Development and characterization of monoclonal antibodies specific for the genus Listeria

Abstract Monoclonal antibodies were obtained by the classic hybridoma technique with lymphocytes of BALB/c mice immunized with formalin killed Listeria monocytogenes cells. Among 1000 hybridomas issued from the fusion, four monoclonal antibodies (mAbs A6 A E4, C10 A F7, G4 A D6, G7 A D5) gave interesting results. By Western-blot analysis with various soluble extracts of different Listeria species, the four mAbs reacted with two major antigens of 38 and 41 kDa, with all Listeria species tested. The mAb A6 A E4 is an IgG2b with κ light chains and reacted only with Listeria antigens without any cross reaction with other organisms tested by ELISA, dot-blotting and Western-blotting. With the same conditions, the three other mAbs reacted with Listeria and with other genus extracts, particularly with Streptococcus and Enterococcus . mAb A6 A E4-reactive antigens are proteins, and glycoprotein immunoassay indicated that the epitope is devoid of carbohydrate moiety. This mAb A6 A E4-reactive protein was neither expressed on cell surface nor released outside the bacteria; immunogold electron microscopy showed that these antigens were localized in the cytoplasma area.     

Chromosomal control of wheat gliadin protein epitopes: analysis with specific monoclonal antibodies

Summary The genetic relationships between small clusters of monomeric alcohol-soluble wheat (Triticum aestivum L.) grain storage proteins (gliadins) were studied using a panel of monoclonal antibodies and immunoblotting, ELISA, and RIA methods. Use of Chinese Spring nullisomic-tetrasomic lines showed that several narrow-specificity antibodies bound specifically to gliadins encoded by genes located on a single chromosome. In at least one case, antibodies bound to genetic blocks of gliadins, indicating that these block members have structural homology. However, often not all gliadins of a block were recognized by an antibody. For broad-specificity antibodies and some narrow-specificity antibodies, structural genes on several chromosomes were important. Studies with several primitive wheat species indicated that, while antibodies usually bound gliadins from the same genome in bread and primitive wheats, antibodies sometimes bound proteins of quite differing mobilities in the two wheat types. Use of antibodies to identify gliadin blocks is simpler than block analysis based on performing crosses, and should be of value in monitoring genotype/end-use quality relationships.     

Generation of monoclonal antibodies detecting specific epitopes in olfactory and respiratory epithelia

Summary Two panels of monoclonal antibodies have been generated, each panel having a distinct specificity for antigens located in the ciliary zone of either the olfactory or respiratory epithelium of rats. Tissue specificity was confirmed in enzyme-linked immunosorbent assays on membrane fractions from various tissues. During ontogeny, the expression of olfactory-specific antigens preceeds that of respiratory-specific antigens; this observation correlates with differences in the genesis of the respective cilia type and confirms that different molecular entities are recognized. A spatial segregation of immunoreactivity in the chemosensory epithelium was observed for one of the olfactory-specific monoclonal antibodies; negative zones were located in the dorsal recess of the nasal cavity and on the tips of the turbinates. Olfactory-specific antibodies reacted with distinct polypeptide bands on Western blots from olfactory ciliary preparations.     

Preparation and characterization of monoclonal antibodies which recognise different gibberellin epitopes

The production and characterization of high-affinity monoclonal antibodies (McAb) to gibberellins (GAs) is reported. Hybrid myelomas were derived from immunisations with conjugates in which immunogenic proteins were linked to GA₁ at carbon-3 and to GA₄ and GA₉ at carbon-17. A series of McAb which display specificities allowing recognition of, and the discrimination between GA₁, GA₂₀, GA₄ and GA₉ is described. These McAb can be used in quantitative immunoassays for underivatised GAs.Abbreviations BSA bovine serum albumin - FCS foetal calf serum - GA_n gibberellin A_n - IFA incomplete Freund's adjuvant - KLH keyhole-limpet haemocyanin - McAb monoclonal antibody (bodies) - PBS phosphate-buffered saline     

Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50–200 by in length and cloning these into the 5 terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.     

