Monoclonal antibodies demonstrate regional specificity in the spinal funiculi of the chick embryo

To explore the extent and significance of chemical diversity of neurons, monoclonal antibodies were generated that bind to the chick embryonic central nervous system with varying degrees of regional specificity. Mice were immunized against a homogenate of optic nerves from 8-day chick embryos. Antibody was screened by an indirect fluorescence immunohistochemical method using frozen sections of embryonic neural tissues. From 3 fusion experiments, 58 lines of hybrydoma were cloned. Twenty-three monoclonal antibodies were studied in detail, and here reported, for their characteristic staining patterns in the spinal cord of 6-day embryos. The majority of the antibodies bound preferentially to different subregions of spinal funiculi, in which glial elements are still undeveloped. At least 6 subregions could be distinguished in the funiculi, probably corresponding to the developing spinal tracts. For 8 of the antibodies, immunoreactive polypeptides were identified in an electrophoretic analysis.     

猫小脑皮质结构的年龄相关性变化

目的：对青年和老年猫小脑皮质结构的年龄性变化进行比较。方法：动用Nissl染色显示小脑皮质神经元,免疫组织化学方法显示胶质纤维酸性蛋白免疫阳性（GFAP-IR）星形胶质细胞和神经丝蛋白免疫阳性（NF-IR）结构。显微镜下观察测量小脑皮质厚度和细胞密度。结果：与青年猫比较,老年猫小脑皮质总厚度及分子层厚度显著下降,颗粒层厚度明显增加,各层神经元密度明显降低;颗粒层中GFAP-IR细胞密度显著增加,阳性反应增强;老年猫蒲肯野细胞（PC）NF免疫阳性树突分支明显减少。结论：衰老过程中小脑皮质神经元丢失和PC中NF阳性树突减少,可能会导致老年小脑皮质接受和整合信息的功能降低,而星形胶质细胞活动增强对皮质神经元可能起保护作用。     

Polyreactivity of human monoclonal antibodies: Human IgM anti-rhesus monoclonal antibodies are frequently polyreactive

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.     

Identification of a novel allergen molecule from Dermatophagoides by monoclonal antibodies

Six different monoclonal antibodies (MAb) have been obtained which bind to components contained in extracts from Dermatophagoides. One out of the six MAb recognized molecules displaying IgE binding ability. This MAb (MADP-1) immunoprecipitated allergenic polypeptides of 42 kDa and 30 kDa from 125I-labeled extracts of D. pteronyssinus and D. farinae respectively. Purified allergen preparations from both extracts have been obtained by affinity chromatography using a column of purified MADP-1 coupled to Sepharose. The purified fractions partially compete the binding of specific IgE contained in sera from sensitized patients to the whole extracts, in a radioallergosorbent inhibition test.     

Conformational changes induced by hapten in murine monoclonal antibodies to dinitrophenyl groups--the analysis by temperature-perturbation spectroscopy

Complement receptor for C3b (CR1) on erythrocytes was investigated in various diseases by immune adherence hemagglutination (IAHA) using aggregated human IgG. In normal controls, 21 out of 312 (6%) revealed defective CR1 reactivity, and there was no difference in the prevalence of defective CR1 reactivity between female (11/157, 7%) and male (10/155, 6%). Among diseases examined significantly high prevalence of defective reactivity of CR1 on erythrocytes was seen in systemic lupus erythematosus (SLE) (22/30, 73%) and malignancy of hematopoietic system, especially in acute myelogenous leukemia (AML)(6/11, 55%).     

Purification of murine IgM monoclonal antibodies by hydroxylapatite chromatography

Summary A simple method is described for the isolation of murine monoclonal immunoglobulin M using hydroxylapatite chromatography.     

Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies

The complement (C)-activating capabilities in human serum of 32 mouse and 10 mouse/human chimeric MoAbs of different isotypes, and their fragments, were tested in vitro. Activation of C via the classical pathway (CP) was performed in 1% factor D-deficient serum in gelatin containing Veronal buffer in the presence of calcium and magnesium (GVB⁺⁺), while activation of the alternative pathway of C (AP) was assessed in 10% Clq-depleted serum in the presence of 5 mm MgCl₂ in GVB⁺⁺. The C-activating ability of MoAbs was expressed relative to the degree of activation of complement by aggregated IgG for the CP and relative to mouse IgG 1 for the AP. All of seven mouse IgG2a MoAbs were potent activators of the CP. The results of CP activation by IgG1, IgG2b and IgG3 isotypes were different for individual MoAbs. Only three (two IgG 1 and one IgG3) of 32 mouse MoAbs were potent activators of the AP. IgG2a and IgG2b were relatively poor AP activators. There were a few MoAbs which activated both the AP and CP. Of 10 chimeric MoAbs, two IgG1, one IgG2 and one IgG4 were poor or non-activators of the CP. On the other hand, IgG2 and IgG4 were good AP activators. IgG3 was the most potent AP activator. Most of the F(ab')₂ fragments were activators of the AP and displayed no activation of the CP. Fc fragments only activated the CP. whereas Fab'did not activate the CP or the AP. These studies suggest that the route of complement activation by class and subclass MoAbs can not always be predicted in advance and based only on their subclass identity.     

