Detection of surface bound complement at increasing serum anticoagulant concentrations

Surface mediated immune complement activation can be detected by a variety of antibody utilizing methods such as ELISA, fluorescence- or radiolabelling techniques, QCM, and ellipsometry. In the present work we investigated how the common anticoagulants heparin, dalteparin, fondaparinux and sodium citrate affected the binding of anti-complement factor 3c (anti-C3c) on a model complement activator surface, immobilised IgG, after incubation in human blood serum.

The results show, as expected, that different anticoagulants affect the antibody binding differently. Increasing amounts of heparin, dalteparin and sodium citrate in normal serum resulted in a decreasing anti-C3c binding. The antibody deposition was not sensitive for the fondaparinux concentration. Surprisingly high concentrations of anti-coagulantia were needed to completely eradicate the antibody binding. Experiments in EGTA–serum showed that anticoagulants interfered directly with both the classical and alternative pathways. Control C3a-des arg ELISA measurements show that the lowered antibody surface binding was not a result of complement depletion in serum. Kallikrein generation by hydrophilic glass surfaces was not affected by high anticoagulant concentrations.     

Methods for research on immune complement activation on modified sensor surfaces

We have developed a methodological system consisting of a new surface sensitive quartz crystal microbalance with dissipation monitoring (QCM-D) sensor surfaces together with different surface modification methods for the investigation of surface associated complement activation in human sera. The QCM-D surface, 10 mm in diameter, was modified by spin-coating of poly(urethane urea) (PUUR) and polystyrene (PS). Some sensor surfaces were also sputtered with titanium (Ti) or modified by hydrophobic self-assembled monolayer (SAM) of an 18-carbon alkane thiol with a ---CH₃ end group. The amount of surface deposited complement protein was investigated by incubation of the modified sensor surfaces in human sera, followed by incubation with antibodies directed against complement factor 3c (C3c). The amounts of bound anti-C3c were then used as an arbitrary measure of surface induced complement activation. The order of complement activation of the different surfaces, as judged by three separate measurements per surface modification, was PUUR>PS=SAM>Ti. The Ti surface had a similar low degree of anti-C3c binding as the negative controls (heat inactivated sera). The novel QCM-D methodology was found to be very simple, accurate, sensitive and well suited as a screening method for complement activation and protein adsorption on different materials. We also compared the sensitivity of QCM-D method with surface plasmon resonance (SPR) for the quantification of protein adsorption and complement activation on gold sensor surfaces. The QCM-D method was equally sensitive as the SPR for the detection of protein adsorption from a solution independently if low flow rate (5 μl/min) was used. A slight increase in sensitivity was found at higher flow rate (30 μl/min). However, we found it difficult to use the SPR method on the Ti, PS and PUUR surfaces due to decreased light penetration of the modified SPR sensor chip.     

Preparation of multilayer plasma protein films on silicon by EDC/NHS coupling chemistry

Crosslinked multilayer protein films were prepared from fibrinogen, albumin, IgG, a combination of fibrinogen and catalase, and blood plasma on silicon by ethyl-dimethyl-aminopropylcarbodiimide and N-hydroxy-succinimide coupling chemistry. The 4–70 nm thick films were placed in blood plasma and the additional protein deposition measured by null ellipsometry after 5 or 60 min of incubation. The activation of the complement system and intrinsic pathway of coagulation were indicated through the subsequent binding of anti-C3c, anti-C3d, anti-properdin and anti-HMWK on top of the surface bound blood plasma. The proportion of Annexin V, Propidium Iodide and 4,6-diamidino-2-phenylindole positive cells, and the secretion of tumor necrosis factor (TNF-) and interleukin-10 (IL-10) were analysed in a monocyte culture. The results show that well known protein coupling techniques can be used for the preparation of protein layers with well controlled thickness. The layers possess low contact activation of blood plasma and induce different release of TNF- and IL-10 in monocyte cultures.     

