Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. To attenuate the risk of alloimmunization, we have pioneered the membrane grafting of methoxypoly(ethylene glycol) (mPEG) to produce immunocamouflaged red blood cells (RBC). Grafting of the mPEG was accomplished using cyanuric chloride activated mPEG (CmPEG; M(r) = 5000), benzotriazole carbonate methoxyPEG (BTCmPEG; M(r) = 2000, 5000 or 20000); or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPAmPEG; M(r) = 5000, or 20000). Because of the heterogeneity of grafting, a crucial tool in developing the stealth RBC is an ability to purify the modified RBC from unmodified (immunogenic) donor cells. As demonstrated, a (5, 4) dextran:PEG aqueous two-phase polymer partitioning system cleanly separated the immunologically silent mPEG-grafted human RBC from control or lightly modified cells. Cell mixing experiments employing varying ratios of mPEG-modified and control RBC confirmed the purification efficacy of the phase partitioning system. Proportional changes in PEG-rich phase partitioning were achieved by increasing either the quantity of surface mPEG or the mPEG molecular weight. The biological viability of purified mPEG-RBC (BTCmPEG; [M(r) = 20000) was demonstrated by their normal in vivo survival at immunoprotective grafting concentrations (相似文献   

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.     

PEGylation of bovine serum albumin using click chemistry for the application as drug carriers

Monomethyl poly(ethylene glycol) (mPEG)-modified bovine serum albumin (BSA) conjugates (BSA-mPEG) were obtained by the mild Cu(I)-mediated cycloaddition reaction of azided BSA (BSA-N(3) ) and alkyne-terminated mPEG. The structure and characteristics of BSA-mPEG conjugates were thoroughly investigated. There were about two PEG chains conjugated onto each BSA molecule as determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) analysis. The intrinsic nonspecific binding ability of BSA was used for adsorption and sustained release of both rifampicn and 5-fluorouracil (5-FU). The helical structures of BSA were preserved to a large extent after modification and drug adsorption on BSA was confirmed via circular dichroism spectroscopy. Drugs adsorbed onto the conjugated formulation to a lesser extent than on BSA due to mPEG modification. The in vitro release of both rifampicin and 5-FU, however, indicated that BSA-mPEG can function as a drug carrier. Overall, the click reaction provided a convenient tool for the pegylation of BSA. The biological activity of the BSA-mPEG conjugates, including the drug transportation capacity and biocompatibility, were largely retained.     

Particle-by-particle quantification of protein adsorption onto poly(ethylene glycol) grafted surfaces

The use and advantage of flow cytometry as a particle-by-particle, low sampling volume, high-throughput screening technique for quantitatively examining the non-specific adsorption of proteins onto surfaces is presented. The adsorption of three proteins: bovine serum albumin (BSA), immunoglobulin gamma (IgG) and protein G, incubated at room temperature for 2 h onto organosilica particles modified with poly(ethylene glycol) (PEG) of increasing MW (2000, 3400, 6000, 10,000 and 20,000 g mol(-1)) and grafted amounts (0.14-1.4 mg m(-2)) was investigated as a model system. Each protein exhibited Langmuir-like, high affinity monolayer limited adsorption on unmodified particles with the proteins reaching surface saturation at 1.8, 4.0 and 2.5 mg m(-2) for BSA, IgG and protein G, respectively. Protein adsorption on PEG-modified surfaces was found to decrease with increasing amounts of grafted polymer. PEG grafting amounts >0.6 mg m(-2) effectively prevented the adsorption of the larger two proteins (BSA and IgG) while a PEG grafting amount >1.3 mg m(-2) was required to prevent the adsorption of the smaller protein G.     

