Receptor analogs and monoclonal antibodies that inhibit adherence of Bordetella pertussis to human ciliated respiratory epithelial cells.

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough. To explore the development of agents that could interrupt adherence, the structure of the receptor on the ciliary surface was investigated. Using an in vitro adherence assay to human ciliated epithelial cells, galactose, lactose, and complex carbohydrates containing lactose eliminated adherence when preincubated with the bacteria. 10(-2) M galactose eluted adherent bacteria from cilia. B. pertussis and its two purified adhesins bound specifically to natural lactose-containing glycolipids in a TLC assay. mAbs to eukaryotic glycoconjugates with specificity for substituted galactose-glucose moieties blocked adherence when preincubated with ciliated cells. The carbohydrates that serve as receptors for B. pertussis on human cilia are galactose-glucose-containing glycolipids. Receptor analogs and anti-receptor antibodies effectively block adherence of B. pertussis to cilia and thus should be considered candidates for therapeutic intervention against disease.

Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl(alpha 1-3)-galactose epitopes.

Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.

Epitope mapping of mAbs to denatured human testicular ACE (CD143).

Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.

MAP1D, a novel methionine aminopeptidase family member is overexpressed in colon cancer.

N-terminal methionine removal is an important cellular process required for proper biological activity, subcellular localization, and eventual degradation of many proteins. The enzymes that catalyze this reaction are called Methionine Aminopeptidases (MAPs). To date, only two MAP family members, MAP1A and MAP2, have been well characterized and studied in mammals. In our studies, we have cloned a full length MAP1D gene. Expression and purification of full length recombinant protein shows that the sequence encodes an enzyme with MAP activity. MAP1D is overexpressed in colon cancer cell lines and in colon tumors as compared to matched normal tissue samples. Downregulation of MAP1D expression by shRNA in HCT-116 colon carcinoma cells reduces anchorage-independant growth in soft agar. These data suggest that MAP1D is a potentially oncogenic, novel member of the MAP gene family that may play an important role in colon tumorigenesis.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Photoaffinity labeling for evaluation of uridinyl analogs as specific inhibitors of rat liver microsomal UDP-glucuronosyltransferases.

The UDP-glucuronosyltransferases (UGT) involved in glucuronidation of endogenous and exogenous toxic compounds transfer the glucuronic acid residue from UDP-glucuronic acid (UDP-GlcUA), to various acceptor groups. A series of compounds that contain N-acyl phenylaminoalcohol derivatives linked to uridine or isopropylideneuridine were tested as UGT inhibitors. The potency of these inhibitors was determined by studying their effect on the photoaffinity labeling of rat liver microsomal UGTs by two photoaffinity probes, [beta-32P]5-azido-UDP-glucuronic acid (5N3UDP-GlcUA) and [beta-32P]5-azido-UDP-glucose (5N3UDP-Glc) and on the enzymatic formation of the two glucuronide conjugates (3-O- and carboxyl-specific) of lithocholic acid. All but one of the compounds tested proved to have an inhibitory effect on UGTs, both in the photoaffinity labeling system and in the enzymatic glucuronidation assay. In the photoaffinity labeling system, the inhibitors containing the isopropylidene moiety were less effective than their unprotected derivatives; however, the protected forms were, with one exception, more potent inhibitors of enzymatic activity. The photoaffinity labeling of UGTs with [beta-32P]5N3UDP-Glc was more susceptible to inhibition by all derivatives than that with [beta-32P]5N3UDP-GlcUA. The effect of one inhibitor, PP50B, on the two enzymatic activities involved in LA glucuronidation was extensively tested. A double-reciprocal plot suggested a competitive inhibition for UDP-GlcUA with an apparent Ki of 35 microM for LA 3-O-glucuronide formation and 94 microM for the carboxyl-linked glucuronide of the same substrate.

Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes.