CK-MB特异性单抗的制备方法及其应用

本研究拟建立肌酸激酶同工酶MB(CK-MB)特异性单克隆抗体(m Ab)的研制方法,对抗CK-MB单抗进行评价分类及性质鉴定,并初步建立CK-MB定量检测试剂。以CK-MB抗原免疫BALB/c小鼠,利用常规单抗制备技术,使用间接和捕获ELISA差异筛选法筛选单抗。利用肌酸激酶同工酶(CK-MM/BB/MB)抗原对所制备单抗的抗原识别表位进行鉴定,另通过免疫印迹法及合成CK-MM、CK-BB差异性的线性表位肽鉴定对所制备的单抗进行评价分类。使用双抗体夹心ELISA方法筛选检测CK-MB抗原的配对m Ab,并初步建立CK-MB定量检测试剂。使用74例临床标本初步评价该试剂与罗氏试剂的检测一致性。最终,我们成功筛选到22株稳定分泌抗CK-MB抗体的杂交瘤细胞株,这些单抗可以分为线性、偏构象的CK-MB和CK-MM或者CK-BB交叉的单抗以及与CK-MB特异反应的偏构象型单抗,并使用偏构象型单抗研制出CK-MB定量检测试剂,该试剂与罗氏试剂相关系数r达到0.930 9。综上所述,本研究建立了研制CK-MB偏构象型特异性单抗的筛选方法,通过对所筛选的单抗进行分析鉴定并建立了CK-MB定量检测试剂,与罗氏试剂检测结果符合率高。     

Development of monoclonal antibodies for monitoring Aedes atropalpus vitellogenesis

Monoclonal antibodies to a mixture of Aedes atropalpus and A. aegypti soluble yolk proteins were produced by hybridomas between the fusion of P3X63.653 myeloma cells and splenocytes of immunized BALB/c mice. Ascites fluid collected from mice innoculated with cloned hybridoma cells contained high specificity and affinity to the soluble yolk proteins of both Aedes species. Seven different hybridoma lines produced antibodies with specificity to both A. atropalpus and A. aegypti and one cell line produced antibodies monospecific to A. aegypti soluble yolk proteins. Monoclonal antibodies specific to A. atropalpus vitellin and vitellogenin were characterized by a combination of gel electrophoresis, western blotting and immunohistochemical staining. An indirect double antibody sandwich enzyme-linked immunosorbent assay was developed using a mixture of the seven hybridoma antibodies to A. atropalpus vitellin for monitoring vitellogenin levels in individual mosquito haemolymph samples. With this procedure, the peak period of vitellogenin synthesis in A. atropalpus was found to be 18 to 30 h after adult eclosion.     

抗酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris)单克隆抗体制备

【背景】基于本实验室已经建立的脂环酸芽孢杆菌检测和鉴定方法工作基础之上,以期建立具有很好的经济价值和实用价值,且更为方便、快捷、准确、特异、灵敏的检测方法。【目的】实现对果汁生产中脂环酸芽孢杆菌从原料到成品的快速检测和鉴定。【方法】采用杂交瘤细胞技术,以Alicyclobacillus acidoterrestris(ATCC49025)免疫BALB/c小鼠,用建立的间接ELISA方法筛选杂交瘤细胞,得到了3株能稳定分泌A.acidocaldarius抗体的杂交瘤细胞株,其中两株为Ig G1亚类,并对其进行生物学特性的鉴定。【结果】得到的两株单抗3F7和9C4是针对不同的抗原位点,且多次传代后稳定性基本保持不变;特异性实验表明两株单抗均不与A.acidocaldarius(NCIMB11725)、Bacillus cereus(ATCC11778)、Bacillus subtilis(ATCC11774)、A.cycloheptanicus(ATCC49029)等发生交叉反应。【结论】3F7和9C4这两株单抗可以进一步用于检测胶体金试纸条的研制。     

Granulocyte/macrophage-colony-stimulating factor augments the induction of antibodies,especially anti-idiotypic antibodies,to therapeutic monoclonal antibodies

