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OBJECTIVE: Based on its expression profile, folate receptor alpha (FRA) is an attractive candidate for targeted diagnostics and therapeutics. However, applicability of these agents in residual or recurrent disease could be influenced by chemotherapy. We evaluated whether chemotherapy modified FRA expression in non-mucinous epithelial ovarian (EOC) and endometrial carcinoma (EC). METHODS: FRA staining was evaluated by immunohistochemistry, using MAb 26B3, in 81 patients (41 EOCs and 40 ECs) and 17 control tissues (5 benign ovarian cysts, 5 normal ovarian, and 7 normal endometrial tissues). Chemotherapy effect was evaluated in 42 patients (30 paired samples at primary and interval debulking surgery and 12 from primary and recurrent disease). FRA expression was assessed using a semi-quantitative staining algorithm, the M-score (range 0-50). RESULTS: Median difference in M-score between tumor and control samples was 27.5 for EOC (95% CI 10.0 to 45.0) and 6.7 for EC (95% CI -6.7 to 21.7). Paired samples from both primary and interval debulking surgery did not differ in FRA expression in EOC (median difference of M-score between paired samples of 0.0 [95% CI -2.6 to 2.6]). Recurrent EOC tumors reflected FRA status at diagnosis (median difference of M-score between paired samples of 3.3 [95% CI -7.0 to 13.6]). CONCLUSIONS: This study shows no significant difference in FRA expression after chemotherapy, strengthening the rationale for FRA targeted diagnostics and therapeutics in FRA expressing tumors, whether newly diagnosed or at recurrence.

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This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

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Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

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Macromolecular interactions observed using surface plasmon resonance technology (BIAcore, Pharmacia) often display kinetic behavior which deviates from the pseudo-first-order time dependence that has been predicted for 1:1 interactions of ligand and ligate. In the present study we reviewed the majors reasons for such deviations, and present results which suggest that the most common source of deviations from the pseudo-first-order kinetic approximation of BIAcore kinetic data is likely to be heterogeneity of the immobilized ligand sites. A simplified analysis of the adsorption stage of BIAcore data is presented in terms of the net observed pseudo-first-order rate constant, kobs, rather than in terms of the association and dissociation rate constants, ka and kd. The analysis is then extended to the determination of the dissociation equilibrium constant for the interaction of ligand and ligate in the solution phase from sensorgrams reflecting competition between soluble and immobilized forms of ligand for ligate.

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Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp 120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

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The use of short peptide affinity tag sequences has become commonplace for the expression and purification of recombinant proteins. Many of these tags are antibody epitopes and detection of tagged proteins via Western blots is straightforward. However, the most common affinity tag used at present for the expression of recombinant proteins is a hexa-histidine, or like sequence, which exhibits strong affinity for Ni(II). The one drawback of histidine-containing affinity tags is the inability to specifically detect such recombinant proteins on Western blots. Here we describe the synthesis and use of biotinyl-nitrilotriacetic acid which, in combination with streptavidin-horseradish peroxidase, allows for the detection of hexa-histidine-tagged recombinant proteins on Western blots. In addition, we describe a surface plasmon resonance technique, employing a solid-phase Ni(II)-nitrilotriacetic acid complex, for the detection and quantitation of hexa-histidine-tagged recombinant proteins in solution. The surface plasmon resonance technique also allows for the oriented immobilization of the recombinant proteins for subsequent ligand interaction studies.

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A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.

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Supported hybrid bilayer membranes (HBM) composed of a monolayer of phospholipid and a monolayer of alkanethiol associated with a thin gold film on glass are useful as model lipid bilayer membranes for studying membrane receptor-ligand and cell-cell binding events by surface plasmon resonance (SPR). Measurements of specific binding of proteins and lipid vesicles to well-defined HBMs have been performed under conditions of continuous flow using a commercial SPR instrument (BIAcore). HBMs are shown to be stable in flow and to block nonspecific adsorption of proteins to the alkanethiol/gold surface. The use of such supported lipid bilayers in flow provides a means of conducting equilibrium and kinetic studies of models of ligand-cell and cell-cell interactions with receptors or ligands in a membrane environment. Compared to the extended dextran polymer layer that is currently used for surface modification of BIAcore "sensor chips," the described HBMs provide a well-defined surface that will permit less ambiguous modeling of these important biological interactions.

