Characterization of a novel monoclonal anti-myelin basic protein antibody: use in immunoblotting and immunohistochemical studies

Monoclonal antibodies (MAbs) to the myelin basic protein (MBP) were produced in CAF1 (BALB/c x A/J) mice immunized with intact bovine MBP. A number of MAbs were obtained, one of which was characterized in detail with respect to its isotype, antigenic determinant on the MBP, the spectrum of antigens with which it reacted in mouse brain, and its immunohistochemical staining characteristics. This monoclonal, GB-1 (an IgG1), recognized an epitope within residues 30-51 of bovine MBP. It also reacted with a family of MBP-related proteins present in brain homogenates of mice from 7-35 days. Immunohistochemically, GB-1 stained myelinated fibers and oligodendrocytes in the rodent CNS. A second monoclonal (GB-2, and IgM) was partially characterized. It reacted with intact MBP when it was immobilized to plastic or nitrocellulose, but it was not found to be useful for immunoblots or immunohistochemistry.     

Monoclonal antibody, KK1, recognizes human retinal astrocytes and distinguishes a subtype of astrocytes in mouse brain

Astrocytes exhibit a diverse morphology and numerous functions in the central nervous system as well as in the retina. In order to obtain markers for the analysis of astrocytes, we prepared monoclonal antibodies that recognized antigens specific to astrocytes. Monoclonal antibody (mAb), designated KK1, reacted with the processes of astrocytes in the nerve fiber layer and the ganglion cell layer in the human retina as detected by indirect immunofluorescence. Normal Müller cells, whose processes are localized vertically in retina, were not labeled by KK1 mAb. In mouse brain, KK1 mAb reacted specifically with astrocytes in the white matter, but not with those in the gray matter. Studies employing a high-resolution confocal laser scanning microscope and double-labeling with KK1 mAb and commercially available anti-glial fibrillary acidic protein (GFAP) mAb (GA5) revealed that KK1 mAb visualized the processes that were not recognized by anti-GFAP rnAb (GA5) in both human retina and mouse brain. In cultured mouse astrocytes. KKI mAb reacted only with anti-GFAP mAb (GA5)-positive cells, but a small percentage of anti-GFAP mAb (GA5)-positive cells were labeled with KK1 mAb. In addition, the subcellular distribution of the KK1 antigen in cultured astrocytes apparently differed from that of GFAP labeled by anti-GFAP mAb (GA5). The antigen that was purified from the normal mouse brain by KK1 mAb-conjugated beads reacted with anti-GFAP mAb(GA5) in immunoblotting. No reactivity of KK1 mAb was observed in immunohistochemical analysis in GFAP − / − mutant mouse brain. These results demonstrate that KK1 mAb specifically recognized an epitope of GFAP that did not react with other anti-GFAP mAb (GA5). Retinal astrocytes and a subtype of astrocytes in the white matter of mouse brain shared the epitope that was recognized by KKI mAb. KKI mAb might be a powerful tool to investigate a subtype of astrocytes.     

Endogenous benzodiazepine-like molecules in the human, rat and bovine brains studied with a monoclonal antibody to benzodiazepines

The anti-benzodiazepine monoclonal antibody 21-7F9 has been used for the identification and study of endogenous benzodiazepine-like molecules in the human, rat and bovine brains. A sandwich radioimmunoassay has been designed for the quantification of the membrane-bound endogenous benzodiazepine-like molecules. The localization of these molecules is not restricted to the brain tissue. They are also present in kidney, liver and spleen as well as in the neuroblastoma × glioma NG108-15 hybrid cells line. Immunoblots show benzodiazepine-like immunoreactivity in the membrane proteins of all these tissues. The membrane-bound benzodiazepine-like molecules are resistant to limited proteolysis of the membranes. Moreover, this treatment increases the binding of the monoclonal antibody 21-7F9 to the membranes, probably by exposing sites that normally are not accessible to the antibody. Immunocyto experiments show that benzodiazepine-like molecules are also present in samples of human cerebella that have been stored in paraffin since 1940, 15 years before the first chemical synthesis of benzodiazepines. The results indicate that the cerebellar benzodiazepine-like molecules recognized by the antibody are the product of biological (not chemical) synthesis. Benzodiazepine-like immunoreactivity has also been detected in NG108-15 cells that have been cultured for 3 months ib serum-free medium. These results suggest that the cells could biosynthesize benzodiazepine-like molecules.     

