人心肌肌钙蛋白Ⅰ单克隆抗体及多克隆抗体的制备

目的:以重组人心肌肌钙蛋白Ⅰ(cTnⅠ)为抗原制备鼠源单克隆抗体(McAb)及兔源多克隆抗体,并鉴定抗体的特性。方法:以纯化的重组人cTnⅠ为抗原免疫BALB/c小鼠,取鼠脾细胞同Sp2/0骨髓瘤细胞融合,利用选择培养基筛选融合的杂交瘤细胞,用有限稀释法分离获得能够稳定分泌抗cTnⅠ的McAb阳性克隆,并利用体内诱生法大规模制备McAb,用辛酸-硫酸铵沉淀法纯化抗体;兔多抗制备以cTnⅠ为抗原常规免疫后取其血清;用间接ELISA和Western印迹鉴定抗体的性质。结果:经ELISA鉴定,筛选出5株能分泌cTnⅠMcAb的杂交瘤细胞株,即C5B2、C5B3、C5B4、C5B1、B1A6,效价最高的B1A6株分泌的McAb为IgG3型,纯化后效价为1∶10000,亲和常数为1.08×10-9mol/L,Western印迹鉴定表明cTnⅠMcAb有良好的特异性;兔多抗纯化后的效价为1∶8000。结论:制备了具有良好特性的cTnⅠMcAb和多克隆抗体。     

Monoclonal antibodies to Mycoplasma pneumoniae antigens]

Approaches to obtaining stable mouse hybridomas, capable of producing monoclonal antibodies (McAb) to M. pneumoniae key antigens, were developed. As the result of hybridization experiments, 7 clones were obtained; of these, 4 clones stably synthesized IgG McAb. Clones H1/H9 and H9/B2 synthesized antibodies to thermolabile, proteinase-sensitive K protein, produced by cytoplasmic membranes of M. pneumoniae cells. The molecular weight of this protein was found to be 90 kD. McAb of clone H1/H9, labeled with horse-radish peroxidase and fluorescein isothiocyanate, specifically reacted with M. pneumoniae antigens in the immunofluorescence test and the enzyme immunoassay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data are prerequisites for the development of diagnostic test systems for the detection of M. pneumoniae antigens in different biological substances obtained from patients with respiratory pathology.     

The surface antigens of Rickettsia prowazekii studied with monoclonal antibodies]

R. prowazekii antigens have been tested with the use of monoclonal antibodies (McAb) to different epitopes of the microorganism. As revealed in these tests, McAb B4/4 and A-3/D, active against species-specific thermolabile antigen, interact with protein having a molecular weight of 90-120 KD. McAb C5/2, active against thermostable group antigen common with that of Rickettsia typhi, interact with LPS-like antigen having a molecular weight of 30 KD. Ultrastructural immunochemical studies have revealed that both R. prowazekii antigens are located on surface structures of rickettsiae, such as the microcapsule and cell wall.     

Monoclonal antibodies to the species-specific and group antigens of Rickettsia prowazekii]

A number of hybridomas to different R. prowazekii determinants were obtained by the hybridization of spleen cells of BALB/c mice immunized with R. prowazekii corpuscular and soluble antigens. Some of the monoclonal antibodies (McAb) reacted with R. prowazekii thermolabile species-specific protein and did not react with R. typhi antigens (McAb of batches B4/4 and A-D3). McAb C5/2 and A3/2 reacted with the group thermostable antigen, common for R. prowazekii and R. typhi. McAb to the species-specific thermolabile antigen belonged to IgG2a. The McAb thus obtained permit the identification of R. prowazekii and R. typhi and the solution of the problem of the intragroup differentiation of rickettsiae belonging to the typhus group.     

Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]

A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA.     

