Application of liposomes to generation of monoclonal antibody to glycosphingolipid: production of monoclonal antibody to GgOse4Cer

Liposomes were applied to the immunization with GgOse4Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized with GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.     

A study of anti-group A streptococcal monoclonal antibodies cross-reactive with myosin

Anti-group A streptococcal monoclonal antibodies were obtained from BALB c/BYJ mice immunized with purified membranes from M type 5 Streptococcus pyogenes. Two of the anti-streptococcal monoclonal antibodies were previously shown to cross-react with muscle myosin. In this study the monoclonal antibodies were reacted with tissue sections of normal human heart and skeletal muscle. Antibody binding was estimated by indirect immunofluorescence and immunoperoxidase techniques. Both of the monoclonal antibodies (36.2.2 and 54.2.8) investigated in this report reacted with heart and/or skeletal muscle sections. When evaluated by immunofluorescence, monoclonal antibody 54.2.8 demarcated the periphery of cardiac striated muscle cells and reacted to a lesser degree with subsarcolemmal components. Monoclonal antibody 36.2.2 failed to react with heart sections, but both of the monoclonal antibodies reacted strongly with skeletal muscle sections. Results similar to those observed with indirect immunofluorescence were obtained with the immunoperoxidase technique. By Western immunoblotting and competitive inhibition assays, monoclonal antibodies 36.2.2 and 54.2.8 both were found to react with the heavy chain of skeletal muscle myosin. However, only 54.2.8 reacted with the heavy chain of cardiac myosin. The specificity of the monoclonal antibodies for subfragments of skeletal muscle myosin indicated that monoclonal antibody 36.2.2 was specific for light meromyosin fragments, whereas 54.2.8 reacted with both heavy and light meromyosin. The data demonstrated that two monoclonal antibodies against streptococci were specific for skeletal muscle and/or cardiac myosin and for subfragments of the myosin molecule. The reactions of the monoclonal antibodies with human tissue sections were consistent with the immunochemical reactions of the monoclonal antibodies with both denatured and native myosin.     

消减免疫法制备克伦特罗单克隆抗体

目的：制备克伦特罗（CL）单克隆抗体。方法：重氮化法制备克伦特罗完全抗原,消减免疫法免疫BALB／c小鼠,常规杂交瘤技术制备抗克伦特罗单克隆抗体。结果：消减免疫法使小鼠获得了对BSA的耐受,单抗筛选过程中获得了高达8．2％的阳性率。最后得到了针对CL的特异性抗体。结论：用消减免疫法制备针对小分子污染物的单克隆抗体,可减少在制备单克隆抗体时筛选的工作量,可增加获得预期抗体的机会。     

Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the binding of the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.     

金霉素单克隆抗体的制备及检测方法的建立

采用羰基二咪唑法,将半抗原金霉素(AM)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制备金霉素免疫抗原AM-BSA和检测抗原AM-OVA,通过紫外光谱扫描检测偶联产物。采用细胞杂交瘤技术,制备抗金霉素单克隆抗体杂交瘤细胞株,建立了金霉素竞争ELISA检测方法,其灵敏度达到50ng/ml,且呈现良好的线性关系(r=0.9812),并且与其他抗生素无交叉反应。     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies

A systematic approach for the determination of epitope specificities of monoclonal antibodies to a complex antigen system is described. After initial screening to identify antigen-binding monoclonal antibodies, one or more of the clones are isolated by limiting dilution cloning, grown in ascites, and the resulting antibodies secreted into the ascitic fluid are affinity purified on Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice were immunized with either purified carcinoembryonic antigen (CEA) or the CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned, resulting in 33 different clones. The antibodies produced by the different cloned hybrids and the remaining uncloned hybrids recognized a total of five different epitopes on CEA. All of the epitopes reside on the protein moiety of the molecule as determined by antibody binding to deglycosylated CEA. The monoclonal antibodies with five different epitope specificities were reacted with tissue sections of normal and cancerous tissues and with peripheral blood smears. Each of the five monoclonal antibodies reacted with tissue sections from colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted with normal liver tissue. Granulocytes in peripheral blood smears bound three antibodies strongly and one antibody weakly, and one antibody was not bound. One monoclonal antibody that reacted with normal liver tissue was not bound by granulocytes. The ability of these five monoclonal antibodies to differentially detect three different CEA-related antigens in normal and malignant tissues may have clinical utility.     

