Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel

Immunoglobulin G (IgG) purification from human plasma with protein A attached supermacroporous poly(hydroxyethyl methacrylate) [PHEMA] cryogel has been studied. PHEMA cryogel was prepared by bulk polymerization which proceeds in aqueous solution of monomer frozen inside a plastic syringe (cryo-polymerization). After thawing, the PHEMA cryogel contains a continuous matrix having interconnected pores of 10–200 μm size. Protein was covalently attached onto the PHEMA cryogel via cyanogen bromide (CNBr) activation. The maximum IgG adsorption on the PHEMA/protein A cryogel was found to be 83.2 mg/g at pH 7.4 from aqueous solutions. The non-specific IgG adsorption onto the PHEMA cryogel was about 0.38 mg/g. The macropore size of the cryogel makes it possible to process blood cells without blocking the column. Higher adsorption capacity was observed from human plasma (up to 88.1 mg/g). Adsorbed IgG was eluted using 0.1 M glycine–HCl buffer (pH 3.5) with a purity of 85%. PHEMA–protein A cryogel was used for repetitive adsorption/desorption of IgG without noticeable loss in IgG adsorption capacity after 10 cycles. PHEMA–protein A cryogel showed several advantages such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–desorption cycles.     

Supermacroporous hydrophobic affinity cryogels for protein chromatography

N-Methacryloyl-l-tryptophan (MATrp) containing poly(2-hydroxyethyl methacrylate) based supermacroporous cryogel [PHEMATrp] was prepared for lysozyme purification form chicken egg white. MATrp was synthesized by reacting methacryloyl chloride with l-tryptophan methyl ester and provided hydrophobic functionality to the cryogel. PHEMATrp cryogel with 60–100 μm pore size was obtained by free radical polymerization of HEMA and MATrp having a specific surface area of 50 m²/g. PHEMATrp cryogel was characterized by swelling studies, FTIR and SEM. The equilibrium swelling ratios of the cryogels were 7.18 g H₂O/g for PHEMA and 6.99 g H₂O/g for PHEMATrp. Lysozyme adsorption experiments were investigated under different conditions in continuous system (i.e., medium pH, flow-rate, protein concentration, temperature, salt type). Lysozyme adsorption capacity of PHEMA and PHEMATrp cryogels from aqueous solutions was estimated as 2.9 and 46.8 mg/g (0.49 and 7.85 mg/mL), respectively. Lysozyme molecules were desorbed with 0.5 M ethylene glycol solution with 91% recovery. It was observed that PHEMATrp cryogel can be used without significant decrease in lysozyme adsorption capacity after five adsorption–desorption cycles. PHEMATrp cryogel was used for the purification of lysozyme from chicken egg white. Purity of lysozyme was estimated by SDS-PAGE. Possible denaturation of purified lysozyme was checked with fluorimetric measurements. Specific activity of the purified lysozyme was found as 43,140 U/mg using Micrococcus lysodeikticus as substrate.     

Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification

The aim of this study is to prepare supermacroporous pseudospecific cryogel which can be used for the purification of plasmid DNA (pDNA) from bacterial lysate. N-methacryloyl-(l)-histidine methyl ester (MAH) was chosen as the pseudospecific ligand and/or comonomer. Poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) [PHEMAH] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Compared with the PHEMA cryogel (50 μg/g polymer), the pDNA adsorption capacity of the PHEMAH cryogel (13,350 μg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The amount of pDNA bound onto the PHEMAH cryogel disks first increased and then reached a saturation value (i.e., 13,350μg/g) at around 300 μg/ml pDNA concentration. pDNA adsorption amount decreased from 1137 μg/g to 160 μg/g with the increasing NaCl concentration. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 90%. The PHEMAH cryogel could be used 3 times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH cryogel disks promise high selectivity for pDNA.     

