ImmunoMethods最新文献

The X-ray crystallographic structures of five antibody-protein antigen complexes are briefly described. The three-dimensional structure of the antibody-antigen interface is compared to the results obtained by two other techniques: analysis of mutant binding data and NMR hydrogen-deuterium exchange.

The combinatorial antibody approach captures the immune repertoire within a library of bacteria or their viruses (phage). Current molecular biology techniques allow the construction of repertoires of a size that at least matches if not surpasses the size of the primary animal repertoire, about 10⁸. Escherichia coli are competent producers of antibody Fab and scFv fragments. To survey repertoires of this size for antibody fragments of the proper specificity and highest affinity, a phage display system that allows an affinity-based selection of clones was developed. These developments have at least three important consequences: (i) they allow direct cloning and expression of human Fab fragments; (ii) they allow the creation of synthetic antibodies; and (iii) they allow the in vitro evolution of antibody specificity and affinity. For the crystallographer, these developments will provide unique opportunities for the study of molecular recognition .c 1993

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.

Three-dimensional structures of antibody and antibody-antigen complexes that have been solved to date exhibit highly conserved folding motifs, and the main structural differences between antibodies are restricted to the interdomain angles and the antigen-binding site. This knowledge has been successfully used to solve a large number of crystal structures of antibody fragments by molecular replacement. This paper reviews approaches that have been used in solving these crystal structures.

The three-dimensional structures of six Fab-peptide complexes have been determined by X-ray crystallography. The peptide antigens range in size from 8 to 19 residues and include a cyclic peptide with unusual amino acid side chains. The structures were all determined by molecular replacement techniques using known Fab structures as models and have been refined to medium or high resolution. These structures not only have improved our general understanding of antibody-antigen recognition but have aided in the determination of the sequence and structural characteristics of peptide epitopes. The peptide structures provide a framework to aid in the design of peptide vaccines or small peptide-like drugs to inhibit viral function.

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.

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 H₂O₂ 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.

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)₂ 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.

Biosensors find application in such widely disparate fields as environmental monitoring and agricultural, chemical, food, and biomedical industries. One of the more prominent examples of a biosensor applicable in all these fields is an immunosensor, i.e., a sensor for either an antigen or an antibody based on the selective binding of antigen-antibody pairs or, in a broader sense, any sensor based on the pairing of a molecular recognition affinity pair. In this paper we review immunomethods with potential for development into a sensor. Issues such as direct versus indirect and homogeneous versus heterogeneous techniques and the relative merit of electrochemical versus optical sensors are given extra attention.