Preliminary crystallographic analysis of the Fab fragment of an antibody against HIV gp120.

Single crystals of the Fab fragment of a murine monoclonal antibody BAT123 (IgG1, kappa) raised against a dominant neutralizing determinant of gp120 of HIV that are suitable for X-ray structural analysis have been obtained. The thick prismatic plate crystals belong to space group P2(1)2(1)2 with unit cell dimensions of a = 177.42 A, b = 37.36 A and c = 73.30 A.     

Preliminary crystallographic study of a complex between the Fab fragment of a monoclonal anti-lysozyme antibody (D1.3) and the Fab fragment from an anti-idiotopic antibody against D1.3

An anti-lysozyme antibody, D1.3, was used as immunogen to obtain syngeneic (Balb/c) monoclonal anti-idiotopic antibodies. The complex between Fab D1.3 and the Fab fragment from the anti-idiotopic antibody E225 has been crystallized. The crystals are monoclinic, space group P2(1), with a = 75.7 A, b = 77.4 A, c = 97.2 A, beta = 111.90 degrees and one molecule of the complex in the asymmetric unit. X-ray photographs show reflections extending to a resolution of about 3 A. Although twinning occurs frequently in the large crystals obtained, this material is suitable for high-resolution X-ray analysis.     

Preliminary crystallographic study of the Fab fragment of a monoclonal antibody directed against a cobra cardiotoxin

We report on the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragments from a murine monoclonal anti-cardiotoxin antibody M gamma 2-3 directed against a cobra cardiotoxin. The Fab fragment has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution, X-ray crystallographic studies. The crystals are monoclinic, space group C2, with a = 161.2 A, b = 40.4 A, c = 96.5 A, beta = 118.3 degrees.     

Crystallization and preliminary crystallographic data of the Fab fragment of an anti-phenylalanine hydroxylase monoclonal antibody

Two crystal habits, one rod shaped and the other square prismatic, of the Fab fragment of a monoclonal anti-phenylalanine hydroxylase antibody have been grown using the method of vapour phase diffusion against polyethylene glycol 6000. The square prisms diffract to better than 2.8 A, belong to the space group P1 and have unit cell parameters a = 41.8 A, b = 50.3 A, c = 114.7 A, alpha = 97.6 degrees, beta = 91.7 degrees, gamma = 91.0 degrees, while the rod-shaped crystals belong to the space group P212121, have unit cell parameters a = 105.6 A, b = 119.8 A, c = 82.2 A and diffract to 3.5 A resolution.     

Preliminary crystallographic study of the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen

Preliminary crystallographic data are given for the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen. This crystalline complex was found by screening a number of Fab-lysozyme complexes prepared from monoclonal anti-lysozyme antibodies produced by hybrids of BALB/c immune spleen cells with a non-secreting mouse hybrid myeloma line. The complex crystallizes in the monoclinic space group P21 with a = 55.5 (+/- 0.1) A, b = 143.5 (+/- 0.3) A, c = 49.1 (+/- 0.1) A, beta = 120 degrees 20' (+/- 10'). X-ray photographs show reflections extending to a resolution of 2.7 A. The crystals are suitable for high-resolution X-ray diffraction studies.     

Preliminary crystallographic studies of an anti-HIV-1 protease antibody that inhibits enzyme activity.

F11.2.32, a monoclonal antibody directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme. The antibody cross-reacts with peptides 36-46 and 36-57 from the protease. Crystals of the Fab have been obtained both in the free state and as complexes formed with the protease peptide fragments, 36-46 and 36-57. Diffraction data have been collected for the free and complexed forms of Fab F11.2.32 and preliminary models for the crystal structures were obtained by molecular replacement.     

Characterization of crystals of an Fab fragment of a murine monoclonal antibody.

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2.9 A resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57.16 A, b = 70.85 A, c = 75.81 A, alpha = 85.11 degrees, beta = 121.28 degrees and gamma = 116.33 degrees. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit.     

2.9 A resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten

The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2.9 A resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (VH, VL, CH1 and CL) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was refined 2.5 A crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (gamma 1, kappa, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19.5% for 12,129 unique reflections between 8.0 and 2.9 A. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0.014 A, and the r.m.s. deviation from ideal bond angles is 3.1 degrees. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments.     

Structural and kinetic studies of the Fab fragment of a monoclonal anti-spin label antibody by nuclear magnetic resonance

Nuclear magnetic resonance has been used to study the structure of the anti-spin label antibody AN02 combining site and kinetic rates for the hapten-antibody reaction. The association reaction for the hapten dinitrophenyl-diglycine (DNP-diGly) is diffusion-limited. The activation enthalpy for association, 5.1 kcal/mol, is close to the activation enthalpy for diffusion in water. Several reliable resonance assignments have been made with the aid of recently reported crystal structure. Structural data deduced from the nuclear magnetic resonance (n.m.r.) spectra compare favorably with the crystal structure in terms of the combining site amino acid composition, distances of tyrosine residues from the unpaired electron of the hapten, and residues in direct contact with the hapten. Evidence is presented that a single binding site region tyrosine residue can assume two distinct conformations on binding of DNP-diGly. The AN02 antibody is an autoantibody. Dimerization of the Fab fragments is blocked by the hapten DNP-diGly. The n.m.r. spectra suggests that some of the amino acid residues involved in the binding of the DNP-hapten are also involved in the Fab dimerization.     

