Immunocytochemical Examination of Neural Rat and Mouse Primary Cultures Using Monoclonal Antibodies Raised Against Pyruvate Carboxylase

Abstract: Pyruvate carboxylase (EC 6.4.1.1; PC) catalyzes the formation of oxaloacetate by energy-dependent fixation of CO₂ to pyruvate. The aim of the present work was to generate antibodies against PC and use them to localize PC in the cells of astroglia-rich and neuron-rich primary cultures derived from the brains of rats and mice. Mouse monoclonal antibodies raised against the enzyme were shown to be monospecific as indicated by immunoblotting. The staining of the cells for PC appeared in grains. These represent mitochondria, as PC is known as a mitochondrial enzyme. Immunocytochemical examination of astroglia-rich primary cultures of rat or mouse brain cells revealed a colocalization of PC with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were GFAP-positive cells showing no specific staining for PC, and vice versa. Also, in neuron-rich primary cultures PC was found only in the ∼10% GFAP-expressing astroglial cells contaminating the neuron-rich primary culture, whereas it was absent from the neurons identified by antibodies against neuron-specific enolase. These results suggest that PC is predominantly an astroglial enzyme and that astroglial cells play an important role in the intermediary and the energy metabolism of the brain.     

Purification of Cytosolic Malic Enzyme from Bovine Brain, Generation of Monoclonal Antibodies, and Immunocytochemical Localization of the Enzyme in Glial Cells of Neural Primary Cultures

Abstract: Cytosolic malic enzyme (EC 1.1.1.40) was purified from bovine brain 5,600-fold to a specific activity of 47 U/mg. The enzyme is a homotetramer with a subunit molecular mass of 60 kDa and an isoelectric point of 6.2. Mouse monoclonal antibodies raised against this enzyme were purified and shown to be monospecific, as indicated by immunoblotting. Immunocytochemical examination of rat astroglia-rich primary cultures at the light microscopic level revealed colocalization of cytosolic malic enzyme with the astroglial marker glial fibrillary acidic protein. Also, a colocalization with the oligodendroglial marker myelin basic protein was found. Neurons in rat neuron-rich primary cultures did not show positive staining. The data suggest that cytosolic malic enzyme is a glial enzyme and is lacking in neurons.     

Monoclonal Antibodies to and Immunoaffinity Purification of Choline Acetyltransferase from Bovine Brain

Three hybridomas producing monoclonal antibodies to bovine brain choline acetyltransferase (ChAT) have been established by fusion of the spleen cells from a mouse immunized with purified enzyme with myeloma NS-1 cells. All three clones produced IgGl antibodies that reacted with enzyme protein denatured with sodium dodecyl sulfate. By using one of the monoclonal antibodies, a rapid and efficient immunoaffinity purification procedure of bovine ChAT has been established. Immunoblot analysis and immunoaffinity purification indicated that bovine ChAT is a single 68-kilodalton protein. The monoclonal antibodies will offer us a good tool to isolate the cDNA clones encoding ChAT.     

应用A蛋白亲和层析法纯化单克隆抗体

应用Protein A亲和层析法,从采集的小鼠腹水中纯化出了抗凝血因子Ⅶ单克隆抗体,用SDS-PAGE和ELISA法分别检测了纯化后单克隆抗体的纯度及效价,结果显示,电泳为两条带,分别为免疫球蛋白G(IgG)的重链和轻链,纯化后的单克隆抗体纯度达到电泳纯,应用间接ELISA法测定腹水效价为1×10-7左右,与未纯化前无差异。结果表明,应用A蛋白亲和层析法能够得到纯度较高的单克隆抗体,适用于高纯度单克隆抗体的制备。     

Immunocytochemical and Biochemical Analysis of Carbonic Anhydrase in Primary Astrocyte Cultures from Rat Brain

