Experiences in the measurement of RBC-bound IgG as markers of cell age

An immunologically mediated pathway has been largely accepted to be one of the mechanisms involved in the clearance of senescent or prematurely damaged RBC. According to this pathway, RBC removal is mediated by binding of naturally occurring IgG to clustered integral membrane proteins, followed by complement deposition. The validation of an immunoenzymatic method for the detection of RBC-bound autologous IgG is presented. The use of RBC-bound IgG as an index related to red cell age was evaluated by measuring IgG binding in RBC treated with the clustering agent ZnCl2, in density fractionated RBC and in a selected group of patients expected to have an altered RBC life span. The immunoenzymatic method for IgG detection resulted to be reproducible (CV = 3.4%). IgG binding to in vitro clustered RBC was found to be enhanced to a very great extent, about 20 times higher with respect to untreated RBC. A slight but significant increase (about 1.8-fold) in membrane-bound IgG was observed in the highest density fraction of normal RBC, which constituted 1% of the total cells. A significantly greater number of RBC-bound IgG was measured in splenectomized beta-thalassemia intermedia patients and in subjects with secondary decreases in the C3 complement fraction concentration.     

Feasibility study of red blood cell debulking by magnetic field-flow fractionation with step-programmed flow

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures, require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation.

Assay for quantitation of mature monocytes in murine haemopoietic tissues

We propose a new assay which allows the extent of production of mature monocytes in murine haemopoietic tissues to be monitored with precision. To develop such a quantitative assay, we chose a T-cell function easily detectable in vivo, namely the ability of T lymphocytes to locally transfer a delayed-type hypersensitivity (DTH) reaction. T cells co-transferred with antigen into footpads of syngeneic mice are mediators of DTH through their ability to recruit phagocytes recently produced in haemopoietic tissues. Therefore, if recipients of DTH-mediating T cells (DTH-T cells) are depleted of phagocytes by lethal irradiation given 36 to 48 h before local transfer of these DTH-T cells mixed with antigen, they no longer respond by a DTH reaction, measured as an increase in footpad thickness, unless they receive i.v. bone marrow cells as a source of recruitable phagocytes. In such conditions, the footpad thickness increase is linearly related to the number of cells injected i.v. Pretreatment of bone marrow cells with the rat IgG2b F4/80 monoclonal antibody abolishes their ability to restore expression of DTH, indicating that monocytes (F4/80⁺ cells) are the phagocytes to be measured. This assay system thus provides a means of measuring levels of recruitable mature monocytes present in haemopoietic tissues. One illustration of the assay is given by the study of the recovery of recruitable phacogytes in bone marrow following a single treatment with cyclophosphamide.     

Phospho‐l‐tyrosine‐agarose chromatography: Adsorption of human IgG and its proteolytic fragments

The behavior of human immunoglobulin G (IgG) and antigen‐binding fragment (Fab fragment) adsorption onto phospho‐l ‐tyrosine immobilized on agarose (P‐Tyr‐agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis–Tris, Tris–HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L^?1 NaP buffer at pH 6.0. The capture of IgG by the P‐Tyr‐agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L^?1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo‐second‐order model. The adsorption isotherm data were well described by the Langmuir–Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P‐Tyr‐agarose from digested human IgG in HEPES 25 mmol L^?1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.     

The effective and specific isolation of antibodies from human serum by using thiophilic paramagnetic polymer beads

A rapid and effective method to specifically isolate immunoglobulin G (IgG) from human serum by thiophilic paramagnetic polymer beads was developed. The thiophilic paramagnetic beads were synthesized with vinyl acetate and divinylbenzene by microsuspension polymerization in the presence of magnetite nanoparticles. Divinylsulfone and 2-mercapto-benzothiazole were subsequently used to modify the surface of these beads, resulting in thiophilic particles that exhibited a high specificity to the antibodies in serum at low salt concentration. The adsorbed IgG was eluted by 0.8 M KCl and 72% of the IgG in the serum was recovered. The purity of the isolated IgG reached 98.4% and the bioactivity was fully maintained (>99%). The high efficiency, mild conditions and simplicity make this technology suitable for the economic purification of antibodies in a large scale.     