Novel thymine-functionalized polystyrenes for applications in biotechnology. 2. Adsorption of model proteins

This paper investigates the adsorption of bovine serum albumin (BSA) and bovine hemoglobin (BHb) model proteins onto novel thymine-functionalized polystyrene (PS-VBT) microspheres, in comparison with polystyrene (PS) microspheres. Maximum adsorption was obtained for both proteins near their corresponding isoelectric points (pI at pH = 4.7 for BSA and 7.1 for BHb). FTIR and adsorption isotherm analysis demonstrated that, although both proteins were physisorbed onto PS through nonspecific hydrophobic interactions, adsorption onto the functionalized copolymers occurred by both physisorption and chemisorption via hydrogen bonding. FTIR analysis also indicated conformational changes in the secondary structure of BSA and BHb adsorbed onto PS, whereas little or no conformation change was seen in the case of adsorption onto PS-VBT. Atomic force microscopy (AFM), consistent with the isotherm results, also demonstrated monolayer adsorption for both proteins. AFM images of BSA adsorbed onto copolymers with 20 mol % surface VBT loading showed exclusively end-on orientation. Adsorption onto copolymers with lower functionality showed mixed end-on and side-on orientation modes of BSA, and only the side-on orientation was observed on PS. The AFM results agreed well with theoretically calculated and experimentally obtained adsorption capacities. AFM together with calculated and observed adsorption capacity data for BHb indicated that this protein might be highly compressed on the copolymer surface. Adsorption from a binary mixture of BSA and BHb onto PS-VBT showed good separation at pH=7.0; approximately 90% of the adsorbed protein was BHb. The novel copolymers have potential applications in biotechnology.     

Improvement of proteolytic resistance of immunoadsorbents by chemical modification with polyethylene glycol

Immunoadsorbents were modified with monomethoxy-polyethylene glycol (PEG; average molecular weights of 5000 (PEG-5000) and 1900 (PEG-1900)) activated with cyanuric acid (activated PEG) by four different methods. In the two methods, anti-BSA antibodies were modified with activated PEG with and without protection of antigen binding sites with BSA and then were coupled to CNBr-activated Sepharose 4B. In the other two methods, Immunoadsorbents, which were prepared by coupling anti-BSA antibodies to CNBr-activated Sepharose 4B, were modified with activated PEG with and without the protection. The effects of PEG modification by these four methods on the binding ratio (the ratio of the numbers of moles of antigen adsorbed to the numbers of moles of binding sites of antibody coupled), the antigen binding property and the resistance to proteolytic digestion of immunoadsorbents were studied. The decrease in the binding ratio by the modification with activated PEG was small enough to use modified immunoadsorbents for industrial purification processes. The resistance to proteolytic digestion of immunoadsorbents was improved by modification with activated PEG. The modification without protection of antigen binding sites gave higher resistance to proteolytic digestion than that with protection, while the former caused larger decrease in the binding ratio of modification. The immunoadsorbents modified with activated PEG-5000 showed higher resistance to proteolytic digestion than those modified with activated PEG-1900.     

氨基PEG化试剂的合成及其修饰能力的测定

分别用氰脲酰氯法及Ｎ－羟基丁二酰亚胺活性酯法合成了蛋白质的氨基ＰＥＧ化试剂ｍＰＥＧｃｃ和ｍＰＥＧ－ＧＳ,并研究了它们对蛋白质的修饰作用。在合成过程中,通过分析反应体系中微量水分的存在对ｍＰＥＧｃｃ合成效率的影响,及溶剂中小分子可活化杂质成分对ｍＰＥＧ－ＧＳ合成产物质量的影响,发现去水剂的存在,可使ｍＰＥＧｃｅ产率提高７倍;二氧六环优于ＤＭＦ,且二氧六环的预处理也很重要。同时,为了测定活化ＰＥＧ修饰蛋白质的效能,首次以ＢＳＡ为模型蛋白,建立起一种测定活化ＰＥＧ修饰能力的方法,应用此方法能直观而又准确地比较各种方法活化的ＰＥＧ对蛋白质的修饰能力,具有普遍的意义。     

Adsorption behavior of bovine serum albumin on lowly activated anionic exchangers suggests a new strategy for solid-phase proteomics

Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.     