Biosynthetic radiolabeling and immunoprecipitation of monoclonal antibodies

Summary A simple method is described for the radiolabeling in vitro and immunoprecipitation of monoclonal antibodies without altering their native structures.     

Developmental changes in concentrations and distributions of neurotrophins in the monkey cerebellar cortex

Neurotrophins are involved in the survival, differentiation, migration and neurite outgrowth of various neuronal populations. Neurotrophins and their receptors are widely expressed in the developing cerebellum of various experimental animals. To gain some insight into the possible roles played by these molecules in monkey cerebellum, we examined the protein levels of BDNF, NT-4/5 and NT-3 and distributions of those neurotrophins and TrkC, a high affinity receptor for NT-3, in the cerebellum of developing macaque monkeys using ELISAs and immunohistochemical methods. We found that the level of BDNF increased during development, while the level of NT-3 was higher during embryonic stages and decreased toward adulthood. The level of NT-4/5 increased from embryonic stages to infant stages and gradually declined with age. Among the three neurotrophins, BDNF immunoreactivity was found in all kinds of cerebellar neurons, including all inhibitory interneurons, throughout the postnatal periods examined, indicating that BDNF may be an essential factor for the maintenance of cerebellar neural functions. The Bergmann glial fibers and the internal part of the external granule cell layer were strongly NT-3 immunopositive at the early postnatal stages, and more weakly immunoreactive toward adulthood. In addition, we found that the premigratory precursors of the granule cells were TrkC immunopositive at early postnatal stages. These findings suggest that NT-3 in Bergmann glial fibers may be involved in the migration of the premigratory granule cells.     

Production and characterization of monoclonal antibodies cross‐reactive with major aflatoxins

Monoclonal antibodies cross‐reactive with four major aflatoxins (AFs) were produced by fusion of P3/NS‐1/1‐AG4–1 murine myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with aflatoxin B3‐hemisuccinate conjugated to bovine serum albumin. Six stable clones were obtained. Isotyping by enzyme‐linked immunosorbent assay (ELISA) revealed that the antibodies produced by all but two of the clones were of the IgG₁ type. Of the remaining clones, one produced IgG₂₁, and the other IgA. Competitive radioimmunoassay using tritiated AFB₁ as the marker ligand revealed that two clones produced antibody that cross‐reacted well with both AFB₁ and AFG₁; one clone produced an antibody that had good specificity toward AFB₁. The relative cross‐reactivities (RCR) of antibody produced by clone 575B8F12 for AFB₁, AFB₂, AFG₁, and AFG₂ were 100, 5, 153, and 6, respectively. The RCR of antibody produced by clone 575G4H7 for the above AFs were 100, 40, 153, and 40, respectively. The RCR of antibody produced by clone 585D4D6 for the above AFs were 100, 40, 152, and 23, respectively. Antibodies produced by the other three clones were inadequate for immunoassay because their affinities for the AFs were 100 times less than the three clones described above.     

Purification of murine monoclonal antibodies by affinity chromatography using protein A-Sepharose

Summary A simple method is described for the isolation of murine monoclonal immunoglobulin G subclasses using protein A-Sepharose affinity chromatography.     

骨桥蛋白单克隆抗体的制备及其特异性分析

为了研制骨桥蛋白(OPN)特异性单克隆抗体(mAb),并鉴定其特异性。本研究在毕赤酵母中表达具有良好免疫原性的重组人OPN蛋白的基础上,用重组人骨桥蛋白(rhOPN)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,间接ELISA筛选杂交瘤细胞,并结合免疫印迹对抗体的特异性进行鉴定,通过竞争抑制试验对单克隆抗体识别的抗原位点进行分析。结果共获得4株能够识别OPN不同抗原位点的mAb,亚类测定显示,3株为IgG1,1株为IgG2a。这些mAb能与重组人OPN特异性结合。本研究的四株抗体中,只有4G2B5能够检出与肿瘤转移密切相关的OPN-c的条带,预示该抗体可用于判断肿瘤预后和高转移肿瘤的临床检测。本研究成功获得了针对骨桥蛋白的特异性mAb,同时为进一步研究OPN蛋白的结构和功能提供了重要工具。     