Inorganic supports coated with N-substituted polyacrylamides: application to biospecific chromatography of proteins

Wide porous glass (WPG) chemically coated with a poly-N-(2-hydroxyethyl)acrylamide layer is proposed as a carrier of biospecific ligands in affinity chromatography. The method of WPG chemical modification includes synthesis of the gamma-aminopropyl derivative followed by chemical adsorption of poly(p-nitrophenyl acrylate). Ester groups of the polyacrylate-coated WPG can be used for coupling the ligands bearing primary amino groups. Condensation of esters with ethanolamine yields a poly-N-(2-hydroxyethyl)acrylamide-coated support with non-specific adsorption properties resembling those of Sepharose 4B. Human IgG immobilized on the polyacrylate support was used for isolation of the first complement component from human serum and for its separation into subcomponents C1r, C1s and C1q by a one-step method. An unbound part of serum may be used as the R1 reagent for determining haemolytic C1 activity. The stepwise elution of C1r, C1s and C1q from the column reflects the course of C1 breakdown after its activation on immune complex formation.     

补体C1q压电免疫传感器的研究

研制了一种可重复使用的压电免疫传感器用于检测补体C1q.比较了聚乙烯亚胺粘附、戊二醛交联法,物理吸附法以及蛋白A法三种固定化抗体蛋白的方法.使用蛋白A法,补体C1q在所测浓度范围内有良好的线性关系.使用过的晶片用0.1 mol/L柠檬酸盐缓冲溶液(pH 3.0)解吸,可重复使用4次.     

Chromatographic and electrophoretic studies of immune complexes in non-A, non-B hepatitis

Immune complexes isolated from two patients with chronic non-A, non-B hepatitis, one patient with acute non-A, non-B hepatitis and one patient with juvenile rheumatoid arthritis were examined by means of a combined chromatographic and electrophoretic method. Both analyses showed the presence of complexes consisting of IgG, IgM, complement c1q factor and albumin; no antigen constituents were detected. The IgG-to-IgM ratio varied from 1:1 to 4:1, suggesting that one could be dealing with complexes of both IgG-IgM and IgG-IgG types. Moreover, the detectable presence of c1q factor might indicate that such complexes were capable of activating complement.     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

A rapid and sensitive immunonanogold resonance scattering spectral probe for complement 3

Gold nanoparticles in size of about 10 nm was used to label goat anti-human complement 3 (anti-C3) to obtain a sensitive and selective immunoresonance scattering spectral probe for C3. It was based on the immune reaction between labeled anti-C3 and C3 in the pH 5.6 Na₂HPO₄–citric acid buffer solutions and in presence of polyethylene glycol (PEG). The resonance scattering (RS) intensity at 560 nm enhanced greatly with C3. Well linear relationships between the enhanced RS intensity (I_RS) and the C3 concentration in the range of 8.33–200 ng ml⁻¹ were obtained, with a detection limit of 2.8 ng ml⁻¹ and the limit of quantification 8.51 ng ml⁻¹ for C3. The convenient and selective and sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.     

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.     

一种新的压电免疫传感器中生物分子固定化方法的研究

生物分子固定化或传感界面设计技术是研制压电免疫传感器的关键之一。本文 结合自组装单分子膜（SAMs）和聚电解质静电吸附组装技术，提出了一种新的压电 免疫传感器中生物分子固定化方法，研制成一种检测补体C_3的压电免疫传感器。 先在石英晶振的金电极表面组装一层胱胺SAMs，再在膜上组装带相反电荷的聚苯磺 酸钠（PSS）单层膜，通过静电吸附作用固定抗体（抗原），实现对相应抗原（抗 体）的检测。利用扫描电镜技术，从形态上考察了晶振组装胱氨SAMs与PSS及固定 补体C_3抗体后的表面形貌。研究了抗体的固定化条件，探讨了传感器采用这种固 定化方法的响应与再生性能，并与戊二醛键合固定法进行比较。结果表明，这种固 定化方法不仅对蛋白质类生物分子的固定化具有普适性，而且对所固定的生物分子 的活性影响小，传感器的响应的频移值大，灵敏度高，选择性和再生性能均较好。     

Evaluation of Amino Acid O-Phosphoserine as Ligand for the Capture of Immunoglubulin G from Human Serum

