A monoclonal antibody inhibiting human placental Fc gamma-receptor activity

Fc gamma receptor (FcR) from human placenta was solubilized using EDTA and 2-mercaptoethanol and purified by affinity chromatography on human IgG-coated Sepharose 4B. BALB/C mice were immunized with FcR and monoclonal antibodies were obtained by growing hybridoma cells following fusion of spleen cells with P3 X 63Ag8 myeloma cells. Using an immunofluorescence technique, the IgG1 monoclonal antibody secreted by clone B1D6 stained the FcR-positive areas in sections of placental tissue. The endothelium of the foetal stem vessels stained more strongly than did the trophoblasts. The antibody also inhibited the haemadsorption to placental tissue of erythrocytes (E) sensitized with IgG antibodies (A), (EA), and inhibited the agglutination of EA by FcR. The data indicate that the monoclonal antibody reacts with the placental FcR at the binding site for IgG, or with an epitope close to the binding site. Apparently, the FcR in different anatomical areas in the placenta have a common antigenic determinant.     

Fc gamma-receptor activity of isolated human placental syncytiotrophoblast plasma membrane.

Fc gamma-receptor activity of isolated human placental syncytiotrophoblast microvillous plasma membrane (StMPM) vesicle preparations has been determined in an immunoradiometric assay using Sepharose-immobilized protein A to separate free 125I-labelled human IgG from membrane-bound 125I-IgG. This receptor assay has been optimalized in terms of buffer pH and molarity, and used to demonstrate that prior 60 min washing of isolated membranes in 3 M KCl to remove extrinsic membrane-bound protein substantially increases the membrane-binding capacity for IgG. Inhibition studies have determined the syncytiotrophoblast Fc gamma-receptor equilibrium constant for association (Ka) as 4.0 x 10(7) M-1 at 37 degrees and the number of available Fc gamma-receptor sites as 1.5 x 10(14) per mg membrane protein.     

A kinetic study of human IgG binding to placental Fc gamma-receptor

Various methods for studying the IgG interaction with its placental receptor have been examined to establish kinetic parameters. IgG binding has an average rate constant k1 of (2.57 +/- 0.94) x 10(7)/M per min. However, high displacement doses (1000 micrograms) of unlabelled IgG resulted in a curvilinear dissociation curve with two binding sites: one with a fast dissociation rate constant, k2 of 0.100/min and a slow one with k2 of 0.010/min. IgG binding was associated with a high average affinity of (3.70 +/- 1.65) x 10(8)/M and a total number of receptor binding sites of (2.07 +/- 0.93) x 10(14) sites/mg of membrane protein, in close agreement with previous results. The stoichiometry of IgG to Fc gamma-receptor was established as a 4:1 binding ratio from log dose-response curves as opposed to a 1:1 binding ratio from previous reports.     

Characterization of a monoclonal antibody (P40) against the 68 kD major allergen of Penicillium notatum

A monoclonal antibody (MoAb P40) against the 68 kD major allergen of Penicillium notatum (P. notatum) was obtained by immunizing the mouse with a crude extract of P. notatum. Analysed by two-dimensional gel electrophoresis and immunoblotting, P40 reacted with two different isoforms of the 68 kD component of P. notatum with pIs of 5.4 and 5.5. In addition to P. notatum, P40 showed positive ELISA activity to Aspergillus fumigatus (A. fumigatus) but not to components of six other fungi including Alternaria porri, Cladosporium cladosporoides, Aureobasidium pullulans, Fusarium solani, Rhizopus arrhizus and Candida albicans. Analysed by ELISA, MoAb P40 also showed positive activity to two (P. frequentans and P. roseopurpureum) of the 10 other Penicillium species and two (A. terreus and A. flavus) of the four other Aspergillus species tested. SDS-PAGE and immunoblotting studies demonstrated P40 positive reactivity to components with MW of about 67 kD in all these Penicillium and Aspergillus species with positive ELISA activity to P40. Furthermore, immunoblotting activity of MoAb P40 to the 67 kD component of A. niger was also observed. The epitope of the 68 kD allergen of P. notatum recognized by MoAb P40 was resistant to treatment of periodate oxidation with concentration of NaIO4 up to 20 mM. This MoAb may thus be useful in the characterization and purification of the 68 kD allergen from crude extracts, and in the molecular cloning of allergen genes.     

