Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain

We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La(3+)) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells.     

High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials.     

Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.     

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening

Hybridoma methods for monoclonal antibody (mAb) cloning are a mainstay of biomedical research, but they are hindered by the need to maintain hybridomas in oligoclonal pools during antibody screening. Here, we describe a system in which hybridomas specifically capture and display the mAbs they secrete: On-Cell mAb Screening (OCMS?). In OCMS?, mAbs displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS? demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG “cap”, as a universal assay for anti-viral mAbs. We produced and characterized OCMS?-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. Lastly, we used OCMS? to assess expression and antigen binding of a recombinant mAb produced in 293T cells. As a novel method to physically associate mAbs with the hybridomas that secrete them, OCMS? overcomes a central challenge to hybridoma mAb screening and offers new paradigms for mAb discovery and production.     

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin's oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.     

Properties of anti-gp41 core structure antibodies, which compete with sera of HIV-1-infected patients

To determine the correlation between the immunoreaction against the core structure of human immunodeficiency virus type (HIV-1) transmembrane protein gp41 epitopes and the disease progression, it is essential to evaluate the anti-core structure antibody epitopes and the humoral immunity against the epitopes. For this purpose we evaluated monoclonal antibodies (mAbs) against the gp41 core structure such as mAbs 50.69, 98.6 and T26, by Western blotting (WB) and flow cytometry. WB showed mAbs 50.69 and 98.6 bound to both monomeric and oligomeric gp41, and mAb T26 exclusively bound to oligomeric gp41. We evaluated the sera from Pneumocystis pneumonia patients (PCP; n=7) and long-term survivors (LTS; n=7). Competition assay with sera and mAbs for binding to H9 cells infected with HIV-1 IIIB virus was done using flow cytometry. The results revealed that PCP sera as well as LTS sera inhibited the binding of all the three mAbs, and the PCP sera inhibited mAb T26 binding more efficiently than LTS. Therefore, PCP patients retain competing immunity to antibodies against not only the shared epitopes of the core structure (binding sites of mAbs 50.69 and 98.6) but also against oligomeric gp41 specific epitope (binding site of mAb T26).     

Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase.

TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.     

Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.     

Interactions between metal ions and carbohydrates. Syntheses and spectroscopic studies of several lanthanide nitrate-D-galactitol complexes

Two complexes of neutral D-galactitol (C(6)H(14)O(6), G) with terbium nitrate, TbGN(I) and TbGN(II), and one complex with samarium nitrate SmGN were synthesized and characterized. From IR, FIR, THz and luminescence spectra the possible coordinations were suggested, and the single-crystal X-ray diffraction results confirm the spectroscopic conclusions. In TbGN(I) (Tb(NO(3))(3)·C(6)H(14)O(6)·3H(2)O), the Tb(3+) is 9-coordinated with three water molecules and six OH groups from two D-galactitol molecules. Nitrate ions do not coordinate to metal ions, which is different from other reported lanthanide nitrate-D-galactitol complexes. In TbGN(II) and SmGN (Ln(NO(3))(3)·C(6)H(14)O(6)), Ln(3+) is 10-coordinated with six OH groups from two D-galactitol molecules and four oxygen from two bidentate nitrate ions, and one nitrate ion is hydrogen bonded. No water exists in the structures. D-Galactitol molecules provide their 1-, 2- and 3-hydroxyl groups to coordinate with one metal ion and their 4-, 5- and 6-hydroxyl groups to coordinate with another metal ion in the three structures. There is still a new topological structure that can be observed for lanthanide-d-galactitol complexes, which indicates that the coordinations between hydroxyl groups and metal ions are complicated.     

Physical characterization and crystallization of the carbohydrate-recognition domain of a mannose-binding protein from rat

A portion of rat mannose-binding protein A (MBP-A), a Ca(2+)-dependent animal lectin, has been overproduced in a bacterial expression system, biochemically characterized, and crystallized. A fragment corresponding to the COOH-terminal 115 residues of native MBP-A, produced by subtilisin digestion of the bacterially expressed protein, contains the carbohydrate-recognition domain (CRD). Gel filtration, chemical cross-linking, and crystallographic self-rotation function analyses indicate that the subtilisin fragment is a dimer, although the complete bacterially expressed fragment, containing the neck and CRD of MBP-A, is a trimer. Crystals of the minimal CRD, obtained only as a complex with a Man6GlcNAc2Asn glycopeptide, diffract to Bragg spacings of at least 1.7 A. Several trivalent lanthanide ions (Ln3+) can substitute for Ca2+, as assessed by their ability to support carbohydrate binding and to protect the CRD from proteolysis in a manner similar to that observed for Ca2+. These assays indicate that Ln2+ binds about 30 times more tightly than Ca2+ to the CRD, and that two Ca2+ or Ln3+ bind to each monomer, a result confirmed by determination of the Ho3+ positions in a Ho(3+)-containing crystal of the CRD. Crystals grown in the presence of Ln3+ belong to different space groups from those obtained with Ca2+ and are therefore not useable for traditional crystallographic phase determination methods, but are well-suited for high resolution structure determination by multiwavelength anomalous dispersion phasing.     