Monoclonal antibody produced against partially purified sporozoite antigen inhibits invasion of cells by sporozoites of avian Eimeria species

Previous studies showed that molecules of in vitro-cultured primary turkey kidney cells bound to 23-, 40-, and 60- to 65-kDa antigens of sodium dodecyl sulfate-solubilized sporozoites of Eimeria adenoeides. Similar binding to antigens of three other species of avian Eimeria, E. tenella, E. acervulina, and E. meleagrimitis, is now reported. Strips containing the most avidly bound sporozoite antigen (approximately 40 kDa) were excised from the sodium dodecyl sulfate-polyacrylamide gels on which E. adenoeides antigens had been electrophoretically separated. The strips were homogenized and injected into mice to produce hybridoma cell lines. Twelve cell lines secreting monoclonal antibodies (McAb) that reacted with E. adenoeides sporozoites were detected. One of these McAb, H11C3, reacted with structures in the anterior tip of sporozoites of E. adenoeides and five other species of avian Eimeria. When included in the inoculation medium, this McAb significantly inhibited invasion of cultured kidney cells by sporozoites of E. adenoeides and E. tenella. In contrast, when the sporozoites were pretreated with McAb H11C3 and then washed free of the antibody, no inhibition of invasion was observed.     

The protective effects of monoclonal antibodies in mice from Naegleria fowleri infection]

Protective effects of monoclonal antibodies against N. fowleri were comparatively studied. BALB/c mice were treated with two types of monoclonal antibodies, Nf 2 and Nf 154, before and after the infection with N. fowleri. The mortality and mean survival times were then compared. Also, direct effect of the monoclonal antibodies on the N. fowleri trophozoites in vitro were observed. In vitro protective effects of the monoclonal antibodies were also studied in cells infected with N. fowleri. The observed results are summarized as follows: 1. Among mice pretreated twice before the infection with monoclonal antibody Nf 2(McAb Nf 2), only 15.8% were killed, and the mean survival time was 17.7 days. This was not much different from the mice pretreated once, as the mortality and mean survival time were 16.7% and 17 days. Those effects were compatible with monoclonal antibody Nf 154(McAb Nf 154). The above findings contrast with the mortality and mean survival time of the control mice, which were 22.7% and 14.6 days respectively. 2. Mice which received twice the McAb Nf 2 following N. fowleri infection incurred a 19.4% mortality rate with 13.6 days survival time; 17.9% and 15.8 days with on time administration, in contrast to the 25% and 14.6 days in the control group. 3. Marked agglutination effect of McAb Nf 2 or McAb Nf 154 were observed on N. fowleri trophozoites. 4. When N. fowleri trophozoites were treated with McAb Nf 2 or McAb Nf 154 combined with comments, the proliferation rate was more significantly suppressed than in that the control. 5. N. fowleri trophozoites treated with McAb Nf 2 or McAb Nf 154 showed an increased number of swollen mitochondria, disfigured cisternae, lipid droplets, and osmiophilic granules in the cytoplasm. 6. A remarkable protective effect of monoclonal antibodies was noticed in CHO cells infected with N. fowleri. More than 90.6% of the infected CHO cells survived, contrasted with 27% of untreated cells. The overall results in this study suggest that N. fowleri treated with monoclonal antibodies against N. fowleri reduce the mortality and prolong the survival time of the mice when the antibodies are administered before the infection. The protective effect of the monoclonal antibodies is surmised being caused by agglutination of the trophozoites.     

Anti-MHC immunity detected prior to intentional alloimmunization

Cell fusion was performed between spleen cells from young BALB/cBy (H-2 ^d) mice which have never been immunized and SP2/0 mouse plasmacytoma cells. A monoclonal H-2-specific cytotoxic IgM antibody was obtained (By-1) which detected a new public biregional H-2 specificity, H-2.m210. The mcAb By-1 reacted strongly with H-2K^d, D^d, and H-2^s antigens, gave weak cross-reactions with H-2K^k, D^q, H-2^r, and H-2^v antigens and was negative with H-2^b, H-2^f, H-2^p, and H-2L^d antigens. A polymorphic reaction pattern was also observed on a panel of lymphocytes from B 10.W strains. The intriguing finding on this reaction pattern was the reactivity on H-2^d cells, including the syngeneic BALB/cBy and truly autologous cells. As shown by capping and immunoprecipitation experiments on H-2^d cells and by studies on H-2^d-transfected mouse L cells, the target molecules for McAb By-1 were H-2K^d and H-2D^d molecules. The BALB/cBy mouse, from whose spleen cells the McAb By-1 was obtained, survived after the fusion experiment, and serum was examined for the presence of cytotoxic H-2-specific antibodies during the rest of its life. At the time of the fusion, no autoreactive serum antibodies were found, but about 4 months later, we found in the serum of this mouse autoreactive H-2-specific cytotoxic IgM antibodies. The serum antibodies followed the same reaction pattern as that of the McAb By-1. As far as we know, this is the first report of autoreactive H-2-specific antibodies in serum of a mouse which has never been immunized and of the first natural autoreactive H-2-specific monoclonal antibody.Abbreviations McAb monoclonal antibody - MHC major histocompatibility complex - H-2 major histocompatibility complex of mice - CTLs cytotoxic T cells - FMF flow microfluorometry - FITC fluorescein isothiocyanate - LPS lipopolysaccharide W.E. coli 0111:134 - PBS phosphate-buffered saline - Iodogen 1,3,4,6,-tetrachloro-3,6-diphenylglycoluril - GAMIg goat-antimouse immunoglobulin - Staph-A Staphylococcus aureus Cowan I     