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).     

High capacity binding of antibodies by poly(hydroxyethyl methacrylate) nanoparticles

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 150 nm in diameter and with a poly-dispersity index of 1.171 were produced by a surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 1779 m(2)/g. Reactive imidazole containing 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was used as a pseudo-specific ligand. IMEO was attached covalently onto the nanoparticles. PHEMA-IMEO nanoparticles were used for the affinity binding of immunoglobulin-G (IgG) from human plasma. To evaluate the degree of IMEO loading, the PHEMA nanoparticles were subjected to Si analysis by using flame atomizer atomic absorption spectrometer and it was estimated as 64.5 mg/g of polymer. The nanoparticles were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). IgG binding onto the PHEMA nanoparticles was found to be 5.2 mg/g. Much higher binding values (up to 843 mg/g) were obtained for the PHEMA-IMEO nanoparticles. IgG could be repeatedly bound and eluted on PHEMA-IMEO nanoparticles without noticeable loss in the IgG binding capacity.     

Dielectrophoretic detection of electrical property changes of stored human red blood cells

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to “freeze” cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0 w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.     

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.     

Combining Low Pressure Liquid Affinity Chromatography and Flow Injection Analysis for the Quantitation of Immunoglobulins

Abstract

A simple, accurate method combining low pressure liquid affinity chromatography and flow injection analysis is described for the quantitation of immunoglobulins in biologicals fluids. The affinity matrix consists of Protein A covalently immobilized to a 2-fluoro-1 methylpyridinium salt activated Fractogel support. Utilizing a 1 cm³ affinity column, optimized binding and eluting buffer flow rates of 0.7 and 1.5 mL min^?1, respectively, and a sample loop size of 100 μL, IgG's can be eluted from the affinity column in about 9 min. Linear standard curves (r > 0.99) were obtained at concentrations up to at least 4.0 and 8.0 mg mL^?1 respectively for human and bovine IgG. Recovery yields are good ranging from 96–100%. The within day CV for human and bovine IgG was found to be less than 3.0% whereas the day to day CV was less than 3.4% (n=10). IgG concentrations of spiked and unspiked bovine plasma samples obtained by the low pressure affinity/flow injection method when compared to those obtained by the radial immunodiffusion agreed to within 4%.     

强阴离子交换色谱结合分子排阻色谱纯化血清中免疫球蛋白和白蛋白

动物血清中免疫球蛋白和白蛋白的等电点分别约为7.8和4.8,根据它们等电点的较大差别,利用Q SepharoseTM-XL强阴离子交换色谱结合分子排阻色谱同时分离纯化这2种蛋白。以0.02 mol/L pH 8.0的Tris-HCl缓冲液平衡离子交换色谱柱并将已稀释10倍的高免疫的兔血清上样,采用pH分段洗脱。在pH 6.0时以0.3 mL/min低流速洗脱得到高纯度的免疫球蛋白,继续在pH 4.0时洗脱,再辅以Sephadex G-75分子排阻色谱可获得纯度大于95%的白蛋白。对纯化后的蛋白进行活性检测,证明所纯化的免疫球蛋白和白蛋白都保持正常的生物活性。蛋白质含量测定说明免疫球蛋白的纯化回收率达到95%以上,而白蛋白的纯化回收率大于90%。该法简便快速,可同时从动物血清中纯化出保持生物活性的免疫球蛋白和白蛋白,纯化效率高。     

Elimination of Human Chorionic Gonadotropin in Sandwich Enzyme Immunoassay for Human Thyroid-Stimulating Hormone in Serum