Carbonic anhydrase (CA) was studied in primary monolayer cultures from neonatal rat cerebral hemispheres with both immunocytochemical and biochemical techniques. In such cultures, which consist predominantly of astrocytes, immunocytochemical staining for CA using antibody raised against the type II enzyme from rat erythrocytes resulted in positive staining of the flat, glial fibrillary acidic protein-positive, astrocytic monolayer. Smaller, process-bearing, round cells that grew on top of the astrocytes stained intensely for CA. We estimated that these cells represented 1% or less of the total cells in the cultures, and they have been identified by others as oligodendrocytes. The intensity of the staining of astrocytes for CA could be increased to that observed in oligodendrocytes when the astrocytes were made to round up and form processes by treatment with 2',3'-dibutyryl cyclic AMP. Enzymatic assays showed that CA activity of the cultures after 3 weeks of growth was 2.5- to 5-fold less than that found for cerebral homogenates from perfused 3-week-old rat brains. However, both activities were totally inhibited by acetazolamide with an I50 of 10(-8) M, confirming that both rat brain and the astrocyte cultures possess the high-activity type II enzyme. CA-II activity was unaffected by treatment of the cultures with a method reported to remove oligodendrocytes. Thus, the immunocytochemical and biochemical studies reported here demonstrate that astroglial cells in primary cultures from neonatal rat brain contain CA-II.     

Inhibition by 2-Deoxyglucose and 1,5-Gluconolactone of Glycogen Mobilization in Astroglia-Rich Primary Cultures

Abstract: The presence of glycogen in astroglia-rich primary cultures derived from the brains of newborn rats depends on the availability of glucose in the culture medium. On glucose deprivation, glycogen vanishes from the astroglial cultures. This decrease of glycogen content is completely prevented if 2-deoxyglucose in a concentration of > 1 m M or 1,5-gluconolactone (20 m M ) is present in the culture medium. 2-Deoxyglucose itself or 3- O -methylglucose, a glucose derivative that is not phosphorylated by hexokinase, does not reduce the activity of glycogen phosphorylase purified from bovine brain or in the homogenate of astroglia-rich rat primary cultures. In contrast, deoxyglucose-6-phosphate strongly inhibits the glycogen phosphorylase activities of the preparations. Half-maximal effects were obtained at deoxyglucose-6-phosphate concentrations of 0.75 (phosphorylase a, astroglial culture), 5 (phosphorylase b, astroglial culture), 2 (phosphorylase a, bovine brain), or 9 m M (phosphorylase b, bovine brain). Thus, the block of glycogen degradation in these cells appears to be due to inhibition of glycogen phosphorylase by deoxyglucose-6-phosphate rather than deoxyglucose itself. These results suggest that glucose-6-phosphate, rather than glucose, acts as a physiological negative feedback regulator of the brain isoenzyme of phosphorylase and thus of glycogen degradation in astrocytes.     

Specific Antiserum and Monoclonal Antibodies Against the Taurine Biosynthesis Enzyme Cysteine Sulfinate Decarboxylase: Identity of Brain and Liver Enzyme

Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, was purified 1,800-fold with a 1% yield from rat liver, where it was found to be 20-fold enriched compared with brain. The final fraction was homogeneous, as ascertained through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase HPLC. An antiserum was raised in the rabbit that (a) quantitatively immunoprecipitated CSD activity and (b) immunolabeled only one band (MW = 51,000) on an immunoblot from liver homogenate. Monoclonal antibodies were also raised that recognized the CSD protein and immunolabeled the same 51-kilodalton protein on an immunoblot from liver homogenate. In a brain extract, two CSD activities had been previously found and named CSDI and CSDII, according to their chromatographic elution patterns. We have compared the properties of CSDI from brain--the most likely enzyme involved in the biosynthesis of taurine in the brain, according to previous investigations-and CSD from liver: Both activities (a) were similarly eluted on ion-exchange and hydroxyapatite chromatographies, (b) showed the same elution pattern on gel filtration with an apparent native molecular weight of approximately 63,000, and (c) were immunoprecipitated in a strictly identical manner by the antiserum against liver CSD. Moreover, this antiserum as well as the monoclonal antibodies immunolabeled a single band (51 kilodaltons) on an immunoblot from brain CSD-enriched fraction or liver fraction. All these data show that CSDI from brain and liver CSD are the same monomeric enzyme. They also indicate that a specific antiserum against rat liver CSD has been raised that can be used for immunocytochemical visualization of CSD-containing cells in the brain.     