The influence of radioiodination on the adsorption of IgG and serum albumin to polystyrene

The adsorption of radioiodinated rabbit IgG and bovine serum albumin (BSA) to polystyrene tubes was investigated. Adsorption isotherms where the proportion of the protein bound was relatively constant over a range of intermediate protein concentrations, and where the proportion bound was protein dependent, were obtained. To investigate the effects of radioiodination, proteins labeled to give a wide range of substitution ratios (0.03 to 3.7 125I/protein molecule) were employed. While labeling did not appear to affect BSA adsorption, the kinetics of IgG binding were altered in a number of ways. The proportion bound in the concentration independent region was decreased even at substitution ratios less than or equal to 0.2. In addition, while all preparations of iodinated BSA, and IgG preparations with less than or equal to 1.6 125I/IgG, gave bimodal adsorption isotherms, with more heavily labeled IgG (greater than or equal to 2.5 125I/IgG) the apparent high affinity binding to the plastic surface was abolished. These results indicate that radioiodination substantially alters the kinetics of the binding of IgG to polystyrene. In addition, the results obtained are discussed with respect to previous relevant and often apparently contradictory findings.     

Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles

The conformational changes in well-characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm-whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the "soft" protein molecules (myoglobin, hemoglobin, and BSA), while they were insingnificant in the "rigid" proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in alpha-helix content upon adsorption. Moreover, the native alpha-helix content, which is given as the percentage of the alpha-helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well-known hypotheses for the confirmational chages in protein molecules upon adsorption. (c) 1992 John Wiley & Sons, Inc.     

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K-ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin-SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K-ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.     

Enhanced lymph node retention of subcutaneously injected IgG1-PEG2000-liposomes through pentameric IgM antibody-mediated vesicular aggregation

An efficient strategy for enhancing the lymph node deposition of rapidly drained liposomes from the interstitial injection site is described. Subcutaneously injected small-sized immuno-poly(ethyleneglycol)-liposomes (immuno-PEG-liposomes), containing 10 mol% mPEG350-phospholipid and 1 mol% PEG2000-phospholipid in their bilayer and where IgG1 is coupled to the distal end of PEG2000, not only drain rapidly from the interstitial spaces into the initial lymphatic system, but also accumulate efficiently among the lymph nodes draining the region when compared with non-PEG-bearing immunoliposomes where IgG is directly coupled to the phospholipid. Liposome deposition among the draining lymph nodes, however, was further enhanced dramatically following an adjacent subcutaneous injection of a pentameric IgM against the surface attached IgG molecules (IgM:IgG, 10:1) without compromising vesicle drainage from the interstitium. This is suggested to arise either as a result of formation of large immuno-aggregates within the lymphatic vessels with subsequent transport to and trapping among the regional lymph nodes and/or following IgM binding to Fc receptors of the lymph node sinus macrophages forming a platform for subsequent trapping of drained IgG-coupled liposomes. This lymph node targeting approach may be amenable for the design and surface engineering of any rapidly drained nanoparticulate system bearing peptides and proteins that can be aggregated with a desired monoclonal pentameric IgM.     

Bioactive surfaces via immobilization of self-assembling polymers onto hydrophobic materials.

Conjugation of proteins to copolymers from poly(acrylic acid) grafted onto PEO-PPO-PEO backbone (Pluronic-PAA) following adsorption of the conjugates onto hydrophobic surfaces is reported. Insulin-Pluronic-PAA conjugates show negligible internalization of insulin into human uterine smooth muscle cells as well as enhancement of mitogenic activity. Glucose-induced release of glycated albumin complexed with a Pluronic-PAA-concanavalin conjugate and adsorbed onto polystyrene nanospheres may provide a model for a glucose-responsive protein delivery system or a heterogeneous diagnostic device.     

New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes

Unmodified and polyethylene glycol (PEG) modified neutral and negatively charged liposomes were prepared by freeze-thaw and extrusion followed by chromatographic purification. The effects of PEG molecular weight (PEG 550, 2000, 5000), PEG loading (0-15 mol%), and liposome surface charge on fibrinogen adsorption were quantified using radiolabeling techniques. All adsorption isotherms increased monotonically over the concentration range 0-3 mg/ml and adsorption levels were low. Negatively charged liposomes adsorbed significantly more fibrinogen than neutral liposomes. PEG modification had no effect on fibrinogen adsorption to neutral liposomes. An inverse relationship was found between PEG loading of negatively charged liposomes and fibrinogen adsorption. PEGs of all three molecular weights at a loading of 5 mol% reduced fibrinogen adsorption to negatively charged liposomes. Protein adsorption from diluted plasma (10% normal strength) to four different liposome types (neutral, PEG-neutral, negatively charged, and PEG-negatively charged) was investigated using gel electrophoresis and immunoblotting. The profiles of adsorbed proteins were similar on all four liposome types, but distinctly different from the profile of plasma itself, indicating a partitioning effect of the lipid surfaces. alpha2-macroglobulin and fibronectin were significantly enriched on the liposomes whereas albumin, transferrin, and fibrinogen were depleted compared to plasma. Apolipoprotein AI was a major component of the adsorbed protein layers. The blot of complement protein C3 adsorbed on the liposomes suggested that the complement system was activated.     

