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.     

Novel monoclonal antibodies with specificity for chelated uranium(VI): isolation and binding properties

A derivative of 1,10-phenanthroline that binds to UO(2)(2+) with nanomolar affinity was found to be a very effective immunogen for the generation of antibodies directed toward chelated complexes of hexavalent uranium. This study describes the synthesis of 5-isothiocyanato-1,10-phenanthroline-2,9-dicarboxylic acid and its use in the generation and functional characterization of a group of monoclonal antibodies that recognize the most soluble and toxic form of uranium, the hexavalent uranyl ion (UO(2)(2+)). Three different monoclonal antibodies (8A11, 10A3, and 12F6) that recognize the 1:1 complex between UO(2)(2+) and 2,9-dicarboxy-1,10-phenanthroline (DCP) were produced by the injection of BALB/c mice with DCP-UO(2)(2+) covalently coupled to a carrier protein. Equilibrium dissociation constants for the binding of DCP-UO(2)(2+) to antibodies 8A11, 10A3, and 12F6 were 5.5, 2.4, and 0.9 nM, respectively. All three antibodies bound the metal-free DCP with roughly 1000-fold lower affinity. The second-order rate constants for the bimolecular association of each antibody with soluble DCP-UO(2)(2+) were in the range of 1 to 2 x 10(7) M(-1) s(-1). Binding studies conducted with structurally related chelators and 21 metal ions demonstrated that each of these three antibodies was highly specific for the soluble DCP-UO(2)(2+) complex. Detailed equilibrium binding studies conducted with three other derivatives of DCP, either complexed with UO(2)(2+) or metal-free, suggested that the antigen binding sites on the three antibodies have significant functional and structural similarities. Biomolecules that bind specifically to uranium will be at the heart of any new biotechnology developed to monitor and control uranium contamination. The three antibodies described herein possess sufficient affinity and specificity to support the development of immunoassays for hexavalent uranium in environmental and clinical samples.     

Cathepsin G binding to human platelets. Evidence for a specific receptor.

We have shown previously that purified human neutrophil cathepsin G is a strong platelet agonist. We now demonstrate that cathepsin G exhibits saturable, reversible binding to human platelets which is characteristic of binding to a specific receptor. At room temperature, cathepsin G displayed apparent positive co-operativity of binding, as indicated by sigmoidal binding curves and a Hill coefficient greater than unity. By contrast, binding curves conducted with native enzyme at 0.5 degrees C displayed a much smaller degree of sigmoidicity, and binding studies performed with phenylmethanesulphonyl fluoride-treated enzyme at 22-25 degrees C exhibited hyperbolic binding curves. The concentrations of cathepsin G required to give half-saturation (S0.5) with inhibitor-treated enzyme or with native enzyme at either room temperature or 0.5 degrees C were all similar, suggesting that sigmoidal binding curves did not result from an alteration in the affinity of the binding sites for cathepsin G. However, platelets bound approximately twice as many molecules of native enzyme as molecules of phenylmethanesulphonyl fluoride-treated cathepsin G per cell. From these observations it can be inferred that the apparent positive co-operativity may in part reflect the exposure of binding sites due to the proteolytic activity of cathepsin G. However, this conclusion is not supported by experiments conducted with subsaturating cathepsin G concentrations, which demonstrated that ligand binding did not show an expected increase at longer time intervals. Measurement of Ca2+ mobilization and cathepsin G binding in the same platelet suspensions demonstrated that elevations in cytosolic free Ca2+ concentration had achieved near-maximal levels in the presence of 15 micrograms of cathepsin G/ml, whereas maximal binding was observed at approx. 35 micrograms/ml, indicating that only a fraction of the total binding sites need be occupied to elicit platelet activation. Pretreatment of platelets with forskolin or phorbol 12-myristate 13-acetate (PMA) decreased cathepsin G binding by approx. 60% and 40% respectively, indicating that the receptor may be desensitized or down-regulated by phosphorylation due to protein kinases. Since forskolin and PMA could diminish receptor availability by activating negative feedback mechanisms, inhibition of negative signal-transduction pathways could conversely play a role in the up-regulation of cathepsin G binding. In any event, these results show that cathepsin G is an agonist that must bind to platelets to initiate processes associated with cell activation, and suggest a role for cathepsin G in platelet function.     

Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185HER2 antibody Fab fragments.

The murine monoclonal antibody 4D5 (anti-p185HER2) inhibits the proliferation of human tumor cells overexpressing p185HER2 in vitro and has been "humanized" [Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W.-L. T., Rowland, A. M., Kotts, C., Carver, M. E., & Shepard, H. M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)] for use in human cancer therapy. We have determined the antigen binding thermodynamics and the antiproliferative activities of chimeric 4D5 Fab (ch4D5 Fab) fragment and a series of eight humanized Fab (hu4D5 Fab) fragments differing by amino acid substitutions in the framework regions of the variable domains. Fab fragments were expressed by secretion from Escherichia coli and purified from fermentation supernatants by using affinity chromatography on immobilized streptococcal protein G or staphylococcal protein A for ch4D5 and hu4D5, respectively. Circular dichroism spectroscopy indicates correct folding of the E. coli produced Fab, and scanning calorimetry shows a greater stability for hu4D5 (Tm = 82 degrees C) as compared with ch4D5 Fab (Tm = 72 degrees C). KD values for binding to the extracellular domain (ECD) of p185HER2 were determined by using a radioimmunoassay; the delta H and delta Cp for binding were determined by using isothermal titration calorimetry. ch4D5 Fab and one of the humanized variants (hu4D5-8 Fab) bind p185HER2-ECD with comparable affinity (delta G degrees = -13.6 kcal mol-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutual conformational adaptations in antigen and antibody upon complex formation between an Fab and HIV-1 capsid protein p24

BACKGROUND: Elucidating the structural basis of antigen-antibody recognition ideally requires a structural comparison of free and complexed components. To this end we have studied a mouse monoclonal antibody, denoted 13B5, raised against p24, the capsid protein of HIV-1. We have previously described the first crystal structure of intact p24 as visualized in the Fab13B5-p24 complex. Here we report the structure of the uncomplexed Fab13B5 at 1.8 A resolution and analyze the Fab-p24 interface and the conformational changes occurring upon complex formation. RESULTS: Fab13B5 recognizes a nearly continuous epitope comprising a helix-turn-helix motif in the C-terminal domain of p24. Only 4 complementarity-determining regions (CDRs) are in contact with p24 with most interactions being by the heavy chain. Comparison of the free and complexed Fab reveals that structural changes upon binding are localized to a few side chains of CDR-H1 and -H2 but involve a larger, concerted displacement of CDR-H3. Antigen binding is also associated with an 8 degrees relative rotation of the heavy and light chain variable regions. In p24, small conformational changes localized to the turn between the two helices comprising the epitope result from Fab binding. CONCLUSIONS: The relatively small area of contact between Fab13B5 and p24 may be related to the fact that the epitope is a continuous peptide rather than a more complex protein surface and correlates with a relatively low affinity of antigen and antibody. Despite this, a significant quaternary structural change occurs in the Fab upon complex formation, with additional smaller adaptations of both antigen and antibody.     

The intrinsic affinity constant (K) of anticapsular antibody to oligosaccharides of Haemophilus influenzae type b

Antibody to the polyribosylribitol phosphate (PRP) capsular polysaccharide of Haemophilus influenzae type b is crucial to host defense. Affinities of antibody elicited by vaccination with PRP and PRP-diphtheria toxoid conjugate were determined using oligosaccharides (OS) from PRP. The affinities of antibody induced by vaccination with PRP to OS of three and four repeat units were similar but greater than the affinity to the two-unit OS. NaBH4 reduction of the three-unit OS did not alter the binding affinity, indicating that the reducing end of the OS did not participate in antibody binding. Over the range of OS concentrations tested, antibody affinity appeared to be homogeneous. Antibody concentration could be determined from binding experiments independently from affinity. Whole serum had 8- to 40-fold less antibody detected by binding analysis than by RIA, but the antibody concentration of an IgG fraction measured by the two methods agreed within a factor of two. We could not account for the discrepancy in concentrations found with whole serum by the presence of IgM or IgA antibody. The average affinity of antibodies of 10 adults vaccinated with PRP was similar to that of antibodies elicited in 14 adults vaccinated with a PRP-diphtheria toxoid conjugate (10.7 x 10(5) vs 7.6 x 10(5) liter/mol, respectively, p greater than 0.05). We conclude that the intrinsic affinity of antibody after vaccination with PRP is low and is not different from that of antibody elicited by PRP diphtheria toxoid conjugate.     