Immunobiological properties of monoclonal antibodies to Mycoplasma hominis antigens

Approaches to obtaining stable mouse hybridomas synthesizing monoclonal antibodies (McAb) to M. hominis key antigens were developed. 4 clones capable of the stable synthesis of McAb of different IgG classes were obtained. Clones A3/2 and A5/D produced antibodies to the thermostable determinant to with a mol. wt. of 80-120 kD, sensitive to sodium periodate and resistant to potassium proteinase. Clone H9/B2 synthesized McAb which interacted with potassium proteinase-sensitive M. hominis thermolabile determinant with a mol. wt. of 80 kD. McAb of clone A3/2, labeled with fluorescein isothiocyanate and horse-radish peroxidase, specifically reacted with M. hominis antigens in the immunofluorescence test and the immunoenzyme assay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data may serve as prerequisites for the development of diagnostic test systems aimed at the detection of M. hominis antigens in different clinical substances.     

抗单纯疱疹病毒单克隆抗体的研究——Ⅱ.单克隆抗体介导细胞毒效应的研究

建立了单克隆抗体(McAb)介导细胞毒作用(ADCC)~(51)Cr释放试验的测定力法。确定了最适工作条件。ADCC测定结果表明,5株抗HSV McAb介导ADCC的活性不同:McAb 1A12、2A8和1G8无ADCC活性;而1D10和2C5两株McAb作1:10稀释时,~(51)Cr释放率分别为27.09％和25.07％,稀释至1:100或1:1000时仍有ADCC活性。结果提示,不同的McAb抗原决定族诱导产生的抗体,在介导ADCC免疫保护作用上有差异,并为McAh治疗临床单纯疱疹病毒感染的可能性提供了实验资料。     

Monoclonal antibodies for the analysis and purification of isopentenyladenine cytokinins

A series of monoclonal antibodies (McAb) have been generated which show a high affinity for the cytokinin, isopentenyladenosine (2iPA). The McAb show different specificities to a range of cytokinins in a radioimmunoassay and are suitable for the analysis of isopentenyladenine (2iP) and related compounds in plant extracts. One McAb, MAC 160, has a high cross-reactivity with several cytokinins and is a good candidate for use in group separation or the analysis of a number of cytokinins simultaneously after separation. When coupled to Sepharose 4B affinity support, the McAb can also be used as immunoaffinity matrices to purify and resolve cytokinins from plant extracts. The McAb have been used to demonstrate the presence of concentrations of 2iP in tissues of the moss, Physcomitrella patens which exceed those in its culture medium.     

Cytotoxicity of monoclonal antibodies to a subset of Giardia isolates

Previous studies showed that some Giardia lamblia isolates differ and can be categorized on the basis of their DNA banding patterns after digestion with endonuclease restriction enzymes, surface antigens, and excretory-secretory (E-S) products. In the present study, monoclonal antibodies (McAb) were produced that reacted with one specific group of Giardia isolates. These McAb recognized a 170,000 dalton antigen, which was present on the surface of these Giardia and released into the medium as an E-S antigen. This antigen was previously characterized and found to distinguish this subgroup of Giardia. In addition, these McAb were cytotoxic only for this subgroup of Giardia. Immobilization occurred immediately, and killing was documented by 7 min. The mechanism(s) of killing remains unknown but was shown to be complement independent and did not occur with Fab'. These McAb identifies certain isolates and can be used to type Giardia.     

