Folate receptor alpha (FRA) expression remains unchanged in epithelial ovarian and endometrial cancer after chemotherapy.

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.

Determination of kinetic rate and equilibrium binding constants for macromolecular interactions: a critique of the surface plasmon resonance literature.

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.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

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.

Site-specific immobilization of antibodies by their oligosaccharide moieties to new hydrazide derivatized solid supports.

This report describes a new method for immobilization of antibodies to solid supports. Antibodies are bound to the solid supports by covalent bonds between aldehydes generated on the carbohydrate side chains of the antibody and hydrazide groups on the solid support. The hydrazone bonds that are formed are stable at least from pH 2-10, permitting the acid elution of antigens from the affinity column. Over 25 mg of affinity-purified rabbit IgG binds per ml of solid support, with most of the bound antibodies retaining biological activity. Advantages of this new affinity support over existing technology are discussed along with procedures for the preparation and use of affinity columns containing monoclonal or polyclonal antibodies.

Measurement of protein interaction bioenergetics: application to structural variants of anti-sCD4 antibody.

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.

Specificity of human IgM monoclonal antibodies from patients with peripheral neuropathy.

Some patients with peripheral neuropathy and gammopathy have IgM monoclonal antibodies that react with the myelin-associated glycoprotein (MAG), some 20-26 kDa glycoproteins present only in the peripheral nervous system (PNS), and some acidic glycolipids that are also PNS-specific. This communication describes an investigation of 18 patients with IgM paraproteinemia and neuropathy to test for the presence of antibodies that react with each of these components. Eleven patients had IgM that reacted with MAG, and in all cases the IgM also reacted with the lower Mr glycoproteins and the acidic glycolipids that are specific for the PNS. With respect to the other 7 patients that did not react with MAG, in no instance did immune-staining of electroblots reveal the presence of reactivity with the 20-26 kDa glycoproteins of the PNS or with any other protein antigen in the PNS or central nervous system (CNS). However, these 7 patients fell into 3 categories with regard to reactivity with acidic glycolipids: three reacted with the acidic glycolipid fraction of both PNS and CNS tissue; two reacted with the acidic glycolipid fraction of the PNS but not the CNS; and two showed no reactivity with the acidic glycolipids from either PNS or CNS.

The species distribution of nervous system antigens that react with anti-myelin-associated glycoprotein antibodies.

The reactivity of monoclonal and polyclonal antibodies directed against human central nervous system (CNS) myelin-associated glycoprotein (MAG) was investigated in a number of animal species. The antibodies included mouse monoclonal antibodies obtained by immunization with human MAG; HNK-1, a mouse monoclonal antibody raised against a human lymphoblastoma and used to identify a subset of lymphocytes with natural killer function; human IgM paraproteins associated with neuropathy; and polyclonal antibodies obtained from rabbits immunized with rat or human MAG. Following polyacrylamide gel electrophoresis of CNS and peripheral nervous system (PNS) tissue from human, bovine, cat, rabbit, guinea pig, rat, mouse, frog, gold fish and chicken, proteins were electrophoretically transferred onto nitrocellulose. The immune-staining of electroblots showed distinct interspecies variation in the reactivity of the antibodies with MAG. In addition, the species distribution of several low molecular weight glycoproteins present in PNS tissue that cross-react with anti-MAG antibodies was determined. These low molecular weight antigens are not present in CNS homogenates or in purified human CNS myelin. It was also shown that IgM from a patient with peripheral neuropathy and paraproteinemia associated with anti-MAG antibodies recognized these low molecular weight antigens. The results suggest that IgM paraproteins, HNK-1 and some mouse monoclonal antibodies react with carbohydrate determinants shared by MAG and several lower molecular weight glycoproteins present only in human, bovine, cat and chicken PNS. Rabbit polyclonal anti-rat MAG antisera and mouse monoclonal antibodies reacting with peptide epitopes of MAG are much more specific for detecting MAG than antibodies reacting with carbohydrate epitopes of human MAG. The results are discussed in relation to human demyelinating peripheral neuropathy associated with IgM paraproteinemia.

A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties.

A novel method is described for the biotinylation of immunoglobulins. The procedure relies on the generation of reactive aldehydes on the carbohydrate moieties of the immunoglobulin by oxidation with sodium periodate and subsequent reaction with biotin hydrazide. The method is simple and specific and results in stable conjugates retaining full immunologic activity. It has been applied successfully to a number of mouse monoclonal antibodies of both IgG and IgM classes, and to human IgM preparations. The procedure may also be applied to conjugation of immunoglobulins with fluorescent dyes.

