重组单抗药物的肽图分析

建立了重组单抗药物的肽图分析方法。在变性条件下向抗体溶液加入还原剂,打开抗体内部所有交联的二硫键,再加入烷基化试剂封闭所有的自由巯基,使抗体分子在溶液中以游离伸展肽链的形式存在。加入胰蛋白酶,将充分伸展的肽链酶解成小的肽段。用反相高效液相色谱层析分析肽图谱。对连续3批中试产品及理化测定对照品进行肽图分析,各样品均能酶解完全,批间肽图谱一致。该方法实用有效,适于进行重组单抗等结构复杂的大分子蛋白药物的肽图检查分析。     

The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide-biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs.     

Arabinogalactan proteins in embryogenic and non-embryogenic callus cultures of Euphorbia pulcherrima

The arabinogalactan protein (AGP) fractions of embryogenic and non-embryogenic callus lines of Euphorbia pulcherrima Willd. ex. Klotzsch were analysed over a cultivation period of 9 weeks using the β -glucosyl Yariv reagent and an anti-AGP antibody (LM2). The amount of AGPs detected with the Yariv reagent increased in embryogenic cultures during the development of somatic embryos. The embryogenic and non-embryogenic callus contained different sets of AGPs characterized with the Yariv reagent and the LM2 monoclonal antibody. AGPs recognized by LM2 are localized primarily in the protodermal cells of globular somatic embryos. The development of somatic embryos of E. pulcherrima appears to be associated with the presence of particular AGPs.     

Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water

U.S. EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1-4 sporozoites or nuclei after staining with 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10-min incubation at 80 degrees C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49% to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.     

Monoclonal antibody covalently coupled to liposomes: Specific targeting to cells

We have evaluated optimal conditions for coupling monoclonal antibody to small unilamellar lipisomes. Coupling of an IgG_2a monoclonal anti-β₂-microglobulin antibody, which reacts with human cells, was examined in detail. Liposomes were composed of dipalmitoyl lecithin and cholesterol, and variable quantities of phosphatidylethanolamine substituted with the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP). They were reacted with antibody derivatized with the same reagent at a 5- to 20-fold molar excess, and activated by mild reduction. This degree of SPDP modification had no effect on the capacity of the antibody to bind to its target antigen. More than 40% of antibody could be reproducibly bound to liposomes, resulting in the coupling of from 1 to 10 antibody molecules per liposome (mean diameter.580 Å). The coupling reaction did not lead to loss of carboxyfluorescein encapsulated within liposomes. At least 80% of liposomes carried nondenatured antibody, as confirmed by precipitation of liposomes and encapsulated carboxyfluorescein by Staphylococcus aureus, strain Cowan I. The liposome-coupled antibody retained its immunological specificity: only cells expressing human β₂-microglobulin bound liposomes in vitro, and the binding was inhibited by the free antibody in solution. Results with antibodies of different antigenic specificity confirm that the technique can be generally applied.     

Production of a monoclonal antibody reacting with vimentin, a structural protein of intermediate filaments

This paper reports on the production of a monoclonal antibody (i-18) reacting with vimentin, the major structural component of intermediate filaments in cells of mesenchymal origin. The antibody was obtained following immunization with hamster fibroblasts and was selected for its ability to bind to the cytoskeleton fraction of the aforementioned cells. It decorated a perinuclear filamentous network characteristic of vimentin filaments in cells of mesenchymal origin of avian through human species. The specificity of the reagent was further ascertained on the basis of the sensitivity of the decorated filaments to colcemide. The strict antibody specificity for cells of mesenchymal versus epithelial origin was confirmed also in vivo on histological specimens from solid tissue. The i-18 monoclonal antibody precipitated a molecule of about 57 Kd from metabolically labelled cellular extracts. The broad cross-reactivity of this monoclonal antibody among different animal species, as well as its strict in vivo mesenchymal tissue specificity makes this antibody a useful reagent for both experimental and diagnostic purposes.     

Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using a novel affinity labeling reagent, acyl adenylate

Large-scale analysis of protein-protein interaction sites is especially needed in the postgenomic era. The combination of affinity labeling with mass spectrometry is a potentially useful high-throughput screening method for this purpose. However, reagents in current use are not ideal as some cause damage to the target molecule and others have poor solubility in physiologic aqueous buffers. In this paper, we describe a novel affinity labeling reagent, acyl adenylate, which is highly soluble in aqueous solutions and reacts in a pH-dependent manner. The adenylate of deoxycholic acid reacts with amino groups on the side chain of a lysine residue and at the N-terminus of proteins/peptides. The reactivity and stability of this reagent were investigated, and it was confirmed that, after formation of a reversible ligand-protein complex under weakly acidic conditions, derivatization with acyl adenylate occurred at the target site under weakly alkaline condition. We further demonstrated the utility of this reagent for affinity labeling using a monoclonal antibody with high affinity for deoxycholic acid. Competitive ELISA indicated that deoxycholic acid was labeled around the antibody ligand binding site, thus enabling the structural elucidation of the ligand-protein interaction. In addition, LC/ESI-MS/MS analysis of the labeled peptide obtained by enzymatic digestion and affinity extraction allowed the identification of the structure surrounding the antigen binding site.     

Lack of antibody induction by Histolyn-CYL, a new skin-testing reagent for histoplasmosis

Histolyn-CYL, a yeast phase skin-test reagent, was administered to 85 histoplasmin-sensitive subjects living in the United States and South America. In a multicenter study, sera were obtained at the time the skin tests were read and again 3 weeks later. In no instance did the skin test induce significant complement-fixing antibody changes or antibodies detected by immuno-diffusion.     

Comparison of commercially available antibody reagents for the cytomegalovirus pp65 antigenemia assay

Background: The Argene Biosoft 1C3 and the Biotest C10,C11 monoclonal antibodies are two of the most commonly used commercially available antibody reagents for the cytomegalovirus (CMV) pp65 antigenemia assay.Objectives: The sensitivities of these two reagents were compared in peripheral blood specimens received for CMV antigenemia testing.Study design: A total of 1149 peripheral blood specimens were processed for CMV antigenemia testing. Duplicate slides were stained with the Biosoft 1C3 and Biotest C10,C11 monoclonal antibodies.Results: A total of 158 specimens gave a positive result by one or both antibodies. One hundred and forty five were positive by the Biosoft antibody and 130 were positive by the Biotest antibody. Positive cell counts were significantly higher on cell preparations stained by the Biosoft antibody (Wilcoxon signed rank, P < 0.001) and the Biosoft antibody detected twice as many low-level positive specimens as the Biotest.Conclusions: The Biosoft antibody reagent was superior to the Biotest reagent for the detection of CMV antigenemia. This is an important factor since early detection is essential for appropriate initiation of preemptive antiviral therapy, particularly in transplant recipients at high risk of CMV disease.     

荧光染料R-藻红蛋白标记小鼠抗人CD4单克隆抗体

以R-藻红蛋白(R-PE)标记小鼠抗人CD4单克隆抗体,形成单色或和用其他荧光染料标记的CD系列单抗组成双色、多色的荧光试剂,应用于流式细胞仪检测分析。用异双功能交联试剂SPDP和SMCC分别活化R-PE和CD4单抗,用DTT使经SPDP活化后的R-PE巯基化,再与用SMCC活化的CD4单抗交联。使用NEM终止交联反应,经Sephacryl S-300柱在AKTA FPLC快速液相色谱系统(简称AKTA)监测下分离纯化。结果用R-PE标记的抗CD4单抗,检测正常人外周血淋巴细胞表面CD4抗原的表达,经流式细胞仪(简称FACS)分析表明,R-PE标记的CD4抗体特异性保持完好,荧光强度较高,还可与用FITC标记的其它CD系列单抗配伍成双标或多标试剂。使用SPDP,SMCC异双功能交联试剂和DTT还原剂,成功地偶联了R-藻红蛋白和CD4单克隆抗体,可应用于流式细胞仪检测分析。     