牛轮状病毒单克隆抗体的制备及其应用

以纯化浓缩的HN-7株牛轮状病毒(牛RV,从河南腹泻犊黄牛粪样中分离)作为免疫原,免疫Balb/c小鼠,取其脾细胞与同系小鼠骨髓瘤细胞SP2/0融合,经克隆化筛选出2株(2C_7和3C_9)具群特异性的单克隆杂交瘤细胞株。2C_7与3C_9的小鼠腹水单克隆抗体(McAb)与牛HN-7、BRV007、BRV014及NCDV,猪Na86,猴SA-11和人Wa株RV细胞培养物的间接ELISA反应效价均达1∶10~5,但它们的HI和NT滴度分别≤1∶8和≤1∶100。McAb的Ig类别和IgC亚类检测结果2C_7为IgG1,3C_9为IgG2b。分别用3C_9 McAb和多克隆抗血清(PcAb)包被微量反应板进行粪样中RV抗原检测,共检测犊黄牛腹泻粪样36份、婴幼儿腹泻粪样40份,McAb和PcAb两系统检测结果的符合率分别达到97.2％(35/36)和100％(40/40)。     

Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells

Glutathione reductase (GR) is an essential enzyme for the glutathione-mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000-fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double-labeling immunocytochemical staining in astroglia-rich and neuron-rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astroglia-rich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron-rich primary cultures, GAP43-positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.     

Monoclonal Antibodies to Rat Brain Acetylcholinesterase: Comparative Affinity for Soluble and Membrane-Associated Enzyme and for Enzyme from Different Vertebrate Species

Seven unique monoclonal antibodies were generated to rat brain acetylcholinesterase. Upon density gradient ultracentrifugation, immunoglobulin complexes with the monomeric enzyme appeared as single peaks of acetylcholinesterase activity with a sedimentation coefficient approximately 3S greater than that of the free enzyme. This behavior is consistent with the assumption of one binding site per enzyme molecule. Apparent dissociation constants of these antibodies for rat brain acetylcholinesterase calculated on the basis of this assumption ranged from about 10 nM to more than 1,000 nM. Some of the antibodies were less able to bind the membrane-associated enzyme that required detergent for solubilization than the naturally soluble acetylcholinesterase of detergent-free brain extracts. Species cross-reactivity was investigated with crude brain extracts from mammals (human, mouse, rabbit, guinea pig, cow, and cat) and from other vertebrates (chicken, frog, and electric eel). Three antibodies bound rat acetylcholinesterase exclusively; one had nearly the same affinity for all mammalian acetylcholinesterases investigated; the remaining three showed irregular binding patterns. None of the antibodies recognized frog and electric eel enzyme. Pooled antibody was found to be suitable for specific immunofluorescence staining of large neurons in the ventral horn of the rat spinal cord and smaller cells in the caudate nucleus. Other potential applications of these antibodies are discussed.     

猪生长激素及其单克隆抗体的制备与纯化

本文介绍一种简便快捷提纯猪生长激素的方法。通过杂交瘤技术,首次获得抗猪生长激素的单克隆抗体,并讨论和比较从腹水纯化单克隆抗体的各种方法。     

Rat 3-Hydroxyanthranilic Acid Oxygenase: Purification from the Liver and Immunocytochemical Localization in the Brain

