Kinetics and locus of failure of receptor-ligand-mediated adhesion between latex spheres. II. Protein-protein bond.

In an extension of the previous paper, we describe the force dependence of break-up of doublets of latex spheres cross-linked by protein G-IgG bonds via the Fc region of the antibody. The receptor, the monoclonal Bear-1 antibody, was either covalently linked to 4.75-microns aldehyde/sulfate (A/S) latex spheres in a one-step reaction, or physically adsorbed to the 4.63-microns carboxyl-modified latex spheres used in Part I of this paper. The spheres were suspended in 19% buffered Dextran 40 containing the ligand, the bivalent recombinant protein G (Gamma-Bind G), and observed in the counter-rotating cone and plate Rheoscope. Break-up of doublets, tracked individually under the microscope, as well as in populations of 50-150 particles, was studied over a range of normal force from 20 to 260 pN. In individual particle studies, the fraction of doublets of spheres with covalently linked IgG breaking up in the first 10 rotations, increased from 16% in the low-force to 63% in the high-force range. In population studies, the fraction broken up increased with duration and magnitude of the applied force, and decreased with increasing ligand concentration. Moreover, doublets of physically adsorbed IgG spheres required significantly lower force than doublets of covalently linked IgG spheres for the same degree of break-up, possibly because of surface detachment of IgG molecules rather than rupture of receptor-ligand bonds. Computer simulation, using the Bell stochastic model of break-up and a Poisson distribution for the number of bonds, described in Part I, showed that the parameters of the protein-protein bond differed significantly from those of the carbohydrate-protein bond studied in Part I of this paper, the former being much more responsive to force than the latter.     

Specific reactivity of lipid vesicles conjugated with oriented anti-lactose antibody fragments

The method previously described (Sinha, D. and Karush, F. (1979) Biochem. Biophys. Res. Commun. 90, 554--560) for the oriented attachment of immunoglobulins to lipid vesicles has been used to confer specific reactivity on liposomes by their conjugation with anti-lactose Fab' fragments derived from rabbit IgG antibody. It is estimated that one-third of the Fab' fragments was irreversibly attached to liposomal membrane, resulting in a membrane concentration of 2 mmol of Fab' per mol of total lipid. The specific reactivity of the modified liposomes was demonstrated by agglutination with a multivalent, lactose-containing diheteroglycan. The availability of virtually all of the binding sites of the attached antibody for reaction with ligand was established by a fluorescence quenching titration with N-(N epsilon-Dnp-L-lysyl)-p-aminophenyl-beta-lactoside. An intrinsic association constant of 8.9 x 10(6) M-1 was found for the attached Fab' compared to a value of 2.8 x 10(6) M-1 for free anti-lactose Fab'. In addition the maximum values for the quenching by bound ligand of the fluorescence of free and attached antibody were the same. It can be concluded that the chemical procedures used to effect attachment of the antibody to the lipid vesicles allow retention of the original structure of the antibody site and its accessibility to external components.     

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.     

Assessment of three simple techniques for the screening of circulating immune complexes: Correlation with a parameter of complement consumption

The sera of 36 normal controls, 45 patients with various diseases and 11 pregnant women were screened for circulating immune complexes using three relatively simple and inexpensive techniques. These included inhibition of agglutination of IgG coated latex particles with a serum having rheumatoid factor activity, polyethylene glycol precipitation and anti-complementary activity test. The circulating immune complexes were detected in a significantly higher proportion of patients as compared to normal controls. In the patients, the presence of circulating immune complexes did not always correlate with clinically detectable immunoinflammatory tissue damage indicating that pathogenic as well as nonpathogenic immune complexes were being detected by the above mentioned techniques. The alpha-1-antitrypsin/C3 ratio, however, correlated well with clinically apparent immuno-inflammation.     

Rheumatoid Arthritis,Rheumatoid Factor,and Tests for Australia or Hepatitis-associated Antigen

False-positive results in tests for hepatitis-associated antigen using latex agglutination techniques may be due to rheumatoid factor in the serum. Possibly the use of IgM antibody in preparing the latex particles might diminish the occurrence of such reactions. No evidence was found for a relation between rheumatoid arthritis and a significant incidence of hepatitis-associated antigen detectable by countercurrent immunoelectro-osmophoresis.     

