Integration takes place due to site-specific recombination between two specific DNA sequences; chromosomal sequence is called attB and IMGEs sequence is called attP. Different IMGEs present in the attP region have different attP structure but all of them are recognized by XerC and XerD enzymes and mediate either reversible or irreversible integration. Cholera toxin phage (CTXΦ), a lysogenic filamentous phage carrying the cholera toxin genes ctxAB, deserves special attention because it provides V. cholerae the crucial toxin and is always present in the dif region of all epidemic cholera isolates. Therefore, understanding the mechanisms of integration and dissemination of CTXΦ, genetic and ecological factors which support CTXΦ integration as well as production of virion from chromosomally integrated phage genome and interactions of CTXΦ with other genetic elements present in the genomes of V. cholerae is important for learning more about the biology of cholera pathogen.     

Specific monoclonal antibodies to the Mycoplasma-type pathogenic agent of grapevine flavescence dorée

Three hybridomas producing monoclonal antibodies specific for the mycoplasma-like organism (MLO), pathogenic agent of grapevine flavescence dorée, were obtained after fusion between spleen cells from a mouse immunized against flavescence dorée MLO and Sp2/O-Ag-14 mouse myeloma cells. These hybridomas were selected in an indirect sandwich ELISA in which antibodies from two different animal species were used (rabbit and mouse). This assay is convenient for anti-MLO monoclonal antibody screening, because of its sensitivity, specificity and good preservation of antigens. The three monoclonal antibodies were examined for reactivity towards the MLO causal agents of 15 plant yellows. None of the three were implicated in a serological relationship between the MLO of these plant yellows and flavescence dorée-MLO. Reactivity of the three monoclonal antibodies was checked towards two other isolates of flavescence dorée. One of the three antibodies detected the wild isolates.     

Preparation and characterization of a monoclonal anti-T helper factor antibody.

Sprague-Dawley rats were immunized by injection of antigen B-specific T helper factor THF) eluted from Sepharose-antigen D adsorbents. Rat spleen cells from animals immunized with THF were fused with a BALB/c tumour cell (P3x63-Ag8.653) to prepare monoclonal anti-THF antibodies. The hybrids produced were screened for anti-THF antibodies by an enzyme-linked immunoassay (ELISA), and we shall describe the characteristics of one of the hybrid (hybridoma 6-2.2 anti-THF) antibodies produced. (i) The monoclonal hybrid 6-2.2 anti-THF antibody blocks water-soluble timothy extract-induced proliferation of timothy-specific T helper cells when these cells were preincubated with an excess of the anti-THF hybrid 6-2.2 antibody; (ii) incubation of timothy-specific T helper or T suppressor cells with an optimal dose of anti-THF 6-2.2 antibody induces significant levels of [3H]-thymidine incorporation in the absence of antigen; (iii) it binds specifically to the idiotypic determinant expressed on THFk, THFd, TSFk, and antigen B-specific IgE in an ELISA; and (iv) it has no effect upon spleen cells from mice primed with ovalbumin or Ascaris suum antigens. In addition, the monoclonal anti-THF 6-2.2 antibody cultured with normal spleen cells in mini-Marbrook chambers induced significant levels of antigen B-specific T suppressor cells. These studies indicate that the monoclonal anti-THF 6-2.2 antibody has anti-idiotypic antibody properties.     

CELLULAR AND MOLECULAR HETEROGENEITY OF H-2Kk ANTIGENS

Up to 70% of mouse spleen cells expressing H-2K^k alloantigens reacted with the monoclonal anti-H-2K^k antibody 11-4.1 in an indirect rosetting assay. When the cells that rosetted with the monoclonal antibody 11-4.1 were removed by density centrifugation, the residual unreactive cells reacted with the polyclonal anti-H-2K^k alloantiserum D23 but not with the monoclonal antibody 11-4.1 indicating the existence of two cell populations. In sequential immunoprecipitation experimens, the glycoprotein fraction of NP40 extracts from H-2K^k cells, after removal of antigens reacting with the monoclonal antibody 11-4.1 still contained structures reactive with the alloantiserum. Therefore, there are at least two antigenically distinct H-2K^k molecules which are differentially expressed on two subpopulations of spleen cells.     