Abstract

A method to eliminate human chorionic gonadotropin (hCG) in the sandwich enzyme immunoassay for human thyroid-stimulating hormone (hTSH) in serum is described. hTSH in serum containing hCG was reacted with dinitrophenyl monoclonal mouse anti-hTSH β-subunit IgG₁, and the complex formed between the dinitrophenyl IgG₁ and hTSH was trapped onto affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. hCG in the test serum was largely eliminated by washing the polystyrene balls. Subsequently, the complex on the polystyrene balls was reacted with affinity-purified rabbit anti-hCG Fab′-peroxidase conjugate followed by washing. The complex of the dinitrophenyl IgG₁, hTSH and the conjugate was eluted with dinitrophenyl-L-lysine from the polystyrene balls, to which hCG had been nonspecifically adsorbed, and was trapped onto clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. Peroxidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls in the absence and presence of hTSH was not significantly affected by the presence of up to 75,000 IU of hCG per liter of serum. As a result, serum hTSH could be sensitively measured with little interference by hCG.     

Characterization of the Fc receptors of the murine leukemia L1210

A glycoprotein extract prepared from the plasma membranes of L1210 cells was passed over columns of Sepharose 4B to which either heat-aggregated human IgG or F(ab')2 fragments has been coupled. The intact IgG column bound 35.7 percent of the applied counts, whereas the F(ab')2 columns bound 2.8 percent. The bound glycoproteins were eluted with citrate buffer (pH 3.2) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three peaks with apparent molecular weights of 65,000 45,000, and 28,000 daltons were identified and purified by electroelution from polyacrylamide gels. The isolated proteins were able to bind to the same sub-classes of mouse IgG myeloma proteins as the intact L1210 cells, indicating that these molecules are related to L1210 surface Fc receptors. Amino acid analyses of the 3 proteins were markedly similar suggesting that the observed molecular heterogeneity might be due to carbohydrate differences. Neuraminidase digestion of the isolated proteins resulted in mobility shifts on polyacrylamide gel electrophoresis which were consistent with the interpretation that either the isolated proteins have considerably different sialic acid contents, or that removal of the sialic acid results in disaggregation of an Fc receptor molecule.     

基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

Avid AL, a synthetic ligand affinity gel mimicking immobilized bacterial antibody receptor for purification of immunoglobulin G.

Avid AL is an affinity gel designed for the purification of immunoglobulin G (IgG). The gel was prepared by first reacting Sepharose with 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine and then with 2-mercaptoethanol. The IgG purified by Avid AL is about 95% pure. The binding parameters of Avid AL for the whole IgG, Fab and Fc fragment and the stability of gel were investigated. The IgG bound to Avid AL can be eluted with an acidic buffer or with a novel neutral buffer containing electron donors. The development of such a mild neutral elution buffer is described. Application of Avid AL in a rapid gram-scale IgG purification was demonstrated. The possible mechanism of IgG binding is discussed.     

Isolation of human serum immunoglobulins with a new salt-promoted adsorbent

In this work a highly acetylated-ethylenediamine-Novarose (HA-EDA-Novarose) gel was synthesized and used as a new agarose-based salt-promoted adsorption chromatography (SPAC) matrix to effectively isolate serum immunoglobulins without the need of denaturing conditions. Samples of human serum in 0.5 M Na2SO4, 10 mM 3-(N-morpholino)-propane-sulfonic acid (MOPS), pH 7.6 were applied to a chromatographic column packed with the SPAC gel. Immunoglobulins (Igs) with affinity for the HA-EDA ligands were specifically adsorbed to the matrix, non-bound serum proteins were readily removed by washing the column with the same feed solution buffer. Bound Igs were effectively and very gently eluted by simply removing the salt from the feed solution buffer. The elution buffer consisted thus of only 10 mM MOPS, at pH 7.6 and no salt. The salt-dependent adsorption capacity of this system was estimated to be 7.3 mg/ml with protein recovery of about 93%. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis analysis, radial immunodiffusion and enzyme-linked immunosorbent assays showed that immunoglobulins G, M and A (IgG, IgM and IgA) were the main components present in the elution fraction. The new SPAC adsorbent was used to purify Igs from human serum and IgG and IgA from non-pure commercially available Igs preparations in a very gentle single step.     