Development and characterization of Ni-NTA-bearing microspheres

BACKGROUND: For ease of purification, proteins are often expressed with a short affinity sequence of five or six adjacent histidine residues (His-tag). This His-tag binds to the metal of metal chelator complexes such as Ni(2+)-nitrilotriacetic acid (Ni-NTA) or -iminodiacetic acid (Ni-IDA). Chromatography resins bearing covalently attached metal chelator complexes are used widely for the easy affinity purification of His-tagged proteins or peptides. Because Ni-NTA microspheres were not commercially available at the beginning of our studies, we prepared and characterized such microspheres to immobilize His-tagged proteins and study their interactions. Our microspheres are of three types: (a) metal chelator complexes bound covalently to polystyrene microspheres, (b) metal chelator complexes bound covalently to silica microspheres, and (c) lipid-linked metal chelator complexes adsorbed to silica microspheres forming self-assembled bilayer membranes where the metal chelators have lateral mobility. METHODS: The microspheres bearing covalently attached Ni-chelator were synthesized by reacting a primary amine-bearing Ni-NTA ligand with carboxy-functionalized microspheres and then loading with Ni(2+). Microspheres with laterally mobile metal chelator were made by incubating glass microspheres with liposomes containing phosphatidylcholine (PC) and the metal chelating lipid 1,2-dioleoyl-sn-glycero-3-[(N (5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]. Binding of a His-tagged enhanced green fluorescent protein (EGFP) was used to characterize these microspheres by flow cytometry for their specificity, sensitivity, capacity and stability. RESULTS: While all micospheres specifically bind His-tagged proteins, the conditions to achieve this are different for the polystyrene- and silica-based spheres. All three types of microspheres bind His-EGFP with saturation occurring at 30-50 nM and an apparent avidity (concentration of half-maximal binding) of approximately 1 to 2 x 10(-8) M at pH 7.4. Binding of His-EGFP is inhibited by imidazole or ethylene-diaminetetraacetic acid (EDTA). Polystyrene Ni-NTA microspheres showed significant nonspecific binding as measured by binding in the presence of imidazole or EDTA or by binding of fluorescent proteins lacking a His-tag. This nonspecific binding of proteins to and aggregation of polystyrene spheres could only be prevented by the inclusion of low concentrations of Tween 20, but not by including bovine serum albumin (BSA), polyethylene glycols, or polyvinylpyrrolidones as blocking agents. In contrast, silica-based microspheres with covalently attached Ni-NTA or silica microspheres bearing adsorbed bilayers that contain Ni-NTA-lipid showed little nonspecific binding in the presence of BSA. Our results on the stability of immobilization indicate that washing destabilizes the binding of His-tagged proteins to Ni-NTA microspheres. This binding consists of two interactions of different affinities. We also demonstrate that limited multiplexed analysis with differently sized silica microspheres bearing the Ni-NTA-lipid is feasible. CONCLUSIONS: The microspheres described are well suited to selectively immobilize His-tagged proteins to analyze their interactions by flow cytometry. The affinity and kinetic stability of the interaction of His-tagged proteins with Ni-NTA are insufficient to use Ni-NTA microspheres in multiplexed analysis formats where different His-tagged proteins are bound to distinct microspheres. Improvements towards this end (improved chelators and/or improved affinity tags) are critical for extending the use of this method. We are currently working on novel chelators to strengthen the stability of immobilization of His-tagged proteins to surfaces. Such improvements would greatly enhance the analysis of interactions of immobilized His-tagged proteins and could make the development of microsphere-based arrays with His-tagged protein/antibody possible.     

Assay of rheumatoid factors by the indirect immunofluorescence method

Sera from 69 patients affected with rheumatoid arthritis were examined for IgM, IgG and IgA rheumatoid factors (RF) by a indirect immunofluorescence method. The results were compared with those obtained from the classical rheumatoid factor latex test. By this technique we have demonstrated antigammaglobulin activity in a high proportion (23%) of sera from latex test seronegative rheumatoid patients. Moreover, by fractionated antisera it was possible to detect also IgG and IgA factors. Indirect immunofluorescence results to be a simple and available technique for detection of RF, also in many "seronegative" patients.     

Specificity of anti-human IgG antibodies in antiglobulin (Coombs) sera.

The agglutination test with latex particles coated with aggregated human IgG was introduced into the evaluation of Coombs serum as an additional test for anti-IgG antibody activity. In Coombs sera prepared by the conventional immunization method employing Freund's adjuvant, latex agglutination titers were found much lower than those of anti-D-coated red cell agglutination. On the other hand, in sera prepared by other immunization methods, such as the one according to Haynes and Chaplin (1971), anti-IgG antibody response was readily observed by IgG-coated latex agglutination. Specificity of anti-IgG antibodies in the latter sera seems to be predominantly directed to aggregated human IgG.     