Hydrazido-derivatized supports in affinity chromatography.

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.

Site-directed immobilization of glycoproteins on hydrazide-containing solid supports.

Methods are described for the preparation and use of solid supports containing hydrazide functions for the immobilization of glycoproteins specifically through the oligosaccharide moieties. The solid supports are prepared from commercial "active ester" agarose by reaction with hydrazine hydrate. Glycoproteins are oxidized with sodium periodate, resulting in the production of aldehydes on the oligosaccharide moieties. Oxidized glycoprotein is then reacted with the hydrazide-derivatized solid support to produce stable hydrazone linkages. Data are presented for the optimization of binding of oxidized glycoprotein to hydrazide-derivatized agarose. Agarose hydrazide/glycoprotein gels were shown to be stable from pH 3 to 10 and activity studies using immobilized avidin show that this method of immobilization results in an increased "specific activity" of bound protein when compared with standard methods of immobilization.

Immobilization chemistries suitable for use in the BIAcore surface plasmon resonance detector.

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.

Molecular imprinting of amino acid derivatives at low temperature (0 degrees C) using photolytic homolysis of azobisnitriles.

Molecular imprints of L-phenylalanine anilide (print molecule) were prepared using a number of free radical initiation systems. Polymers were prepared using azobisnitriles as either thermal initiators or photoinitiators at temperatures ranging from 0 to 60 degrees C and evaluated in the high-performance liquid chromatographic mode for enantioselectivity. The results show that preparation of molecular imprints at 0 degrees C using photolytic homolysis of azobisnitriles significantly increases enantioselectivity and allows separations of the enantiomers of the print molecule to be performed at room temperature. This compares to previous reports of molecular imprints where separations needed to be performed at elevated temperatures (80-90 degrees C). The present method is also easier to perform and less time-consuming than those previously described.

Molecular recognition in synthetic polymers. Enantiomeric resolution of amide derivatives of amino acids on molecularly imprinted polymers.

Molecular imprints were prepared using L-phenylalanine anilide as the print molecule and methacrylic acid as the functional monomer. Methacrylic acid interacts ionically with the primary amine of the print molecule and via hydrogen bonding with the amide function. In the HPLC mode such polymers were shown to exhibit efficient enantiomeric resolution of a racemic mixture of the original print molecule. Enantiomeric resolution was shown to be dependent on the ratio of methacrylic acid to print molecule in the pre-polymerization mixture and specific for the presence of both print molecule and functional monomer. Further analyses showed the importance of both the primary amino and amide functions in the correct stereochemistry for recognition and enantiomeric resolution of compounds on such polymers. Other amide derivatives of amino acids including p-nitroanilides, beta-naphthylamides and amides were recognized by such polymers, and enantiomeric resolution was obtained for amide derivatives of amino acid ranging from alanine to tryptophan on a single polymer. The implications of these findings with respect to the mechanism of recognition and the ability to predict enantiomeric resolution of molecules on molecularly imprinted polymers will be discussed.

Inability of second trimester human amniotic fluid cell cultures to aromatize C19-steroids.

Amniotic fluid (AF) and fibroblast (F) cell types, derived from human amniotic fluid by amniocentesis in the second trimester, were cultured using media of varying composition and their ability to transform C19-steroids to oestrogens was investigated by fluorimetry, radioimmunoassay, tritium release and measurement of cytochrome P450. Although the cells were shown to be metabolically active by the presence of mitotic figures, the incorporation of tritiated leucine into protein and the production of the beta-subunit of hCG, no evidence of aromatization was obtained despite the inclusion in the medium of a number of steroids known to be transformed to oestrogens in several in vivo and in vitro preparations. The addition of reported stimulants of aromatase activity, viz. hCG, FSH, diethylstilbestrol, prostaglandins E2 and F2 alpha and dibutyryl cyclic AMP, was without effect.

Interpretation of deviations from pseudo-first-order kinetic behavior in the characterization of ligand binding by biosensor technology.

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.

Specific conjugation reactions of the oligosaccharide moieties of immunoglobulins.

Methods for the specific conjugation of both polyclonal antibodies and monoclonal antibodies via their carbohydrate moieties are described. Mild oxidation of the immunoglobulin with sodium periodate produces reactive aldehydes on the carbohydrate moieties. Subsequent reaction with hydrazide derivatives of biotin, fluorescent dyes, or enzymes produces stable antibody conjugates which retain full immunological activity. In addition, immunoaffinity supports can be prepared in the same manner using solid supports containing a hydrazide function.