Comparative studies on the interaction of proteins with a polydimethylsiloxane elastomer. II. The comparative antigenicity of primary and secondarily adsorbed IgG1 and IgG2a and their non-adsorbed counterparts

The antigenicity of bovine IgG1 and IgG2a adsorbed on a polydimethysiloxane (PEP) elastomer, on a widely used polystyrene (Imm 2, Dynatech) or immobilized as biotinylated proteins to streptavidin covalently bound to polystyrene (SA-PS) was compared using various monoclonal (mAbs) and polyclonal antibodies (pAb) to bovine IgG. The IgGs were either adsorbed as native proteins or pre-denatured with 6M Guanidine-HCl (Gu-HCl) or 6 M Gu-HCl/0.1% 2-mercaptoethanol. In special situations, bovine and human IgG was immobilized by secondary adsorption to an albumin monolayer adsorbed on either PEP or Imm 2. Results indicate that pre-denaturation of IgGs with 6 M Gu-HCl/2-mercaptoethanol destroys all antigenicity whereas those IgGs pretreated with 6 M-GuHCl are indistinguishable in their antigenicity from the IgGs adsorbed to either PEP or Imm 2 without such treatment. When immobilized on SA-PS, Gu-HCl-treated IgGs were significantly less detectable, especially when tested using mAbs. In general, IgGs adsorbed on PEP or Imm 2 were less antigenic than when immobilized on SA-PS. However, two monoclonals specific for the IgG2a(A2) allotypic variant, favored the adsorbed protein and one polyclonal best recognized the IgG2a(A1) variant adsorbed on Imm 2 rather than when adsorbed on PEP or immobilized on SA-PS. Both IgG1 and IgG2a, bound by apparent protein-protein interactions to an albumin monolayer, were significantly more detectable than when directly adsorbed on either Imm 2 or PEP. Using ¹²⁵l-antibody or its Fab fragment to reduce steric hindrance in detection, we observed the same differences in detectability as when measured by enzyme-linked immunosorbent assay. Failure to identify a steric hindrance effect and the preference of some antibodies for adsorbed allotypic variants, support the concept of adsorption-induced conformational change (AICC). We conclude that proteins adsorbed as a monolayer on the PEP elastomer used to form the envelope of silicone breast implants are conformationally altered, but not necessarily to the same extent or the same manner as when adsorbed on polystyrene. The significantly great antigenicity of secondarily adsorbed IgG suggests that it may be present in near native conformation. © 1997 John Wiley & Sons, Ltd.     

Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).     

Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).     

Solid-phase PEGylation of recombinant interferon alpha-2a for site-specific modification: process performance, characterization, and in vitro bioactivity

'Solid-phase' PEGylation, in which a conjugation reaction attaches proteins to a solid matrix, has distinct advantages over the conventional, solution-phase process. We report a case study in which recombinant interferon (rhIFN) alpha-2a was adsorbed to a cation-exchange resin and PEGylated at the N-terminus by 5, 10, and 20 kDa mPEG aldehydes through reductive alkylation. After PEGylation, a salt gradient elution efficiently purified the mono-PEGylate of unwanted species such as unmodified IFN and unreacted PEG. Mono-PEGylation and purification were integrated into a single, chromatographic step. Depending on the molecular weight of the mPEG aldehyde, the mono-PEGylation yield ranged from 50 to 65%. Major problems associated with the solution-phase process such as random or uncontrollable multi-PEGylation and post-PEGylation purification difficulties were overcome. N-terminus sequencing and MALDI-TOF mass spectrophometry confirmed that the PEG molecule was conjugated only to the N-terminus. A cell proliferation study indicated reduced antiviral activity of the mono-PEGylate compared to that of the unmodified IFN. As higher molecular weight PEG was conjugated, in vitro bioactivity and antibody binding activity, as measured by a surface plasmon resonance biosensor, decreased. Nevertheless, trypsin resistance and thermal stability were considerably improved .     