Immunoassays for haptens such as short peptides or drugs are usually based on the principle of competition for a limited number of binding sites on antibody molecules. Owing to the small size of these antigens it has been thought that two specific antibodies cannot simultaneously bind a hapten. However, antisera containing so called anti-metatypic antibodies have been reported (Voss et al. (1988) Mol. Immunol. 25, 751-759) that bind to hapten-mAb complexes in a reaction where conformational changes on the primary antibody are important. Here, we report on monoclonal antibody pairs able to form ternary complexes with the octapeptide angiotensin II. The first mAb (mAb1) is conventional and binds angiotensin II with high affinity (Kd 10(-11) M). The secondary (anti-metatypic) mAbs (mAbs2s) recognize the immune complex consisting of angiotensin II bound to mAb1, but only poorly recognize mAb1 alone. An immunization technique involving tolerization with uncomplexed mAb1 was used to generate mAb2s. None of the mAbs2s were able to bind angiotensin II by themselves but all efficiently bound the complex of angiotensin II and mAb1. All mAb2s stabilized the angiotensin II-mAb1 complex and one mAb2 distinctly improved the specificity of the assay for angiotensin II. By either labelling mAb1 and immobilizing mAb2 (or vice versa) two-site immunometric assays with detection limits of 1 pg/ml angiotensin II have been established. The kinetics of the complex formation was investigated by fiber optic biospecific interaction analysis (FOBIA), a system allowing real time observation of binding events on the surface of a glass fiber. The association rate towards the liganded conformation of mAb1 was higher than towards the free mAb1. By contrast, the mAb2s dissociated at similar rates from complexed and uncomplexed mAb1.

Monoclonal antibodies in analysis of cathepsin G-digested proteolytic fragments of human plasma fibronectin.

Proteolytic fragments of fibronectin, obtained by digestion with cathepsin G, were transferred electrophoretically from sodium dodecyl sulphate (NaDodSO4) polyacrylamide gels to nitrocellulose sheets and used as antigens for monoclonal antibodies. All 9 monoclonal antibodies tested reacted with undenatured intact fibronectin or its fragments applied directly to nitrocellulose sheets. Two of the clones did not react with the NaDodSO4-treated transferred material suggesting reactivity with conformational determinants. Distinct fragments of fibronectin could be detected by several of the antibodies. None of the monoclonal or the polyclonal antibodies used reacted with the Mr = 40,000 or Mr = 30,000 gelatin-binding fragments of fibronectin. However, one of the monoclonal antibodies reacted specifically with their precursor Mr = 64,000 fragment, but apparently with its gelatin-nonbinding segment. The apparent non-immunogenicity of the gelatin-binding domain is conspicuous, suggesting that it may be highly conserved in evolution. The present method, combination of controlled proteolytic cleavage with electrophoretic transfer, provides an effective means for characterization of monoclonal antibodies raised against proteins.

Glycosphingolipid-blotting: an immunological detection procedure after separation by thin layer chromatography.

A method for detecting glycosphingolipids (GSL) in situ after thin layer chromatography is described. The separated GSL are transferred by diffusion to nitrocellulose. The replica is incubated with poly- or monoclonal antibodies and bound antibodies are detected with second antibodies coupled to peroxidase. Advantages of the procedure are its speed, the non-radioactive detection method, and its suitability for screening applications. In addition, small scale affinity purification of antibodies from the replicas is possible. The presence of Forssman antigen in mouse tissues and the reaction of monoclonal antibodies with human GSL is demonstrated.

Cytokine immunotrapping: an assay to study the kinetics of production and consumption or degradation of human interferon-gamma.