3-Hydroxyanthranilic acid oxygenase (3HAO; EC 1.13.11.6), the biosynthetic enzyme of the endogenous excitotoxin quinolinic acid, was purified to homogeneity from rat liver and partially purified from rat brain. The pure enzyme is a single subunit protein with a molecular weight of 37-38,000. Kinetic analyses of both pure liver and partially purified brain 3HAO revealed an identical Km of 3 microM for the substrate 3-hydroxyanthranilic acid. Evidence for the identity of liver and brain 3HAO was further provided by physicochemical (electrophoretic behavior, heat sensitivity) and biochemical (pH dependency, activation by Fe2+) means. Antibodies were produced against the pure liver enzyme and the identity of liver and brain 3HAO substantiated immunologically in immunotitration and Ouchterlony double-diffusion experiments. Immunohistochemical studies using purified anti-rat 3HAO antibodies were performed on tissue sections of perfused brains and demonstrated a preferential staining of astroglial cells. Notably, the cellular localization of 3HAO in the brain appears to be in part distinct from that of quinolinic acid phosphoribosyltransferase, the catabolic enzyme of quinolinic acid. Pure rat 3HAO and its antibodies can be expected to constitute useful tools for the further elucidation of the brain's quinolinic acid system.     

Metabolism of Angiotensin Peptides by Neuronal and Glial Cultures from Rat Brain

The degradation pattern and rate of [Ile5]-Angiotensin (Ang) I, II, and III were studied in neuron-enriched and glia-enriched cells in primary cultures from rat brain. Metabolites were separated by HPLC, and their identities were evaluated by comparison of their retention times with those of synthetic Ang peptide fragments and by analysis of their amino acid composition. Major metabolites were identified as des-Asp1-[Ile5]-Ang I, des-Asp1-[Ile5]-Ang II, [Ile5]-Ang II (3-8) hexapeptide, [Ile5]-Ang II (4-8) pentapeptide, and [Ile5]-Ang II (5-8) tetrapeptide. Glia-enriched cells degraded [Ile5]-Ang I and [Ile5]-Ang III significantly faster than neuron-enriched cells, whereas no difference between the two types of cells was found in the degradation rate of [Ile5]-Ang II. Although the half-lives of [Ile5]-Ang I and [Ile5]-Ang III in neuron-enriched cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were not significantly different, neuron-enriched cultures from WKY rats metabolized [Ile5]-Ang II about 2.6 times faster than neuron-enriched cells derived from SHR.     

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity ^1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection ³. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop ^4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.The principle and protocol of this methodology have been described in the literature ^7,8. Additionally, alternate methodologies to isolate primary microglia are well described ^9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia ¹². However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture ^9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.     

抗人肺癌细胞单克隆抗体的制备及其特性的研究

用人肺鳞癌细胞LTEP-78细胞系免疫Blab/c小鼠获得3株抗人肺癌细胞的单克隆抗体杂交瘤系。其中BLTI-01株经六次克隆化培养,体外传代8个月以上。BLTI-01与白细胞抗原及血型抗原基本上无交叉反应;与骨髓细胞无交叉反应;与癌胚抗原和胎甲球蛋白不相关;与肺鳞癌、肺腺癌细胞系及部分其它肿瘤细胞呈阳性反应。     

Mitochondrial Malic Enzyme: Purification from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization in Neurons of Rat Brain

Abstract: To elucidate the cellular location of mitochondrial malic enzyme in brain, immunocytochemical studies were performed. For this purpose, mitochondrial malic enzyme was purified to apparent homogeneity from bovine brain and used for the immunization of rabbits. Subjecting the antiserum to affinity purification on immobilized antigen as an absorbent yielded a purified immunoreactive antibody preparation, which was characterized by probing cytosolic and mitochondrial fractions of bovine and rat brain in western blotting. As neither crossreactivity with cytosolic malic enzyme nor immunoreactivity against other proteins could be observed, the antibody preparation was found suitable for immunocytochemistry. By using sections of perfusion-fixed rat brain, considerable resolution was achieved at the light-microscopic level. Distinct and specific staining of neurons was observed; in contrast, no staining of astrocytes and possibly unspecific staining within the nuclei of oligodendrocytes were obtained. From these data, it is concluded that mitochondrial malic enzyme is located in neurons; however, in astrocytes, the enzyme appears to be either lacking or present at a much lower level. A protective role against oxidative stress in neurons is proposed for mitochondrial malic enzyme.     