Expression and characterization of a recombinant Fab fragment derived from an anti-human alpha-fetoprotein monoclonal antibody

Alpha-fetoprotein (AFP) is a well-known molecular marker indicating the development of cancer as well as fetal abnormalities such as open neural tube defects. Accordingly the measurement of serum AFP is important for the diagnosis of hepatocellular carcinoma (HCC) and other abnormalities. Monoclonal antibodies (McAb) to AFP were produced to develop an immunoassay kit, and to study the possibility of an antibody (Ab) therapy. The immunoglobulin genes were cloned from hybridoma cells, and expressed in E. coli as an Fab soluble into a culture medium. The Fab of anti-AFP McAb exhibited binding to AFP and similar affinity compared to the original IgG. This recombinant antibody can be studied further for in vivo imaging and immunotherapeutics.     

Preliminary crystallographic studies on duck ovotransferrin

Crystals of duck ovotransferrin and duck apo-ovotransferrin have been grown from polyethylene glycol solutions. For both crystals, the space group is P2(1)2(1)2(1), the unit cell dimensions for the ovotransferrin are a = 49.6 A, b = 85.6 A, c = 178.7 A and for the apo-ovotransferrin a = 77.6 A, b = 98.8 A, c = 127.0 A, giving four molecules in the unit cell.     

Preliminary crystallographic studies on bovine lactoferrin

The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.     

Allosteric binding properties of a monoclonal antibody and its Fab fragment

Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.     

Preliminary crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies

We report on the preparation, crystallization, and preliminary x-ray crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies obtained from the secondary response to this hapten. The Fab fragment from one of these (NQ10/12.5) has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution x-ray crystallographic studies. These crystals are monoclinic, space group C2, with a = 129.2 A, b = 79.4 A, c = 57.7 A, beta = 96.2 degrees, and one Fab/asymmetric unit. Determination of the three-dimensional structure of Fab NQ10/12.5 should help clarify the role of somatic mutation in the maturation of an immune response. This antibody and an anti-lysozyme antibody also under study apparently use the same germ-line encoded VK and a similar VH gene, respectively, as the idiotypic anti-oxazolone antibodies characteristic of the primary response. A comparative study of the two structures should shed light on the role of the pairing of heavy and light chains in the antigen-binding function of antibodies.     

Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2.     

Generation and characterization of biotinylated recombinant Fab antibody fragment against cortisol

This paper describes the in vivo generation method of biotinylated recombinant Fab antibody fragments. The original molecular vectors for Escherichia coli Fab fragments expression were designed. In vivo biotinylated recombinant antibody Fab fragment against cortisol was generated. The kinetic parameters of interaction of these antibody fragments with cortisol-BSA complex were measured via the biolayer interferometry method. An equilibrium dissociation constant of this interaction K_D is 5.48 × 10^–10 M. The interaction is reversible and it could be competitively inhibited by free cortisol addition. These results could be used in generating of immunoassays for quantitative cortisol determinations. The in vivo biotinylation system under review is universal and suitable for expression of any biotinylated Fab fragments in the E. coli system.     

Crystal structure of the alpha1beta1 integrin I domain in complex with an antibody Fab fragment

The alpha1beta1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human alpha1beta1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the alpha1beta1 integrin and the Fab fragment of humanized AQC2 antibody. The structure of the complex shows that the antibody blocks the collagen binding site of the I domain. An aspartate residue, from the CDR3 loop of the antibody heavy chain, coordinates the MIDAS metal ion in a manner similar to that of a glutamate residue from collagen. Substitution of the aspartate residue by alanine or arginine results in significant reduction of antibody binding affinity. Interestingly, although the mode of metal ion coordination resembles that of the open conformation, the I domain maintains an overall closed conformation previously observed only for unliganded I domains.     

Crystallization and preliminary X-ray diffraction studies of a monoclonal antibody Fab fragment specific for an influenza virus haemagglutinin and of an escape mutant of that haemagglutinin

Preliminary crystallographic data are given for two molecules involved in the interaction between the humoral immune response and the influenza virus. These molecules are the Fab fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong 1/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose structure is known; they diffract to 3.4 A resolution. Crystals of the Fab fragment are trigonal with space group P3(1)21 (or P3(2)21) and diffract to 2.6 A resolution. The unit cell dimensions are a = b = 98.9 A, c = 89.2 A. A native data set has been collected for both proteins.