Production and characterization of murine monoclonal antibodies to Histoplasma capsulatum yeast cell antigens

Four monoclonal antibodies (MAbs) were produced by immunizing mice with a disrupted yeast cell homogenate of Histoplasma capsulatum. MAbs 1 and 2 reacted only with the yeast cell antigens of H. capsulatum and Blastomyces dermatitidis, whereas MAbs 3 and 4 showed broader cross-reactivity. MAb 3 cross-reacted with B. dermatitidis, Paracoccidioides brasiliensis, Sporothrix schenckii, and Candida albicans, and MAb 4 cross-reacted with B. dermatitidis, C. albicans, Coccidioides immitis, Aspergillus fumigatus, and Mycobacterium tuberculosis. All four MAbs exhibited unique specificity when reacted with three different strains of H. capsulatum (G217B, A811, and P-IN). MAb 1 belonged to the IgG2b subclass, MAb 3 belonged to the IgG1 subclass, and MAbs 2 and 4 belonged to the IgG3 subclass. MAbs 1, 2, and 3 formed bands in the Western immunoblot assay; the two dominant distinct bands had apparent molecular masses of 72 and 62 kilodaltons.     

Preparation and characterization of murine monoclonal antibodies toSalmonella typhimurium K-antigen

Monoclonal antibodies toS. typhimurium K-antigens representing IgG1 immunoglobulins with a “kappa”-type light chain are prepared and characterized. They are highly active in indirect enzyme immunoassay with purified K-antigens and whole fixedS. typhimurium cells and virtually do not cross-react with O-antigen of the same bacterial species or withE. coli antigenic structures. The kinetics of binding and ability to agglutinate whole bacterial cells are studied and epitope analysis is carried out, which shows that antigenic determinants ofS. typhimurium K-antigen qualitatively differ from those of O-antigen of the same bacterium. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 6, pp. 633–635, June, 1994 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Rapid production of monoclonal antibodies to T lymphocyte functional antigens

Brief immunization of rats with mouse lymphoid cells was combined with the rat/mouse hybridoma technology and functional hybridoma screening to yield a rapid method for the production of monoclonal antibodies (MAb) against functionally important T lymphocyte cell surface antigens. Two protocols were used. In one, rats were immunized once with mouse thymocytes followed by fusion and screening of the hybridomas for interference with the thymocyte co-stimulator (interleukin 1) assay. The resultant hybridomas included producers of MAbs against the L3T4-antigen (inhibitory), the Ly-1-antigen (stimulatory), and the Thy-1-antigen (inhibitory?). In the second protocol, rats were immunized twice with a T cell hybridoma. The resultant hybridomas were screened for inhibition of polyclonal T cell activation, induced by an anti-Thy-1 (MAb G7). A panel of MAbs against the Thy-1 antigen with different reactivity profiles was generated by this procedure. Most of the MAbs were of the IgM class. Short-term immunization may lead to less selection of response to highly immunogenic determinants than a protocol involving several boosters. Thus, this alternative may be useful for producing MAbs against rare or weakly immunogenic cell surface molecules, as suggested by the ease with which we were able to make MAbs against the L3T4-molecule.     

The isolation and characterization of murine monoclonal antibodies directed to polymorphic epitopes on HLA antigens

Twenty-two murine monoclonal antibodies directed to distinct polymorphic epitopes on HLA class I or class II antigens have been isolated and characterized using a simple protocol for fusion and hybridoma selection. Thirteen MAbs directed to class I antigens are reported here for the first time. The majority of these MAbs reacted with multiple specificities, often revealing a surprising sharing of epitopes. MAbs directed against single classically defined alloantigenic specificities were also obtained.     

抗人滋养细胞单克隆抗体的制备及其免疫生物学特性鉴定

研制抗人滋养细胞单克隆抗体并分析其免疫生物学特性。方法采用滋养细胞免疫Ｂａｌｂ／ｃ小鼠;用细胞ＥＬＩＳＡ法鉴定ｍＡｂ的特异性;用补体依赖的溶细胞作用测定ｍＡｂ的细胞毒性。结论利用细胞免疫法所获该组ｍＡｂ所针对的抗原表位,可能是诱发免疫性流产的滋养细胞膜特异性抗原。     

Preparation and characterization of monoclonal antibodies to rotavirus

By utilizing a strain of cultivable simian rotavirus (SA-11) as an immunizing antigen, we prepared 4 clones of mouse-mouse hybridoma, namely C127, C139, C172, and C214 which secreted monoclonal antibodies against the immunogen itself, SA-11 and also against other group A strains such as Wa and S2. Western blot analyses revealed that all of these antibodies are directed against VP6, a 42 kDa major inner capsid protein of group A rotavirus. Competitive experiments suggested that C127, C172 and C214 recognized three distinct epitopes on VP6, while C139 appeared to react with an epitope at or near the same epitope recognized by C172. We developed a two-step ELISA with excellent sensitivity and specificity for rotavirus detection by utilizing C127 and/or C214 as a capture antibody and rabbit anti-rotavirus conjugated with horseradish peroxidase as a probe. Also, when both monoclonal C127 capture antibody and polyclonal rabbit anti-rotavirus-HRP were incubated with rotavirus simultaneously in a one-step assay, equivalent sensitivity and specificity were observed. The data show that these generated anti-rotavirus antibodies can be utilized effectively as reagents for the detection of human rotaviruses in stool specimens.     