Effect of Dexamethasone and Nitrogen Mustard on the Production of Rheumatoid Factor in Rheumatoid Arthritis

The effect of dexamethasone and nitrogen mustard on the production of rheumatoid factor, as measured by sensitized sheep cell and latex agglutination tests, was studied in 19 patients with classical rheumatoid arthritis. Dexamethasone was given orally in a daily dose of 6-8 mg. which was slowly reduced after a two-week period. Nitrogen mustard was infused in the usual therapeutic dose of 0.3 mg./kg. The level of circulating rheumatoid factor decreased, following administration of each agent, after a latent period of 10 days. The effect was most marked at around 30 days. Dexamethasone was more potent than nitrogen mustard. Both drugs together caused transient disappearance of rheumatoid factor in one patient.It is concluded that dexamethasone and nitrogen mustard have the capacity to suppress the formation of the macroglobulins associated with rheumatoid arthritis.     

The preparation of an IgG diagnostic agent based on stained polyacrolein latexes for use in the latex agglutination reaction

IgG diagnosticum for measuring the concentration of 131I-labeled IgG antibodies to enteric antigen beta 1MA by the latex agglutination inhibition (LAI) test has been prepared on the basis of polyacrolein latexes. A method for the titration of anti-IgG antibodies with the use of the above diagnosticum has been developed, based on the late, agglutination (LA) test. The optimum conditions for the microtitration variant of the LA and LAI tests have been defined. High sensitivity, specificity and simplicity of analysis with the use of latex IgG diagnosticum have been demonstrated. The newly developed methods have been successfully used in laboratory trials of a new diagnostic radiopharmaceuticals for the assay of 131I-labeled antibodies in this preparation and for the detection of side effects of immunization on the recipients.     

Charge-directed targeting of antimicrobial protein-nanoparticle conjugates

Use of antimicrobial enzymes covalently attached to nanoparticles is of great interest as an antibiotic-free approach to treat microbial infections. Intrinsic properties of nanoparticles can also be used to add functionality to their conjugates with biomolecules. Here, we show in a model system that nanoparticle charge can be used to enhance delivery and increase bactericidal activity of an antimicrobial enzyme, lysozyme. Hen egg lysozyme was covalently attached to two types of polystyrene latex nanoparticles: positively charged, containing aliphatic amine surface groups, and negatively charged, containing sulfate and chloromethyl surface groups. In the case of bacterial lysis assay with a Gram-positive bacteria Micrococcus lysodeikticus, activity of lysozyme conjugated to positively charged nanoparticles was approximately twice as large as that of free lysozyme, while lysozyme conjugated to negatively charged nanoparticles showed little detectable activity. At the same time, when assayed using a low-molecular weight oligosaccharide substrate, lysozyme attached to both positively and negatively charged nanoparticles showed slightly lower activity than free enzyme. A possible explanation of these results is that lysozyme attached to negatively charged nanoparticles cannot be effectively targeted to the bacteria because of the electrostatic Coulombic repulsion from the negatively charged bacterial cell walls, whereas lysozyme conjugated to positively charged nanoparticles was targeted better than free enzyme due to stronger electrostatic attraction to bacteria. Zeta potential measurements confirmed the validity of this hypothesis. Thus, nanoparticle charge is an important factor that can be used to control targeting and activity of protein-nanoparticle conjugates.     

Large-scale validation of a latex agglutination test for diagnosis of tuberculosis

Large-scale validation of a simple latex agglutination test for the diagnosis of tuberculosis is described. Soluble antigens extracted from a non-pathogenic saprophytic mycobacterium, Mycobacterium w, which shares antigenic determinants with Mycobacterium tuberculosis, were covalently linked to carboxylated polystyrene latex beads. Batch to batch reproducibility of coated latex was ensured. Latex reagents were standardized to overcome non-specific agglutination. Reagents of the test are stable for 1 year at 4 degrees C. A total of 1,058 serum samples of pulmonary and extrapulmonary tuberculosis patients or patients with other pulmonary diseases and healthy controls living in endemic areas were tested. Sensitivity of 94% for pulmonary tuberculosis and 87% for extrapulmonary tuberculosis was obtained. Specificity is 92.2% for healthy controls and patients with other respiratory diseases. We conclude that the latex agglutination test can be utilized for mass screening for both pulmonary and extrapulmonary tuberculosis where diagnosis by existing methods is much more difficult.     