With the aim of determining the rate of cytokine production, we have investigated immunoassay conditions which prevent consumption/degradation. These assays, termed cytokine immunotrapping assays (CITA), are based on early capturing of cytokines secreted during cell culture by immobilised or soluble mAbs and a recently described chemiluminescent immunoassay. Here we describe assay conditions using IFN-gamma as a prototype cytokine. For production of IFN-gamma, PBMC, purified CD4+ or CD8+ T cells, or diluted whole blood were cultured with different T cell stimulating agents. Polystyrene macrobeads precoated with an anti-IFN-gamma mAb were put in culture and after a defined incubation period, a dimethyl acridinium ester (DMAE)-labelled second anti-IFN-gamma mAb and sodium azide were added into the culture for additional 24 h. The beads were washed and chemiluminescence signals determined in a luminometer. Trapping experiments were also performed with the beads or the soluble mAbs alone. Irrespective of the configuration, IFN-gamma concentrations measured in trapping conditions were always higher (3-20-fold) than in conventional cultures. By using the best trapping combination, i.e. both bead-mAb1 and DMAE-mAb2 added at the start of culture (single step), it was possible to detect IFN-gamma production as early as 2 h. Also, IFN-gamma secreted by less than 500 PBMC or whole blood cells could be detected within 24 h. When purified CD4+ or CD8+ cells were used instead of PBMC, a reduction of the trapping effect was observed. Conversely, addition of monocytes to purified T cells increased the trapping factor suggesting that a substantial amount of IFN-gamma was consumed or degraded both by CD14+ cells as well as T cells in culture. Preliminary results show that this assay is also suitable for the early detection of IL-1 and IL-4 which are known to be more tightly regulated. Thus, the new principle described here is expected to be useful in clinical settings where both the time and amounts of material are limited to investigate the role of cytokines in particular disease.

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies.

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.

Quantitation of interferon-induced Mx protein in whole blood lysates by an immunochemiluminescent assay: elimination of protease activity of cell lysates in toto.

Due to the rapidly expanding usage of interferons and its costliness of therapy, it is important to evaluate the clinical efficacy of the various interferons. Directly assaying circulating interferon is technically quite difficult. Here, we present an alternate method to evaluate interferon therapy by assaying a unique protein, called Mx protein, which is a 78 kDa cytoplasmic protein selectively induced by type-1 interferon in human leukocytes. The current assay is a two-site chemiluminescent immunoassay, designed to detect Mx protein in whole blood lysates. Since the Mx protein once solubilized, is highly susceptible to proteolysis in whole blood lysates, we have devised a new procedure both to maximize its solubility and virtually eliminate its proteolytic degradation. A mouse monoclonal antibody conjugated to the derivatized-paramagnetic particles and an acridinium ester-labeled antibody serve as the solid phase capture and detector antibodies, respectively. This assay is applicable to both manual and automated modes with a detection limit of Mx protein at 20 ng/ml whole blood. Availability of a reliable assay for Mx protein should facilitate the clinical evaluation of many of the newly constructed type-1 interferons.

Monoclonal antibody based enzyme-linked and chemiluminescent assays for the human interleukin-1 receptor antagonist. Application to measure hIL-1ra levels in monocyte cultures and synovial fluids.

Interleukin-1 receptor antagonist (IL-1ra) has the potential to counteract at least part of the biological effects of interleukin-1. The outcome of an inflammatory reaction may therefore be determined by the balance between IL-1 and IL-1ra, rather than by IL-1 alone. We have developed an immunoassay to address this issue as well as to assess the effects of anti-inflammatory agents on the expression of IL-1 and IL-1ra in vitro or in body fluids. Recombinant human IL-1ra was expressed in an E. coli system, purified to homogeneity, and used to derive monoclonal antibodies in mice as well as polyclonal antibodies in rabbits. A sandwich ELISA was constructed with F(ab')2 fragments of a high affinity monoclonal antibody and the rabbit serum as a source of secondary antibody. The assay required no sample treatment to avoid interference by rheumatoid factor. The measuring range was 0.020-2 ng/ml. By labelling a second monoclonal antibody with an acridinium ester, a chemiluminescence assay with a wider measuring range (0.050-15 ng/ml) was generated. In accord with published data, we found that IL-1ra was secreted by human monocytes stimulated with LPS, Zymosan, IL-1 alpha, or human IgG. After an induction phase of ca. 4 hours and depending on the stimulus, IL-1ra accumulated linearly for periods up to 96 h. IL-1ra levels in synovial fluids of 19 patients suffering from various inflammatory joint diseases were compared with the cytokine levels of IL-1 beta, IL-6, IL-8, and TNF-alpha. Highest positive correlations were found with IL-8 and IL-1 beta. In normal blood donors IL-1ra serum levels were 150-800 pg/ml (Median: 387 pg/ml). Owing to its sensitivity and large measuring range the newly developed assays appear to be suitable for measuring IL-1ra in cell cultures as well as in biological fluids.