Monoclonal antibodies against human plasma protein C and their uses for immunoaffinity chromatography

Human protein C, isolated by conventional multistep methods, was used for immunization of mice. Monoclonal antibodies were prepared and screening of antibodies to human protein C was achieved using an immunoblotting technique. Five monoclonal anti-protein C antibodies were compared as affinity ligands. Different parameters were studied (adsorption capacity, specificity of adsorption, possibility of desorption under mild conditions) and two antibodies were selected. One antibody allows preparation of highly purified protein C in a single-step procedure from a fraction of plasma containing high levels of coagulation factors whereas the other can be used for preparation of protein C deficient plasma.     

Antigenic determinant recognized by a monoclonal antibody to human myelin basic protein

From an examination of electroimmunoblots and peptide maps, a mouse monoclonal antibody to human myelin basic protein MBP was shown to react with the amino acid sequence Ala-Ser-Asp-Tyr-Lys-Ser which is located in the C-terminal half of MBP. Although a completely different immunization schedule was used by Sires et al. (1981) they obtained a monoclonal antibody reacting with the same determinant. In contrast to results with other monoclonal antibodies to globular proteins (Todd et al. 1982) this monoclonal antibody seems to react with a sequential rather than a topographical determinant.     

The axon reaction in motor and sensory neurones of mice studied by a monoclonal antibody marker of neurofilament protein

Following unilateral sciatic nerve crush in mice, changes in the neurofilament content of neuronal perikarya were studied, using a monoclonal antibody to neurofilament protein (RT97). In the spinal cord, anterior horn motor neurones, normally unstained, showed a positive staining reaction with immunoperoxidase on the operated side. This reaction was short lived and maximal on the 11th post-operative day. In spinal ganglia, the proportion of positively staining sensory neurones showed an earlier but otherwise similar increase. In both cases, the response was well defined and contrasted with the changes on Nissl staining, which were markedly different in the two populations of neurones. In the nerve crush region, although regenerating axons were visible with silver staining only 5 days post-operatively, neurofilament protein was not demonstrated in these axons until several days later, after the peak perikaryal increase. These results suggest that an increase in perikaryal neurofilament protein is a consistent and quantifiable event following distal axon trauma, possibly indicating either synthesis of protein subunits or repolymerization of neurofilaments prior to their transport distally down the regenerating axons. The findings may be useful in identifying neurones with distal axon lesions in experimental and other neuropathological material.     

Characterization of gp 50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody

Several cell lines secreting monoclonal antibodies (Mabs) against a major forebrain synaptic membrane (SM) glycoprotein, gp 50, have been raised. Western blots show that the Mabs react with a polypeptide doublet of Mrs 49 and 45 kDa. These polypeptides exist solely in a concanavalin A (Con A) binding form. Removal of the Con A receptors by digestion with endo-beta-N-acetylglucosaminidase H (endo H) lowers the Mrs of the glycoprotein doublet to 36.5 and 34 kDa. Western blots of 2D polyacrylamide gels indicate that gp 50 exists in several isoforms. Solid phase radioimmunoassay (RIA) and Western blots of brain subcellular fractions show the antigenic material to be concentrated in the SM fraction, but to be present in much lower amounts in synaptic junctions and postsynaptic densities. Gp 50 appears to be brain specific. Regional distribution studies show that it is present in all brain regions but is two-fold concentrated in cerebellum, brainstem and midbrain compared to forebrain. Immunocytochemical studies of several brain regions show that gp 50-like immunoreactivity is neuron specific and is concentrated in selected neuronal species, particularly granule cells. In both cerebellar and hippocampal granule cells gp 50-like immunoreactivity is localized in the perikarya and primary dendrites. Though immunocytochemistry did not show staining of synaptic regions this may be due to masking of the reactive epitope. The results are discussed in terms of the molecular properties of gp 50 and its subcellular localization in brain tissue.     

抗人脑胶质瘤单克隆抗体的制备及识别抗原的提取

目的：制备新的抗人脑胶质瘤单克隆抗体（monoclonal antibody,McAb)，并提取其识别抗原。方法：以人脑恶性胶质瘤细胞系SHG－44为抗原免疫Balb/c小鼠，通过杂交瘤技术获得稳定分泌抗胶质瘤McAb的杂交瘤细胞株。以酶联免疫吸附测定及免疫组化等方法研究McAb的特性。通过亲和层析法提取该McAb识别的抗原。结果：得到1株稳定分泌抗体、效价高的杂交瘤细胞株，命名为SU－2000。鉴定表明该McAb属IgGl亚类，效价高，特异性高。识别的抗原位于胞膜，可能属于神经外胚层抗原；通过亲和层析法成功地提取出该抗原，十二脂硫酸3钠－聚丙烯酰胺凝胶电泳证实提取的抗原纯度高，分子量约为75kDa。结论：SU－2000 McAb及其识别抗原可进一步用于相关实验研究。     