Modulation of antibody pharmacokinetics by chemical polysialylation

Chemical coupling of a variety of polymers to therapeutic proteins has been studied as a way of improving their pharmacokinetics and pharmacodynamics in vivo. Conjugates have been shown to possess greater stability, lower immunogenicity, and a longer blood circulation time due to the chemicophysical properties of these hydrophilic long chain molecules. Naturally occurring colominic acid (polysialic acid, PSA) has been investigated as an alternative to synthetic polymers such as poly(ethylene glycol) (PEG) due to its lower toxicity and natural metabolism. Antibodies and their fragments are a good example of the types of proteins which benefit from pharmacokinetic engineering. Here, we chemically attached differing amounts and differing lengths of short (11 kDa) and longer (22 kDa) chain colominic acid molecules to the antitumor monoclonal antibody H17E2 Fab fragment. Different coupling ratios and lengths were seen to alter the electrophoretic mobility of the Fab fragment but have a minor effect on the antibody immunoreactivity toward the placental alkaline phosphatase (PLAP) antigen. Polysialylation generally increased Fab fragment blood half-life resulting in higher tumor uptake in a KB human tumor xenograft mouse model. One H17E2 Fab-PSA conjugate had over a 5-fold increase in blood exposure and over a 3-fold higher tumor uptake with only a marginal decrease in tumor/blood selectivity ratio compared to the unconjugated Fab. This conjugate also had a blood bioavailability approaching that of a whole immunoglobulin.     

Ligation of low-density lipoprotein receptor-related protein with antibodies elevates intracellular calcium and inositol 1,4, 5-trisphosphate in macrophages

We have probed the signaling characteristics of the macrophage low-density lipoprotein receptor-related protein (LRP) with monoclonal antibody 8G1, its Fab and F(ab')(2) fragments directed against the ligand binding heavy chain, and monoclonal antibody 5A6 directed against the membrane-spanning light chain of LRP. Ligation of LRP with 8G1, its Fab and F(ab')(2) fragments, or 5A6 increased intracellular Ca(2+) levels two- to threefold. Prior ligation of LRP with 8G1 did not affect the increase in [Ca(2+)](i) observed on subsequent ligation of LRP with lactoferrin, P. exotoxin A, or lipoprotein lipase. Binding to LRP by 8G1, its Fab and F(ab')(2) fragments, or 5A6 increased inositol 1,4,5-trisphosphate (IP(3)) levels by 50 to 100%. Incubation of macrophages with guanosine 5', 3'-O(thio)-triphosphate (GTP-gamma-S) before treatment with antibody potentiated and sustained the 8G1-induced increase in IP(3) levels. Treatment of macrophages with guanyl-5'-yl thiophosphate prior to GTP-gamma-S treatment abolished the GTP-gamma-S-potentiated increase in IP(3) levels in 8G1-treated macrophages. Antibody-induced increases in IP(3) and [Ca(2+)](i) in macrophages on ligation of LRP were pertussis toxin sensitive. Binding of 8G1 or its Fab or F(ab')(2) fragments to LRP stimulated macrophage protein kinase C (PKC) activity as evaluated by histone IIIs phosphorylation by about two- to sevenfold. Staurosporin inhibited the anti-LRP antibody-induced increase in PKC activity. Ligation of LRP with 8G1 increased cellular cAMP levels about twofold. Preincubation of macrophage with the LRP-binding protein receptor-associated protein suppressed the 8G1-induced increase in cAMP levels. Thus, binding of antibodies directed against either chain of LRP triggers complex signaling cascades.     

