Band 3 protein: structure, flexibility and function

The oxidation of antibody carbohydrate residues is a common approach used for site-specific antibody immobilization or modification. In this study a flow injection analysis system (FIA) was developed for monitoring antibody oxidation. Antibodies were oxidized with periodate and the resulting aldehyde groups were labeled with Lucifer yellow CH (LyCH). The labeled antibodies were then injected onto an FIA system where the amount of LyCH label was determined by absorbance measurements at 428 nm and the amount of antibody was determined using an on-line bicinchoninic acid protein assay. The analysis time was 2 min per 20 microliters sample injection. The limits of detection for rabbit immunoglobulin G (IgG) and LyCH were 1 x 10(-8) and 4 x 10(-7) M, respectively. The dynamic ranges for IgG and LyCH extended to 2 x 10(-5) and 7 x 10(-3) M. The within-run precision was +/- 5% or less for both analytes. Studies with known LyCH/antibody mixtures indicated that the FIA system had greater accuracy than manual methods at high LyCH levels. One specific application studied for this system was its use in monitoring the time course of periodate-antibody oxidation.     

Evaluation of protein A and protein G as indicator system in an ELISA for detecting antibodies in mink to Pseudomonas aeruginosa

A modified, indirect enzyme-linked immunosorbent assay (ELISA) was developed and applied in the detection of mink antibodies to Pseudomonas aeruginosa. In this assay, peroxidase conjugated protein A and protein G were evaluated as indicator systems for detecting antigen-antibody complexes. It was found that protein A has a strong affinity for mink immunoglobulins. In contrast, protein G showed no such affinity. The affinity of protein A for mink immunoglobulins was further demonstrated by immunoprecipitation assays.     

Development of an automated turbidimetric immunoassay for quantification of bovine serum immunoglobulin G

OBJECTIVE: To develop an automated turbidimetric immunoassay (TIA) for measurement of bovine IgG. SAMPLE POPULATION: 24 bovine serum samples. PROCEDURE: IgG concentration was determined by use of the TIA, and results were compared with those of the radial immunodiffusion (RID) method. Variables were determined, using commercially available reagents and a clinical biochemical analyzer. For the TIA, polyclonal goat anti-bovine IgG (Fc specific) serum, bovine IgG calibrator serum, and polyethylene glycol reaction buffer were used. Sample concentrations were determined by the instrument, using the linear regression method of least squares. The accuracy of this assay was validated by referencing to a purified bovine IgG standard and by recovery of control standards. Parallelism was documented by assay linearity and serial sample dilution linearity. Interference resulting from hemolyzed samples was examined. RESULTS: The TIA method correlated positively (r = 0.9957) and significantly (P < 0.05) with the RID method, yielding a regression equation with slope of 0.78708 and y-intercept of 1.02102. Bias attributable to hemolysis was not observed. CONCLUSIONS: The TIA method is automated, accurate, and precise for bovine serum IgG quantification. CLINICAL RELEVANCE: This assay provides sample results in approximately 10 minutes and may be used as an alternative to the manual RID method.     

An optical biosensor for real-time chromatography monitoring: breakthrough determination

The use of an optical biosensor for immunorecognition of protein products during affinity chromatography is discussed to provide rapid data describing the loading and subsequent breakthrough, followed by elution and fraction collection. The optical biosensor works by following in real-time the interaction of soluble ligate with an appropriate ligand attached to the optically active surface. The initial rate of interaction between soluble ligate and immobilized ligand has been shown to correlate well with ligate concentration. This method of analysis has also been shown to agree well with ELISA, the traditionally employed technique for immunoassay of protein products lacking, for example, catalytic activity. Forward prediction, using models of the breakthrough fitted to the real-time data, has enabled the column saturation point to be determined before it has been reached, thus enabling appropriate action to ensure minimal loss of protein product while improving column utilization efficiency. The biosensor, operated within a flow injection analysis regime, has been demonstrated to provide concentration data within 10 s, with a total assay turnaround of 30 s.     