Synthesis of versatile thiol-reactive polymer scaffolds via RAFT polymerization

Well-defined polymer scaffolds convertible to (multi)functional polymer structures via selective and efficient modifications potentially provide an easy, versatile, and useful approach for a wide variety of applications. Considering this, a homopolymer scaffold, poly(pyridyldisulfide ethylmethacrylate) (poly(PDSM)), having pendant groups selectively reactive with thiols, was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Soluble polymers with controlled molecular weights and narrow PDIs were generated efficiently. The versatility of the scaffold to generate random co- and ter-polymers combining multiple functionalities with controlled-composition was shown by separate and simultaneous conjugation of different mercapto-compounds, including a tripeptide in one-step. Conversion of water-insoluble scaffold to peptide-containing water-soluble copolymers was observed to yield nanometer-size particles with narrow polydispersity. The overall results suggest that the well-defined PDSM homopolymer scaffold generated via RAFT polymerization can be a versatile building block for generation of new structures having potential for drug delivery applications via a straightforward synthetic approach.     

Surface modification of silicone intraocular implants to inhibit cell proliferation

Photo-cross-linkable polymers bearing cinnamic, sulfonate, and carboxylate groups were synthesized by radical polymerization leading to randomly distributed copolymers. These polymers were used to coat silicone intraocular lenses in order to reduce posterior capsule opacification, also named "secondary cataract". We previously demonstrated that polymers containing both carboxylate and sulfonate groups inhibit cell proliferation, and formulations with the ratio R = COO-/(COO- + SO3-) equal to 0.64 provided the highest inhibitory effect. Ionic polymers with this formulation were synthesized to contain a monomer with pendant siloxane groups in order to get compatibility with the silicone matrix of the intraocular lenses. Anchorage of the ionic polymer at the surface of the silicone implant was achieved by a cycloaddition reaction of the photosensitive groups according to two options. These modified silicone surfaces grafted onto intraocular lenses were shown to inhibit cell proliferation to 60%.     

Biodegradable nanoparticles for direct or two-step tumor immunotargeting

In this study, selective cancer cell targeting of biodegradable poly(lactic acid) (PLA) nanoparticles (NPs) has been investigated in vitro. SKOV-3 (HER2 positive) ovarian cancer and Daudi (CD20 positive) lymphoma cell targeting was mediated by anti-HER2 (trastuzumab, Herceptin) and anti-CD20 (rituximab, Mabthera) monoclonal antibodies (mAbs), respectively. The mAb against nonexpressed antigen serving on each cell as isotype matched irrelevant control. Two different targeting approaches have been studied, a direct method using antibody-labeled NPs (mAb-NPs) and a pretargeting method using the avidin-biotin technology. For the direct protocol, fluorescent PLA-NPs were prepared including 10% 1-pyrenebutanol (PB)-labeled PLA in the NP-preparation (PB-NP). Thiol groups were covalently bound to the PB-NP, and the resulting thiolated PB-NP were coupled with the two mAbs using a bifunctional cross-linker. The effective targeting of cells by mAb-PB-NP was shown by flow cytometry analysis. Clearly anti-HER2-PB-NP specifically bound to the SKOV-3 cells and not to the Daudi cells, while anti-CD20-PB-NPs bound to Daudi cells but not to SKOV-3 cells. Specific mAb-PB-NP binding to tumor cells produced a mean 10-fold or higher signal increase compared to irrelevant IgG-PB-NPs. For the pretargeting protocol, plain PLA-NPs were also thiolated and NeutrAvidin-Rhodamine Red-X (NAR) coupled to the functionalized PLA-NPs with sulfo-MBS. The two-step method was evaluated in vitro by incubating SKOV-3 cells first with biotinylated mAbs followed by NAR-NPs. The relative fluorescence associated to the specific binding of NPs produced a 6-fold increase in flow cytometry signal compared to nonspecific binding. In conclusion, these experiments have shown that NPs covalently coupled with antibodies or NAR can specifically and efficiently bind to cancer cells in both a pretargeting and a direct approach, suggesting that functionalized NPs may be a useful drug carrier for tumor targeting.     