戊型肝炎病毒衣壳蛋白中和表位间的构象诱导

重组蛋白NE2包含了戊型肝炎病毒(HEV)衣壳蛋白(pORF2)的aa394～606片段.在NE2上已鉴定出了2个HEV中和表位,并获得了3个识别中和表位的单克隆抗体(MAb)8C11、13D8和8H3.这3个MAb间的交叉阻断ELISA实验发现,8C11和13D8可以彼此完全阻断,8H3对8C11和13D8均不能阻断,而8C11非但不能阻断8H3,反而显著增强了8H3与抗原的结合.用生物传感器进行的抗体与抗原结合的动力学分析也证实了这一现象.这些结果提示,在NE2上8H3表位区域受到抗原上某些结构的掩盖,而8C11与NE2的结合引起了抗原空间结构的改变,导致了掩盖8H3表位的结构的去除和8H3表位的充分暴露.免疫捕获RT-PCR发现,8C11同样可以显著增强8H3对天然HEV病毒的捕获能力,提示这种结合诱导的衣壳蛋白空间构象改变在天然HEV病毒颗粒上同样存在.     

Modulation of arginine decarboxylase activity from Mycobacterium smegmatis. Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme

Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km = 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function. Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.     

Chloride concentration dependency of the electrogenic activity of halorhodopsin

The capacitive photoelectric current responses of the halorhodopsins from Halobacterium salinarum (shR) and from Natronobacterium pharaonis (phR) were studied using membrane fragments adsorbed onto a thin polyester film. The electric current of shR was not much affected by ionic strength or cations present in the medium (Na+, K+, Li+, Mg2+, or Ca2+), but was greatly influenced by the Cl- concentration. It increased biphasically as the Cl- concentration increased from 0 to 5 M, then decreased and almost vanished at around 10 or 12 M. Apparent Kd's of about 0.1 and 6 M were deduced for the Kd of Cl- uptake sites. We had to assume a sigmoidal increase of Cl- binding with a Hill coefficient of about 8 at the cytoplasmic, Cl- release site(s). The half-maximum Cl- concentration for the sigmoidal binding was about 7.5 M. The electric current of phR had a maximum around 30 mM Cl- and biphasically decreased at higher Cl- concentrations. The apparent Kd for the Cl- uptake site was 5 mM. The biphasic decrease in the transport activity was explained by assuming a sum of simple hyperbolic type binding (Kd = 0.2 M) and sigmoidally increasing binding with a Hill coefficient of 10 on the cytoplasmic side. The half-maximum concentration of the latter cooperative binding was 5.6 M. This great difference between the apparent affinity of the release site of shR and that of phR can explain the previously reported difference between the Cl- dependency of their photocycles. These results also suggest that there may be multiple Cl- binding sites in the Cl- transport pathway. A simple sequence of Cl- transport steps based on a multiion channel model is proposed.     

Human erythrocyte membrane band 3 protein influences hemoglobin cooperativity. Possible effect on oxygen transport

Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete. For example, the 43-kDa chymotryptic fragment of the cytoplasmic portion of red cell membrane Band 3 protein and its corresponding N-terminal 11-residue synthetic peptide lower the oxygen affinity of hemoglobin but effects on cooperativity are unclear. Using highly purified preparations, we also find a lowered Hill coefficient (n values <2) at subequivalent ratios of Band 3 fragment or of synthetic peptide to Hb, resulting in an oxygen affinity that is moderately decreased and a partially hyperbolic shape for the O2 binding curve. Both normal HbA and sickle HbS display this property. Thus, the determinant responsible for the Hb cooperativity decreases by the 43-kDa fragment resides within its first 11 N-terminal residues. This effect is observed in the absence of chloride and is reversed by its addition. As effector to Hb ratios approach equivalence or with saturating chloride normal cooperativity is restored, and oxygen affinity is further lowered because the shape of the oxygen binding curve becomes completely sigmoidal. The relative efficiencies of 2,3-diphosphoglycerate (DPG), the 43-kDa Band 3 fragment, and the 11-residue synthetic peptide in lowering cooperativity are very similar. The findings are explained based on the stereochemical mechanism of cooperativity because of two populations of T-state hemoglobin tetramers, one with bound effector and the other with free (Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139-237). As a result of this property, hemoglobin at the membrane inner surface in contact with the N-terminal region of Band 3 could preferentially bind O2 at low oxygen tension and then release it upon saturation with 2,3-diphosphoglycerate in the interior of the red cell. Membrane modulation of hemoglobin oxygen affinity has particularly interesting implications for the polymerization of hemoglobin S in the sickle red cell.     

Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast-displayed repertoires diversified by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.     

Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation

The radiolabelled monoclonal antibody, 5G11, directed against native thrombospondin, has been used to assess the surface expression of secreted thrombospondin on human blood platelets. Emphasis has been placed on studying the role of fibrinogen in this process. Unstimulated platelets bound low amounts of 5G11 (about 2000 molecules/platelet). Binding increased 2-fold and 5-7-fold after stimulation of platelets with ADP or thrombin (or ionophore A23187) respectively. Unstimulated platelets from patients deficient in alpha-granule proteins (gray platelet syndrome) bound baseline levels of 5G11. However, binding was not increased after activation. Thrombospondin expression on thrombin-stimulated normal platelets was for a large part divalent-cation-dependent and was not affected by AP-2, a monoclonal antibody to GPIIb-IIIa complexes. However, binding of 5G11 was some 50% lower when platelets were stimulated in the presence of Fab fragments of a polyclonal rabbit antibody to fibrinogen. This suggested either a direct binding of thrombospondin to surface-bound fibrinogen or a steric inhibition due to a close proximity of the two proteins. The fact that binding of 5G11 was at the lower limit of the normal range to the stimulated platelets of an afibrinogenaemic patient specifically lacking detectable fibrinogen favoured the latter explanation. Thus, a major fibrinogen-independent pathway for thrombospondin expression must exist.     

Structure of an anti-Lewis X Fab fragment in complex with its Lewis X antigen

The Lewis X trisaccharide is pivotal in mediating specific cell-cell interactions. Monoclonal antibody 291-2G3-A, which was generated from mice infected with schistosomes, has been shown to recognize the Lewis X trisaccharide. Here we describe the structure of the Fab fragment of 291-2G3-A, with Lewis X, to 1.8 A resolution. The crystallographic analysis revealed that the antigen binding site is a rather shallow binding pocket, and residues from all six complementary determining regions of the antibody contact all sugar residues. The high specificity of the binding pocket does not result in high affinity; the K(D) determined by isothermal calorimetry is 11 microM. However, this affinity is in the same range as for other sugar-antibody complexes. The detailed understanding of the antibody-Lewis X interaction revealed by the crystal structure may be helpful in the design of better diagnostic tools for schistosomiasis and for studying Lewis X-mediated cell-cell interactions by antibody interference.     

Fluorescence study of steroid hormone binding activity of Helix pomatia agglutinin

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin, found in the reproductive gland of a Roman snail. The present study has shown that HPA, in addition to its carbohydrate binding capacity possesses a hydrophobic binding activity. This protein binds with high affinity (k(D)=1.9-2.4 microM) steroid hormones: testosterone and progesterone, identified as putative ligands for the animal lectin HPA. Additionally, we have found that this lectin also interacts with adenine (k(D)=5.4+/-0.5 microM) and arylaminonaphthalene sulfonate TNS (k(D)=12+/-0.3 microM). Binding of HPA to hormones and adenine was accompanied by a significant increase of the intrinsic Trp fluorescence (up to 50%), characterizing the conformational changes in the lectin molecule. The hyperbolic shape of the binding curves indicated one high affinity site for the two steroid hormones and adenine, and more than one hydrophobic site for TNS, showed by the sigmoidal curve fit and Hill coefficient of (n(H)=1.5+/-0.2). Hormones and adenine compete for an identical binding site, suggested to occupy the central hydrophobic cavity of the HPA hexamer. Fluorescence resonance energy transfer (FRET) was applied to calculate the intramolecular distance between TNS and Trp chromophores.     

Antibody-antigen complex formation with immobilized immunoglobulins.

We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays.     

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.

In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

Isolation and characterization of cytoplasmic NADP+-malic enzyme from Lupinus luteus leaves

NADP⁺-dependent malic enzyme (L-malate : NADP⁺ oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH₄)₂SO₄ and Sephadex G-25 chromatography, followed by purification on DEAE-cellulose and Sephadex G-200 columns. The enzyme was purified 122-fold. The enzyme affinity towards L-malate was found to be significantly higher with Mn²⁺ than with Mg²⁺. The Hill coefficient for Mg²⁺ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP⁺ followed a hyperbolic curve. K_m values and Hill coefficients for NADP⁺ were similar with both Mn²⁺ and Mg²⁺. The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg²⁺. The enzyme had 4-fold higher affinity towards Mn²⁺ than towards Mg²⁺, the K_m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.