Production of a monoclonal antibody against the 128 kDa (CagA) protein of Helicobacter pylori

Helicobacter pylori is recognised as an important factor in gastroduodenal pathology. The 128 kDa CagA protein has been established as a useful marker of H. pylori strains associated with more severe forms of disease. A mouse monoclonal antibody raised against the CagA protein has been produced and characterised as belonging to the IgG1 subtype. It identified the protein in all clinical isolates (10/10) from this laboratory and in two NCTC reference strains (NCTC 11637 and NCTC 11961). No cross-reacting proteins were detected in H. pylori L2, a well characterised strain known not to contain the cagA gene, or in four Helicobacter sp. from non-human sources (H. canis, H. mustelidae, H. muridarum and H. acinonyx). The monoclonal antibody was used to develop an antigen capture ELISA system for detecting the presence of antibodies to the CagA protein in human serum samples.     

Immunological Detection of Lipopolysaccharide Antigens of Thermophilic Campylobacters Captured on Polymyxin-Coated Polyester Cloth

Cholate-extracted lipopolysaccharide (LPS) antigens from thermophilic campylobacters were captured on polymyxin-coated polyester cloth. The captured antigens were detected by sequential reactions with rabbit anli-Campylobacter antibody, anti-rabbit IgG peroxidase conjugate and chromogenic peroxidase substrate. A polyclonal rabbit antibody elicited against a single Campylobacter jejuni strain detected the reference strains of the twenty most frequently isolated thermophilic campylobacters in the Lior serotyping scheme. Moreover, LPS antigens of six C. jejuni Penner serotypes fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting were recognized by four antisera prepared against homologous and heterologous Penner serotypes. The results suggest the potential application of polymyxin-cloth enzyme immunoassay for rapid detection of thermophilic campylobacters where monoclonal antibodies can be raised to possible common LPS epitopes.     

Application of an enzyme-linked immunosorbent assay for detecting mold contamination in agricultural commodities and comparison with conventional assays

Mold contamination in agricultural commodities including grains, grain-based foods, fruits, and vegetables was investigated by two analytical methods. One method employed a mixed monoclonal antibody sandwich ELISA for detection of Aspergillus, Penicillium and Fusarium genera-specific antigens. The detection limit of ELISA for these antigens was 100 ng/g. The other protocol was HPLC-based detection of ergosterol, the predominant sterol in most molds. Recoveries of ergosterol from grains and grain-based foods at spiking levels of 50-1000 ng g^-1 ranged between 82 and 86%. The detection limit of the HPLC method for ergosterol was 40 ng g^-1. Ninety-two samples (59%) and 116 (75%) among 155 samples tested positive with antigens and ergosterol by the ELISA and the HPLC method, respectively. The cause of conflicting data between both detection methods found in nine dried persimmon samples were solved by using the conventional direct plating method that showed the prevalence of other genera with rarity of Aspergillus, Penicillium and Fusarium. The sandwich ELISA method offers a rapid assay for selectively tracing the potential mycotoxigenic mold, and also is a preliminary screening method for mycotoxin analysis.     

Use of the fluorescence activated cell sorter to enrich dendritic cells from mouse spleen

Dendritic cells are a specialized but trace population of antigen presenting cells that always have been enriched by multi-step procedures over a period of 1 or more days in tissue culture. Here we describe the isolation of dendritic cells from fresh mouse spleen suspensions using the FACS and a monoclonal antibody, N418, to the p150/90 member of the leukocyte integrin family (Metlay et al., 1990). By two color fluorescence activated cell sorter (FACS) analyses, the trace N418+ subset expressed most of the surface markers, including the 33D1 antigen, that are characteristic of dendritic cells isolated by other methods. An exception was that small amounts of Fc receptors, CD4 and F4/80 antigen were detected initially, but these diminished upon culture. In functional assays, sorted N418+ cells from fresh spleen were at least 30 times more active than N418- cells in presenting antigen to T cells. The assays were stimulation of the primary mixed leukocyte reaction and presentation of exogenous protein antigens to sensitized populations of lymph node T cells. The viability and MLR stimulating function of the sorted populations both were increased upon exposure to the cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF). These results indicate that dendritic cells can be enriched from fresh isolates of mouse spleen using the FACS, and that when this is done, many of the distinctive features of dendritic cells - phenotype, APC function, and sensitivity to appropriate cytokines - are apparent.     

