Murjne Anti-Idiotypic Monoclonal Antibodies to Syngeneic Antihuman High Molecular Weight-Melanoma Associated Antigen Monoclonal Antibodies: Development,Characterization, and Clinical Applications

Following a discussion of the rationale underlying the selection of human melanoma to test the usefulness of anti-idiotypic monoclonal antibodies in the therapy of solid tumors, the development of the anti-idiotypic monoclonal antibody MoAb) MF11–30 is described. This antibody recognizes a private idiotope within the antigen-combining site of the immunizing antihuman high molecular weight melanoma-associated antigen MoAb 225.28. The results of a phase I clinical trial with the MoAb MF11–30 in patients with advanced melanoma are described. The lack of toxic effects and the minor responses in six patients suggest that these studies should be extended to a larger number of patients with an emphasis on the analysis of the mechanisms underlying the clinical response.     

Production and Characterization of Monoclonal Antibodies Against Myelin-Associated Glycoprotein

Monoclonal antibodies against myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen lymphocytes from BALB/c mice immunized with human myelin-associated glycoprotein purified from CNS myelin. Three groups of antibodies were identified: IgG antibodies recognizing the polypeptide moiety and IgG and IgM antibodies recognizing the carbohydrate moiety of the intact molecule. Properties of these antibodies were examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the immunostaining technique using human CNS and peripheral nerve myelin, and ganglioside fractions isolated from human brain and peripheral nerve, and with immunohistochemical staining of human peripheral nerves. Part of human peripheral blood mononuclear cells was stained with the antibodies against the carbohydrate moiety, but not with IgG antibodies recognizing the polypeptide moiety. Natural killer activity was partially reduced after treatment of human peripheral blood lymphocytes with an IgM antibody and complement in vitro. The possibility that anti-myelin-associated glycoprotein antibodies might play a role in the pathogenesis of demyelinating diseases through modification of natural killer activity is discussed.     

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.     

Preparation and Characterization of Monoclonal Antibodies to Serotonin Binding Protein

Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 X 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44-10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44-10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44-10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with MAB 44-10 were located immunocytochemically in sections of rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

高亲和力抗有机磷杀虫剂单克隆抗体的制备与鉴定

制备抗有机磷杀虫剂单克隆抗体。采用人工合成带有特殊功能基团的半抗原,将其与载体蛋白偶联,以半抗原偶联物为免疫原,免疫BALBc小鼠,取免疫小鼠的脾细胞与小鼠骨髓瘤细胞融合,HAT选择、有限稀释法克隆化,建立分泌抗机磷杀虫剂单克隆抗体的杂交瘤细胞系。ELISA间接法和ELISA竞争抑制法检测抗体滴度,非竞争抑制法检测抗体亲和常数。结果获得9株分泌抗机磷杀虫剂单克隆抗体的杂交瘤细胞系,其中7株为型特异性,2株为组特异性;Ig亚型均为IgG1;亲和常数为407×108±043M和127×109±024M。结论为人工人工合成的、含特殊功能基团的半抗原,与载体蛋白偶联后,做免疫原,可用于制备高滴度的抗有机磷杀虫剂单克隆抗体,这种单抗可用于机磷杀虫剂残留物的免疫化学检测 。     

Y盒结合蛋白1单克隆抗体的研制、表位测定及其免疫学应用

建立稳定分泌抗人Y盒结合蛋白1单克隆抗体(anti-YB-1 mAb)的杂交瘤细胞株,鉴定其表位与免疫学应用。将重组YB-1蛋白免疫BALB/c小鼠,取脾细胞与Sp2/0骨髓瘤细胞融合。经ELISA法筛选鉴定、定株后采用腹水诱生法制备anti-YB-1 mAb;Protein G亲和层析法纯化mAb,ELISA法测定mAb效价、亚型及相对亲和力。采用抗原表位预测法鉴定anti-YB-1 mAb识别表位所在区域。Western blot和免疫组化鉴定mAb识别内源性YB-1的特异性。经筛选鉴定获得2株稳定分泌anti-YB-1 mAb的杂交瘤细胞(1-D9,3-E8);腹水抗体效价均≥1×10-6,亚型均为IgGl;1-D9和3-E8单抗识别表位分别位于(134-160aa)与(266-303aa)肽段。Western blot、免疫组化结果证实anti-YB-1 mAb能特异性识别内源性YB-1。该研究为YB-1免疫学定性、定量检测方法的建立、肿瘤靶向抗体治疗及进一步探讨YB-1的生物学功能奠定了基础。     

Production and Characterization of Monoclonal Antibodies to the Major Peripheral Myelin Glycoprotein P0

Several monoclonal antibodies were generated against the major glycoprotein P0 of human peripheral nervous system myelin. Antibodies were selected for their reactivity with P0 in Western blots. The antibodies were of the immunoglobulin G subclass and reacted with the glycopeptidase F-treated P0, indicating that the reactive epitope resides in the protein backbone. In fresh frozen and paraffin-embedded sections of central and peripheral nervous system of rat and human, P0 antibody 592 reacted with myelin sheaths of peripheral, but not central, nervous system.     

