Output grating couplers on planar optical waveguides as direct immunosensors

We demonstrated the feasibility of using integrated optical output grating couplers in direct immunosensing. We monitored as functions of time, first the adsorption of an antigen (Ag) on the waveguide's surface, and subsequently, the binding of the corresponding antibody (Ab), i.e. the formation of the immuno-complex Ag-Ab. The Ag was human immunoglobulin G (h-IgG), and the Ab was rabbit anti-h-IgG. We also studied the adsorption of avidin. The refractive indices nF', thicknesses dF', and surface coverages gamma of the adsorbed adlayers and of the immuno-complex Ag-Ab, respectively, were determined.     

A stoichiometric analysis of bovine serum albumin-gossypol interactions: a fluorescence quenching study.

The interaction of gossypol with bovine serum albumin, human serum albumin and n-bromosuccinimide-modified bovine serum albumin has been followed by fluorescence quenching measurements. The presence of a high affinity site (association constant K = 2.2 x 10(6) M-1) for gossypol on bovine serum albumin and human serum albumin is indicated. The stoichiometry of binding for the high affinity site was evaluated using Job's method of continuous variation, thereby suggesting the formation of 1:1 complex. Modification of the tryptophan residues on bovine serum albumin does not affect the binding of gossypol to either high or low affinity site of albumin.     

Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.     

Covalent linkage of ruthenium polypyridyl compounds to poly(L-lysine), albumins, and immunoglobulin G.

A series of ruthenium polypyridyl complexes has been covalently bound to poly(L-lysine), bovine serum albumin, human serum albumin, ovalbumin, and immunoglobulin G using different binding methods. The conjugation ratios and the luminescence properties of the bioconjugates are reported. All conjugates show nonsingle-exponential decay curves. Quenching of the emission by oxygen has been studied.     

Lymphoblastoid cell-produced immunoglobulins: preparative purification from culture medium by hydroxylapatite chromatography

Hydroxylapatite (HA) is a chromatography matrix capable of separating nucleic acid as well as protein species. HA adsorbs biomolecules as a function of the extent and distribution of their surface charge. HA has been evaluated for its ability to differentially retain immunoglobulin molecules, which are planar relative to the generally globular serum proteins, particularly albumin, contained in tissue culture medium. HA chromatography provides a single-step method to purify and concentrate immunoglobulin proteins secreted by lymphoblastoid cells into culture medium from the vast excess of serum proteins used to supplement the medium. A human lambda light-chain protein was recovered in 5% of the applied volume of medium, and was separated from 95% of the total protein. More than 75% of a hybridoma-produced complete immunoglobulin was recovered essentially free of serum protein contamination. HA appears to provide a valuable alternative chromatographic medium for the purification of immunoglobulin proteins secreted by lymphoblastoid cells.     

Association of an albumin antigen with phosphatidylcholine liposomes alters the nature of immunoglobulins produced during the immune response against the antigen

Human serum albumin has been injected intravenously in rabbits either free in solution or associated with liposomes. Blood samples were obtained from the rabbits at various time intervals after injection, and two different antibody determinations were performed in each sample. Whereas a haemagglutination technique was applied for determination of predominantly IgM anti-human serum albumin antibodies, a second technique, using antigen-coated Sepharose beads and horseradish peroxidase-conjugated anti-rabbit IgG, was used to detect the IgG anti-human serum albumin antibodies. Liposomes appeared to enhance strongly the IgM response against human serum albumin. No such marked differences were found, however, between the IgG responses against liposome-associated or free human serum albumin. The conclusion is drawn that the immunoadjuvant effect of liposomes during the primary immune response against an albumin antigen is mainly due to an enhanced IgM antibody production.     

The effect of bovine and human serum albumins on the mechanical properties of human erythrocyte membranes

Crystalline bovine serum albumin increased the mechanical resistance of fresh human erythrocytes to lysis by hydrodynamic shear forces. A saturation effect suggests that the bovine alubmin molecules are adsorbed on to a finite number of “attachment sites” on the erythrocyte surface, possibly by displacing human proteins already occupying these sites. A heterogeneous fraction of human serum albumins does not exhibit the same marked protection effect, nor displace adsorbed bovine albumin molecules from the erythrocyte surface. The precise nature and extent of the interaction between any given concentration of either human or bovine serum albumin and the intact erythrocyte membrane depends upon the chronological age of the cell concerned.     

