Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

Phenotypic analysis of lymphoproliferative disorders is now considered mandatory for accurate classification which is the basis for optimum patient management. This is presently carried out in most cases using a range of antibodies recognizing B and T-cell antigens effective in paraffin sections, and an antibody to CD3 is currently a key member of such panels, indicating T-cell phenotype. Current antibodies to CD3 are polyclonal with the inherent disadvantages of this type of reagent compared to monoclonal antibodies. In this study, we have used a recombinant fusion protein representing part of the epsilon subunit of the CD3 molecule to generate a novel monoclonal antibody (NCL-CD3-PS1) effective in paraffin sections. The antibody has been characterized biochemically and by immunohistochemistry using a wide range of normal and pathological tissues. Lineage and phenotype specificity have been supported in our study and results from other laboratories are awaited with interest.     

Characterization of HPC-1 antigen, an isoform of syntaxin-1, with the isoform-specific monoclonal antibody, 14D8

We raised polyclonal and monoclonal antibodies against rat recombinant HPC-1/syntaxin 1A lacking a transmembrane domain. The polyclonal antibody recognized two major bands at 35 and 40 kDa from rat brain membranes. A hybridoma clone designated 14D8, however, recognized only one band at 35 kDa. A polyclonal antibody detected recombinant syntaxin 1B, as well as HPC-1/syntaxin 1A on an immunoblot, whereas 14D8 recognized recombinant HPC-1/syntaxin 1A, but not syntaxin 1B. Therefore, 14D8 is specific for HPC-1/syntaxin 1A. Using this monoclonal antibody, we investigated the expression of HPC-1/syntaxin 1A in the rat hippocampal membranes. HPC-1/syntaxin 1A was present even in the embryonic d 19 (E19) hippocampal membranes, and it increased during the next two postnatal wk. Pyramidal cell axons were intensely stained with the 14D8 monoclonal antibody, suggesting that HPC-1/syntaxin 1A was not restricted to the presynaptic terminal. Furthermore, we investigated the phosphorylation of HPC-1/syntaxin 1A in the rat brain membranes. HPC-1/syntaxin 1A affinity-purified on a 14D8 IgG-coupled column was recognized by antiphophoserine antibody, but not by antiphosphotyrosine and phosphothreonine antibodies.     

Autoimmune reactions in patients with M-component and peripheral neuropathy.

A study of 17 patients with autoimmune axonal or demyelinating peripheral neuropathy in combination with M-component is described. The M-component was associated with MGUS (monoclonal gammopathy of undetermined significance) in 12 patients, CLL in one patient, WaldenstrÖm's disease in one patient, and myeloma in three patients. Immunohistological examination with direct and indirect fluorescence showed binding of antibodies to nerve structures of the same class and light chain as seen in the M-component. In five cases of IgM M-component, the demyelinating neuropathy was caused by binding of the IgM M-protein and complement C3b to myelin-associated glycoproteins (MAG). In 12 cases with axonal neuropathy, binding of IgG to the connective tissue of the peri- and endoneurium was found in 50% of cases, IgM in five cases, and IgD in one case. None of the patients had central nervous system (CNS) symptoms. The clinical and therapeutic difficulties are discussed; only two patients with an acute course responded to immunosuppression. A marked co-expression of other autoimmune phenomena is interpreted in the light of cross-reactions between the autoantibody and similar tissue autoantigens.     

抗蝮蛇毒磷酸二酯酶单克隆抗体的制备及鉴定

从蝮蛇毒中提取磷酸二酯酶,用此酶免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞Fo融合,以间接ELISA检测杂交瘤细胞培养上清液和小鼠腹水中的特异性抗体,其效价分别为1∶128和1∶51 200.抗原阻断试验结果表明,此抗体对蛇毒磷酸二酯酶具有特异性.该杂交瘤细胞株定为G_8,该株单抗属鼠IgG_(2a)亚型,经体外持续培养6个月,其分泌抗体性能稳定.     

胃癌单克隆抗体-丝裂霉素C结合物的制备及其细胞毒特征

作者报道胃癌单克隆抗体MG11-丝裂霉素C(MMC)结合物的制备及其对肿瘤细胞的杀伤作用。首先用戊二酐处理MMC,于MMC上引入羧基,MMC衍生物与N羟基琥珀酰亚胺及2,2′-二环已基碳二亚胺反应,得到MMC活性酯,后者与抗体反应将MMC引入抗体中。经测定,每克分子抗体中引入约6～7克分子药物,结合物对人胃癌细胞KATⅢ具有较强的选择性杀伤作用,在0.56μg/ml水平(药物浓度)对肿瘤细胞杀伤牢达60％,优于游离药物(51％)及无关抗体结合物(9.3％),提示选用的胃癌单抗对MMC具有较好的导向作用。     

丁型肝炎病毒中国SZ株的cDNA克隆及基因变异分析

以四川地区１例无症状携带丁型肝炎病毒（ＨＤＶ）和乙型肝炎病毒（ＨＢＶ）患者的外周血为标本，采用逆转录－ＰＣＲ技术，获得了ＨＤＶ全基因组的ｃＤＮＡ克隆（ＨＤＶＳＺ９３株），全长１６８４ｂｐ。与世界其他七个地区株相比，其核酸同源性为８１．８％～９５．４％；丁肝病毒抗原蛋白编码区核酸同源性为８８．９％～９６．１％，氨基酸同源性为８６，４％～９３．０％。另从１例慢性重型肝炎患者血清内克隆了丁肝病毒抗原蛋白（ＨＤＡｇ）编码基因（ＨＤＶ－ＳＺ９２株），与ＳＺ９３株相比，亦存在若干变异。对这些不同地区、不同病情ＨＤＶ株变异的可能意义进行了讨论。     