The increased flexibility of CDR loops generated in antibodies by Congo red complexation favors antigen binding

The dye Congo red and related self-assembling compounds were found to stabilize immune complexes by binding to antibodies currently engaged in complexation to antigen. In our simulations, it was shown that the site that becomes accessible for binding the supramolecular dye ligand is located in the V domain, and is normally occupied by the N-terminal polypeptide chain fragment. The binding of the ligand disrupts the beta-structure in the domain, increasing the plasticity of the antigen-binding site. The higher fluctuation of CDR-bearing loops enhances antigen binding, and allows even low-affinity antibodies to be engaged in immune complexes. Experimental observations of the enhancement effect were supported by theoretical studies using L lambda chain (4BJL-PDB identification) and the L chain from the complex of IgM-rheumatoid factor bound to the CH3 domain of the Fc fragment (1ADQ-PDB identification) as the initial structures for theoretical studies of dye-induced changes. Commercial IgM-type rheumatoid factor (human) and sheep red blood cells with coupled IgG (human) were used for experimental tests aimed to reveal the dye-enhancement effect in this system. The specificity of antigen-antibody interaction enhanced by dye binding was studied using rabbit anti-sheep red cell antibodies to agglutinate red cells of different species. Red blood cells of hoofed mammals (horse, goat) showed weak enhancement of agglutination in the presence of Congo red. Neither agglutination nor enhancement were observed in the case of human red cells. The dye-enhancement capability in the SRBC-antiSRBC system was lost after pepsin-digestion of antibodies producing (Fab)2 fragments still agglutinating red cells. Monoclonal (myeloma) IgG, L lambda chain and ovoalbumin failed to agglutinate red cells, as expected, and showed no enhancement effect. This indicates that the enhancement effect is specific.     

Binding of affinity cross-linked oligomers of IgG to cells bearing Fc receptors.

The binding of covalently cross-linked oligomers of rabbit IgG antibodies to tumor cells resembling macrophages and lymphocytes and to normal spleen cells has been measured. With all cells trimeric IgG binds with greater affinity than does the dimer, which in turn binds more tightly than does the monomer. However, as the oligomers increase in size, the individual monomeric subunits bind with decreasing energy. The macrophage tumor line. P388D1, binds oligomers with greater affinity than does the lymphocyte line, AKTB-1, but the differences in affinities are not great, differing by at most a factor of 5 in equilibrium constant. Normal spleen cells bind oligomers in the same concentration range as the tumor cells. The kinetics of binding do not occur as single first or second order reactions and suggest a multistage mechanism for oligomer binding. The presence of large concentrations of monomeric IgG tends to weaken oligomer binding and increases the exchange rate of bound oligomer from the cells. Since plasma and extracellular fluid also contain large concentrations of monomeric IgG, it is suggested that many immune complexes will bind weakly to the types of cells examined here and will rapidly exchange with IgG from the external medium.     

Recognition of Promoter DNA by Subdomain 4.2 of Escherichia Coli σ70: A Knowledge Based Model of -35 Hexamer Interaction With 4.2 Helix-Turn-Helix Motif

Abstract

The dye Congo red and related self-assembling compounds were found to stabilize immune complexes by binding to antibodies currently engaged in complexation to antigen. In our simulations, it was shown that the site that becomes accessible for binding the supramolecular dye ligand is located in the V domain, and is normally occupied by the N-terminal polypeptide chain fragment. The binding of the ligand disrupts the β-structure in the domain, increasing the plasticity of the antigen-binding site. The higher fluctuation of CDR-bearing loops enhances antigen binding, and allows even low-affinity antibodies to be engaged in immune complexes. Experimental observations of the enhancement effect were supported by theoretical studies using L λ chain (4BJL-PDB identification) and the L chain from the complex of IgM-rheumatoid factor bound to the CH3 domain of the Fc fragment (1ADQ-PDB identification) as the initial structures for theoretical studies of dye-induced changes. Commercial IgM-type rheumatoid factor (human) and sheep red blood cells with coupled IgG (human) were used for experimental tests aimed to reveal the dye- enhancement effect in this system. The specificity of antigen-antibody interaction enhanced by dye binding was studied using rabbit anti-sheep red cell antibodies to agglutinate red cells of different species. Red blood cells of hoofed mammals (horse, goat) showed weak enhancement of agglutination in the presence of Congo red. Neither agglutination nor enhancement were observed in the case of human red cells. The dye-enhancement capability in the SRBC-antiSRBC system was lost after pepsin-digestion of antibodies producing (Fab)2 fragments still agglutinating red cells. Monoclonal (myeloma) IgG, L λ chain and ovoalbumin failed to agglutinate red cells, as expected, and showed no enhancement effect. This indicates that the enhancement effect is specific.     