Brownian dynamics study of ion transport in the vestibule of membrane channels

The Immunoglobulin (Ig) binding capacity of Toxoplasma gondii tachyzoites was investigated using fluorescence flow-cytometry analysis. Polyclonal mouse, human and rat immunoglobulins without specific anti-Toxoplasma activity bound to parasites in a concentration-dependent manner, saturating them at circulating serum concentrations. The immunoglobulin class and subclass specificity of binding was investigated using irrelevant monoclonal antibodies. IgM, IgA and IgG reacted with the parasite membrane. The attachment of mouse IgM to the parasite surface was hampered by mouse IgG1, IgG2a, IgG2b and IgG3. The binding of mouse IgG was proportionally reduced with increasing concentrations of mouse monoclonal IgM. The binding of murine immunoglobulin was diminished when in presence of human IgG. Purified Fc- but not Fab portions of immunoglobulins, fixed to parasites. Using labelled calibrated beads, the Ig binding capacity of parasites was estimated to be 6900 +/- 500 sites per tachyzoite. The Kd of the T. gondii Fc Receptor (FcR) activity was determined at 1.4 +/- 0.1 microM (mean +/- SEM). Such FcR activity was reduced by phospholipase C, trypsin and pronase treatment of the parasites. These data show a low affinity FcR activity on T. gondii tachyzoites which recognizes Ig of different species and isotypes and is likely supported by a glycosyl-phosphatidylinositol (GPI)-anchored surface protein of the parasite.     

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori

Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG1, IgG3 and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.     

Separation of equine IgG subclasses (IgGa, IgGb and IgG(T)) using their differential binding characteristics for staphylococcal protein A and streptococcal protein G

Equine IgG possesses four well-defined subisotypes, designated IgGa, IgGb, IgGc and IgG(T) on the basis of their increasing anodal mobility in electrophoresis. However, the preparation of IgGa and IgGb reference proteins has not previously been reported. Certain bacterial cell wall proteins, termed protein A and protein G, have been used for purification of IgG subisotypes from the serum of domestic animals which, combined with other techniques utilising differences in the physico-chemical properties of the proteins, has allowed the purification of Ig isotypes. This paper describes purification of the subisotypes of equine IgG. Purification of IgGa and IgGb was achieved by the separation of a 'fall-through' peak from ion-exchange chromatography consisting of IgGa and IgGb into two fractions (peaks C and D) by FPLC protein A and protein G affinity chromatography. Peak C consisted of IgGb and peak D consisted of IgGa exhibiting slightly faster cathodal migration than peak C in IEP analysis. Affinity chromatography using protein A and G columns also indicated that there may be two different components of IgG(T); one with a low affinity for protein G and the other having a greater affinity for protein G.     

Proton nuclear magnetic resonance sequential assignments and secondary structure of an immunoglobulin light chain-binding domain of protein L

The 1H NMR assignments have been made for the immunoglobulin (Ig) light chain-binding B1 domain of protein L from Peptostreptococcus magnus. The secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone coupling constants, and slowly exchanging amide protons. The B1 domain was found to be folded into a globular unit of 61 amino acid residues, preceded by a 15 amino acid long disordered N-terminus. The folded portion of the molecule contains a four-stranded beta-sheet spanned by a central alpha-helix. The fold is similar to the IgG-binding domains of streptococcal protein G, despite the fact that the binding sites on immunoglobulins for the two proteins are different; protein G binds IgG through the constant (Fc) part of the heavy chain, whereas protein L has affinity for the variable domain of Ig light chains.     

Evaluation of three immunoassays for detection of Toxoplasma-specific immunoglobulin G and M

A time-resolved immunofluorometric assay of the IgG specific for Toxoplasma gondii has been compared with two commercial methods, one automated enzyme linked fluorescent assay (VIDAS) and one automated colorimetric enzyme immunoassay (BEIA). The coefficients of variation were 3.4% and 7.4% within-assay (n = 12), and 9.2% and 8.1% between-assay (n = 10) for two sera at low and high concentrations of specific IgG. The regression lines obtained with 96 samples were y = 1.04x + 2.1 and y = 0.98x - 1.1. We also compared a time-resolved capture fluoroimmunoassay for Toxoplasma-specific IgM with a commercial immunoenzymatic assay (BEIA TOXO-M). The sensitivity and specificity were 100%, calculated from assays of 78 samples.     