CD40单克隆抗体的制备、筛选及功能鉴定

CD40是肿瘤坏死因子受体超家族成员之一,属于Ⅰ型跨膜糖蛋白,其自然配体是CD40L。CD40-CD40L信号通路在许多重要的生理和病理过程中都起到关键的调节作用。基于其在激活免疫应答中的重要作用,在肿瘤免疫治疗中,具有激活CD40-CD40L信号的CD40激动剂抗体可用于提高抗肿瘤免疫应答;在移植排斥和炎症反应中,CD40-CD40L信号也起重要作用,阻断CD40信号通路是目前治疗风湿性关节炎、肠炎、系统性红斑狼疮、多发性硬化和糖尿病的一个重要靶点。制备CD40阻断性抗体或激动剂抗体不仅可用于检测CD40分子表达,还可以开发用于炎症性疾病或肿瘤治疗的免疫疗法。本研究表达CD40胞外段蛋白,通过免疫制备14株CD40单克隆抗体,筛选发现全部可作为western blotting检测CD40蛋白表达的抗体,9株CD40抗体可作为一抗用于流式细胞术检测人CD40表达,1株CD40抗体能阻断人CD40L和CD40相互作用。有意义的是,8株CD40抗体能激活人CD40分子信号通路,另外有10株能激活小鼠脾脏细胞CD40下游基因的表达,7株能上调小鼠脾脏B细胞表面共刺激分子CD86的表达。本研究制备一系列CD40抗体,可用于CD40检测、CD40信号的阻断或激活。     

Structural and functional characterization of a human IgG monoclonal antiphospholipid antibody

Antiphospholipid antibodies (aPL) are likely involved in the pathogenesis of the antiphospholipid syndrome (APS). This study analyzes the structural and functional characteristics of a human monoclonal aPL (HL7G) from the IgG2 subtype with λ light chains generated from a patient with primary APS and recurrent cerebral microemboli. DNA encoding the variable region of heavy and light chains of the antibody was sequenced, analyzed, and compared to HL5B a previously described monoclonal aPL from the same patient. Both antibodies are derived from the same germline genes. HL7G had similar but more extensive somatic mutations in the CDR1 and 2 regions than HL5B, indicating that both antibodies are closely related and derived by a T cell-dependent antigen driven process. In ELISA assays HL7G bound to cardiolipin and several other phospholipid antigens in the absence of protein cofactors. Different from HL5B this aPL bound to β2-glycoprotein I (β2GPII). This suggests that reactivity of aPL against β2GPI is determined by only few specific amino acid exchanges. HL7G was able to induce tissue factor (TF) as one of the procoagulant effects of aPL. Our data suggest that the binding specificity of aPL is only of limited value to predict the biological effect and the pathophysiological impact of the antibodies.     

Confirming human antibody responses to a therapeutic monoclonal antibody using a statistical approach

Confirmatory assays for immunogenicity testing typically involve testing a sample in the presence or absence of excess drug. A decrease in assay signal in the presence of drug is taken to indicate the presence of anti-drug antibodies (ADAb) and the sample is confirmed positive. While there is widespread acceptance of the principle, there are currently no published guidelines for determining how much the signal should be reduced for a sample to be confirmed positive. In this report we address this issue using a novel approach employing a Student's t-test. The basic premise is to assess if an observed decrease in signal in the presence of drug is greater than what might be expected to occur as a result of the normal variation in the system. A key component of the method involves being able to capture and measure all of the normal variation. This requires a modification of commonly employed methods of sample preparation. We validated the method and tested samples from a clinical study. In addition, we reanalyzed the data to see what would have been the outcome had we used two other common approaches for confirmatory assays, one based on a minimum percent decrease in signal to confirm positivity (arbitrarily set at 25%), and one requiring a minimum drop in signal, set by a low quality control (QC) sample. The t-test approach proved superior over a wide range of assay signals and appeared to result in fewer false negatives.     

一株识别CD40分子新位点的单克隆抗体研制及生物学特性鉴定

目的：研制识别CD40分子新位点的单克隆抗体（mAb）。方法：以转人CD40转基因细胞L929-CD40为免疫原，常规免疫6—8周龄的雌性BALB／c小鼠；采用B淋巴细胞融合技术，将免疫小鼠脾脏细胞与Sp2／0融合，以L929-CD40转基因细胞为抗体筛选阳性细胞，经免疫荧光标记分析对杂交瘤进行反复筛选和多次的克隆化培养；采用快速定性试纸法及竞争抑制结合试验分析了该mAb的亚类及抗原识别位点；采用MTT法分析mAb在体外对肿瘤细胞的促增殖效应、ELISA法测定细胞因子分泌以及PI染色分析细胞周期。结果：获得1株持续、稳定分泌鼠抗人CIM0mAb的杂交瘤细胞株（命名为286），该mAb能特异性地识别人CD40分子并较好的识别肿瘤细胞株H08910表达的CD40分子并能够体外促进肿瘤细胞增殖。结论：成功地研制1株识别CD40新位点mAb，该mAb能够很好地识别肿瘤细胞表达的CD40分子并具有体外促进肿瘤细胞生长的作用。     

Purification of tick-borne encephalitis virus by affinity chromatography using monoclonal antibody

The application of a simple technique for purification of tick-borne encephalitis (TBE) virus is described. TBE virus was grown in chick embryo cell (CE) cultures and the virus was concentrated by differential centrifugation. Final purification was made by the filtration through Sepharose column to which monoclonal antibodies to TBE virus had been bound. The method was effective in eliminating avian retroviruses.     