The isolation of monoclonal antibodies to Cryptococcus neoformans antigens

In this work conditions for the reproduction of hybridoma technology, specially adapted to C. neoformans, for obtaining monoclonal hybridomas (McAb) to diagnostically significant antigens of C. neoformans, the infective agent of cryptococcosis, are presented. The advantages of using the short-time cycle of stimulation of mouse B lymphocytes with low doses of C. neoformans capsular polysaccharide and the effectiveness of the hybridization of mouse spleen cells with myeloma cells, line Sp2/0, are shown. Four lines of stable hybridomas, producing McAb to different epitopes of C. neoformans surface antigens, have been obtained. The specific activity of McAb has been studied in the indirect immunofluorescence assay, the cytochemical and solid-phase enzyme immunoassays (EIA). McAb 3E2, Cr2 and 2G9 have been shown to be suitable for use in diagnostic EIA systems.     

The detection of the herpes simplex virus 1 and 2 antigens in clinical specimens by using monoclonal antibodies

The possibility of using monoclonal antibodies (McAb), obtained earlier, for the detection of herpes simplex virus (HSV) in clinical specimens taken from sick and infected persons was studied. The examination of 90 persons revealed that the mixture of McAb 4A and 2C could effectively detect the presence of HSV antigen in the indirect immunofluorescence assay (IFA) directly in cells contained in cytological preparations (smears, scrapes, impressions) obtained from different organs of patients. The search of optimum combinations of McAb for the detection of HSV antigens by the method of the solid-phase enzyme immunoassay (EIA) was carried out. This study, made on purified HSV used as an experimental model, revealed that the maximum sensitivity could be achieved with the use of two McAb (4f6 and 7c4) out of three McAb (4f6, 7c4 and 3d10). The approbation of both variants of EIA on clinical specimens taken from 99 patients (blood clots, seminal fluid, scrapes of cervical canal cells, peripheral blood lymphocytes) showed that the addition of McAb 3d10 made it possible to detect 8 more positive specimens. 754 specimens from 337 patients were studied with the use of McAb-based EIA, and in 204 of these patients (61%) HSV antigen was detected. The results obtained with the use of our McAb were compared with the data obtained with certified commercial test systems. The coincidence of the EIA data with those obtained with the use of the Murex Wellcozyme HSV test system (UK) was registered in 75% of cases (in 15 out of 20 cases). The coincidence of the IFA data with those obtained with the use of the Sanofi test system (France) was observed in all 19 cases (100%).     

Plasmodium yoelii: characterization of a protective idiotype during malarial infection in mice

We have previously identified and characterized a monoclonal antibody (McAb 302) with potent passive protective activity in mice infected with Plasmodium yoelii, a murine malarial parasite which depends on antibodies for resolution. To further study the appearance and regulation of this antibody during infection, we prepared syngeneic monoclonal antibodies specific for idiotopes present on McAb 302. Three hybridomas were established which synthesized antibodies that bound only to the homologous idiotype but which did not recognize isotypic specificities. All three of these antibodies were found to recognize distinct 302 idiotopes and two of these were shown to be specific for determinants associated with the antibody combining site of McAb 302. One of these monoclonal anti-idiotypic antibodies was used to develop an enzyme-linked immunosorbent assay for the 302 idiotype. When serum samples taken at different times from mice undergoing a primary infection with P. yoelii were tested in this assay, the 302 idiotype could not be detected even though the host was mounting a significant humoral response to the 230-kDa antigen recognized by McAb 302. These studies suggest that the idiotype of the protective McAb 302 is not a predominant one involved in the resolution of a P. yoelii infection and that only some idiotypes of antibodies directed to relevant plasmodial antigens possess significant biological activity. Therefore, protective immunization with plasmodial antigens may require the elicitation of selected idiotypes. Attempts to alter the course of P. yoelii infection by preimmunization with monoclonal or polyclonal anti-idiotypic reagents were unsuccessful.     