Kinetic characterization of the interaction of biotinylated human interleukin 5 with an Fc chimera of its receptor alpha subunit and development of an ELISA screening assay using real-time interaction biosensor analysis

The interaction of biotinylated human interleukin 5 ([BT]hIL5) with immobilized receptor was measured with a real-time biosensor, and these results were used as a basis for configuring an ELISA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5R alpha-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor alpha subunit to the constant domain (Fc) of immunoglobulin G), were produced by the expression of cloned vectors in Drosophila schneider (S2) cells. Initial attempts to develop a screening assay by direct immobilization of soluble IL5 receptor to microtiter plates proved unsatisfactory and led to use of the Fc chimera attached by oriented immobilization via protein A. Hence, shIL5R alpha-Fc was bound to protein A covalently immobilized on a carboxymethyl dextran (CM-5) biosensor chip. Specific binding was demonstrated of [BT]hIL5 to protein A/shIL5R alpha-Fc receptor complex. The binding was high affinity (Kdapp = 6 nM), reversible and saturable. The affinity of [BT]hIL5 was similar to that determined with the biosensor assay for unmodified hIL5. The observed kinetics of the interactions of Fc chimera with protein A (slow dissociation) and of [BT]hIL5 with immobilized Fc chimera (faster dissociation) were favorable for subsequently establishing a microtiter plate based ELISA assay. In the latter, Fc chimera was immobilized to the plate via protein A as in the biosensor experiment. Binding of [BT]hIL5 to immobilized Fc chimera in the ELISA was concentration dependent and was competed by both hIL5 and shIL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

单标准合并带注入FIA光度法测定铝合金中硅、铜和铁

以一种新设计的注入试样与试剂的FIA光度法系统,应用微机控制单标准FIA梯度校正技术,测定了铝合金中的硅、铜和铁。并将这种方法与常规FIA法和反相FIA法在测试性能上进行了比较。实验结果表明,这种方法的灵敏度,分析速度,通用性是令人满意的。     

Development of multicomponent immunoassay and micro-localization analysis by laser-photoacoustic and microscopic image analyzer]

Laser photoacoustic spectroscopy (PAS) has a potential to be developed as a sensitive and solid-phase analytical method and was applied to enzyme immunoassay. The test sample for immunoassay was prepared by adsorbing multi-component immunoglobulins on a nitrocellulose membrane filter. Human lambda- and kappa-chains, which are used as a principal indication of malignant lymphoreticular disease, and immunoglobulin G were used as model proteins, and PAS immunoassay was applied to the individual detection of these three proteins in the urine. Furthermore, in order to develop a sensitive analysis for particular biological components in tissues or cells, laser photoacoustic microscopy (PAM) and video intensified microscopy (VIM) were developed. PAM was shown to be applicable to the detection and quantification of human lambda-chain in a micro-region of the tissue sections of the human fetal spleen and pancreas. VIM was applied to the detection of stimulation-response processes in a cell. By using neutrophils which are stimulated by many substances and produce active oxidants as the results, dynamic changes in the stimulation-response process in a living cell were visualized as fluorescence or chemiluminescence images by the VIM system.     

Fractionation of fowl immunoglobulins

Serum, Na2SO4-precipitated serum immunoglobulins and bile from 12-week-old fowls, and serum from day-old chicks, were fractionated by Sephadex G-200 gel filtration, DEAE Sephadex A-50 ion exchange chromatography and ultracentrifugation through 10-40 per cent sucrose gradients. Elution of IgM, IgG, IgA and albumin was monitored by examination of fractions in agar gel diffusion against antisera specific to these proteins. Serum and bile from 12-week-old fowls contained IgM and IgA in two molecular sizes and a single molecular size of IgG. Day-old chick serum contained IgM estimated to be 7S, a polymerised form of IgG in addition to the normal 7S component, and a small molecular weight protein antigenically related to IgA. Most of the albumin in bile was of lower molecular weight than serum albumin, while heavy forms of albumin were detected in ultracentrifugation of bile and day-old chick serum.     

Direct determination of procainamide and N-acetylprocainamide by capillary zone electrophoresis in pharmaceutical formulations and urine

A method is described for the simultaneous determination of sixteen organochlorine pesticides in drinking water using automated solid-phase extraction followed by high-volume (80 microliters) capillary column gas chromatography using electron capture detection. The fully automated extraction method followed by high-volume injection permits rapid sample analysis compared to previously described procedures since no further pre-concentration of the analytes is necessary after they have been eluted from the octadecyl solid-phase extraction cartridge. The lowest detectable concentrations of the pesticides are between 1-5 ng l(-1), relative recoveries range from 92-105% in tap water spiked at 100 ng l(-1) and the relative standard deviations are in the range 5-12%.     