A novel blocking monoclonal antibody recognizing a distinct epitope of human CD40 molecule

CD40, a member of the tumor necrosis factor receptor superfamily, is an important costimulatory molecule during the immune response. Here, we report a blocking mouse antihuman CD40 monoclonal antibody, mAb 3G3, of which the specificity was verified by flow cytometry and Western blot. It was shown by competition test that 3G3 bound to a different site (epitope) of CD40 from the reported CD40 mAbs, including clone mAb89, 3B2, and 5C11. It was also found that mAb 3G3 could inhibit homotypic aggregation of Daudi cells induced by the agonistic anti-CD40 mAb 5C11. Furthermore, mAb 3G3 effectively inhibited the proliferation of peripheral blood mononuclear cells in mixed lymphocyte reaction assay. Finally, a sensitive and specific soluble CD40 (sCD40) ELISA kit was established by matching mAb 3G3 with 5C11, and it was found that the levels of sCD40 in sera from patients with immune disorders such as hyperthyroidism, chronic nephritis, and rheumatoid arthritis were obviously higher than those from normal individuals. Thus, this blocking anti-CD40 mAb provides a novel tool for the study of CD40.     

Purification of woodchuck hepatitis surface antigen using a monoclonal antibody raised against the antigen

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.     

Solubilization of human peripheral nerve Fc gamma receptors and purification of a functional 40 kDa receptor

Extracts from myelinated and unmyelinated nerves, prepared using Tris-HCl buffer with EDTA and ME, contained functionally active receptors for the Fc region of IgG (FcR). This was evident from the results obtained in indirect haemagglutination and rosette inhibition assays. Using a monoclonal antibody, a functional active 40-kDa FcR was purified from the nerve extracts. The receptor was a single-chained glycoprotein with low affinity for native IgG, apparently belonging to the FcRII family. In addition, peripheral nerve extracts contain FcR not recognised by the monoclonal antibody.     

Purification of human basophils using mouse monoclonal IgE.

A method was developed to purify human blood basophils using monoclonal mouse IgE. Enriched suspensions of basophils were sensitized with IgE anti-trinitrophenol (TNP) hapten which was prepared from hybridoma culture supernatants. Rosettes were induced with TNP-coupled red cells and separated from contaminating cells on Percoll gradients. Basophils were dissociated from red cells using cold hypotonic lysis. Using this technique the percentage of basophils was increased from less than 1% to an average of 83.5 +/- 7.8% with a recovery of 58.0 +/- 12.7%. Final purities were highest when enriched sensitized suspensions contained greater than or equal to 10% basophils. Using a radioenzymatic assay, purified basophils were shown to contain an average of 1.3 +/- 0.3 pg histamine. Anti-human, but not anti-mouse IgE, released histamine at an optimal dilution. Using this technique, in 3-4 h, we are able to prepare highly purified suspensions of basophils for further study.     

The human mononuclear phagocyte high-affinity Fc receptor, FcRI, defined by a monoclonal antibody, 10.1

The properties of the mononuclear phagocyte (Mph) high-affinity Fc receptor, FcRI, were investigated using a novel monoclonal antibody (mAb) designated 10.1. This receptor was shown to be a protein of 71 kDa, presented chiefly on monocytes and the myeloid cell lines U937 and HL60. mAb 10.1 inhibited the binding to Mph of erythrocytes opsonized with rabbit IgG or human IgG3. It also blocked T cell proliferation induced by murine CD3 mAb of the IgG2a but not the IgG1 subclass. These results suggest that rabbit IgG, human IgG3 and murine IgG2a all bind to FcRI in a similar manner and that mAb 10.1 reacts with an epitope on FcRI near to the binding site for the Fc region of IgG. In addition, although it is well known that FcRI has a high affinity for both monomeric human IgG1 and IgG3, we show in this study that while erythrocytes opsonized with human IgG3 bind to Mph, equivalent cells opsonized with IgG1 surprisingly do not. These results define further the nature of the constraints on the interaction between Mph FcRI and particular IgGs.     