Production and Characterization of Monoclonal Antibodies to Peripheral and Central Nervous System Myelin

Monoclonal antibodies against P0, myelin basic protein, or myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso-type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin-associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin-associated glycoprotein. Antibodies were characterized in solid-phase radioimmunoassay for their species cross-reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin-producing cells, since in both cases the concentration of myelin proteins is low.     

Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase

Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.     

番茄花叶病毒单克隆抗体的制备及检测应用

用番茄花叶病毒(ToMV)免疫的BAL B/c鼠脾细胞与SP2/0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ToMV单克隆抗体(Mab)的杂交瘤细胞,其中2株能同时检测ToMV和烟草花叶病毒(TMV),各单克隆抗体腹水ELLSA效价在1∶32 000～1∶1 024 000之间。经TASELISA测定,4株单克隆抗体检测病汁液的稀释度均能达到1∶2 000倍以上。4株单克隆抗体与其他病毒无交叉反应。Westernblot分析表明,其中两株与ToMV176kD的外壳蛋白亚基有特异反应,而另两株无反应,推测它们是针对构象决定簇的抗体。     

A Library of Monoclonal Antibodies to Torpedo Cholinergic Synaptosomes

Abstract: A library of monoclonal antibodies was generated to the cholinergic synaptosome. The immunogen was a preparation of highly purified synaptosomes from Torpedo electric organ. One hundred forty-one hybridoma cell lines were generated from the fusion of a single mouse. Tests reveal these cells produce antibodies with a vast range of neuronal specificities. The initial screen for specificity of antibody production was solid phase radioimmune binding to the original, highly purified synaptosome preparation. Subsequent tissue specificity tests have indicated that most antibodies are synaptosome-specific amongst the fish tissues tested: brain, liver, and purified synaptic vesicles. Less than 11% cross-react with liver. Many antibodies cross-react with frog and rat CNS. Localization within the frog and rat nervous tissue has revealed a vast array of antibody staining patterns. Some antibodies stain in a synaptic fashion. A few stain a restricted set of mammalian CNS neurons. Others define a broader set of CNS neurons. These Torpedo antibodies promise to be valuable probes with which to describe the molecular cell biology of the nervous system, of neurons in general, and of cholinergic neurons in particular.     

Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

抗FLAG标签单克隆抗体的制备、鉴定及初步应用

用碳化二亚胺法合成FLAG完全抗原,利用杂交瘤技术制备出5株分泌抗FLAG标签单克隆抗体的杂交瘤细胞株。经鉴定,腹水效价均高于1：106 ,且5株单抗与其它融合蛋白标签无交叉反应性,并在免疫亲和层析实验中取得了满意的效果,为含标签的融合蛋白的纯化和研究应用提供了重要的工具。     

Characterization of Mono- and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.     

Production, Characterization, and Use of Monoclonal Antibodies Against Acremonium coenophialum

Monoclonal antibodies were produced using intact mycelium of the fungus Acremonium coenopbialum as the immunogen. During the initial stages of characterization, the antibodies were found to react to A. coenophialum, A. loliae, Epichloe typhina , and also to cross-react with some other fungi normally associated with tall fescue. Careful selection of the ELISA system in which the antibodies were used eliminated reactions to all but the two Acremonium spp. and E. typhina. Asa result it was possible to detect Acremonium spp. (presumably A. coenophialum ) sensitively and unambiguously in leaf tissue of tall fescue.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

The 84-kDa Form of Human Matrix Metalloproteinase-9 Degrades Substance P and Gelatin

Abstract: Matrix metalloproteinase-9 (MMP-9) is secreted from cells and, once activated, is thought to degrade collagen in the extracellular matrix. Because collagen is not readily localized where neurons have been shown to produce MMP-9 in the human brain, the ability of this enzyme to degrade bioactive peptides was investigated with representative tachykinin peptides [substance P (SP), neurokinin A, neurokinin B, and kassinin]. Latent MMP-9 (94 kDa) was purified from the human cell line HL-60 and converted to an intermediary active form (84 kDa) with p -aminophenylmercuric acetate. This active form of MMP-9 degraded SP with a k _cat/ K _m of 170 m M ⁻¹ min⁻¹, which is 30-fold greater than the previously reported value for a representative collagen-derived peptide. The major digestion products were identified as SP_1–6 and SP_7–11, which were derived from cleavage of the Gln⁶-Phe⁷ bond. Minor products were also generated from cleavage of the Gly⁹-Leu¹⁰ bond. The other representative tachykinin peptides were cleaved at rates >10-fold slower than that of SP. The 84-kDa peptidase was also active as a gelatinase. Longer treatment of MMP-9 with p -aminophenylmercuric acetate caused the conversion of the 84-kDa enzyme to the established 68-kDa active form; however, the rate of SP degradation did not increase. Because MMP-9 is produced by neurons of the CNS, these results suggest a possible regulatory role for the enzyme in intercellular communication by altering the availability of bioactive peptides.     