B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage-associated antigens

Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double-) immunofluorescence staining of single cell suspensions from lymphoid organs. HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compartment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes. HIS22 and HIS24 detected B lineage-associated antigens expressed by major subpopulations of B cells. HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone. In accordance with this, substantial proportions of sIg+ cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively). In bone marrow the vast majority of cytomplasmic mu+ pre-B cells were HIS14+ and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis. The antigens recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: none of the mAb bound to plasma cells. As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22, or HIS24. In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig- cells. HIS22 did not bind to Ig- peripheral lymphocytes. Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches. As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen. In addition, the antigens recognized by HIS14, HIS22 and HIS24 co-capped with the leukocyte common antigen. This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen.     

Studies of murine malaria antigens using monoclonal antibodies

A panel of ten monoclonal antibodies made againstPlasmodium chabaudi andPlasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs toP. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted withP. chabaudi antigens. Of the MAbs toP. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

抗赭曲霉毒素A单克隆杂交瘤细胞系的建立及特性

用杂交瘤技术制备了4株稳定产生抗赭曲霉毒素A单克隆抗体的杂交瘤细胞株,命名为1A9、5F2、5G5和6G11。1A9、5G5和6G11的Ig亚类为IgG1,5F2的亚类为IgG2a。抗体腹水效价为500000～1000000。6G11检测纯毒素的线性范围为2～500ng/ml,最低检出量为1ng/ml。交叉反应的结果还表明,该单抗与共试的其他结构类似物无反应,具有较高的特异性。     

抗人尿激酶单克隆抗体制备和鉴定

用人高分子量尿激酶(HMW-UK)为抗原,通过杂交瘤技术获得了两株阳性杂交瘤细胞(2B8、3A2)。采用葡萄球菌 A 蛋白(SPA)亲和层析或 Water's650快速蛋白分离系统纯化抗 UK 单克隆抗体(McAb).3A2McAb 为 IgG 1亚类,特异地识别 HMW-UK 和低分子量 LMW-UK,抑制 UK 活性,表明3A2McAb 所作用的位点为 UK 的 B 链,即活性中心。EIA 测定3A2McAb 与 UK 的亲和常数为8.43×10~8M~(-1)。2B_8McAb 亦为IgG_(?)亚类,特异地识别 HMW-UK 及其氨基端1～135氨基酸片段(ATF),不抑制活性,EIA 测定2B_8McAb 与UK 的亲和常数为2.8×10~9M~(-1)。2B_8McAb 可用于导向溶栓的研究以及 UK 与其受体间反应的研究。     

Preparation and characterization of polyclonal and monoclonal antibodies to polyamines

In order to develop an immunocytochemical method suitable for the study of the cellular localization and intracellular distribution of polyamines we have prepared and characterized antibodies to polyamines. Artificial immunogens were prepared by coupling putrescine, spermidine and spermine to a carrier protein. Immunogens containing bovine serum albumin as a carrier protein were used to immunize rabbits (polyclonal antibodies) and mice (for the production of Mabs). The specificity of the antibodies was tested in an ELISA system utilizing antigens synthesized from thyroglobulin and one of the polyamines. Polyclonal antibodies to putrescine, spermidine and spermine were obtained. However, these antibodies showed a variable degree of cross-reactivity to the polyamines not used for immunization. Two hybridoma cell lines were developed. The first, MPut88, selectively produces a Mab to putrescine, the second, MSpm/d88 produces a Mab which recognizes spermine and spermidine but does not react with putrescine.     

Generation and serological characterization of murine monoclonal antibodies against O antigens from Acinetobacter reference strains.

O-antigen-specific monoclonal antibodies were generated against Acinetobacter strains from international type culture collections and characterized by enzyme immunoassay and Western and colony blotting. The antibodies aid in the further completion of an O-serotyping scheme for Acinetobacter and, due to their high specificity, are especially useful to all working with these strains.     