Effect of experimental conditions on strong biocomplimentary pairing in high-performance monolithic disk affinity chromatography

The effect of flow-rate on quantitatively determined binding parameters for several biocomplementary pairs in affinity mode high-performance monolithic disk affinity chromatography (HPMDAC) has been investigated using frontal analysis approach. Affinity interactions were evaluated from linearized adsorption isotherms and dynamic dissociation constants of the complexes K(diss.) and the theoretical adsorption capacities Q(max) were calculated. HPMDAC isolation of a typical protein trypsin from both buffered solution and artificial mixture as well as biospecific extraction of antibodies against bovine serum albumin and recombinant protein G from such complex mixtures as blood serum and cellular lysate were examined. Immobilized counterparts soybean trypsin inhibitor, bovine serum albumin, and human immunoglobulin G were used in chromatographic experiments. The maximum adsorption capacities obtained at different flow-rates were compared with those determined at static conditions. The dependence of quantitative parameters on the surface density of immobilized ligands has also been explored. Finally, a series of experiments was carried out to evaluate the dependence of dynamic affinity binding on temperature for two complementary pairs.     

Comparison of kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin with that bound to rat serum albumin.

It has been stated by McDonagh, Palma & Lightner [(1982) J. Am. Chem. Soc. 104, 6867-6871] that complexing of bilirubin with serum albumin has a marked species-dependent influence on bilirubin photoisomerization in vitro and in vivo. Therefore the kinetics for the quantitatively important reaction: (Formula: see text) of the photochemical interconversion between bilirubin and its photoisomers bound to human or rat serum albumin in aqueous solution, assayed by h.p.l.c., was used to elucidate the observed species-dependent difference. The relative rate constants for bilirubin bound to human serum albumin, except for k4, the rate of interconversion from (ZZ)-bilirubin into (EZ)-bilirubin, proved to be considerably larger than those for bilirubin bound to rat serum albumin. In accordance with these rate constants, the formation of photoisomers of bilirubin bound to human serum albumin, except for (EZ)-bilirubin, is very rapid and much greater than that for bilirubin bound to rat serum albumin.     

Antiamyloidogenic Effects of Ellagic Acid on Human Serum Albumin Fibril Formation Induced by Potassium Sorbate and Glucose

Oxidative stress has the main role in protein conformational changes and consequent direct involvement in different kind of diseases. Potassium sorbate as a widespread industrial preservative and glucose are two important oxidants that can be involved in oxidative stress. In this study the effect of ellagic acid as a phenolic antioxidant on amyloid fibril formation of human serum albumin upon incubation of potassium sorbate and glucose was studied using thioflavin T assay, surface tension, atomic force microscopy, Amadori product, and carbonyl content assays. The thioflavin T assay and atomic force microscopy micrographs demonstrated the antiamyloidogenic effect of ellagic acid on the human serum albumin fibril formation. This antioxidant also had the repair effect on surface tension of the modified human serum albumin (amyloid intermediates), which was destructed, caused by potassium sorbate and glucose. This mechanism takes place because of potent carbonyl stress suppression effect of ellagic acid, which was strengthening by potassium sorbate in the presence and absence of glucose.     

A general method for isolation of individual polyribosomes and pure mRNAs by immunosorption technique

A highly specific procedure has been developed for the isolation of individual polyribosomes by immunosorbents containing immobilized antigen-antibody complexes. Polyribosomes synthesizing immunoglobulin IgGl and serum albumin were quantificated and isolated in the native state from MOPC 21A plasmacytoma and rat liver cells. For albumin polyribosomes, the presumptive messenger RNA was demonstrated; it contained three components: 20S, 16S and 9S.Abbreviations IgG murine immunoglobulin - RSA rat serum albumin - RGG rat gamma-globulin - EDTA ethylene diamine tetraacetate - SDS sodium dodecyl sulphate     

Adducts of glycosylated serum albumin with amino acids

Glycosylation of human serum albumin was conducted by its long incubation with the excess either of D-glucose or D-glucose-6-phosphate at 37 degrees C. The glycosylated fractions were isolated by the cation-exchange chromatography on CM-cellulose. The quantity of glucose bound covalently with protein was determined by thiobarbituric acid. The glucose-modified human serum albumin forms stable adducts with amino acids. These complexes are, evidently, produced as a result of the Schiff's base formation between the carbonyl group of the ketoamine adduct of glucose with protein and primary amino group of amino acid further followed by the Amadori rearrangement.     

Thickness and density of protein films by optical mixing spectroscopy.