The amino acid ortho-phosphoserine (OPS) immobilized on agarose gel was evaluated as a ligand for adsorption of polyclonal human immunoglobulin G (IgG) from human serum in the presence of low ionic strength buffers. Screening of buffer systems showed sodium phosphate as the buffer that exhibited higher IgG purity values. Through breakthrough curve analysis for agarose-OPS (feeding of 31.93?mg of total protein per mL of gel), a purification factor of 5.4 with an IgG purity of 89?% was obtained (based on IgG, IgM, IgA, HSA, and Trf nephelometric analysis). IgG adsorption equilibrium studies showed that these data followed the Langmuir-Freundlich model, with cooperativity parameter (n) equal to 1.74, indicating the presence of positive cooperativity, probably due to multipoint interactions. The maximum IgG binding capacity was 24.2?mg?mL^?1, near the value for the bioaffinity ligand protein A. The agarose-OPS adsorbent provides an attractive alternative for capturing of IgG from human serum.     

Application of surface biopassivated disposable poly(dimethylsiloxane)/glass chips to a heterogeneous competitive human serum immunoglobulin G immunoassay with incorporated internal standard

A microfluidic platform for a heterogeneous competitive immunoassay of human immunoglobulin G (IgG) employing Cy5-human IgG as tracer and Cy3-mouse IgG as internal standard was developed. The device consisted of microchannels made of poly(dimethylsiloxane) and glass which were patterned with antibodies against human IgG and mouse IgG. Electrokinetic sample transport was employed in order to exploit the small difference between the net mobilities of analyte and tracer, thereby achieving favorable conditions for the performance of the competitive immunoreaction. The overall quality of the disposable chip and performance of the immunoassay were controlled by monitoring the fluorescence of bound tracer and bound internal standard. Analyses with an insufficient internal standard response were discarded, and immunoassay data evaluation was based on the ratio of tracer and internal standard fluorescence. Using synthetic samples in the range from 0 to 80 microg/mL IgG and alkaline running conditions, a concentration-dependent response with reproducible Cy5/Cy3 signal ratios (average relative standard deviation of 6.8%) was obtained. Chips stored with solution in the channels at 4 degrees C over a two-month period were found to perform like freshly prepared chips, whereas chips stored dry at -20 degrees C and rehydrated prior to use could not be employed. The analysis of patient sera showed that the immunoassay platform behaved differently in the presence of serum-based samples. Using the same conditions as for the synthetic samples, no concentration dependence was noted. With a large excess of tracer, however, an IgG concentration dependence was observed, permitting distinction of samples of patients with normal IgG serum levels (8-16 mg/mL) from those with elevated IgG concentrations (>16 mg/mL).     

Serum protein immunogenicity: implications for liver xenografting

The objectives of this study were threefold: (i) assess immunogenicity of donor plasma proteins following hepatic xenotransplantation, (ii) identify potential immunogens, and (iii) consider the implications of antibody formation against these plasma proteins in xenograft survival. We studied liver and heart xenografts in a concordant combination, hamster to rat. All grafts were examined at necropsy for evidence of rat immunoglobulin G (IgG) deposition. Cardiac xenografts were placed in recipients who had, or had not, been sensitized with hamster serum. Hepatic xenografts were placed in naive recipients to see if antibodies to hamster serum proteins could be eluted from the rejecting organ. Sera of immunized rats were examined for the presence of anti-hamster antibodies by immunoelectrophoresis and by Western blotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of hamster serum. Antibodies in sera of immunized rats were compared with those eluted from rejecting livers. Candidate antigens were identified by tandem mass spectrometry, sequence analysis, and reference to protein databases. Results showed that sera of immunized rats recognized a minimum of four different antigens in hamster serum by immunoelectrophoresis, and a minimum of seven by the more sensitive SDS-PAGE Western blot. IgG eluted from rejecting livers bound three of seven candidate antigens recognized by sera of the immunized animals. Sequence analysis searches revealed proteinase inhibitors in each of the three SDS-PAGE bands common to the above samples. All of these candidate proteinase inhibitor immunogens share a common catabolic fate, uptake via the lipoprotein-related protein (LRP/alpha 2-macroglobulin receptor (CD91). Sensitization to hamster serum proteins hastened cardiac xenograft rejection in 30-50% of recipients (depending on sensitization protocol). Vascular deposition of rat IgG occurred in all rejecting xenografts. Antibody binding to proteinase inhibitors could disturb their functional activity and contribute to the pathogenesis of delayed xenograft rejection.     