The detection of two surface antigens on human lung macrophages using monoclonal antibodies

Monoclonal antibodies (McAb), designated AMH1 (IgM, lambda) and AMH2 (IgG1, Kappa), against specific surface antigens of human lung macrophages were produced by the fusion of the NS-1 plasmacytoma cell line with spleen cells from BALB/c mice immunized with bronchoalveolar lavaged (BAL) cells obtained from selected smoking subjects. The screening and characterization of these McAb were carried out employing cellular radioimmunoassay, flow cytofluorography, and immunohistochemical methods. These two antibodies specifically reacted with macrophages in the alveolar spaces and BAL fluids. AMH1 did not react with peripheral blood cells including freshly separated monocytes, cultured monocytes, lymphocytes, granulocytes, and platelets. In addition, AMH1 did not react with peritoneal exudate cells or pleural exudate cells. On the other hand AMH2 showed the dull-positive reaction with some monocytes and pleural exudate cells among above-mentioned cells. These two McAb seemed to detect cell surface antigens that are expressed by highly differentiated or mature macrophages compared to OKM1. These antibodies will allow not only better characterization of immune cells but also assessment of maturity of lung macrophages.     

Synthesis of monoclonal antibodies to tissue-specific antigens of human skin and thymus epithelium in polyclonal activation by pertussis toxin

Following the immunization of BALB/c mice with B. pertussis toxin, monoclonal antibodies (McAb) to the antigens of human epithelium, both dermal (the basal, superbasal or all epidermis levels) and thymic (the epithelium of the medullary zone, the cortical and medullary epithelium around Hassall's corpuscles), have been obtained. McAb have been obtained as the result of the polyclonal activation of autoreactive B-cells with B. pertussis toxin. McAb thus obtained can be used for the determination of the corresponding antigens in the epithelial tissues of the thymus and other organs in man, as well as for the diagnosis of tumors, histogenetically related to integumentary tissues of the epidermal genesis.     

抗黄曲霉毒素B1单克隆抗体的制备及特性

用杂交瘤技术制备了5株产生抗黄曲霉毒紊B₁单克隆抗体的杂交瘤细胞株。对其中之一AFB1－2H8进行了较系统的研究。AFB1一2H8属IgC3。纯化腹水抗体效价约5×10⁶。ELISA检测标准毒素的线性范围为0．5～50ng／ml。最低检出量为0．01ng／ml。该单抗与参试的其它黄曲霉代谢物的交叉反应系数为0～0．21,该抗体有较大的应用价值。     

Isolation of monoclonal antibodies to antigen Lyt-3.2

A hybridoma producing monoclonal antibodies (McAb) NATF9.9 (F9) was obtained from fusion of murine myeloma X63 and splenocytes of AKR mice immunized with a single intravenous injection of 5 X 10(7) thymocytes of CBA mice. F9 McAb were cytotoxic for 80% thymocytes, 10% splenocytes, 20% lymph node cells, 85% cortical and 32% medullary thymocytes of CBA, C57BL/6, BALB/c, DBA/2 and SJL but not for the cells of C58 and AKR mice. F9 McAb reacted only with T cells and did not react with B cells and EL4 thymoma cells (Thy-1.2+, Lyt-1+2-3-). The proportion of F9+ cells accounts for about 40% among T lymphocytes of the lymph nodes and spleen as tested by flow-type cytometry. Lymph node cells treated with F9 McAb plus complement completely lost their reactivity with rat anti-Lyt-2 McAb and only partly (by 30%) with anti-Lyt-1 McAb. The reactivity pattern of F9 McAb attests to their specificity for Lyt-3.2 antigen.     

Brain-associated glycoproteins expressed in bovine heart purkinje fibres

Summary The Purkinje fibres of bovine heart were investigated immunohistochemically by use of monoclonal antibodies with specificity against the glycoproteins Thy-1 and Gp120, expressed in human brain. The existence and expression in bovine tissues (brain and thymus) of antigens displaying similar properties and immunochemical crossreactivity with monoclonal antibodies against the human antigens were confirmed.Both these antigens, as identified by use of anti Thy-1 and anti-Gp120 monoclonal antibodies were found to be associated with the membranes of the impulse conduction system. The presence of the antigens was seen in areas facing other conduction cells. No parts of the cells facing the basal membrane of the fibres were stained. The continuous staining between the cells was different from that of desmosome related proteins. These findings may have physiological and functional implications and are interesting in relation to recent evidences suggesting that the conduction tissue might be a derivative of the neural crest.