Neutralization of interleukin-1 beta activity in vivo with a monoclonal antibody alleviates collagen-induced arthritis in DBA/1 mice and prevents the associated acute-phase response.

Interleukin-1 (IL-1) has been implicated in the development and progression of a variety of acute and chronic inflammatory diseases. Due to its pro-inflammatory and tissue-degrading activities, IL-1 is regarded as a major mediator of chronic inflammatory joint diseases, including rheumatoid arthritis in man, adjuvant arthritis in rats and collagen-induced arthritis in mice. However, conclusive experimental evidence for the crucial role of IL-1 in the development of joint destruction has not been presented as yet. In the present study, we investigated the effect of a neutralizing monoclonal mouse antibody against mouse IL-1 beta (IgG1 isotype) on the development and progression of collagen-induced arthritis in DBA/1 mice. The antibody was injected intraperitoneally 3 times a week, either from day 3 or from day 21 after primary immunization, to day 60. In the positive control group an arthritis incidence of 80% was observed after 60 days. The injection of a control antibody of the same isotype did not influence the incidence of arthritis, whereas injection of anti-IL-1 beta from day 21 reduced the arthritis incidence to about 30%. Injection of anti-IL-1 beta starting at day 3 totally prevented both the development of arthritis and the associated increase of the acute phase protein serum amyloid P (SAP). Anti-collagen antibody titers, which increased significantly after immunization, were not influenced by the injection of anti-IL-1 beta antibodies, in spite of the suppressive effect on arthritis development.(ABSTRACT TRUNCATED AT 250 WORDS)

An assay for the detection of interleukin-1 synthesis inhibitors: effects of antirheumatic drugs.

The monokines interleukin-1 beta and alpha (IL-1) play a central role in the connective tissue destruction of many chronic inflammatory diseases. A high capacity screening assay for the detection of inhibitors of IL-1 biosynthesis has been established. Normal human monocytes were obtained by leukapheresis and elutriation. IL-1 beta and alpha biosynthesis was stimulated with LPS, and cell-associated and secreted IL-1 beta and IL-1 alpha were measured by specific immunoassays (ELISA). The mean total IL-1 beta (cell-associated and secreted) production in 18 different donors was 11 ng/10(6) cells (range 1.2-28.8). Secreted IL-1 beta represented 31 to 86% of the total IL-1 beta. More IL-1 alpha than IL-1 beta was produced but, unlike IL-1 beta, IL-1 alpha was poorly secreted. The steroids prednisolone and dexamethasone, gold (sodium aurothiomalate) and chloroquine were potent inhibitors of the IL-1 production. Mean IC50 values of 180 nM (range 2.5 nM-1 microns), 10 microM (range 6-20 microM) and of 75 microM were found for prednisolone, gold and chloroquine, respectively. Above 5 microM, the non-steroidal anti-inflammatory compounds indomethacin and BW755C increased IL-1 beta biosynthesis. Nordihydroguaiaretic acid inhibited the level of the secreted form of IL-1 beta, but tended to increase the cell-associated level. D-Penicillamine (up to 6 mM), cyclosporin A (up to 1 microM) and methotrexate (up to 12 microM) inhibited neither cell-associated nor secreted IL-1 beta levels. This high capacity assay, which is insensitive to classical NSAIDs, may serve in the detection and characterization of new classes of anti-inflammatory compounds.

A photoaffinity labelling study of the messenger RNA-binding region of Escherichia coli ribosomes.

