ImmunoMethods最新文献_第7页

Crystallization of Antibodies and Antibody-Antigen Complexes 抗体和抗体-抗原复合物的结晶

ImmunoMethods Pub Date : 1993-12-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1051

Stura Enrico A., Fieser Gail G., Wilson Ian A.

{"title":"Crystallization of Antibodies and Antibody-Antigen Complexes","authors":"Stura Enrico A., Fieser Gail G., Wilson Ian A.","doi":"10.1006/immu.1993.1051","DOIUrl":"10.1006/immu.1993.1051","url":null,"abstract":"<div><p>Although many antibodies have been crystallized, the number of structures determined in both their complexed and unliganded forms remains relatively small. With the recent improvements in the use of molecular replacement (MR), the structure determination of Fabs and Fab-complex structures can proceed more rapidly, but crystallization often remains a major obstacle. Substantial improvements in methodologies have helped with the success rate in the crystallizations of Fabs and Fab-antigen complexes that are beyond previous expectations. Crystallization and structure determination have been directed mainly toward Fab fragments. The reason for this choice remains linked both to the ease with which the structure of Fabs can be determined and to the difficulties that have been presented by the crystallization of whole immunoglobulins. Such difficulty is currently believed to be due to flexibility or conformational heterogeneity of the IgG as well as the added heterogeneity from the glycosylation of the Fc fragment. Fabs share some of the same problems mainly because of the degree of heterogeneity that is the result of the proteolytic cleavage used to fragment the immunoglobulins, of the flexibility in elbow regions, and in some cases of glycosylatlon. A systematic approach to the cleavage, purification, and analysis of the resultant product can yield inmunoglobulin fragments amenable to crystallization. A rational screening of crystallization conditions with extensive use of seeding can In most cases enable progress from small microcrystalline aggregates to large X-ray-quality crystals. Such methodologies have become so effective that Fabs are now being used as tools to aid in the crystallization of other molecules that have been found difficult to crystallize by themselves.</p></div>","PeriodicalId":79341,"journal":{"name":"ImmunoMethods","volume":"3 3","pages":"Pages 164-179"},"PeriodicalIF":0.0,"publicationDate":"1993-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/immu.1993.1051","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"51492603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 23

Monoclonal Fab Fragments from Combinatorial Libraries Displayed on the Surface of Phage 噬菌体表面显示组合文库中的Fab单克隆片段

ImmunoMethods Pub Date : 1993-12-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1050

Burton Dennis R., Barbas III Carlos F.

引用次数: 5

Crystal Structures of Intact IgG Antibodies 完整IgG抗体的晶体结构

ImmunoMethods Pub Date : 1993-12-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1054

Edmundson Allen B., Guddat Luke W., Andersen Kim N.

{"title":"Crystal Structures of Intact IgG Antibodies","authors":"Edmundson Allen B., Guddat Luke W., Andersen Kim N.","doi":"10.1006/immu.1993.1054","DOIUrl":"10.1006/immu.1993.1054","url":null,"abstract":"<div><p>Crystal structures of intact IgG immunoglobulins have provided new insight into the interrelations of the Fab arms responsible for antigen binding and the Fc region mediating effector functions such as complement fixation and receptor attachment. Human immunoglobulins lacking the \"hinge\" region critical for segmental flexibility between the Fabs and the Fc (e.g., Dob and Mcg IgG1 proteins) exhibit Fc units that are represented by interpretable modules of electron density. In contrast, two human \"cryoglobulins\" (Kol IgG1 and Zie IgG2 molecules) with normal hinge regions have mobile Fc units that do not contribute to the X-ray diffraction patterns. A murine IgG2a antibody (Mab 231) has longer and less restricted Fab-Fc connectors, which act more like flexible tethers than hinges. These tethers permit movements of the Fc into crystal packing arrangements in which they are secured in a single orientation. The ensuing order gives rise to well-defined electron density for the Fc. In the process the inherent symmetry between the two halves of the molecule is lost. Half molecules are related by a crystallographic twofold axis of rotation in each of the human crystalline immunoglobulins. All crystals show some disorder, but the Kol, Mcg, and Mab 231 have been solved to sufficiently high resolution (3.1 to3.5 Å) to trace the polypeptide chains and correlate structural and functional relationships in considerable detail.</p></div>","PeriodicalId":79341,"journal":{"name":"ImmunoMethods","volume":"3 3","pages":"Pages 197-210"},"PeriodicalIF":0.0,"publicationDate":"1993-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/immu.1993.1054","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"51492716","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8

Structure Determination of Antibodies and Antibody-Antigen Complexes by Molecular Replacement 用分子置换法测定抗体和抗体-抗原复合物的结构

ImmunoMethods Pub Date : 1993-12-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1052

Brünger Axel T.

引用次数: 9

Author Index for Volume 3 第3卷作者索引

ImmunoMethods Pub Date : 1993-12-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1057

引用次数: 0

X-Ray Crystallographic Studies of Antibody–Peptide Complexes 抗体-肽复合物的x射线晶体学研究

ImmunoMethods Pub Date : 1993-12-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1055

Stanfield Robyn L., Wilson Ian A.

引用次数: 24

Fiber-Optic Immunosensors Based on Continuous Reagent Delivery 基于连续试剂输送的光纤免疫传感器

ImmunoMethods Pub Date : 1993-10-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1045

Agayn Venetka, Walt David R.