Characterization of baboon testicular antigens using monoclonal anti-sperm antibodies

Sperm antigens that appear during spermatogenesis in the baboon were identified by using three monoclonal antibodies generated in culture from mice immunized with baboon caudal epididymal spermatozoa. Antibodies BSA1 and BSA2 recognize trypsin-sensitive 84,000 and 45,000 dalton determinants that are restricted to the tail and anterior acrosomal regions of the sperm, respectively, as determined by Western blot and immunofluorescence techniques. The tail antigen absent in 2- and 3-yr-old baboon testes first appears in spermatid cells at about 4 yr of age. In contrast, the acrosomal antigen recognized by BSA2 is present in 3-yr-old primitive testicular germ cells. In the mature testis, the 45,000 molecular weight determinant is predominantly localized in the nucleus of late pachytene spermatocytes and round spermatid cells as observed via the avidinbiotin immunoperoxidase method. Antibody BSA3 reacted only with sailidase-treated sections of adult testis. This trypsin-resistant determinant, not expressed on testicular sperm, is recognized by antibody BSA3 only on epididymal sperm, thus indicating a post-testicular sperm modification.     

Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.     

Regulation of the BALB/c anti-p-azophenylarsonate antibody response by monoclonal anti-idiotype. II. Idiotope expression in serum and its regulation

Five murine monoclonal antibody (mAb) anti-idiotypes (id), shown in the accompanying report by binding studies to be reactive with five different id on a single member of the 5AF6 family of BALB/c antibodies against the p-azophenylarsonate (Ar) hapten, were used to examine the distribution of their recognized id among anti-Ar in BALB/c and other mouse strains immunized with keyhole limpet hemocyanin-Ar (KLH-Ar). Differences in id expression in BALB/c and other strains substantiate that all five monoclonal anti-id reacted with different id. This suggests that the anti-id repertoire for a single antibody molecule may be extensive. Two of the anti-id reacted with id that were found in virtually all KLH-Ar immunized BALB/c mice, but constituted only a subset (approximately 33%) of the antibodies representing the 5AF6 family. The other three anti-id reacted with id infrequently expressed among BALB/c anti-Ar. Other mouse strains producing 5AF6 family anti-Ar antibodies also produced antibodies recognized by mAb 2CB8 and 6BA1; however, the three id infrequently expressed in BALB/c mice were produced in higher quantities and in a greater percent of mice. Monoclonal anti-id were capable of suppressing a portion but not all of the 5AF6 family of anti-Ar antibodies. Four of the five anti-id suppressed a greater fraction of the 5AF6 family than that id represented in a normal immune response, suggesting that suppression was mediated via an id other than that recognized by these monoclonal anti-id. Overall, the results indicate that an extensive repertoire of anti-id can be produced against a single id antibody, but suppression induced by treatment with these anti-id in this model is presumably mediated via another as yet unidentified id determinant(s).     

玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立

玉米赤霉烯酮具有较强的生物毒性,检测谷物中的玉米赤霉烯酮在食品和饲料安全中具有重要的作用.将玉米赤霉烯酮与牛血清白蛋白的偶联物免疫BALB/c小鼠制备单克隆抗体,并建立基于单克隆抗体的酶联免疫法作为检测玉米赤霉烯酮的方法.结果共筛选到4株抗玉米赤霉烯酮单克隆抗体,3株抗体亚类为IgG1,1株为IgG2b.选择其中的一株杂交瘤细胞2C9制备小鼠腹水,纯化后测定了抗体效价为1/40 000.以此单抗建立的间接竞争ELISA方法,其半数抑制率(IC50)为1.90 ng/mL,检测限(IC10)为0.051 ng/mL,检测区间(IC20-IC80)为0.115-13.900 ng/mL;且对玉米赤霉烯酮有很好的特异性.回收率检测在样品含1.46-93.80 μg/kg时回收率为96.5％-113.0％.本实验建立的检测方法可用于多种谷物及饲料样本中玉米赤霉烯酮的检测.     