Sensitive enzyme immunoassay of antibodies to HIV-1 p17 antigen using indirectly immobilized recombinant p17 for diagnosis of HIV-1 infection

Recombinant p17 (rp17) antigen of HIV-1 and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized onto polystyrene beads in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption; biotinyl-rp17 and biotinyl-MBP-rp17 were immobilized indirectly onto streptavidin-coated polystyrene beads; and 2,4-dinitrophenyl (DNP)-MBP-rp17 was immobilized indirectly onto (anti-DNP) IgG-coated polystyrene beads. These directly and indirectly immobilized antigens were incubated with urine samples containing antibody IgG to p17 antigen and subsequently with rp17-beta-D-galactosidase conjugate or (anti-human IgG gamma-chain) Fab'-beta-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene beads was assayed by fluorometry. When rp17-beta-D-galactosidase conjugate was used, signals (fluorescence intensities for bound beta-D-galactosidase activity) were much higher with the indirectly immobilized antigens than those with the directly immobilized antigens. By experiments using (anti-human IgG gamma-chain)Fab'-beta-D-galactosidase conjugate, the binding of rp17-beta-D-galactosidase conjugate to antibodies against p17 antigen bound to directly immobilized rp17 antigen was shown to be seriously limited as compared with that to antibodies against p17 antigen bound to indirectly immobilized DNP-MBP-rp17. When rp17-beta-D-galactosidase conjugate and serum samples were used, serum interference was much less with indirectly immobilized DNP-MBP-rp17 than with directly immobilized rp17 antigen, and the sensitivity of enzyme immunoassay for antibody IgG to p17 antigen using indirectly immobilized DNP-MBP-rp17 was 1,000- to 3,000-fold higher than that of enzyme immunoassay using directly immobilized rp17 antigen and Western blotting for p17 band. This sensitive enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as conventional methods for antibodies to HIV-1 and earlier than Western blotting for p17 band.     

Screening of compounds interacting with HIV-1 proteinase using optical biosensor technology

A high-resolution optical biosensor assay for screening of low-molecular-weight compounds, using an immobilized protein target, has been developed. HIV-1 proteinase was immobilized on the sensor surface by direct amine coupling and a variety of inhibitors and noninteracting reference drugs were applied to the sensor surface in a continuous flow of buffer. The procedure did not require intrinsic reporter groups, substrates, inhibitors, or other ligands for detection. By using a reference protein, the signal could be corrected for the relatively large background signal caused by differences in dimethyl sulfoxide concentration between running and sample buffers. Substances binding with high affinity (Ki in nM range) required efficient regeneration of the sensor surface and washing of the injection system between sample cycles to get consistent results. Analysis was simplified by using report points, extracted during both association and dissociation phases, and a simple graphical display of data. The optimized assay could correctly distinguish HIV-1 inhibitors from other compounds in a randomized series, indicate differences in their interaction kinetics, and reveal artifacts due to nonspecific signals, incomplete regeneration, or carryover. The method is expected to be generally applicable to secondary screening of low-molecular-weight compound libraries with proteins as targets.     

Molecular cloning of a novel putative G protein-coupled receptor (GPCR21) which is expressed predominantly in mouse central nervous system

Agarose gel affinity electrophoresis has been used to demonstrate interactions between autologous IgG and specific erythrocyte membrane proteins. These binding phenomena are here further examined by combining affinity electrophoresis with affinity chromatography, absorption experiments, and immunoblotting. It is demonstrated that the interactions are highly dependent on polyreactive IgG binding favored by the low ionic strength conditions of the electrophoretic assay. Thus, about 25% of normal IgG under low ionic strength conditions bound to the purified cytoskeletal protein, spectrin, immobilized on Sepharose. This IgG reacted in affinity electrophoresis in a polyspecific fashion with the same array of membrane proteins as before the low ionic strength-affinity chromatography. Further, the binding seen in affinity electrophoresis, including the interaction with spectrin, was completely abolished by preabsorption of the IgG with spectrin-devoid membranes. The charge characteristics of an IgG subclass might be responsible for the observed binding. However, the observed precipitate formation suggested an interaction involving at least two binding sites on each molecule and the binding appears to require structurally intact IgG because reductive treatment with dithiothreitol diminished the reactivity considerably. Conclusively, under the conditions of affinity electrophoresis with ligand present in the gel, electrostatic interactions are amplified. The degree of binding of IgG to erythrocyte membrane proteins that take place under these conditions does not reflect binding which would occur to the same extent under physiological ionic strength conditions.     