The preparation of bacterial luciferase conjugates for immunoassay and application to rubella antibody detection

Bacterial luciferase has been modified with the thiolating reagent S-acetylmercaptosuccinic anhydride and covalently crosslinked to either Staphylococcus aureus protein A or anti-human immunoglobulin G (IgG) with the heterobifunctional reagent m-maleimidobenzoic acid N-hydroxysuccinimide ester. The conjugates retain enzymatic light-emitting activity and have the ability to bind IgG antibody. The ability of these conjugates to detect human IgG has been demonstrated by application to rubella immunity screening. Rubella antibodies are isolated from serum on the surface of rubella antigen-coated tubes and subsequently determined by light emitted from bound conjugate. In a preliminary study, the bioluminescent immunoassay has been compared to a commercial rubella antibody radioimmunoassay and found to be comparable in the ability to determine rubella immunity.     

Development and Use of a Polyvalent Conjugate to Differentiate Histoplasma capsulatum and Histoplasma duboisii from Other Pathogens

Previous investigations have demonstrated the existence of five Histoplasma capsulatum serotypes. Available specific fluorescent-antibody reagents stain only four of the five serotypes. Antibodies produced against the most complete H. capsulatum serotype were labeled with fluorescein isothiocyanate to develop a reagent specific for H. capsulatum that was reactive with all the known serotypes. The unadsorbed reagent not only stained all the H. capsulatum serotypes, but it also stained cultures of Blastomyces dermatitidis, H. duboisii, several Candida species, and a variety of other fungi. Adsorption of the conjugate with antigens of C. albicans produced a reagent that intensely stained only H. capsulatum, H. duboisii, and B. dermatitidis. Differentiation of B. dermatitidis from the Histoplasma species was accomplished by application of a B. dermatitidis specific fluorescent antibody to antigens positive with the H. capsulatum reagent. At present, differentiation of H. capsulatum from H. duboisii may be accomplished only by animal inoculation. Our data substantiate the antigenic relationships hypothesized earlier, and they indicate that H. capsulatum shares at least two antigens with the other fungi that were studied.     

A liposome-PCR assay for the ultrasensitive detection of biological toxins

We describe an ultrasensitive immunoassay for detecting biotoxins that uses liposomes with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as a detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time PCR. Assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods.     

Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry

A monoclonal antibody against histone 2B (anti-H2B) was used as a reagent to stain isolated chromosomes for analysis using flow cytometry. Chromosome suspensions were treated with a mouse monoclonal antibody specific for the histone 2B (clone HBC-7) and then with a fluorescein-labeled goat anti-mouse-IgM antibody. The chromosomes were also stained for DNA content with either Hoechst 33258 or propidium iodide. The amount of antibody and the amount of DNA-specific stain bound to each chromosome were measured simultaneously using flow cytometry. The order of the steps in the staining protocol is important. Propidium iodide prevents anti-H2B from binding to chromosomes, and therefore must be added only after antibody labeling is completed. In contrast, the addition of Hoechst 33258 before antibody labeling reduces antibody binding by only 20%–30%. Binding of anti-H2B was proportional to the DNA content of both human and Chinese hamster chromosomes. Human chromosomes bind an average of three to four times more anti-H2B than do Chinese hamster or mouse chromosomes of the same DNA content. This was determined by analyzing mixtures of human and Chinese hamster chromosomes and human and mouse chromosomes. The results demonstrate that it is possible to label the proteins of chromosomes in suspension with fluorescent antibodies and to use these reagents for the analysis of chromosome structure by flow cytometry.     

Comparison of two methods of preparing enzyme-antibody conjugates: application of these conjugates for enzyme immunoassay.