鼠抗人OX40功能性单克隆抗体的研制及其生物学特性的鉴定

目的：研制功能性鼠抗人OX40单克隆抗体。方法：以转人OX40的转基因细胞L929-OX40为免疫原，常规免疫6—8周龄的雌性BALB／c小鼠；采用B淋巴细胞融合技术，将免疫小鼠脾脏细胞与SP2／0融合，以L929-OX40转基因细胞及PHA活化的T细胞为抗体筛选阳性细胞，经免疫荧光标记分析对杂交瘤进行反复筛选和多次的克隆化培养；采用快速定性试纸法及竞争抑制结合试验分析了该单抗的亚类及抗原识别位点；采用MTT法分析单抗在体外对T细胞的促增殖效应以及ELISA分析活化T细胞分泌的细胞因子。结果：获得1株持续、稳定分泌鼠抗人OX40单克隆抗体的杂交瘤细胞株(命名为7E11)，该单抗能特异性地识别人OX40分子和介导有效的共刺激信号，体外促进活化的T细胞增殖和细胞因子的分泌。结论：成功研制成一株能分泌功能性鼠抗人OX40单克隆抗体的杂交瘤，该抗体特异性地识别人OX40分子并具有在体外协同刺激T细胞的作用。     

A functional monoclonal antibody recognizing the human alpha 1-integrin I-domain

αβ1 heterodimer is a member of the integrin receptor superfamily that has been described to be involved in cell-matrix binding through its interaction with collagens, fibronectin and laminin. The αl integrin belongs to a subset of I-domain containing integrins that includes α_M, α_L, α_X and α2. In this study we describe an anti-αl mAb (FBI2) that recognizes an epitope located in the human αl I-domain, since the mAb can bind to human, but not to rat, recombinant I-domain GST fusion protein. FBI2 mAb efficiently and specifically inhibits the binding of activated human lymphocytes to laminin, collagen and fibronectin. These data support the notion that the αl I-domain itself has an important role in receptor-ligand binding. In particular, we show that al inte-grin-dependent lymphocyte adhesion to fibronectin is I-domain mediated, at variance with the RGD-dependent adhesion which seems to be mediated by the βl rather than the αl integrin chain. Lastly, the overexpression of the αl-integrin by stromal cells and blood vessels of solid tumors may suggest a role for this integrin in tumor biology.     

A highly sensitive radioimmunoassay for human growth hormone using a monoclonal antibody

Biozzi-strain mice were immunized with a highly purified preparation of 20K variant of hGH. Spleen-cells were fused with SP2/0Ag14 myeloma cells. Clone productions were screened for specificity toward 20K and 22K hGH and for the affinity constant of antibody-antigen reaction. For the selected monoclonal antibody, Ka was 1.02.10(11) L/M using 22K hGH as both tracer and reference preparation. No cross reactivity was found with PRL and other pituitary hormones; hPL reactivity was 0.002 percent that of hGH. According to these antibody characteristics, a highly sensitive RIA system was developed and used for specific GH measurement in human serum. Using logit-log co-ordinates, the slope of the standard curve was -1.099 and the minimum detected dose was 0.5 uIU/ml. Excellent correlation (r = 0.9575) was found between assay data in this system and those of a conventional RIA method using specific polyclonal rabbit antiserum. The International Reference preparation (66/217) could adequately be used to calibrate the monoclonal antibody system since the in house internal 22K GH standard and international one were equally well recognized by the monoclonal antibody.     

鼠抗人OX40L功能性单克隆抗体的研制及其生物学特性的鉴定

目的　鼠抗人OX4 0L分子功能性单克隆抗体的研制及其生物学特性的鉴定。方法以高表达人OX4 0L分子的转基因细胞L92 9/OX4 0L为免疫原 ,常规免疫BALB/c小鼠 ,采用B淋巴细胞杂交瘤技术进行细胞融合 ,并以L92 9/OX4 0L为阳性抗体筛选细胞 ,L92 9/mock为阴性抗体筛选细胞 ,经免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养 ,筛选出特异分泌鼠抗人OX4 0L分子单克隆抗体的杂交瘤细胞株 ;采用Westernblot、Ig亚型快速定性试纸法、间接免疫荧光法、竞争结合抑制试验和3 H TdR增殖试验等对单抗进行生物学特性的鉴定。结果　成功获得 3株持续、稳定分泌鼠抗人OX4 0L单克隆抗体的杂交瘤细胞株 ,命名为 9H10、4C12和 1G1。对单抗的生物学功能的研究结果表明 ,3株单抗均能识别活化B细胞和成熟DC表达的OX4 0L分子 ,且能抑制成熟DC对T细胞的促增殖作用 ,并与阻断型抗人B7 1单抗具有协同作用。结论　获得的 3株分泌鼠抗人OX4 0L功能性单克隆抗体的杂交瘤 ,所分泌的抗体具有特异性地识别人OX4 0L分子并能阻断OX4 0 /OX4 0L共刺激信号及抑制DC对T细胞的激发作用。