PRODUCTION AND CHARACTERIZATION OF AN ANTI-IDIOTYPIC SINGLE CHAIN FV THAT RECOGNIZES AN ANTI-DNA ANTIBODY

A well-characterized recombinant anti-idiotype to an anti-DNA antibody can be useful for studies of the regulation of anti-DNA-producing B cells. Using a hybridoma technique, a monoclonal anti-idiotypic antibody, designated O2F3, was obtained, and its scFv gene was constructed. O2F3 single chain Fv (scFv) was produced against an idiotope of a monoclonal anti-DNA antibody, 3D8, that was obtained from an autoimmune-prone mouse, MRL-lpr/lpr. Here we describe the production and in vitro characterization of the O2F3 scFv, and compare it with its parent monoclonal antibody, O2F3 IgM. To characterize O2F3 scFv and O2F3 IgM, we generated recombinant 3D8 fragments, including 3D8 scFv, 3D8 V_H, and 3D8 V_L, that were used as antigens in several assays. ELISA and Western blot analysis showed that both O2F3 scFv and O2F3 IgM recognized a conformational determinant formed by the association of the variable region heavy and light chains of the 3D8 antibody, suggesting that O2F3 scFv retained a similar binding pattern to its parent O2F3 antibody. The idiotope recognized by O2F3 was shown by competitive ELISA to be outside of the DNA binding site of the 3D8 antibody. This characterized O2F3 scFv could be applied for the regulation of anti-DNA antibody production and the manipulation of recombinant antibody-based proteins to which toxins, enzymes, and chemical agents can be connected.     

Rapid detection and serotyping of adenovirus by direct immunofluorescence

Four fluorescent antibody reagents were evaluated for their suitability for the identification of adenovirus isolates by immunofluorescence. The antibodies used in the reagents consist of monoclonal antibodies against adenovirus type 3 (Ad3), Ad4, Ad8, and adenoviruses of subgroup C (Ad1,2,5,6), serotypes known to occur in outbreaks of disease. Most of the monoclonal antibodies employed were reactive against type-specific antigens found on the hexon protein. Reagents employing two noncompeting anti-hexon antibodies were more sensitive than reagents prepared with only one monoclonal antibody, although both types of reagents exhibited a high degree of specificity. Five hundred and seventeen adenovirus isolates (359 of which had previously been typed by other methods) and 46 nonadenovirus isolates were examined with all four type-specific reagents in parallel with an adenovirus group-specific reagent. The results indicate that direct typing of adenovirus isolates is feasible, leading to significant savings in time compared to other typing methods and should contribute to the management of certain adenovirus infections, particularly during outbreaks. J. Med. Virol. 51:198–201, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of two panels of monoclonal antibodies for determination of Chlamydia trachomatis serovars.

A panel of monoclonal antibodies was developed for serovar typing of clinical isolates of Chlamydia trachomatis. The panel could distinguish all 15 established serovars from one another, although the hybridomas of the panel were developed by fusions of myeloma cells and spleen cells from mice immunized with antigen derived from the urogenital serovars D to L3. The typing assay was based on a dot enzyme immunoassay, and the monoclonal antibodies that were included in the panel reacted strongly in this assay. A collection of 289 clinical isolates from The Netherlands was typed. The observed serovar frequency distribution was 51 isolates of serovar D (17.6%), 103 isolates of serovar E (35.6%), 62 isolates of serovar F (21.5%), 28 isolates of serovar G (9.9%), 14 isolates of serovar H (4.8%), 2 isolates of serovar I' (0.7%), 20 isolates of serovar J (6.9%), and 9 isolates of serovar K (3.1%). These results were confirmed by typing these isolates with a panel of monoclonal antibodies purchased from the Washington Research Foundation, Seattle. No strain variation was observed within serovar D with both panels. However, restriction fragment length polymorphism analysis of the gene encoding the major outer membrane protein showed that 32 isolates were similar to the prototype D and 17 were similar to the variant D-. The two others showed a new restriction pattern. Our panel of monoclonal antibodies contained one monoclonal antibody that divided the serovar G isolates into two groups. This differentiation was confirmed by restriction fragment length polymorphism analysis, confining this difference to a known sequence variation in variable domain IV. These data support the subdivision of serovar G into serovars G (prototype strain UW-57) and Ga (prototype strain IOL-238).     

Lymphocytes of persons with the acquired immunodeficiency syndrome and related conditions express reactivity with the monoclonal antibody 4D12 reflective of in vivo lymphocyte activation

We observed that lymphocytes obtained from healthy persons generally expressed infrequent reactivity with the monoclonal antibody 4D12, an antibody raised against a cell line infected by the human T-lymphotropic virus type I. As had been observed previously, persons bearing HLA-B5 cross-reactive antigens and certain other allotypes had frequent lymphocyte reactivity with 4D12. Lymphocytes obtained from persons infected by the human immunodeficiency virus were highly reactive with 4D12 as were lymphocytes obtained from persons with other viral or bacterial infections. Flow cytometric studies revealed greater 4D12 reactivity by larger lymphocytes, and in vitro studies demonstrated that lectin-stimulated lymphocytes acquired 4D12-reactive antigens. There was also a significant correlation between expression of 4D12-reactive antigens and the presence of the interleukin-2 receptor as recognized by the monoclonal antibody anti-Tac. Thus, the monoclonal antibody 4D12 recognizes a lymphocyte surface antigen frequently expressed among persons with various acute and chronic infections. This antigen is a marker of lymphocyte activation.     