The value of immunohistological screening in the production of monoclonal antibodies

This paper describes an immunoperoxidase technique for labelling cryostat tissue sections which is routinely used in the authors' laboratories both in the initial screening of hybridoma culture supernatants, and also during the subsequent cloning and growth of antibody-secreting cell lines. The technique can readily be performed on 100 samples in less than 3 h and is free of non-specific background labelling. The staining pattern of a monoclonal antibody on a single tissue section allows semiquantitative assessment of its reactivity against a wide variety of tissue constituents and is thus inherently much more informative than conventional screening techniques (such as binding assays) which yield only a single numerical value for each test performed. In consequence it is often possible to identify the probable specificity of a new monoclonal antibody at the primary screening stage. A further important advantage of immunohistological screening is that it detects antigens on cells or other tissue structures which do not readily enter suspension and also antibodies against nuclear and cytoplasmic antigens.Examples of monoclonal antibodies analysed by immunohistological screening include antibodies against C3b receptor, HLA-DR, factor VIII-related antigen, human syncytiotrophoblast, dendritic reticulum cells and a proliferation-associated cell surface glyco-protein.     

Establishment of hybridomas secreting monoclonal antibodies to Chlamydia psittaci

Eleven stable anti-Chlamydia hybrid clones by fusions between X63-Ag8653 myelomas and immune splenocytes from Chlamydia psittaci immunized F1 (C57BL6×BALB/c) mice have been established which react with the 12 reference Chlamydia strains (seven C. trachomatis and five C. psittaci. Ten of these monoclonal antibodies are directed against the genera-specific epitope (40,000 MW component) for which prolonged immunization seems to be responsible.     

Expression of myelin basic protein (MBP) epitopes in human non-neural cells revealed by two anti-MBP IgM monoclonal antibodies

Two monoclonal antibodies (1H6.2 and 45.30) were raised against MBP purified from human brain under experimental conditions that allowed MBP to retain binding to surrounding myelin lipids (human lipid-bound MBP (hLB-MBP)). 1H6.2 and 45.30 MoAbs were selected on the basis of their different binding properties to: hLB-MBP, human lipid-free-MBP (hLF-MBP) and bovine lipid-free-MBP (bLF-MBP). Although the isotype of both MoAbs was IgM, their specificity, as tested in ELISA assays against chemical haptens and unrelated protein antigens, was restricted to MBP. 1H6.2 and 45.30 MoAbs stained MBP from human brain white matter tissue extracts, as well as bLF-MBP, in Western blot assays. Both MoAbs stained oligodendrocytes and myelin in immunohistochemical analysis of white matter from human brain. Tissue sections from human peripheral nerves were labelled by 1H6.2 only, however, demonstrating that the MoAbs recognize two different epitopes. Epitopes recognized by 1H6.2 and 45.30 MoAbs were also expressed by a wide array of human non-neural cells of either normal or pathological origin, as evidenced by cytofluorimetric assays. In particular, MBP epitopes (MEs) were expressed by lymphoid cells as well as by cells which play a pivotal role in immune homeostasis and in the immune response, such as thymic epithelial cells and professional antigen-presenting cells. Both MoAbs were efficiently internalized by cells from a human B cell line, suggesting trafficking of MEs along the endocytic pathways. These findings support hypotheses regarding the role of MEs expressed by non-neural cells in establishing self-tolerance and/or in triggering the immune response against MBP antigen.     

Reduction of CAG expansions in cerebellar cortex and spinal cord of DRPLA

Recent studies have identified an unstable expansion of a CAG repeat in a gene located on chromosome 12 as a cause of dentatorubropallidoluysian atrophy (DRPLA). To investigate whether the somatic heterogeneity may relate to the selective neuronal damage caused by the disease, genomic DNA from various tissues of an autopsied patient with DRPLA was examined to compare possible variations of expanded CAG repeats for the disease. Although the size of the expanded CAG repeat from many organs was almost the same as that from peripheral lymphocytes, those from cerebellar cortex and spinal cord were unexpectedly reduced and numbers of peaks within an expanded allele were relatively strict. These results suggest that the CAG repeat is not simply expanded in the genome of the tissues that are most involved in DRPLA, but that another mechanism might be responsible for the specific neuronal death.