A photoaffinity labelling study of the messenger RNA-binding region of E. coli ribosomes has been made, using oligoadenylic acids as mRNA analogs. The oligonucleotides, of chain length 6 to 8 and thus several nucleotides longer than oligonucleotides previously employed for this purpose, carried a radioactive photolabile aromatic azide reagent bound covalently to the 3'-terminal ribose moiety. The synthesis of the reagent, p-azidobenzoyl-(3H)-glycylhydrazide, is described. The derivatized oligonucleotides were shown to be functional messengers. They stimulated the binding of the cognate aminoacyl-tRNA, lysyl-tRNA: their binding was reciprocally stimulated by lysyl-tRNA; and they competed with underivatized oligoadenylates for ribosomal binding sites. When the 70 S ribosomal binding complex was irradiated, the photolabile reagent reacted covalently with both RNA and proteins of the 30 S subunit and with tRNA, but not with the 50 S subunit. The 16 S RNA appeared to be labelled at more than one site. Of the proteins, S3 and S5 reacted with the reagent with high specificity; and the possibility was not eliminated that S4 may have been labelled to a minor degree. Functional studies in other laboratories have implicated S3 and S5 in the decoding process, but these proteins were not labelled by any of the previously reported mRNA affinity labelling analogs. The results reported here therefore indicate that S3 and S5 not only affect the decoding process, but are located in the mRNA-binding region of the ribosome, presumably to the 3' side of the decoding site.

Chemiluminescent and enzyme-linked immuno assays for sensitive detection of human IFN-gamma.

We have produced and characterized 4 mAbs to human IFN-gamma and established sensitive, non-radioactive immuno-assays. The first two assays use microtiter plates as the solid phase and enzymes or chemiluminescence (acridinium ester) for development. The use of chemiluminescence instead of peroxidase increased the sensitivity of the assay by a factor of about 75. The third and the fourth assays utilize polystyrene beads as the solid phase and enzymes or acridinium ester for development. The use of beads also increased the sensitivity of detection. The most sensitive IFN-gamma detection was achieved by the combination of bead with acridinium ester. In this configuration we were able to detect about 0.2 pg/ml IFN-gamma (1/250th of a unit). These chemiluminescent immunoassays (CLIA) appear to be more sensitive than existing ELISAs or radioimmunoassays and may find new application areas.

Acridinium ester labelled cytokines: receptor binding studies with human interleukin-1 alpha, interleukin-1 beta and interferon-gamma.

As a consequence of environmental protection and legal restrictions, increasing efforts are made to avoid radioactivity. One alternative is the labelling of ligands with chemiluminescent acridinium esters such as 2,6-dimethyl-4-(N-succinimidyloxy-carbonyl)phenyl 10-methylacridinium-9-carboxylate methosulphate (DMAE-NHS). When exposed to hydrogen peroxide in a basic solution, the DMAE-moiety decays with emission of a short-lasting chemiluminescent flash. With the goal of replacing the radioactive label in protein ligands with a DMAE label, and of increasing the efficiency by using microtitre plate technology for DMAE detection, we compared the receptor binding properties of iodinated interleukin-1 alpha (125I-IL-1 alpha), interleukin-1 beta (125I-IL-1 beta) and interferon-gamma (125I-IFN-gamma) with the corresponding DMAE-labelled ligands. The luminescence signal was assessed in a single-tube luminometer and in the prototype of a chemiluminescent microtitre plate reader. Derivatization of the three proteins with DMAE-N-hydroxy-succinimide resulted in photon yields of up to 100,000 counts per femtomole. As shown by Scatchard analysis, no significant loss of receptor binding affinity was observed, which might have been expected as a consequence of the chemical modification of the proteins. The use of DMAE labelling of proteins has the following advantages as compared to iodination: (i) the coupling reaction and binding assay can be performed in a normal laboratory, (ii) since there is no radiolysis, the DMAE-labelled proteins remain stable, (iii) the detection sensitivity may be improved as a consequence of higher specific activity of the DMAE label.(ABSTRACT TRUNCATED AT 250 WORDS)

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients.

Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.