Localization of IpaB protein in Escherichia coli K-12 MC1061 strain carrying Shigella sonnei form I plasmid pSS120

We partially purified antigens which reacted with shigellosis convalescent-monkey antisera. Hybridomas, which were constructed from mice immunized by the antigens, produced monoclonal antibodies recognizing IpaB protein. Using the monoclonal antibody against IpaB, evidence indicating that IpaB proteins were localized on the cell surface of invasive bacteria (Escherichia coli K-12 MC1061 harboring pSS120 plasmid) was obtained by immunoelectron microscopy.     

Production and immunological characterization of a monoclonal antibody to Trichophyton quinckeanum: interaction with phosphorylcholine-bearing components

A monoclonal antibody (Tq-1) that interacts with the phosphorylcholine (PC)-bearing antigens of Trichophyton quinckeanum was produced by fusion of myeloma cells (JKAg-8) with spleen cells of BALB/c mice immunized with an alum-precipitated fraction of T. quinckeanum cytoplasmic antigen. It was characterized as an IgM class antibody by immunodiffusion using anti-Ig heavy chain specific reagents, ELISA using immunoglobulin-specific peroxidase-conjugated antibodies, and by gel filtration chromatography; it showed high affinity for Staphylococcus aureus protein-A. Interaction of Tq-1 with PC-like antigens of T. quinckeanum was demonstrated by inhibition studies using ELISA, immunoblotting, immunoprecipitation and immuno electron microscopy techniques. The binding activity of Tq-1 antibody with a range of dermatophyte proteins was completely inhibited by prior incubation with PC hapten. Moreover, dermatophyte antigens reacting with the monoclonal antibody reacted strongly with sera from chronically infected mice. Dermatophyte antigens derived from both young (24 h) and old (20 d) cultures reacted with Tq-1 and this binding was inhibited by PC, suggesting that Tq-1 target antigen PC appears at an early stage during fungal growth and remains throughout its life.     

Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

BALB/c mice were immunized with purified White spot syndrome virus (WSSV). Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E. coli. in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish. Western-blot suggested that all these monoclonal antibodies were against the conformational structure of VP28. The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling. These monoclonal antibodies could be used to develop immun-ological diagnosis methods for WSSV infection.     

Immunoprotective murine monoclonal antibodies specific for the outer-core polysaccharide and for the O-antigen of Escherichia coli 0111:B4 lipopolysaccharide (LPS)

The antigen specificity of two immunoprotective monoclonal antibodies derived from mice immunized with Escherichia coli 0111:B4 bacteria and boosted with purified lipopolysaccharide (LPS) were investigated. One of the antibodies, B7, was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunostaining to bind to the O-antigen containing LPS species, whereas the other antibody, 5B10, reacted with both O-antigen containing homologs and the O-antigen-deficient LPS. 5B10 did not bind to LPS from E. coli J5, an Rc mutant of E. coli 0111:B4 that lacks both the O-antigen and outer core sugars. 5B10 did not cross-react with LPS from several other E. coli strains. Thus 5B10 appeared to recognize a type-specific epitope in the outer core of LPS exclusive of Rc determinants. The monoclonal antibody specific for the polymeric O-antigen is of the IgG3 subclass, and the monoclonal antibody 5B10 specific for the outer core of LPS is an IgG2a. Although B7 and 5B10 were equally able to protect mice from a lethal challenge of E. coli 0111:B4 organisms, the outer core-specific IgG2a antibody was much more efficient at mediating the binding of human complement C3 than the O-antigen-specific IgG3 monoclonal antibody.     

Production and some properties of monoclonal antibodies against human pancreatic secretory trypsin inhibitor.

Hybridomas that secrete monoclonal antibodies against human pancreatic secretory trypsin inhibitor (PSTI) were established by fusion of spleen cells obtained from mice immunized with PSTI with mouse NS-I-Ag 4/1 myeloma cells. One of three resulting monoclonal antibodies (KN-1) was found to recognize the N-terminal moiety of the inhibitor, while the others (KN-2 and KN-3) reacted with other as yet undefined parts of the molecule. Trypsin inhibitory activity of PSTI treated with KN-1 monoclonal antibody was the same as that of PSTI itself, thus indicating no relationship between the N-terminal moiety of the PSTI molecule and its inhibitory activity. We further examined the applicability of one of the monoclonal antibodies (KN-1) for immunohistochemical study of human pancreatic cancer tissue including the normal as a model, and found granular staining of the cytoplasm of the normal acinar and duct cells and also of that of adenocarcinoma cells in formalin-fixed, paraffin-embedded tissue sections.     