An unexpected complication following immunoadsorption with a staphylococcal protein a column

Extracorporeal immune adsorption with staphylococcal protein A (SPA) columns can remove immune complexes and immunoglobulins in the treatment of a variety of diseases. We present the case of an elderly man with neuropathy associated with monoclonal gammopathy, treated by 3 on-line SPA procedures. At the completion of these treatments his neuropathy relapsed, progressing to near-total paralysis. Return to a baseline clinical status required several months. The reason for this severe relapse is not clear. Possible explanations include SPA activation of T-lymphocytes, with release of gamma interferon and increased antigen recognition, or removal of an anti-idiotype control mechanism. We advise caution in the application of immunoadsorption to conditions in which it has not yet been evaluated. © 1992 Wiley-Liss, Inc.     

Epstein-Barr virus latent and replicative gene expression in oral hairy leukoplakia

Oral hairy leukoplakia is an epithelial lesion of the tongue associated with productive infection by Epstein-Barr virus (EBV). However, no data concerning the pattern of EBV latent gene expression have been reported, and it remains unresolved whether true latent infection occurs in basal cell layers of oral hairy leukoplakia. We have studied six cases of oral hairy leukoplakia using monoclonal antibody immunohistology for EBV latent--EB nuclear antigen (EBNA) 1, EBNA 2 and latent membrane protein 1 (LMP 1); immediate-early (BZLF1); and replicative (EA, VCA, MA) proteins, and for the EBV-receptor (CD21 antigen). EBV DNA was demonstrated by nucleic acid in situ hybridization. Mid- to upper-zone keratinocytes contained EBV DNA and co-expressed EBNA 1, EBNA 2 (5 of 6 cases), LMP 1, BZLF1 protein, EA, VCA and MA. No EBV genome or gene expression could be demonstrated in basal or parabasal cells. Spinous keratinocytes were labelled by anti-CD21 antibodies HB5 and B2, but did not express the EBV-receptor as defined by reactivity with OKB7. The co-expression of latent and replicative infection-associated antigens is striking, indicating possible functional roles for latent proteins during the productive cycle. Our results suggest that oral hairy leukoplakia is caused by repeated direct infection of upper epithelial cells with virus from saliva or adjacent replicatively infected cells, rather than by a latent EBV infection of basal epithelial cells with a differentiation-dependent switch to productive infection as previously proposed.     

A novel lamina lucida component of epithelial and endothelial basement membranes detected by LH39 monoclonal antibody.

The murine monoclonal antibody, LH39 was characterized in this study and appeared to bind to a novel basement membrane epitope. This antigen was expressed in the epithelial basement membrane of human tissue derived from all three germ cell layers and in basement membranes surrounding small blood vessels within the stroma of all organs examined. LH39 antigen could be first detected in fetal skin at the dermo-epidermal junction at 7 weeks estimated gestational age but was not present in the dermal vasculature until 16 weeks. When tested against tissue from a range of lower mammalian species, LH39 antigen appeared to be primate-specific. The epithelial basement membrane zone in organotypical cultures, where there is de novo synthesis of basement membrane components, contained abundant LH39 antigen in contrast to other basement membrane components, type IV collagen, laminin, and type VII collagen. Ultrastructural localization of LH39 epitope, using immunogold electron microscopy on unfixed freshly frozen tissue, was to the lamina lucida. No cross-reactivity could be detected between LH39 and laminin, fibronectin, and collagens I, III, IV, and V using the ELISA assay. In vitro studies with a range of proteolytic enzymes suggested that the antigen was non-collagenous in nature. LH39 precipitated a polypeptide with a molecular weight of 185 kD from extracts of metabolically labelled cultured keratinocytes, and polypeptides of 185 and 200 kD from the culture medium. The tissue distribution of LH39 antigen suggested that it may be an epitope within anchoring filaments. Potential applications of this antibody include the study of benign and malignant human vascular disorders, diseases and tumours associated with angiogenesis, epithelial neoplasms, and conditions of tissue regeneration and repair, such as wound healing.     

Effect of anti-interferon-γ and anti-interleukin-2 monoclonal antibody treatment on the development of actively and passively induced experimental allergic encephalomyelitis in the SJL/J mouse

SJL/J mice challenged with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) developed only mild chronic-relapsing experimental allergic encephalomyelitis (EAE) with very low incidence. However, treatment of challenged mice with anti-infeferonγ (IFN-γ) monoclonal antibody (mAb) determined severe disease in all cases. Similarly, in passive EAE, the addition of anti-IFN-γ to the in vitro MBP-activated cells at the time of transfer led to significant disease exacerbation in all recipients. The disease enhancing effect was observed only when the mAb was given at the time of active challenge or of passive transfer, but not at later times. Anti-interleukin-2 (IL-2) antibody had only a marginal effect in the active induction, but drastically reduced the manifestations of passive EAE, even when mixed with a disease-enhancing dose of anti-IFN-γ. These findings support the notion that IL-2 is required for disease induction whereas IFN-γ plays a disease-limiting role early in the development of EAE.