{"title":"Fiber-Optic Immunosensors Based on Continuous Reagent Delivery","authors":"Agayn Venetka, Walt David R.","doi":"10.1006/immu.1993.1045","DOIUrl":"10.1006/immu.1993.1045","url":null,"abstract":"<div><p>Immunoassay technology has advanced tremendously during the past 30 years and has provided indispensable tools for clinical and environmental analysis. The immunoassays in use are highly sensitive, with some achieving femto- and attomolar detection limits. These assays possess a variety of drawbacks due mainly to the demand for trained technical personnel and intensive sample manipulations. The introduction of fiber optics offers the potential for miniaturization and simplification. Some examples of fiber-optic immunosensors have been described in the literature. Most of these face the usual immunosensor limitation of single-use detection; hence, they are not appropriate for continuous use. The main limitation of all immunosensors lies in the strong antibody-antigen binding, which requires either extensive washing for long periods of time or use of chaotropic reagents for dissociation. Several examples of regenerable fiber-optic sensors are based on displacement of a labeled by an unlabeled antigen or by an antibody developed especially to possess decreased binding affinity. In our laboratory, research is concentrated on developing a continuous fiber-optic sensor based on prolonged delivery of reagents from slow-release polymers. In this paper we review the achievements in the area of continuous sensing with fiber-optic immunosensors, describe the use of slow-release polymers to deliver reagents necessary for the immunoassay, and comment on the advantages and the limitations of the technique.</p></div>","PeriodicalId":79341,"journal":{"name":"ImmunoMethods","volume":"3 2","pages":"Pages 112-121"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/immu.1993.1045","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"51492169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

Immunoelectrochemical Biosensors for Detection of Saxitoxin and Brevetoxin 免疫电化学生物传感器检测蛤蚌毒素和Brevetoxin

ImmunoMethods Pub Date : 1993-10-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1047

Carter Robert M., Poli Mark A., Pesavento Maria, Sibley Deborah E.T., Lubrano Glenn J., Guilbault George G.

{"title":"Immunoelectrochemical Biosensors for Detection of Saxitoxin and Brevetoxin","authors":"Carter Robert M., Poli Mark A., Pesavento Maria, Sibley Deborah E.T., Lubrano Glenn J., Guilbault George G.","doi":"10.1006/immu.1993.1047","DOIUrl":"10.1006/immu.1993.1047","url":null,"abstract":"<div><p>Prototype immunoelectrochemical sensors for marine biotoxins, brevetoxin and saxitoxin, have been developed using chemical immobilization techniques on a solid support membrane. Briefly, PbTx-3 antigen was conjugated to BSA by succinylation and linked covalently through the lysine moiety of BSA to an immobilon membrane. The membrane was then incubated with antibody to PbTx-3, which had been conjugated to the enzyme glucose oxidase by the glutaraldehyde method. A noncovalent, antibody-antigen reaction occurred and the resulting sensor reacted to challenges of a suitable substrate (β-D-glucose) by detection of H<sub>2</sub>O<sub>2</sub> amperometrically. Maximum signal occurred when Immobilized PbTx-3 or STX electrodes were incubated with antibody-enzyme without competition from free PbTx-3 or STX. Competition reactions between free marine biotoxins and the immobilized biotoxin in the presence of enzyme-labeled antibody will decrease the signal in a manner inversely proportional to the free brevetoxin or saxitoxin concentration.</p></div>","PeriodicalId":79341,"journal":{"name":"ImmunoMethods","volume":"3 2","pages":"Pages 128-133"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/immu.1993.1047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"51492957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 35

Strategies for the Immobilization of Antibodies 抗体固定化策略

ImmunoMethods Pub Date : 1993-10-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1043

Schramm Willfried, Paek Se-Hwan, Voss Gundula

{"title":"Strategies for the Immobilization of Antibodies","authors":"Schramm Willfried, Paek Se-Hwan, Voss Gundula","doi":"10.1006/immu.1993.1043","DOIUrl":"10.1006/immu.1993.1043","url":null,"abstract":"<div><p>The immobilization of antibodies in two-dimensional (physical adsorption and immobilization via binding proteins) and that in three-dimensional (chemical binding to polypeptide strands) coverage of solid matrices have been compared. Methods of optimizing exposure of the immunoglobulin paratope after binding to surfaces to permit unimpaired antibody-antigen complex formation have been investigated. At comparable antibody densities, a larger number of paratopes are available for complex formation when antibodies are immobilized by means of binding protein (Protein A, Protein G, secondary antibodies, and F(ab)<sub>2</sub> fragments of secondary antibodies) compared to physical adsorption. Using a primary coat of binding protein, reproducible immobilization of antibodies at densities up to two orders of magnitude lower than densities produced by physical adsorption and chemical immobilization is achieved. Binding of antibodies by means of polypeptide strands can be used to obtain multi-layers of IgG molecules. This method of immobilization gives the IgG molecules some flexibility in lateral positioning which leads to formation of clusters and increased attachment to the solid matrix, impairing the paratopes′ ability to form complexes. However, flexible attachment can permit separation of the IgG molecules by treatment with detergents (e.g., Tween) or with charged molecules (e.g., fluorescein) or by introducing binding proteins as spacers. The implications for engineering organized multilayers of antibodies for optical biosensors are reviewed.</p></div>","PeriodicalId":79341,"journal":{"name":"ImmunoMethods","volume":"3 2","pages":"Pages 93-103"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/immu.1993.1043","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"51492058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 37

Immunosensors with Commercial Potential 具有商业潜力的免疫传感器

ImmunoMethods Pub Date : 1993-10-01 Epub Date: 2002-05-25 DOI: 10.1006/immu.1993.1048

Madou Marc, Joseph Jose

引用次数: 6