Preparation of a novel monoclonal antibody specific for myelin basic protein phosphorylated on Thr

Phosphorylation is one of a number of post-translational modifications resulting in charge microheterogeneity of myelin basic protein (MBP). This phosphorylation is claimed to destabilise the compact myelin sheath by decreasing the interaction of membrane bilayers, thereby creating or maintaining pockets of cytoplasm. To further investigate and localise MBP phosphorylation to discrete regions of the myelin sheath we raised a monoclonal antibody with specificity for a known phosphorylation site in MBP. A synthetic peptide was made by Fmoc peptide chemistry and phosphorylation of Thr⁹⁸ was achieved on the resin by the global phosphorylation methodology, utilising dibenzyl-N,N-diethylphosphoramidite phosphitylation and t-butylhydroperoxideoxidation. The peptide coupled to tuberculin was used to immunise mice for monoclonal antibody production. The selected hybridoma (Clone P12) secreted an IgG2a antibody which reacted strongly with the phosphorylated immunogen and with phosphorylated fractions of bovine MBP obtained by ion exchange chromatography. The antibody had minimal reactivity with the unphosphorylated peptide; the same peptide phosphorylated at another site Ser¹⁰²; a preparation of unphosphorylated MBP obtained by ion exchange chromatography; and with an irrelevant phosphorylated protein (histone). Similar phosphorylation state-specific monoclonal antibodies could be made to recognise other specific phosphorylation sites in MBP or other proteins. It is planned to use these antibodies to quantify and locate the extent of MBP phosphorylation in normal and multiple sclerosis myelin.     

抗磷脂抗体、活化蛋白C抵抗与脑梗死的关系研究

目的探讨抗磷脂抗体（APA）、活化蛋白C抵抗（APCR）与脑梗死之间的关系及其临床意义。方法对157例脑梗死（CI）患者和82例正常对照组（NC）分别采用ELISA法检测ACA-IgG、IgM、IgA,血浆部分凝血活酶时间-狼疮抗凝物法（PTT-LA）筛选狼疮抗凝物（LA）,活化的部分凝血活酶时间±活化蛋白C（APTT±APC）检测APCR。结果CI组血清中ACAIgG、IgM、IgA以及LA的阳性率均明显高于NC组（P＜0．05）；CI组总APA阳性率（25．5％）显著高于NC组（4．9％）（P＜0．005）；CI组中APCR阳性率[5．1％（8/157）]与NC组[1．2％（1/82）]没有显著性差异（P＞0．05）；APA阳性脑梗死患者中APCR阳性率[7．5％（3/40）]与APA阴性脑梗死中APCR阳性率[4．3％（5/117）]也没有显著性差异（P＞0．05）。结论抗磷脂抗体与脑梗死密切相关,促使脑梗死患者血液呈高凝状态,是脑梗死发生的危险因素之一；APCR的发生在脑梗死患者中未见增加,APA不是导致获得性APCR的主要原因。     

Proliferative potential of meningiomas determined with the monoclonal antibody Ki-67

Summary In 30 meningiomas we investigated the proliferation rate of various subtypes with the monoclonal antibody Ki-67. Frozen sections were incubated with Ki-67 antibody using a modified Alkaline Phosphatase anti-Alkaline Phosphatase (APAAP)-technique and evaluation of proliferation rate was done by cell counting. Meningiomas of the meningiotheliomatous, fibrous and angioblastic subtype without atypical histological findings contained 1% or less proliferating cells. In recurent tumors, in transitional and in anaplastic meningiomas there is a marked increase of proliferating cells up to 20%. The distribution of marked cells varies in recurrent tumors and anaplastic meningiomas, and a focal proliferation of tumor cells was seen in meningiomas from transitional type. Immunohistological labelling of proliferating cells in meningiomas may allow a more precise prediction of the proliferation potential of each meningioma.     

Reactivity of a human monoclonal anti-GM1 and anti-GD1b IgM antibody with human neurons in cultures

A serum containing a monoclonal IgM λ with anti-GM1 and anti-GD1b activity was obtained from a patient with upper motor neuron syndrome. By indirect immunocytochemical techniques with double staining, the patient's IgM strongly stained membranes of neurons in primary cultures of fetal central and peripheral nervous system. It was cytotoxic for neurons in two human neuroblastoma established cell lines in a complement-dependent chromium release assay. These results are in keeping with the hypothesis of a direct pathogenetic role of such monoclonal anti-GM1 and GD1b IgM antibodies.     