Imatinib (Gleevec, Glivec) tumour tissue analysis by measurement of sediment and by liquid chromatography tandem mass spectrometry

The analysis of the signal transduction inhibitor imatinib in patient tumour tissue using LC and MS/MS is described. The anticancer agent is eluted over RP-C18 within 2 mm together with its internal standard STI571-d8. Calibration curves were prepared in red blood cells (RBC). For quantitative isolation of the RBC, measurement of sediment was applied. There were no indications of signal suppression by substances originating in the biological matrix. The limit of determination in tumour tissue was in the range of those recorded for RBC and plasma. The assay is selective and sensitive, with its robustness favouring the experimental application in clinical oncology and its routine use in animal experiments. The LOD was 4.5 ng per gram in tumour tissue.     

Photoinduced elimination of senescent microglia cells in vivo by chiral gold nanoparticles

Parkinson''s disease (PD) is an age-related neurodegenerative disease, and the removal of senescent cells has been proved to be beneficial for improving age-associated pathologies in neurodegeneration disease. In this study, chiral gold nanoparticles (NPs) with different helical directions were synthesized to selectively induce the apoptosis of senescent cells under light illumination. By modifying anti-B2MG and anti-DCR2 antibodies, senescent microglia cells could be cleared by chiral NPs without damaging the activities of normal cells under illumination. Notably, l-P⁺ NPs exhibited about a 2-fold higher elimination efficiency than d-P⁻ NPs for senescent microglia cells. Mechanistic studies revealed that the clearance of senescent cells was mediated by the activation of the Fas signaling pathway. The in vivo injection of chiral NPs successfully confirmed that the elimination of senescent microglia cells in the brain could further alleviate the symptoms of PD mice in which the alpha-synuclein (α-syn) in cerebrospinal fluid (CFS) decreased from 83.83 ± 4.76 ng mL⁻¹ to 8.66 ± 1.79 ng mL⁻¹ after two months of treatment. Our findings suggest a potential strategy to selectively eliminate senescent cells using chiral nanomaterials and offer a promising strategy for alleviating PD.

The apoptosis pathways of senescent microglia cells induced by chiral NPs under the irradiation of 808 nm laser in the brain of PD mice.     

Effect of the conserved oligosaccharides of recombinant monoclonal antibodies on the separation by protein A and protein G chromatography

Glycosylation of the conserved asparagine residue in CH2 domains of IgG molecules is an important post-translational modification. The presence of oligosaccharides is critical for structure, stability and biological function of IgG antibodies. Effect of the glycosylation states of recombinant monoclonal antibodies on protein A and protein G chromatography was evaluated. Antibodies lacking oligosaccharides eluted later from protein A and earlier from protein G columns than antibodies with oligosaccharides using a gradient of decreasing pH. Interestingly, different types of oligosaccharides also affected the elution of the antibodies. Antibodies with high mannose type oligosaccharides were enriched in later eluting fractions from protein A and earlier eluting fractions from protein G. While antibodies with more mature oligosaccharides, such as core fucosylated biantennary complex oligosaccharides with zero (Gal 0), one (Gal 1) or two (Gal 2) terminal galactoses, were enriched in earlier eluting fractions from protein A and in the later eluting fractions from protein G. However, analysis by enzyme-linked immunosorbent assay (ELISA) revealed that antibody binding affinity to protein A and protein G was not affected by the absence or presence of oligosaccharides. It was thus concluded that the elution difference of antibodies with or without oligosaccharides and antibodies with different types of oligosaccharides were due to differential structural changes around the CH2–CH3 domain interface under the low pH conditions used for protein A and protein G chromatography.