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Over the past year, the BIAcore system (which is based on the surface plasmon resonance phenomenon) has become increasingly popular for the study of macromolecular interactions. This biomolecular interaction analysis system allows the detection of macromolecular interactions in real time and in a label-free mode. The real-time detection properties of this technique suggest its potential in the generation of data relating to the kinetics of interaction of biomolecules.

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Mammalian peripheral nervous system (PNS) myelin contains several glycoproteins with molecular weights of 19 to 28 kDa, including the major 28 kDa P0 glycoprotein and a recently cloned protein called PMP-22. Some glycoproteins in this M(r) range in humans, cats and some other mammals react with HNK1, a mouse monoclonal antibody that identifies a carbohydrate epitope shared between the immune system and a number of adhesion proteins in the nervous system. A variety of antibodies to P0, PMP-22, and the carbohydrate determinants reacting with HNK1 were used to characterize immunochemically these 19 to 28 kDa glycoproteins of cat PNS myelin. The HNK1-reactive components include P0 and two slightly smaller 23 to 26 kDa proteins that are immunologically related to P0. However, HNK1 reacts most strongly with a lower molecular weight glycoprotein that does not react with the antibodies to P0 and was identified as PMP-22. Since the carbohydrate structure reacting with HNK1 is generally expressed on adhesion molecules, this result suggests that PMP-22 may function in cell-cell or membrane-membrane interactions. Furthermore, the related human anti-MAG monoclonal IgM antibodies from patients with neuropathy also react strongly with PMP-22, suggesting that it may be a target antigen in the pathogenesis of this disease. Purified PNS and CNS myelin from bony fish (toadfish and trout) were also shown to contain major glycoproteins, in the same 19 to 28 kDa M(r) range, that react very strongly with HNK1. It is shown that fish myelin has major proteins of this size that are immunologically and structurally related to mammalian P0, and it is demonstrated here that one of the strongly HNK1-positive proteins reacted well with an antiserum raised to bovine P0. The presence of high levels of the adhesion-related HNK1 epitope on these major myelin proteins of fish suggests that this carbohydrate structure may have played a role in the molecular evolution of myelin.

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Surface plasmon resonance (SPR) is a label-free, real time, optical detection method which has recently been commercialized as the BIAcore (Pharmacia). The technique relies on the immobilization of one of the interactants, the ligand, onto a dextran-coated gold surface. The second interactant, the ligate, is then injected across the surface and the interaction of the soluble ligate with the immobilized ligand is observed continuously and directly. The process of dissociation of bound ligate may also be observed directly after the sample plug has traversed the layer. Thus, the data generated contain information on the kinetic rate and equilibrium binding constants for the interaction under investigation. Historically, data from this instrument have been analyzed in terms of linear transformations of the primary data and requires that data from several ligate concentrations be analyzed to determine a single value for the association and dissociation rate constants. Here we discuss the analysis of untransformed BIAcore data by nonlinear least squares methods. The primary data are analyzed according to the integrated rate equations which describe the kinetics of the interaction of soluble ligate with immobilized ligand and the dissociation of the formed complex from the surface, respectively. Such analyses allow the direct determination of the association and dissociation rate constants for each binding experiment and, further, allow the analysis of data over a wider concentration range with lower associated errors compared to previously described methods. Through the use of modeling these interactions, we also demonstrate the limitations in determining the dissociation rate constant from the association phase of the interaction, thereby requiring that the dissociation process be analyzed. Indeed, the dissociation phase should be analyzed first to yield a relatively precise and unambiguous value of the dissociation rate constant, kd, which can then be used to constrain the analysis of the association phase to yield a better estimate of the association rate constant, k(a). We further demonstrate that, at least for the interaction investigated, the apparent rate and equilibrium binding constants determined using SPR are concentration independent and can be determined with good reproducibility.