The adsorption of protein films on polystyrene latex spheres was studied by optical mixing spectroscopy. With this technique, we show that both the hydrodynamic thickness of protein films and their optical density can be measured. Thus, we found that films of the glycoproteins isolated from the human erythrocyte membrane were four times as thick as films of either human serum albumin or bovine serum albumin for about the same surface coverage. This result suggests an end-on orientation for the adsorbed glycoprotein molecules, which is consistent with the model proposed by others for the orientation of these molecules at the surface of the red blood cell itself.     

The preparation of bacterial luciferase conjugates for immunoassay and application to rubella antibody detection

Bacterial luciferase has been modified with the thiolating reagent S-acetylmercaptosuccinic anhydride and covalently crosslinked to either Staphylococcus aureus protein A or anti-human immunoglobulin G (IgG) with the heterobifunctional reagent m-maleimidobenzoic acid N-hydroxysuccinimide ester. The conjugates retain enzymatic light-emitting activity and have the ability to bind IgG antibody. The ability of these conjugates to detect human IgG has been demonstrated by application to rubella immunity screening. Rubella antibodies are isolated from serum on the surface of rubella antigen-coated tubes and subsequently determined by light emitted from bound conjugate. In a preliminary study, the bioluminescent immunoassay has been compared to a commercial rubella antibody radioimmunoassay and found to be comparable in the ability to determine rubella immunity.     

Determination of the Surface tension of proteins. I. Surface tension of native serum proteins in aqueous media

The desorption patterns of serum proteins in hydrophobic chromatography suggest that serum proteins that remain immersed in an aqueous medium and do not become in a protein-air interface are very hydrophilic. Contact angle measurements on fairly thick layers of hydrated serum proteins, formed on ultrafiltration membranes, yield surface tensions that correlate well with the degree of hydrophilicity derived from desorption data obtained by hydrophobic chromatography. For further confirmation the absorptivity of four human serum proteins was measured with respect to surfaces of different polymers of various surface tensions, for solution in aqueous solvents of different surface tensions. The surface tension of the solvent from which a dissolved protein adsorbs to precisely the same extent onto all solid substrates (regardless of their surface tensions) is equal to the surface tension of that protein. The surface tensions found by the contact angle (first value given) and by the protein adsorption methods (second value given) were. in erg/cm2; alpha 2-macroglobulin, 71.0, 71.0; serum albumin, 70.5, 70.2; immunoglobulin M, 69.5, 69.4; immunoglobulin G, 67.4, 67.7.     

Effect of the binding of bilirubin to either the first class or the second class of binding sites of the human serum albumin molecule on its photochemical reaction.

The kinetics of the photochemical changes of bilirubin were studied at a constant concentration of bilirubin bound either to the first class or to the second class of binding sites of the human serum albumin molecule. The more the bilirubin binds to the first class of binding sites in the human serum albumin molecule, the more readily geometric photoequilibrium to give (ZE)-bilirubin takes place. The more the bilirubin binds to the second class of binding sites or allosterically transformed binding sites induced by added SDS, the more readily structural photoisomerization, i.e. the formation of (EZ)-cyclobilirubin, takes place. When the serum bilirubin concentration is at low, safe, values bilirubin binds exclusively to the first class of binding sites and serves as an antioxidant [Onishi, Yamakawa & Ogawa (1971) Perinatology 1, 373-379]; at these concentrations human serum albumin protects bilirubin from irreversible photodegradation by only allowing readily reversible geometric photoisomerization. As the serum bilirubin concentration increases to high, and potentially dangerous, values, bilirubin binds to the second class of binding sites, and under these conditions human serum albumin seems to promote the photocyclization of bilirubin. During irradiation human serum albumin seems to act by retaining low, useful, concentrations of bilirubin while facilitating irreversible photoisomerization of excess bilirubin.     

The N-terminal sequence of the heavy chain of rabbit immunoglobulin IgG

The absence of an N-terminal amino acid with a free alpha-amino group from the heavy chain of rabbit immunoglobulin IgG has been confirmed and no evidence could be found of a blocking formyl, acetyl or propionyl group. The N-terminal amino acid appears to be pyrrolid-2-one-5-carboxylic acid (PCA) in all molecules. A mixed amino acid sequence follows in the approximate proportions: PCA-Ser-Val-Glu-Glu-Ser-Gly-Gly-Arg, 50%; PCA-Ser-Leu-Glu, 20%; PCA-Glu(NH(2)), 20%. The heavy chains of a purified antibody, namely anti-(human serum albumin), and of immunoglobulin IgG from a rabbit homozygous at the allotypic loci both showed a similar mixed N-terminal sequence.     

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.     

Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates

A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.     

Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates.

A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.