A group of 86 patients with advanced colorectal carcinoma were treated with the mouse (m) (IgG2A) or chimeric (c) monoclonal antibody (mAb) 17-1A. Prior to therapy, no patient had detectable levels of antibodies to mAb17-1A. All mmAb17-1A-treated patients (n=76) developed antibodies against both idiotypic and isotypic determinants. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of anti-idiotypic (ab₂) as well as anti-isotypic antibodies. Of the mmAb17-1A-treated patients, 16 developed type I allergic reactions. These patients had significantly higher concentrations of anti-(mouse Ig) antibodies than patients without type I reactions. Of these 16 patients, 5 had received mmAb17-1A alone; they constituted 9% of this group (5/56). The remaining 11 patients had been given mmAb17-1A together with GM-CSF, and represented 55% of this treatment group (11/20). The difference was statistically significant (P<0.001). Of 10 patients, 9 (90%) treated with cmAb17-1A and GM-CSF developed ab₂. The ab₂ concentration in this patient group was significantly lower compared to those treated with mmAb-17A. Anti-(mouse Ig) antibodies caused clinical symptoms requiring therapeutic intervention in fewer than 10% of the patients treated with mmAb17-1A alone. With the addition of GM-CSF, the antibody concentration as well as the frequency of allergic side-effects calling for medical action increased significantly. Significantly more patients with a high ab₂ concentration (at least 15g/ml) 1 month after completion of mAb therapy responded to mAb treatment as compared to those with a low ab₂ concentration (P<0.05). Moreover, patients with a high ab₂ concentration (at least 15 g/ml) had a median survival time of 15 months while those with a lower concentration survived for a median time of 9 months (P=0.01).     

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, ) at ratios of 1.2–35 mol dye/mol antibody and 9.2.27 (IgG_2a, ) at 0.6–6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.This work was supported by funds of the Division of Urologic Surgery, Department of Surgery, University of Pittsburgh; by an NSF Center Grant (MCB-8920118) to the Center for Light Microscope Imaging and Biotechnology, Carnegie-Mellon University; and by intramural funds of the VAMC     

An extended set of monoclonal antibodies to pectic homogalacturonan

Three novel rat monoclonal antibodies, designated LM18, LM19 and LM20, were isolated from screens for binding to Arabidopsis thaliana seed coat mucilage. The binding of these antibodies to mucilage subject to enzyme and high pH pre-treatments and to a series of model homogalacturonan-rich pectins with defined levels of methyl-esterification indicated their recognition of pectic homogalacturonan epitopes. The binding capacities of these monoclonal antibodies to cell walls in sections of tobacco stem pith parenchyma were also differentially sensitive to equivalent treatments with high pH buffers and pectate lyase. The epitopes bound by these antibodies display some similarities and some differences to the epitopes recognized by the previously isolated and established pectic homogalacturonan probes JIM5 and JIM7.     

Monoclonal antibodies specific to plant tubulin

Summary Tubulin was isolated from mung bean seedling by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4 B) and ion exchange (DEAE-Sephacel) chromatography. Using SDS-PAGE together with blotting with subunit-specific antitubulins, mung bean tubulin has been shown to consist of two -tubulin subunits, MBT₂ and MBT₃, of which MBT₃ is a minor component, and one -tubulin, MBT₁.Monoclonal antibodies were produced by fusing mouse myeloma cells and spleen cells from a Balb/c mouse immunized with mung bean tubulin. Antibody producing cell lines were identified by an ELISA assay and immunofluorescence microscopy and subsequently cloned by limiting dilution.The properties of monoclonal antibody (K₄E₇G₃) were examined by Western blot analysis and indirect immunofluorescence studies. K₄E₇G₃ reacts with MBT₂ and MBT₃ -tubulin subunits of mung bean tubulin, but not with MBT₁ -tubulin nor with the - and -subunits of sheep brain tubulin. Peptide fragments transferred onto nitrocellulose papers were treated with K₄E₇G₃ and with other monoclonal antibodies that are known to be specific to the -subunit of yeast tubulin and - or -subunit of mammalian brain tubulin. MBT₂ and MBT₃ are shown to be similar but not identical and are quite different from MBT₁ and the -subunit of sheep brain tubulin. K₄E₇G₃ reacts with peptide fragments in MBT₂ and MBT₃ that are not found in digests of brain tubulin, and that are either not reactive or only weakly reactive to the antibodies to yeast and brain -tubulin. It is concluded that K₄E₇G₃ and another monoclonal antibody, K₂D₇B₈, which has similar properties, are relatively specific for plant -tubulin.In indirect immunofluorescence studies on a wide range of plant cells, the epitopes recognised by these monoclonal antibodies are shown to be present in all types of microtubule array that were investigated. The spindle, preprophase band, phragmoplast and interphase microtubules were clearly observed in onion and mung bean root tip cells. Reactions with spindle microtubules ofFunaria spore mother cells and with the blepharoplast and flagella microtubules of fern spermatozoa are also seen. However, studies using several animal cell lines have shown that K₄E₇G₃ and K₂D₇B₈ do not give positive immunofluorescent localization of animal microtubules, correlating with the inability of K₄E₇G₃ to react with brain tubulin subunits on Western blot analysis.     