Mode of complement activation by acidic heteroglycans from the leaves of Artemisia princeps PAMP.

The mode of action of the anti-complementary acidic heteroglycans, AAF-IIb-2 and IIb-3 which consisted of rhamnogalacturonan core and arabinogalactan moieties, purified from the leaves of Artemisia princeps PAMP (Japanese name = Gaiyo) were investigated. The anti-complementary activities of AAF-IIb-2 and IIb-3 were reduced partially in the absence of Ca2+ ions. A marked consumption of C4 was observed to have occurred when serum was incubated with both polysaccharides in the presence of Ca2+ ions. AAF-IIb-2 showed more potent C4 consumption than IIb-3. After the incubation of the serum with AAF-IIb-2 in the absence of Ca2+ ions, a cleavage of C3 in the serum was detected by immunoelectrophoresis. AAF-IIb-2 showed more significant consumption of the complement than IIb-3 when rabbit erythrocytes were used in the assay system in the absence of Ca2+ ions. These results indicate that AAF-IIb-2 activates the complement via both the alternative and classical pathways, whereas IIb-3 mainly activates the complement via the classical pathway. The absorption of serum with Protein A-Sepharose results in a decrease of the activity of AAF-IIb-2 and IIb-3. However, the decrease of the activity was restored by the replacement of the immunoglobulin G (IgG) fraction after its recovery from the Protein A-Sepharose. These results suggest that IgG dependent mechanisms are both involved in the anti-complementary activity of AAF-IIb-2 and IIb-3.     

Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.     

The application of DNA-cellulose chromatography in the isolation of immunoglobulin M and complement component C4b-binding protein from human serum

By utilising the ability of some anionic serum proteins to bind to DNA-cellulose, at physiological pH and ionic strength, two such proteins have been purified and identified as immunoglobulin M (IgM) and complement component C4b-binding protein (C4BP). The method entails ammonium sulphate precipitation followed by QAE-Sephadex, DNA-cellulose and Sephacryl S-300 chromatographic steps. Fractionation of the anionic proteins eluting from QAE-Sephadex over a selected range of ionic strength has afforded IgM which was characterised electrophoretically and immunochemically. A mixture containing IgM complexed with C4BP has also been obtained by utilizing fractions eluted from QAE-Sephadex at higher ionic strength. C4BP can be isolated from this mixture by using immunoaffinity chromatography to remove IgM.     

Purification of ascitic fluid-derived murine monoclonal antibodies by anion-exchange and size-exclusion high-performance liquid chromatography

Ascitic fluid-derived murine monoclonal antibodies (MoAbs) of immunoglobulin (Ig) M and IgG isotypes (IgG1 and IgG2a subisotypes) were previously prepared against an isolate of Actinobacillus sp (CAs8C) for the purpose of identifying and characterizing outer membrane antigens on this bacterium. An attempt was made to purify these MoAbs by anion-exchange and size exclusion high-performance liquid chromatography (HPLC). Hybridomas producing the IgG1 and IgG2a MoAbs posed unique difficulties in that they also secreted irrelevant IgG2b MoAbs that were present in the ascitic fluids. Anion-exchange chromatography (Protein-Pak DEAE-5PW column), with a simultaneous change in gradients of pH and ionic strength, was used to purify IgG and as a first step in the purification of IgM. There was good separation of IgG2b from IgG2a, but not from IgG1. Size-exclusion chromatography (Protein-Pak 300 SW column) was required to complete the purification of IgM. The presence of MoAbs in the HPLC fractions was confirmed by discontinuous gradient polyacrylamide gel electrophoresis (denatured and either reduced or non-reduced conditions) and the enzyme-linked immunosorbent assay. HPLC-purified MoAbs were free from transferrin and albumin and retained their specificity for As8C.     