Establishment and evaluation of a new chemiluminescent enzyme immunoassay for carcinoembryonic antigen adapted to the fully automated ACCESS system

We have established a new chemiluminescent enzyme immunoassay for carcinoembryonic antigen (CEA), designated ACCESS CEA, which is adapted to the fully automated ACCESS immunoassay analyzer. The assay is based on a one step sandwich-type method using two monoclonal antibodies, one of which is immobilized on micrometer-size paramagnetic particles and the other is conjugated to alkaline phosphatase. Ten microliters of calibrators or sera are incubated for 5 minutes at 37 degrees C with the particles and with the alkaline phosphatase conjugate. The particles are then magnetically separated and washed to remove unbound components. Time needed to obtain the first result is less than 15 minutes. The assay range was 0.04-1000 micrograms/l of CEA, and the possible high-dose hook effect was prevented at CEA concentrations up to 100000 micrograms/l in this working range. The coefficient of variation (CV) for intra-assay precision was 3.0 to 4.7%, and inter-assay CV was 3.4 to 5.6%. The sample carryover was less than 0.001%. The analytical recovery ranged from 98 to 104% and a dilution linearity was demonstrated. No interference was detected in any sample with levels up to 300 mg/l for bilirubin, 12000 mg/l for haemoglobin, 50000 mg/l for human serum albumin, 8500 mg/l for triacylglycerol, and 500000 IU/l for rheumatoid factor. The ACCESS CEA assay also showed very homogeneous reactivity with purified CEA preparations from different tumours and could discriminate CEA from four CEA-related normal antigens tested. Serum samples (n = 362) from patients with malignant or non-malignant disease, as well as from healthy individuals, were analyzed by the ACCESS CEA assay and by the established IMx CEA assay. The CEA values determined by the ACCESS CEA assay were in good agreement with those determined by the IMx CEA assay, and the ACCESS CEA assay significantly increased the sensitivity and specificity of tumour diagnosis as compared with the IMx CEA assay.     

Streptococcal protein H forms soluble complement-activating complexes with IgG, but inhibits complement activation by IgG-coated targets

Protein H, a surface protein of Streptococcus pyogenes interacting with the constant Fc region of IgG, is known to be released from the streptococcal surface by a cysteine proteinase produced by the bacteria. Poststreptococcal glomerulonephritis and rheumatic fever are conditions in which immune complexes and autoimmune mechanisms have been suggested to play pathogenetic roles. The present study demonstrates that addition of protein H to human serum produces complement activation with dose-dependent cleavage of C3. The activation was IgG-dependent and the result of complexes formed between IgG and protein H. These complexes were size heterogeneous with molecular masses of 400 kDa to 1.4 MDa. Using complement-depleted serum reconstituted with complement proteins, the activation by protein H was found to be dependent of the classical, but independent of the alternative pathway of complement. In contrast to results of experiments based on soluble protein H.IgG complexes, complement activation was inhibited by protein H when IgG was immobilized on a surface. The interaction between C1q and immunoglobulins represents the first step in the activation of the classical pathway, and protein H efficiently inhibited the binding of C1q to IgG immobilized on polyacrylamide beads. Protein H reduced C3 deposition on the IgG-coated beads and inhibited immune hemolysis of IgG-sensitized erythrocytes. Finally, significantly less C3 was deposited on the surface of protein H-expressing wild-type streptococci than on the surface of isogenic mutant bacteria devoid of protein H. The results demonstrate that protein H.IgG complexes released from the streptococcal surface can produce complement breakdown at the sites of infection, whereas complement activation on bacterial surfaces is inhibited. This should have important implications for host-parasite relationships. In addition, soluble protein H.IgG complexes might contribute to immunological complications of streptococcal infections.     

Thermodynamic stability of immunoglobulins and allosteric interactions with ferritin and protein A: distinct properties of the two antibodies of IgG2a subclass

The two anti-ferritin monoclonal antibodies of mouse IgG2a subclass, G10 and F11, are described that have similar affinity to human spleen ferritin and identical protein A-binding affinity. Antigen binding was shown to change significantly the protein A-binding parameters of the IgG2a antibodies. Antigen-induced conformational changes result in enhanced protein A-binding affinity of the G10 antibody while reduced affinity of the F11 antibody. Antigen binding does not change inherently low affinity of the anti-ferritin IgG1 antibody C5 to protein A. Differential scanning calorimetry revealed that the enthalpy and Gibbs free energy of denaturation for G10 was respectively by 19 and 29% higher than the corresponding parameters for F11. The lower structural energetics of F11 is associated with the lack of a calorimetrically revealed folding unit that may be responsible for distinct interaction between the antigen-binding and protein A-binding sites. This work provides experimental demonstration of the fact that functionally significant interactions between the two spatially remote recognition sites in antibodies of the same heavy chain isotype can be modulated by relatively small structural variations that also result in different thermodynamic stability.