Preparation and characterization of antisera and monoclonal antibodies to haloperidol

For the first time a library, of monoclonal antibodies (MoAbs) to the butyrophenone haloperidol (D-2 antagonist) has been prepared. Synthesis of a haloperidol derivative suitable for chemical coupling to a protein carrier via oxobutyric acid produced an immunogen which was used to develop two polyclonal antisera and twelve MoAbs specific for the hapten. Our library of MoAbs can be grouped into three classes; 1) high affinity and specificity for free 3H-haloperidol, 2) moderate affinity with significant cross-reactivity to other butyrophenone ligands, and 3) a group which binds poorly to free 3H-haloperidol but instead recognizes the ligand only when it is coupled to carrier protein. Clone (189(2)-6) was found to have the highest equilibrium binding affinity (Kd = 4 nM) and is far more specific than the currently available antisera to haloperidol. This MoAb has significantly lower affinity for all of the common metabolites of haloperidol. This capability makes 189(2)-6 a candidate for further development with regard to use in clinical radioimmuno-assays of therapeutic drug levels. In addition, one of the anti-haloperidol Moabs (190(2)-6) binds more tightly to spiperone than to haloperidol and displays a qualitative correlation in the rank order of neuroleptic binding affinity for a limited series of analogs when compared to membrane bound D-2 receptor binding.     

Preparation and characterization of monoclonal antibodies to human neuron-specific enolase

Neuron-specific enolase (NSE) has been increasingly recognized as a marker for neuroendocrine tumors including small cell carcinoma of the lung (SCCL). To prepare monoclonal antibodies (MAbs) specific for human NSE, we first developed a simple method of purifying NSE by direct chromatofocusing of a crude extract of human brain tissue. BALB/c mice were then immunized with our preparation of NSE, and MAbs against NSE were generated utilizing a hybridoma technique. The antibodies were screened against both NSE and non-neuronal enolase (NNE) by a solid-phase radioimmunoassay (SPRIA). After cloning and subcloning of hybridomas, two groups of anti-NSE MAbs were identified by SPRIA. One group reacted specifically with NSE but not with its isoenzyme NNE, irrespective of whether antigens were glutaraldehyde fixed or unfixed. A second group reacted with both NSE and NNE when the latter were glutaraldehyde fixed, but surprisingly with neither antigen in the absence of fixation. Group I antibodies were further characterized by immunoblotting, and by immunocytochemistry of normal brain and liver sections and sections of SCCL. The results further supported the specificity of group I antibodies for NSE. These MAbs have potential utility in the diagnosis and management of neuroendocrine tumors, and in further understanding the biology of NSE.     

Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens

We have prepared 2 mouse monoclonal antibodies which react with differentiation antigens on guinea pig lymphoid cells. Monoclone 5AB2 recognizes an antigen expressed on both T and B lymphocytes and absent on macrophages. It has proven useful in the preparation of populations of antigen presenting cells which are free of T and B lymphocytes. The second monoclonal, 8BE6, is specific for peripheral T cells and 10% of thymocytes. It reacts with a 68,000 dalton molecule which is also expressed on the guinea pig B cell leukemia, EN-L2C. 8BE6 has proven to be lytic for peripheral T cells in the presence of rabbit complement and has been used to deplete T cells from heterogenous cell populations.     

Preparation and characterization of monoclonal antibodies to substance P.

A series of mouse hybridomas that produced monoclonal antibodies reactive with Substance P was prepared in an attempt to find a surrogate NK-1 receptor. When the binding profile of one particular antibody, SubP14.36.1, was compared in a rank order plot with NK-1 receptors against various agonists and antagonists of Substance P, a high correlation was found between these two binding structures. In addition, this monoclonal antibody inhibited a functional assay for Substance P. This result indicated that the pharmacological effects of this peptide can be blocked by an antibody (Ab).     

抗人gp130分子单克隆抗体的制备和鉴定

ｇｐ１３０可与许多细胞因子受体（ＩＬ－６Ｒ,ＩＬ－１１Ｒ,ＯＳＭＲ,ＬＩＦＲ,ＣＮＴＦＲ和ＣＴ－１Ｒ）组成受体复合物,并在这类细胞因子的信号转导中的起着重要的作用。方法以痘苗病毒为载体构建获得的可溶性ＣＤ１３０（ｓＣＤ１３０）为免疫原,通过融合的方法。得到４株稳定分泌抗ＣＤ１３０单克隆抗体（ｍＡｂ）Ｌ２,Ｌ４ａ,Ｌ４ｂ和Ｌ５）的杂交瘤细胞株。结果本组ｍＡｂ对靶细胞具有识别特异性,不但可识别相对分子     

Multiple assay characterization of murine monoclonal antimelanoma antibodies

Selected murine monoclonal antimelanoma antibodies have been extensively evaluated using multiple radioimmunoassay methods, immunoprecipitation and immunohistochemistry. Use of this approach has permitted more complete definition of the specificity of these reagents and provided information regarding the nature and distribution of the respective tumor associated antigens (TAA). Several patterns of reactivity were identified. Some of the reagents were highly reactive with melanomas but also with a variety of tissues of nonmelanoma origin. Others were less highly reactive but of greater specificity for melanoma. Finally, certain of the reagents were poorly reactive in the assays utilized or demonstrated assay-dependent reactivity. None of the included monoclonal antibodies appeared to detect TAA restricted in distribution solely to melanoma.