High Performance Liquid Chromatography of Mouse Monoclonal Antibodies on Spherical Hydroxyapatite Beads

Abstract

High performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite was applied for the simple purification of monoclonal antibodies (mAbs) secreted into mouse ascitic fluid. Sixteen mAbs including all four subclasses of IgG and IgM were separated successfully from serum albumin a major contaminant in the crude mAb preparation by a 30 min-linear gradient of phosphate ion concentration from 0.01M-0.3M at pH 7.2. Not only IgG mAbs but also IgM mAbs were quantitatively eluted from the column. Each antibody had a different retention time (apparent capacity factor of 2.24–4.14) in the chromatography and no relation was found between the retention time and the type of immunoglobulin (class or subclass). A monomeric form of IgM was also resolved successfully from IgM (pentamer) after its reduction with dithiothreitol; the monomer form of IgM was eluted from the column by a lower concentration of phosphate ion than was the pentamer. These results indicate that HPLC on the hydroxylapatite beads will be useful for the purification and characterization of mouse mAbs.     

The effect of NaCl on the adsorption of human IgG onto CM-Asp–PEVA hollow fiber membrane-immobilized nickel and cobalt metal ions

Over the past decade, immobilized metal-affinity adsorbents have attracted increasing interest for purification of natural and recombinant immunoglobulin G (IgG). In this work, nickel and cobalt metal ions complexed with CM-Asp (carboxymethylaspartate) immobilized on poly(ethylenevinyl alcohol) (PEVA) hollow fiber membranes were evaluated for purification of human IgG from serum. The buffer system and NaCl had important effects on human serum protein adsorption in both adsorbents. Efficient purification of IgG was accomplished in sodium phosphate buffer without NaCl at pH 7.0. Under this condition, the electrostatic interactions are important for adsorption. The Ni(II)-CM-Asp–PEVA had a protein adsorption capacity of 17.5 mg of IgG mL^?1 fiber when human serum diluted was loaded in crossflow filtration mode and the eluted IgG had a purity of 82.6 % (based on total protein and IgG, IgM, HSA, and Trf nephelometric analysis). Fitting the experimental IgG adsorption data to the Langmuir and Langmuir–Freundlich models showed that Ni(II)-CM-Asp and Co(II)-CM-Asp had Langmuirean and non-Langmuirean behavior, respectively, with positive cooperativity for IgG-Co(II)-CM-Asp binding, probably due to multipoint interactions (n = 2.12 ± 0.31). Thus, these membranes can be considered as alternative adsorbents for the purification or depletion of IgG from human serum.     

Improved Sensitive Sandwich Enzyme Immunoassay for Secretory Immunoglobulin a in Human Serum

Abstract

To overcome the IgA interference in enzyme immunoassay for serum secretory IgA (SIgA), a new specific, simple and more sensitive sandwich enzyme immunoassay, fully free of the serum IgA interference, was developed. SIgA standards or samples were added into the wells of polystyrene plate coated with rabbit IgG antibody to human secretory component. After incubation, the wells were washed and then, horseradish peroxidase-labeled Fab′ fragment of goat IgG antibody to human α-chain was added and incubated. The wells were washed again to remove the unbound labeled antibody, and the enzyme activity specifically bound to the well was assayed using 3,3′, 5,5′ - tetramethylbenzidine and H₂O₂ as substrate. The enzyme reaction was stopped by addition of 2M H₂SO₄. The SIgA concentration was determined from the absorbance at 450 nm. The minimun detectable sensitivity was 6ng/ml. The assay system had very good selectivity overcoming the interference of IgA. As the result of high sensitivity, only small amount of sample (2 μ1 for serum) was needed for analysis. In this assay, no cross reactivity was found with purified human IgG, mIgA, IgM or free secretory component (FSC). The recovery of SIgA mixed with human sera or biles was 99.6–108.1%. The coefficients of within-assay and between-assay variation were 5.8–9.3% and 6.2–9.2% respectively. It correlated well with a liquid phase competitive radioimmunoassay for human serum SIgA (r=0.96, n=30, P<0.0l). The level of SIgA in normal human serum was 8.04±3.60 (SD) μg/ml (n=117) and increased significantly in patients with choledocho- lithiasis (57.35±49.70 μg/ml, n=15, P<0.0l). SIgA concentrations in bile samples were also determined by the 2 4′ assay under the condition that FSC did not, interfere with the assay.