Characterization of a monoclonal antibody to myosin specific for mammalian and human type II muscle fibers

We have characterized a monoclonal antibody (McAb), ALD-19, generated against slow myosin from chicken anterior latissimus dorsi (ALD) muscle for use in studies of human and animal muscle fiber types. This McAb bound selectively to the 200 kDa myosin heavy chain band in immunoblots against chicken, rat and human myosins and showed selective staining of A bands in the myofibrils. The reactivity of ALD-19 with various myosin types was quantitated by radioimmunoassays. Fiber type analysis revealed unexpected specificity of McAb ALD-19 for type II mammalian muscle fibers. This antibody should, therefore be useful for identification and quantification of normal type II fibers in human muscle biopsy specimens.     

Immunochemical characterization of a monoclonal antibody specific for Alzheimer''s disease associated protein

In this study, the monoclonal antibody PHF-1 which recognizes epitopes unique to Alzheimer's disease associated proteins (ADAP) has been characterized. Crossed affinity immunoelectrophoresis was used to estimate the binding constant for the interaction of PHF-1 with ADAP and to estimate the fraction of PHF-1 reactive protein. The binding constant of PHF-1 was determined to be 1.3 x 10(-8) M. Furthermore, the effect of dephosphorylation on the electrophoretic pattern of the PHF-1 reactive protein and the ensuing changes in its immunoreactivity were demonstrated.     

Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies

This study describes the identification and differentiation of neonatal rat retinal cells in monolayer cultures. A panel of monoclonal antibodies was used as a molecular probe of both cell type and developmental stage. Previously described cell-type specific monoclonal antibodies were used to label rod photoreceptors, horizontal cells, amacrine cells or ganglion cells. Two new antibodies that react with rat retina are described. The first, RET-G7, reacts with a cytoplasmic antigen of Muller glia, astrocytes and some horizontal cells. The second, RET-B2, reacts with bipolar cells and photoreceptor inner segments. Two main findings are presented. The first is that each of the major subclasses of retinal neurons have been unambiguously identified in these cultures. The morphology of some subclasses was very characteristic. All photoreceptors, as defined by reactivity with antibody RET-P1, were small spherical cells with one or fewer processes. Horizontal cells, as defined by reactivity with antibody B-1, were large with a characteristic multipolar network of processes. Bipolar and amacrine cells, on the other hand, were of similar size and could only be distinguished on the basis of immunocytochemical labeling. The second finding is that while RET-B2 antigen appeared on bipolar and photoreceptor cells after about 5 days in culture, several Muller cell and photoreceptor antigens were not expressed in monolayer cultures. The results suggest that the expression of some molecules in culture is the result of properties intrinsic to the cells whereas expression of others depends upon extrinsic factors or cell interactions that may not be present in monolayer cultures.     

Immunohistochemical staining of the human forebrain with monoclonal antibody to human choline acetyltransferase

Monoclonal antibody to human choline acetyltransferase (ChAT) was successfully produced from a mouse hybridoma cell line. The antibody was found to be of the IgM molecular species. By using this monoclonal antibody, immunohistochemical staining for ChAT was obtained on human brain sections. Only large sized cells were stained in the putamen and the substantia innominata. The specificity of the staining was comparable to that with polyclonal rabbit antibody to human ChAT produced by standard immunization procedures. No staining was observed when mouse monoclonal antibodies prepared against other human or bacterial antigens, or when normal mouse IgM, was employed.     

Characterization of novel glycoprotein components of synaptic membranes and postsynaptic densities, gp65 and gp55, with a monoclonal antibody