Production and Characterization of Monoclonal Antibodies to Bovine Brain Succinic Semialdehyde Reductase

Abstract: Monoclonal antibodies against bovine brain succinic semialdehyde reductase were produced and characterized. A total of nine monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which two inhibited the enzyme activity and three stained cytosol of rat spinal cord neurons as observed by indirect immunofluorescence microscopy. When unfractionated total proteins of bovine brain homogenate were separated by gel electrophoresis and immunoblotted, the antibodies specifically recognized a single protein band of 34 kDa, which comigrates with purified bovine succinic semialdehyde reductase. Using the antisuccinic semialdehyde reductase antibodies as probes, we investigated the cross-reactivities of brain succinic semialdehyde reductases from some mammalian and an avian species. The immunoreactive bands on western blots appeared to be the same in molecular mass—34 kDa—in all animal species tested, including humans. The result indicates that brain succinic semialdehyde reductase is distinct from other aldehyde reductases and that mammalian brains contain only one succinic semialdehyde reductase. Moreover, the enzymes among the species are immunologically very similar, although some properties of the enzymes reported previously were different from one another.     

尿激酶型纤溶酶原激活剂单克隆抗体的生产及其在抗原纯化中的应用

从ＣＨＯ工程细胞培养上清初步纯化的ｕＰＡ免疫ＢＡＬＢ／ｃ小鼠,通过杂交瘤技术制备单克隆抗体细胞株,取其中一株３８－１－７株作高密度大量培养,细胞密度达１３．２×１０６／ｍＬ时,抗体滴度为１∶６１．４４×１０４。用自制的ｕＰＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱纯化抗体。抗体滴度提高２４３倍。纯化后的抗体与活化的Ｓｅｐｈａｒｏｓｅ４Ｂ珠交联,制成ＩｇＧ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析抗体柱,亲和力常数：１．２８×１０９（ｍｏｌ／Ｌ）－１,交联率：８３．５％。直接从培养上清纯化ｕＰＡ,纯度为９６．３％,回收率：８１．６％±１９,纯化倍数：５０倍左右,比活１．１１±０．２９×１０５。试验结果表明该法效果好,方法简单、操作方便、值得进一步研究和应用。     

Production and Preliminary Application of Monoclonal Antibodies Against Paulownia Witches' Broom MLO

Monoclonal antibodies against mycoplasma-like organisms (MLO) associated with paulownia witches’broom (PWB) were produced by using partially purified preparations from diseased paulownia. Splenic cells from immunized mice were fused with sp2/0murine myeloma cells. Screened by indirect ELISA using partially purified PWB-MLO and healthy paulownia extracts as detecting antigens, two hybridoma clones that stably secreted specific antibodies against PWB-MLO were obtained from 459 clones of four successful fusions. The monoclonal antibodies were isotyped and determined to be immunoglubin classes IgG2a and IgG3. Antibody titers of ascitic fluids were both over 1.6 × 104 assayed by indirect ELISA. Priliminary application on several specimens proved that they were the monoclonal antibodies against PWB-MLO.     

抗人beta-HCG单克隆抗体的制备及化学发光 检测方法的建立

目的：制备人绒毛膜促性腺激素（beta-HCG）单克隆抗体,建立人beta-HCG双抗体夹心CLIA 检测方法。方法：用人beta-HCG 抗原 免疫小鼠,通过细胞融合、筛选后得到杂交瘤细胞株,然后将细胞株扩大培养并纯化上清液获得抗体,测定抗体亲和力、特异性及 表位,最后建立双抗体夹心CLIA方法。结果：获得4 株抗人茁-HCG的杂交瘤细胞株(beta-1-1、beta-2-1、beta-3-1、beta-4-1)。用beta-1-1 和 beta-2-1 建立的双抗体夹心法的检测范围为0.5 mIU/mL-800 mIU/mL,灵敏度0.23 mIU/mL,检测结果的相对偏差均在± 10 %内,回 收率在90 %以上。结论：本研究最终成功制备了抗人beta-HCG mAb,建立了定量检测人beta-HCG 的双抗体夹心CLIA 方法,为 beta-HCG 检测及疾病的诊断奠定基础。