Trypsin purification by affinity binding to small unilamellar liposomes

A novel protein purification process using affinity-ligand-modified liposomes and membrane ultrafiltration is described. The feasibility of the process was tested using trypsin as the model protein and p-aminobenzamidine (PAB) as the affinity ligand for trypsin. The affinity liposomes were prepared by covalently attaching PAB to the surface of small unilamellar liposomes via the hydrophilic spacer arm diglycolic acid. The liposomes were comprised of dimyristoyl phosphatidyl choline, cholesterol, and dimyristoyl phosphatidyl ethanolamine to which the diglycolic acid was attached. The equilibrium binding constant between trypsin and immobilized PAB was shown to be dependent on the PAB density of the liposome surface. Bound trypsin was eluted from the liposomes by the trypsin inhibitor benzamidine. Trypsin was purified from a trypsin/chymotrypsin mixture and from one of its naturally occurring sources, porcine pancreatic extract. A recovery yield from the crude mixture of 68% was obtained with a trypsin purity of 98%. The affinity-modified liposomes were stable in the complex mixture and retained their trypsin binding capacity after multiple adsorption/elution cycles over a 30-day period.     

S-layer-coated liposomes as a versatile system for entrapping and binding target molecules

In the present study, unilamellar liposomes coated with the crystalline bacterial cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were used as matrix for defined binding of functional molecules via the avidin- or streptavidin-biotin bridge. The liposomes were composed of dipalmitoyl phosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4 and they had an average size of 180 nm. For introducing specific functions into the S-layer lattice without affecting substances encapsulated within the liposomes, crosslinking and activation reagents had to be identified which did not penetrate the liposomal membrane. Among different reagents, a hydrophilic dialdehyde generated by periodate cleavage of raffinose and a sulfo-succinimide activated dicarboxylic acid were found to be impermeable for the liposomal membrane. Both reagents completely crosslinked the S-layer lattice without interfering with its regular structure. Biotinylation of S-layer-coated liposomes was achieved by coupling p-diazobenzoyl biocytin which preferably reacts with the phenolic residue of tyrosine or with the imidazole ring of histidine. By applying this method, two biotin residues accessible for subsequent avidin binding were introduced per S-layer subunit. As visualized by labeling with biotinylated ferritin, an ordered monomolecular layer of streptavidin was formed on the surface of the S-layer-coated liposomes. As a second model system, biotinylated anti-human IgG was attached via the streptavidin bridge to the biotinylated S-layer-coated liposomes. The biological activity of the bound anti-human IgG was confirmed by ELISA.     

Effect of Peg Homopolymer and Grafted Amphiphilic Peg-Palmityl on the Thermotropic Phase Behavior of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine Bilayer

Abstract

Phospholipids covalently attached to polyethylene glycol (PEG-PE) are routinely used for the preparation of long-circulating liposomes. The common preparation procedure for long-circulating liposomes involves use of organic solvent. Although there is a plethora of studies describing the interaction of PEG-PE with bilayers, little is known about the effects of PEG homopolymers and single chain amphiphilic PEG on liposome structure. In the present investigation the interaction of PEG homopolymer and amphiphilic PEG-palmityl conjugate with large multilamellar liposomes composed of 1,2-dipalmitoyl-sn-glycero-phosphocholine was investigated utilizing differential scanning calorimetry. Vesicle and aggregate sizes were determined by dynamic light scattering. DSC thermograms revealed interaction of PEG homopolymer with DPPC when the two are premixed in organic solvent. The data suggest that PEG interacts with the phospholipid acyl chains deep in the bilayer. Several questions are raised regarding the suitability of the current procedure for preparation of long-circulating liposomes which utilizes organic solvent. Incorporation of only 2 mol% 5 kDa PEG-palmityl conjugate completely solubilized DPPC liposomes. Packing geometry of the lipid anchor, irrespective of the polymer molecular weight, is suggested to be the primary factor for successful grafting of hydrophilic polymers on liposomes. Pure PEG-palmityl formed self-assembled organized structures of potential use in the delivery of poorly soluble drugs.     

A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes.

A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.