Physicochemical characterization of poly(ethylene glycol)-modified anti-GAD antibodies.

Monoclonal antibodies against glutamic acid decarboxylase (anti-GAD) were modified with poly(ethylene glycol) (PEG), and the resulting conjugates were characterized. Monoclonal anti-GAD antibodies were purified from ATCC HB184 hybridoma cells by either cell culture supernatant or ascites fluid from BALB/c mice. Polyclonal rabbit IgG antibodies were also used as a model protein. Polyclonal rabbit IgG or purified anti-GAD was modified by PEG (MW = 5000 or 20000 Da) through either the lysine residues or through the carbohydrate moiety. Lysine modification was performed in PBS (pH 7.4) or 0.1 M borate (pH 9.2) by adding a molar excess (5-80) of a succinimidyl activated propionic acid terminated mPEG (SPA-PEG) while stirring at room temperature. Carbohydrate modifications were performed in PBS (pH 6.2) by first oxidizing the antibody with sodium periodate followed by incubation with hydrazide-terminated PEG followed by reduction with sodium cyanoborohydride. The degree of modification was assessed by 1H NMR or TNBS (trinitrobenzenesulfonic acid). Circular dichroism (CD) spectra were obtained for lysine-modified rabbit IgG at various degrees of modification ranging from 5 to 60 PEG per antibody. Binding was assessed using an ELISA method with GAD or rabbit anti-mouse-IgG (H+L) coated plates. The TNBS and 1H NMR analysis of the modified antibody showed reasonably similar results from 5 to 60 PEG per antibody. The 1H NMR method showed greater sensitivity at low modifications (below 20:1) and was fairly linear up to about 60 PEG per antibody. The CD spectra of the polyclonal rabbit IgG showed only small differences at variously modified antibody. The binding affinity of anti-GAD is lower for all PEG modifications with respect to unmodified anti-GAD. Modifications at pH 7.4 show lower binding to GAD than modifications at pH 9.2. Binding to GAD or anti-mouse-IgG is decreased as the degree of modification is increased. Lysine modifications showed lower binding to GAD or anti-mouse-IgG than carbohydrate modifications. Binding to GAD or anti-mouse-IgG is lower for PEG20000-modified anti-GAD with respect to PEG5000-modified anti-GAD.     

Sequential adsorption of F(ab')(2) and BSA on negatively and positively charged polystyrene latexes

The aim of the present work is to study the sequential adsorption of F(ab')(2) and bovine serum albumin (BSA) molecules adsorbed onto positively and negatively charged polystyrene latexes. Cationic and anionic latexes were prepared by emulsifier-free emulsion polymerization. Adsorptions of F(ab')(2) on both latexes at a low ionic strength and different pHs were performed. The cationic latex showed a higher adsorption of F (ab')(2) molecules over a range of pH, which could be due to the formation of multilayers. Sequential adsorption of anti-CRP F(ab')(2) and monomeric BSA were performed at two different pre-adsorbed F(ab')(2) amounts on both types of latex. Displacement of F(ab')(2) occurred only when the preadsorbed amounts were larger than a certain critical value, which depends on the adsorption pH. A greater displacement of larger preadsorbed amounts might be the result of a weaker contact between the protein molecules and the polystyrene surface. The displacement of F(ab')(2) previously adsorbed onto both latexes occurred due to pH changes, an increase of ionic strength and the presence of BSA molecules. The effect caused by these three factors was studied independently. The main factors in the desorption of F(ab')(2) on the anionic latex are the changes in pH and ionic strength, whereas on the cationic latex the desorption is mainly caused by the increase of the ionic strength and the presence of BSA. The colloidal stability of the immunotatex was improved by BSA adsorption, especially on cationic latex. (c) 1995 John Wiley & Sons, Inc.