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Surface plasmon resonance (SPR), a label-free, real time optical detection principle, has been investigated for its potential to detect and quantitate macromolecular ligand-ligate interactions. As model systems, the interactions of the HIV-1 envelope glycoprotein, gp120, and the monoclonal antibody L-71, with a soluble form of the T-cell receptor CD4 (sCD4), were investigated. In an effort to demonstrate potential analytical applications of this technology, operational characteristics of the SPR instrumentation (BIAcore, Pharmacia) including stability of the sensing surface and reproducibility in the measurement of such macromolecular interactions were investigated. In addition, the ability to detect and quantitate sCD4 directly from unfractionated cell culture supernatants, such as Streptomyces lividans, was investigated. The results demonstrate that SPR has potential in quantitating macromolecular interactions in both purified and crude samples and that the reproducibility in, and sensitivity of, such determinations is comparable to other techniques.

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Surface plasmon resonance detectors, such as the BIAcore instrument produced by Pharmacia, show promise for the detection and quantitation of macromolecular interactions in a label-free mode. Such detectors rely on the covalent immobilization of one of the interacting species onto the sensing surface. To date, the only published chemistry for this purpose is reaction of primary amino-containing ligands with an N-hydroxysuccinimide (NHS) ester-activated surface. In an effort to increase the versatility of the BIAcore with respect to immobilizing ligands, we undertook an investigation of activation chemistries compatible with this system. Using readily available reagents, we demonstrated that the carboxylated dextran-coated sensing surface could be easily converted to functions other than NHS-esters, including amine-activated, hydrazine-activated, and sulfhydryl-activated surfaces. In addition, use was made of the streptavidin/biotin interaction to probe chemical modifications of the sensing surface, by employing specifically modified biotin derivatives.

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Analytical affinity chromatography (AAC) was used to detect and quantitate the self-association of p24gag, the major structural capsid protein of human immunodeficiency virus (HIV-1). p24gag was immobilized on a hydrophilic polymer (methacrylate) chromatographic support. The resulting affinity column was able to interact with soluble p24, as judged by the chromatographic retardation of the soluble protein upon isocratic elution under nonchaotropic binding conditions. The variation of elution volume with soluble protein concentration fit to a monomer-dimer model for self-association. The soluble p24-immobilized p24 association process was observed using both frontal and zonal elution AAC at varying pH values; the dissociation constant was 3-4 x 10(-5) M at pH 7. That p24 monomer associates to dimers was determined in solution using analytical ultracentrifugation. The solution Kd was 1.3 x 10(-5) M at pH 7. AAC in the zonal elution mode provides a simple and rapid means to screen for other HIV-1 macromolecules that may interact with p24 as well as for modulators, including antagonists, of HIV p24 protein assembly.

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A rapid, simple assay for aldehydes generated by oxidation of saccharide units in glycoproteins, using dyes containing hydrazide functionalities, is described. The assay is used, in conjunction with tests of biological activity, to predict oxidation conditions that will result in a maximum of active protein coupled to a hydrazide chromatographic support. Glycoproteins are labeled with Lucifer Yellow CH or Texas Red Hydrazide, and the extent of labeling is determined. Using the assay, it is shown that the efficiency of coupling to Affi-Prep Hydrazide is proportional to oxidation.

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Potentiometric measurements have been applied to the detection of enantiomeric separations on molecularly imprinted polymers. A flow-through column electrode, based on the use of polymers imprinted against L-phenylalanine anilide, is described. The electrode consisted of a glass column in which the polymer was packed and where the end frits constituted the electrodes. The flow stream potential across the column can be continuously recorded as solvent is pumped through the system. The column resolved the enantiomers of phenylalanine anilide as detected by both UV absorption and potentiometric measurements and the recorded signals could be correlated with the concentration of phenylalanine anilide. The calibration graphs obtained for the UV absorption of phenylalanine anilide were linear over the concentration range investigated, whereas the potentiometric signal was shown to be exponentially linear with concentration. The application of molecular imprints to the preparation of supports suitable for chromatographic separations of enantiomers and for the preparation of specific electrodes is discussed.

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Many chemistries have been developed for the immobilization of ligands onto insoluble matrices for subsequent use in affinity systems. One such chemistry which has received little attention involves the use of hydrazido-derivatized solid supports. Hydrazine derivatives are strong nucleophiles which will react with a number of functional groups including aldehydes which may be generated on the oligosaccharide moieties of glycoconjugates by specific oxidation reactions. This paper presents a brief overview of the chemistries involved and the uses of hydrazido-derivatized solid supports for the site-directed immobilization of glycoconjugates. Specific examples from the literature on the uses of affinity matrices prepared by this method are cited.

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