Poly(alkylene phosphates): from synthetic models of biomacromolecules and biomembranes toward polymer-inorganic hybrids (mimicking biomineralization)

Syntheses of poly(alkylene phosphates), with repeating units having two or three methylene groups and phosphoryl groups and mimicking backbones of biomacromolecules, are reviewed. Two major methods elaborated in this laboratory, namely, ring-opening polymerization and transesterification, are described. The resulting polymers were used as carriers of cations (Ca2+ and Mg2+) in membrane processes and in controlling the crystallization of CaCO3, in a process related to biomineralization.     

Mapping epitopes on the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum using fusion proteins.

Epitopes for a number of monoclonal antibodies (mAbs) binding (Ca(2+)-Mg2+)-ATPase purified from skeletal muscle sarcoplasmic reticulum have been defined by studying binding to fusion proteins generated from cDNA fragment libraries. Comparison of these results with those of previous studies of binding of mAbs to proteolytic fragments of the ATPase have allowed the definition of the epitopes to within approx. 100 residues and for one (mAb 1/2H7) to within 45 residues. The experiments suggest considerable exposure of the nucleotide binding domain of the ATPase on the top surface of the protein. Those mAbs that were found to inhibit steady-state ATPase activity were found to bind to epitopes in the nucleotide binding domain of the ATPase.     

C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities

Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new β-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.     

Characterization of reduced expression of glycine N-methyltransferase in cancerous hepatic tissues using two newly developed monoclonal antibodies

Glycine N-methyltransferase (GNMT) is a protein with multiple functions. Recently, two Italian siblings who had hepatomegaly and chronic elevation of serum transaminases were diagnosed to have GNMT deficiency caused by inherited compound heterozygosity of the GNMT gene with missence mutations. To evaluate the expression of GNMT in cell lines and tissues from hepatocellular carcinoma (HCC) patients, we produced two monoclonal antibodies (mAbs) 4-17 and 14-1 using two recombinant GNMT fusion proteins. M13 phage peptide display showed that the reactive epitopes of mAbs 4-17 and 14-1 were amino acid residues 11-15 and 272-276 of human GNMT, respectively. The dissociation constants of the binding between GNMT and mAbs were 1.7 x 10(-8) M for mAb 4-17 and 1.8 x 10(-9) M for mAb 14-1. Both mAbs can identify GNMT present in normal human and mouse liver tissues using Western blotting (WB) and immunohistochemical staining assay (IHC). In addition, WB with both mAbs showed that none of 2 hepatoblastoma and 5 HCC cell lines expressed GNMT. IHC demonstrated that 50% (13/26) of nontumorous liver tissues and 96% (24/25) of HCC tissues did not express GNMT. Therefore, the expression of GNMT was downregulated in human HCC.     

Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies.

A monoclonal antibody (3D6) was produced which reacted only with Brucella sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B. melitensis B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated Escherichia coli and Yersinia enterocolitica sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B. melitensis B115 than with that of Escherichia coli. This mAb was also used in immunogold electron microscopy with whole Brucella cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B. melitensis B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for Brucella OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of monoclonal antibody binding with the <Emphasis Type="Italic">C</Emphasis> domain of human angiotensin converting enzyme

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied, and the inhibition of the enzyme by the mAb 4A3 was found. The dissociation constants of complexes of two mAbs, 1B8 and 2H9, with tACE were 2.3 ± 0.4 and 2.5 ± 0.4 nM, respectively, for recombinant tACE and 4.7 ± 0.5 and 1.6 ± 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) 1B3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 for Leu that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.     

Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions.

Monoclonal antibody (mAb) probes were used to investigate the expression of lipopolysaccharide (LPS) on four Escherichia coli strains, grown under a variety of conditions in batch culture which mimicked some of the in vivo environmental conditions of an infected host. Techniques of silver staining, immunoblotting, whole cell ELISA and flow cytometry were all used to monitor the expression of LPS on the bacteria and the binding of the anti-LPS mAbs. Growth in heat-inactivated sheep serum and magnesium-depleted conditions demonstrated increased expression of LPS core and subsequent increased binding of anti-core mAbs. Magnesium-depleted conditions also resulted in decreased production of O-polysaccharide material. Iron-depleted bacteria showed only minor changes in LPS expression, although increased binding of anti-core mAbs was observed. Nitrogen-deficient/high-carbon conditions, chosen to promote capsule production, resulted in increased expression of O-polysaccharide and decreased binding of anti-core mAbs.