Specificity of monoclonal antibodies generated against arabinoxylans of cereal grains

Xylooligosaccharides substituted by arabinose have been produced by degradation of wheat flour arabinoxylans with an endoxylanase. These oligosaccharides were coupled to carrier proteins (KLH and BSA) and three monoclonal antibodies were isolated. The specificity of antibody recognition was studied using arabino-xylo-oligosaccharides exhibiting different pattern of substitution by arabinose.

ELISA competition tests and molecular modelling suggest that the conformation adopted by beta-(1→4) linked xylose residues is an antigenic determinant recognized by the different antibodies. Arabinose was not specifically involved in the interaction of antibody and epitope.     

Domain structure of neurofilament subunits as revealed by monoclonal antibodies

Monoclonal antibodies have been prepared against purified neurofilament (NF) subunits (NF68, NF150, and NF200). From 25 fusions, several hundred strongly positive antibodies have been obtained. Among them are antibodies against the specific subunits as well as antibodies recognizing common antigenic determinants. These have all been characterized according to the following properties: ELISA (enzyme-linked immunosorbant assay) testing against each subunit, immunoblots against enriched neurofilament preparation, immunoblots of cyanogen bromide or chymotrypsin-treated neurofilaments, immunofluorescence with PC12 cells, and immunohistochemistry of cerebellum. Whereas the antibodies against the NF68 and NF150 appear to react with single cyanogen bromide fragments, the antibodies against the NF200 react with multiple cyanogen bromide fragments. These data are consistent with the hypothesis that the NF200 is partially composed of several repeated structural determinants. Furthermore, all of the antibodies that react with the NF200 recognize the solubilized "sidearm" domain from limited chymotryptic digestions. The locations of the common and variable domains of the three subunits are discussed in light of these results.     

百合无症病毒单克隆抗体的制备及检测应用

用百合无症病毒(Lilysymptomlessvirus,LSV)免疫的BALBC鼠脾细胞与SP20鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗LSV单克隆抗体(MAb)的杂交瘤细胞(2A2、5H9、5H2和5E12),并分别制备它们的单抗腹水。4株单克隆抗体腹水间接ELISA效价达10-6,5H9和5E12的抗体类型及亚类均为IgG1,而2A2和5H2均为IgG3,4株单克隆抗体的轻链均为κ链。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)检测LSV的方法。病叶作1300倍稀释、提纯LSV病毒浓度为18ngmL(每孔的病毒绝对量为1.8ng)时,该方法仍能检测到病毒。利用ACP-ELISA检测了田间样品,发现LSV在百合上发病很普遍。     

Preparation and identification of anti-rabies virus monoclonal antibodies

To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection,anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotei...     

Mouse monoclonal antibodies specific for endothelial cells

Summary Balb/c mice were immunized with a human endothelial cell pool. Spleen cells were then fused with a NS-0 hybridoma cell line. A number of hybridomas secreted antibodies that reacted with the immunizing endothelial cell pool as well as with every other tested umbilical cord vein~derived human endothelial cell. These monoclonal antibodies also stained pig, rabbit and ox aortic endothelial cells indicating their specificity for this cell type. Five of 16 monoclonal antibodies additionally reacted with human fibroblasts (HFIB). The produced monoclonal antibodies did not recognize FVIIIRAG or MHC determinants. They can therefore be regarded as additional and reliable markers for endothelial cells in vitro.     

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies

Abstract Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis , is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.     

Principles of the therapeutic use of monoclonal antibodies in oncology

Monoclonal antibodies have reached the stage of therapeutic agents, mostly in oncology, as illustrated by their wide use in lymphoma, breast cancer or colorectal cancer. The unravelling of their mechanisms of action and their interactions with their cellular receptors allows us to engineer new classes of therapeutic antibodies with increased efficacy. The identification of some of the tumour escape mechanisms may also help to define new approaches for patient selection and immunomonitoring. The present review addresses these various aspects.