A monoclonal antibody, mab SMgp65, which recognises two major glycoprotein components of isolated forebrain synaptic subfractions has been raised. The mab has been used to study the cellular and subcellular localisation of these novel glycoproteins and for the partial characterisation of both molecular species. Western blots show that the mab reacts with two diffuse glycoprotein bands (gp) of apparent Mr 65,000, gp65, and 55,000, gp55. Both glycoproteins are membrane-bound, only detectable in CNS tissue and exist solely in a concanavalin A (con A) binding form. Digestion with endoglycosidase H lowers the Mr of both glycoproteins by some 5-7 kDa. Gp65 and gp55 are enriched in synaptic membrane (SM), light membrane (LM) and microsomal fractions. However, whilst gp65 is enriched in isolated postsynaptic densities (psds) gp55 is conspicuously absent from this fraction. Regional distribution studies show a marked variation in the level of gp65. Gp65 is concentrated in several forebrain regions notably cerebral cortex, hippocampus and striatum, is present only in low levels in cerebellum and is barely detectable in pons and medulla. In contrast gp55 is present in all regions studied, but is most concentrated in cerebellum. Immunocytochemical studies show intense staining of regions rich in gp65, but no staining of regions deficient in this glycoprotein. This suggests that the mab recognises gp65, but not gp55 in fixed tissue sections. Exposure of tissue sections to Triton X-100 increases the intensity of gp65-like immunoreactivity, but does not alter its pattern of subcellular distribution. Higher resolution studies show the immunoreactivity to be localised to subsets of neurites, many being axonal. The reaction deposits also extend into the synaptic region of the immunoreactive neurones. Cultured cerebellar granule cells, but not astrocytes express gp55. The results are discussed in terms of the molecular properties and localisation of these two novel glycoproteins.     

A monoclonal antibody to mammalian neurofilament protein stains somata and dendrites in gymnotid fish

Monoclonal antibody N210 (mabN210) recognizes the 210 kdalton neurofilament protein in mammals and gives characteristic immunocytochemical staining of neurofilament-rich processes. For example, in the cerebellum it recognizes myelinated axons and the calyx formed by basket cell axon collaterals. The distribution of mabN210 immunoreactivity was studied in the gymnotid brain (Apteronotus albifrons). In contrast to the mammalian distribution, mabN210 immunoreactivity was not found in most axons of the gymnotid brain. Instead, deposits of reaction product were present in the somata and dendrites of most neurons and were especially dense in those neurons with extensive dendritic trees, the Purkinje cells, pyramidal cells of the electrosensory lateral line lobe, the crest cells of the nucleus medialis and the pyramidal cells of the tectum. Electrosensory lateral line lobe pyramidal cells are known to contain few, if any, neurofilaments in their dendrites. Western blots of whole gymnotid brain proteins demonstrated that mabN210 recognizes two polypeptides apparent molecular weights 60 and 19 kdaltons. These proteins are thus antigenically similar to neurofilament protein and their expression in the gymnotid brain may be related to the peculiar dendritic branching pattern of Purkinje cells and similar cell types.     

Monoclonal antibody to brain cytoplasmic tetrodotoxin-sensitive protein detects an epitope associated with lymphocytes and involved in lymphocyte activation

We have previously derived a monoclonal antibody, BIP-4, which is specific to a mammalian brain protein representing a type of sodium channel. Here we show that this antibody detects an epitope associated with lymphocytes and that it triggers a proliferative response of the cells. BIP-4 epitope can be detected on both human peripheral blood and murine splenic mononuclear cells. Surface immuno-globulin-negative (i.e. resting T) lymphocytes are neither bound by the antibody nor proliferate to it. Proliferative response exerted in 7-day cultures of murine splenic mononuclear cells by recombinant interleukin-2 was blocked by BIP-4 antibody. We conclude that the epitope shared by a type of brain sodium channel protein and lymphocyte surface is involved in some, yet unrecognized, step of immune cell activation.     

Effect of the tricyclic antidepressant desipramine on protein kinase C in rat brain and rabbit platelets in vitro

Abstract  Protein kinase C (PKC), which participates in cellular responses to various stimuli such as hormones, neurotransmitters and growth factors, is essential for cell proliferation and differentiation. Desipramine, which is a tricyclic antidepressant, inhibited PKC activity in concentrations starting from 0.1 mmol/L in rat brain and its inhibitory effect on PKC activity did not involve competitive inhibition with calcium. However, rabbit platelets incubated with desipramine showed a biphasic dose—response change in PKC activity in vitro . The stimulatory effect of desipramine on PKC activity in rabbit platelets was observed over a concentration range of 0.5–2.0 mmol/L, and an inhibitory effect on PKC activity in platelets began to be seen at a concentration of 3.0 mmol/L desipramine. The stimulatory effects of desipramine and calcium on PKC activity in platelets appear to be occurring by the same mechanism. Several lines of evidence indicate that neurotransmitter uptake is linked to PKC activation. The present study supposes that the inhibitory effect of desipramine on neurotransmitter uptake may, at least in part, be associated with its inhibitory or stimulatory effect on PKC.