Two methods of preparing enzyme-antibody conjugates were evaluated. High yields of conjugate were obtained with both methods. The first procedure utilizes the homobifunctional crosslinking reagent N,N′-o-phenylenedimaleimide. Sulfydryl residues were introduced into second antibodies by reaction with methyl-mercaptobutyrimidate. The modified antibodies were reacted with N,N′-o-phenylenedimaleimide, excess reagent was removed by gel filtration, and the activated antibodies were cross-linked to β-galactosidase. Up to 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of enzyme activity. The second method utilizes the heterobifunctional meta-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The antibodies were reacted with MBS, excess reagent was removed by gel filtration and the activated antibodies were crosslinked to β-galactosidase. Typically, approximately 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of both enzyme and antibody activity. Conjugates prepared using these two procedures were used as labels in an immunoassay system and were able to detect approximately 5 to 10 ng of first antibodies. The MBS procedure was simpler to perform, could more easily be adapted to large-scale work, and gave more reproducible results, and the conjugates produced were able to detect slightly lower concentrations of first antibody.     

Use of colloidal gold in diagnostic surgical pathology

Colloidal gold immuno-electron microscopy is a powerful tool for defining antigenicity at the subcellular level. Such studies permit correlation with cell fractionation studies. They also allow one to assess the specificity of a particular antibody. The most useful reagent for immuno-electron microscopy is colloidal gold stabilized by a binding protein, either staphylococcal protein A or immunoglobulin. This method permits highly discrete labeling, and the system is useful for most antibodies used in diagnostic pathology.     

A monoclonal antibody to somatostatin with potent in vivo immunoneutralizing activity

The spleen from a Robertsonian mouse with high titer and affinity antiserum after being immunized with somatostatin-14 conjugated to keyhole limpet hemocyanin was fused with FOX-NY cells. Hybridomas were cloned by limiting dilution, subcloned, and ascites was produced from the highest affinity close in pristine-primed Balb/c mice. Ascites fluid contained approximately 20 mg/ml IgG and bound 50% of 1 fmol 125I-[Tyr1]-somatostatin at a final dilution of 1:10,000,000. Binding of this IgG1 antibody, CURE.S6, was inhibited by 50% at 40 pM concentrations of either somatostatin-14 or somatostatin-28, but was not inhibited by [D-Trp8 -somatostatin at 1000-fold higher concentrations. The antibody produced very intense specific immunohistochemical staining of somatostatin endocrine cells in the stomach and pancreas and of intestinal somatostatin neurons with extremely low background staining. Intravenous injection of 2 mg purified antibody in urethane-anesthetized rats resulted in 300-fold increase in plasma GH within 15 min. CURE.S6 is a high affinity monoclonal antibody directed at the biologically active somatostatin ring structure. This antibody is useful for in vivo immunoneutralization of exogenous and endogenous somatostatin in the rat and also is an excellent reagent for immunohistochemical localization of somatostatin.     

Detection of mouse hepatitis virus antibody by protein A-ELISA in 6 prevalent inbred strains or outbred stocks of mice.

Protein A was applied as a reagent for the secondary reaction in ELISA (protein A-ELISA). Mouse hepatitis virus antibody in 6 prevalent mouse strains or stocks reared in a MHV-contaminated room was effectively detected by protein A-ELISA, whereas significant strain differences in the antibody detection rate were demonstrated using the complement fixation test. C57BL/6 mice were particularly reactive in the protein A-ELISA test.     

Automated Complement Fixation with Low Reagent Consumption

A microvolume automated (continuous-flow) system for the detection of complement-fixing antigen or antibody was evaluated. The model complement fixation test used was the detection of deoxyribonucleic acid antibody, and comparisons were made with a manual procedure. An "automatic two-dilution" feature of the apparatus was not used because of the clinical need for antibody titers. The system proved useful for the detection and estimation of anti-deoxyribonucleic acid antibody in patients with systemic lupus erythematosus and, indeed, accomplished this with low reagent consumption, a useful feature not available in "older" continuous-flow complement fixation systems.     

Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.