Characterization of Cross-Reactive Anti-DNA Autoantibodies in Murine Lupus

The role of crossreactive anti-DNA autoantibodies in the pathogenesis of Systemic Lupus Erythematosus (SLE) and its counterpart in the mouse (murine lupus) remains undefined. Five murine monoclonal anti-DNA autoantibodies tested in ELISA and immunofluorescence assays were found to cross-react with a variety of both nucleic acid and non-nucleic acid antigens. These included double stranded DNA (dsDNA), single stranded DNA (ssDNA), transfer RNA (tRNA), and the murine thymoma cell lines WEHI-22, WEHI-7, and EL-4. The majority of the autoantibodies reacted with all antigens tested; none of the autoantibodies reacted with only one antigen. To determine if the multiple reactivities demonstrated by these hybridoma-derived monoclonal anti-DNA autoantibodies accurately reflects the in vivo, autoimmune environment, the same assays were used to measure the reactivities of autoantibodies secreted directly from unfused autoimmune spleen cells cultured in vitro. These spleen cell-derived autoantibodies were found to display reactivities very similar to those demonstrated by the monoclonal anti-DNA autoantibodies indicating that the hybridoma process itself does not appear to select and amplify reactivities which are not present in vivo. Initial molecular characterization of F11, a monoclonal anti-DNA autoantibody crossreactive with both dsDNA and ssDNA, revealed that it utilizes the same VH gene segment as an anti-DNA autoantibody specific for ssDNA. F11 was also found to utilize similar VH, D, and JH gene segments as an antibody directed against the hapten polymer (Glutamic acid⁶⁰,Alanine³⁰, Tyrosine ¹⁰)n (GAT). Thus, the same Ig gene segments used to encode crossreactive anti-DNA autoantibodies can also be utilized by anti-DNA autoantibodies displaying strict antigen specificity as well as by antibodies directed against exogenous antigens.     

Maternal antigenic stimulation actively produces suppressor activity in offspring

Pregnant B10.A(3R) and B10.A(5R) mice were immunized with heterologous erythrocyte and protein antigens and the active immune responsiveness of their offspring was investigated by the plaque-forming cell (PFC) assay. In offspring derived from mothers which were stimulated with optimal amounts of antigens and which had fully developed antibody-forming cells, there was a clear-cut suppression in development of specific PFC over a significant period after delivery. In order to characterize the suppressor cell population, spleen cells were prepared from offspring whose mothers were immunized and thereafter treated by anti I-Jk or anti I-Jb monoclonal antibody and complement (C') followed by adoptive transfer to normal corresponding mouse strain. Only the group that received 3R spleen cells treated with anti I-Jb monoclonal antibody and C' had no suppressed PFC. To clarify the suppressor site in offspring, precursor B-cells of experimental offspring responded as hapten specific antibody forming cells by employing homologous hapten and heterologous carrier antigens. These results suggest mechanisms for suppression in offspring whose mothers were stimulated during pregnancy.     

抗成骨肉瘤mAb的制备、鉴定及其细胞毒效应

目的 建立抗人成骨肉瘤mAb杂交瘤细胞系并对其抗体特性进行研究。方法 利用我室自建的骨肉瘤细胞系（OS——9607）免疫Balb/c小鼠,取小鼠脾细胞与骨髓瘤细胞Sp2/0进行融合,经筛选和克隆化后,建立杂交瘤细胞系,以免疫组化和Western Blot等方法研究抗体特性,以MTT法研究细胞毒作用,结果 得到1株能够稳定分泌抗体,效价高的杂交瘤细胞系3D9,免疫组化结果显示着色区主要分布在细胞核上,相应抗原在成骨肉瘤标本和成骨肉瘤细胞系高度表达（83%）,正常组织未见表达,WesternBlot显示3D9相应分子量为Mr54000。MTT法示该抗体对成骨肉瘤细胞的杀伤率明显高于对照组。结论 得到的杂交瘤细胞系分泌的mAb体具有成骨肉瘤细胞特异亲和性,并有一定的细胞毒效应,可能成为新的成骨肉瘤生化标志,在肿瘤的免疫治疗方面有广阔的应用前景。