Protection of mice against Clostridium chauvoei infection by anti-idiotype antibody to a monoclonal antibody to flagella

Abstract Polyclonal rabbit anti-idiotypic antibody (anti-Id) against the protective monoclonal antibody specific to the flagella of Clostridium chauvoei was produced, purified, and characterized. Anti-Id inhibited the binding of its related monoclonal antibody to the flagellar antigen, suggesting that the anti-Id bore an internal image of the flagellar antigen. When mice were immunized with anti-Id intraperitoneally, the survival rate increased significantly, compared with mice immunized with normal rabbit IgG ( P < 0.01), and specific anti-flagellar antibodies were induced.     

Monoclonal antibodies to hemagglutinin-neuraminidase and fusion glycoproteins of Newcastle disease virus: relationship between glycosylation and reactivity.

Eighteen hybridoma lines obtained by immunization of mice with Newcastle disease virus (NDV) lentogenic strain La Sota or velogenic strain Italien produced hemagglutinating monoclonal antibodies. The 18 monoclones were divided into four groups according to their reactivity toward native hemagglutinin neuraminidase protein (HN), nonglycosylated HN precursor, and heat-denatured HN blotted on nitrocellulose membranes. Only group II reagents were reactive toward their targets in all conditions tested. They were considered sequence-specific antibodies. Group I antibodies did not require glycosylation but lacked reactivity towards the denatured glycosylated antigen. Monoclonal antibodies from group III recognized only the native HN. Group IV was made up of a single monoclone that lacked reactivity with NDV Italien but recognized the La Sota strain in hemagglutination inhibition and enzyme-linked immunosorbent assays. Five hybridoma lines produced monoclonal antibodies which neutralized viral infectivity but failed to inhibit hemagglutination. One monoclonal antibody obtained after immunization of mice with NDV La Sota showed a low neutralization index versus NDV Italien. Four monoclonal antibodies derived from mice immunized with NDV Italien showed higher neutralization indices towards this strain. Neither the denatured F protein nor its nonglycosylated precursor was reacted against by the five monoclonal antibodies.     

Structural similarities among enzyme pterin binding sites as demonstrated by a monoclonal anti-idiotypic antibody

BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.     

Mapping the anatomy of the immunodominant domain of the human immunodeficiency virus gp41 transmembrane protein: peptide conformation analysis using monoclonal antibodies and proton nuclear magnetic resonance spectroscopy.

Thirty-six monoclonal antibodies from mice and three from rats were raised against a peptide corresponding to the immunodominant domain of the transmembrane gp41 protein of human immunodeficiency virus (HIV) type 1 (LGLWGCSGKLIC; amino acid residues 598 to 609). Of these, three monoclonal antibodies from the mice and one from a rat also reacted with the corresponding peptide derived from the HIV type 2 transmembrane gp41 protein (amino acid residues 593 to 603; NSWGCAFRQVC). Immunochemical studies using a variety of synthetic peptides indicated that the cross-reactivity was due to antibody binding to CSGKLIC of HIV type 1 or CAFRQVC of HIV type 2. Single amino acid substitutions for a cysteine at either the amino or the carboxy end of the peptide interrupted antibody binding, indicating that the site recognized was the Cys-XXXXX-Cys loop. Similar results were obtained when the 11-mer HIV type 2 gp41 peptide (amino acids 593 to 603) was inoculated into mice to raise monoclonal antibodies. In this instance, of 30 monoclonal antibodies developed, 4 reacted with both HIV type 1 and HIV type 2 peptides. The conformation of a seven-residue peptide, CSGKLIC, corresponding to residues 603 to 609 of the gp41 immunodominant epitope of HIV-1 was investigated by proton nuclear magnetic resonance spectroscopy. The immunologically active form of CSGKLIC contains an intramolecular disulfide bond and maintains a preference for a folded conformation, apparently including a type I reverse turn about the residues SGKL. No such preference is observed for the reduced form of the peptide, which contains two thiol groups. The presence of the disulfide bond is thus integral to the formation of the structure of the loop in solution. In agreement with this finding, elimination of the possibility of loop formation by substitution of S for C at the amino or carboxy termini of the 7-mer is accompanied by the failure of antibody binding to this peptide.     

Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody.

An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.