天然属性抗LDL及oxLDL IgM亚类抗体的制备与鉴定

目的：制备天然属性抗低密度脂蛋白（LDL）及抗氧化低密度脂蛋白（oxLDL）IgM亚类抗体。方法：给予Babl/c小鼠高胆固醇饮食,4周后取脾细胞直接与SP2/0细胞融合,以纯化的LDL及oxLDL为抗原,对阳性杂交瘤细胞生长孔进行间接ELISA筛选。鉴定杂交瘤上清的免疫球蛋白亚类,进而采用免疫沉淀和免疫印迹法对获得的抗体进行免疫学反应性鉴定。结果：杂交瘤细胞分泌的抗LDL及抗oxLDL的天然抗体通过ELISA法被筛选出来,可以与LDL或oxLDL发生高亲和力结合,经过4次克隆化,最终获得2株稳定分泌天然抗LDL的抗体,命名为5G8和2H7,及1株稳定抗oxLDL的抗体,命名为3A6,3株抗体均属于IgM亚类,无交叉反应,可以满足免疫印迹、免疫沉淀等实验要求。结论：成功制备了抗LDL及抗oxLDL IgM亚类抗体,为研究天然抗体在体内脂质代谢和相关心脑血管疾病如动脉粥样硬化等发生发展中的作用提供了重要的研究工具。     

Functional Studies with Substance P Analogues: Effects of N-Terminal, C-Terminal, and C-Terminus-Extended Analogues of Substance P on Nicotine-Induced Secretion and Desensitization in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Substance P (SP) and SP analogues, including C-terminal, N-terminal, and C-terminus-extended analogues, have been investigated for their ability to modulate nicotine-induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line luciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10^-5M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C-terminus-extended analogue SP-Tyr¹²-NH₂, 65% by (Ala³)SP-NH₂, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 ×; 10^-5M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr¹²-NH₂ > (Ala³)SP-NH₂ > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala³)SP-NH₂ protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (all at 3 ×; 10^-5M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr¹²-NH₂ at 3 × 10^-5M did not protect against nicotinic desensitization. Cyclo-SP(3-9), Ac-SP(3-9)-NH₂, SP(3-9), and SP(3-6) had neither inhibitory nor facilitatory effects on secretion. Of the 20 SP analogues extended at the C terminus by one amino acid, there were only three that protected against nicotinic desensitization, whereas the majority inhibited nicotine-evoked catecholamine secretion. The present work indicates that for inhibition of nicotine-evoked secretion, both the C terminus and N terminus of SP are necessary. For the protection against nicotine-induced desensitization, the C terminus of SP is important. This suggests that the two mechanisms, inhibition of nicotine-evoked secretion and protection against nicotinic desensitization, are regulated independently.     

Production of Monoclonal Antibodies Against Neurofilament-Associated Proteins: Demonstration of Association with Neurofilaments by a Coimmunoprecipitation Method

Abstract: A panel of monoclonal antibodies (MAbs) was produced against mouse brain proteins that bind to the tail domain of the neurofilament (NF) heavy (200-kDa) subunit (NF-H) in vitro. An in vivo association of the MAb ligands with NFs was confirmed by examining reactivity of the MAbs with immunoprecipitated NF-H complexes. Using this method we demonstrated association of the ligands of three of the MAbs with NFs. In contrast, glial fibrillary acidic protein and an unknown 97-kDa brain protein were not associated with NFs by this criterion. An 80-kDa doublet that coimmunoprecipitated with NF-H complexes, recognized by MAb 223, was shown by immunocytochemistry and immunoblotting to be synapsin Ia and Ib. Using a complementary approach, we confirmed an association of synapsin with NFs by demonstrating that immunoprecipitated synapsin I complexes contained NF-H and NF medium (160-kDa) subunits. MAbs 63 and 105 recognized a more complex set of proteins that had predominantly synaptic localizations. These data suggest that NFs may provide important support for attachment and/or transport of synaptic proteins in brain.