Protein A immobilized polyhydroxyethylmethacrylate beads for affinity sorption of human immunoglobulin G

Protein A immobilized polyhydroxylmethyacrylate (PHEMA) microbeads were investigated for the specific removal of HIgG from aqueous solutions and from human plasma. PHEMA microbeads were prepared by a suspension polymerization technique and activated by CNBr in an alkaline medium (pH 11.5). Protein A was then immobilized by covalent binding onto these microbeads. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The maximum protein A immobilization was observed at pH 9.5. Up to 3.5 mg protein A/g PHEMA was immobilized on the CNBr activated PHEMA microbeads. The maximum HIgG adsorption on the protein A immobilized PHEMA microbeads was observed at pH 8.0. The non-specific HIgG adsorption onto the plain PHEMA microbeads was low (about 0.167 mg of HIgG/g PHEMA). Higher adsorption values (up to 6.0 mg of HIgG/g PHEMA) were obtained in which the protein A immobilized PHEMA microbeads were used. Much higher amounts of HIgG (up to 24.0 mg of HIgG/g PHEMA) were adsorbed from human plasma.     

Recognition of novel amphiphiles with many pendent mannose residues by Con A.

Novel amphiphiles which carry many mannose residues as side chains were prepared by telomerization of N-methacryloylaminopropyl D-mannopyranoside (alpha:beta = 20:1), N-methacryloylaminohexyl D-mannopyranoside (alpha:beta = 20:1), or 3-(2-methacryloylaminoethylthio)propyl D-mannopyranoside (alpha:beta = 4:1) using a lipophilic radical initiator. The mannose-carrying amphiphiles incorporated in liposomes were recognized by a lectin from Canavalia ensiformis (Con A), which was proven by the increase in turbidity of the liposome suspension after mixing with Con A. The interaction between sugar residues on the liposome surface and the lectin was largely affected by the degree of polymerization (DP) and the surface density of the amphiphile in the liposomes. The distance between the sugar residues and the polymer main chain did not affect the specific recognition by the lectin significantly in the liposome system, whereas it appreciably affected the recognition in the water-soluble polymer system. The association constants (Ka) of the amphiphiles (DP approximately 18) with Con A (0.3-2.2 x 10(6) M-1 at 25 degrees C) were much larger than that of alpha-methyl D-mannopyranoside (8.2 x 10(3) M-1) due to the "cluster effect ". The positive entropy change (20-52 J/mol K) for the binding of Con A to mannose residues on the liposome surface showed that the recognition in the liposome system was largely promoted by the release of water molecules from both the sugar residues on the liposome surface and the binding site of Con A.     

Heparin-immobilized polyhydroxyethylmethacrylate microbeads for cholesterol removal: a preliminary report

Heparin-attached polyhydroxyethylmethacrylate (PHEMA) microbeads were investigated for specific removal of cholesterol from human and rabbit plasma. PHEMA microbeads were prepared by a suspension polymerization technique and activated by cyanogen bromide (CNBr) in an alkaline medium (pH 11.5). Heparin was then immobilized by covalent binding onto these microbeads. Cholesterol adsorption onto PHEMA microbeads containing two different amounts of immobilized heparin, i.e., 57.3 and 122.7 mg/g, from both hypercholesterolaemic human and rabbit plasma was investigated. The non-specific cholesterol adsorptions on the plain PHEMA microbeads were 0.47 mg/g and 0.30 mg/g from human and rabbit plasmas, respectively. About 35% and 32% of the cholesterol was removed from human and rabbit plasmas, respectively, when the heparin-immobilized PHEMA microbeads were used.     

In vitro and in vivo binding of snowdrop (Galanthus nivalis agglutinin; GNA) and jackbean (Canavalia ensiformis; Con A) lectins within tomato moth (Lacanobia oleracea) larvae; mechanisms of insecticidal action

When fed in semi-artificial diet the lectins from snowdrop (Galanthus nivalis: GNA: mannose-specific) and jackbean (Canavalia ensiformis: Con A: specific for glucose and mannose) were shown to accumulate in vivo in the guts, malpighian tubules and haemolymph of Lacanobia oleracea (tomato moth) larvae. Con A, but not GNA, also accumulated in the fat bodies of lectin-fed larvae. The presence of glycoproteins which bind to both lectins in vitro was confirmed using labelled lectins to probe blots of polypeptides extracted from larval tissues. Immunolocalisation studies revealed a similar pattern of GNA and Con A binding along the digestive tract with binding concentrated in midgut sections. Binding of lectins to microvilli appeared to lead to transport of the proteins into cells of the gut and malpighian tubules. These results suggested that both lectins are able to exert systemic effects via transport from the gut contents to the haemolymph across the gut epithelium. The delivery of GNA and Con A to the haemolymph was shown to be dependent on their functional integrity by feeding larvae diets containing denatured lectins. Con A, but not GNA, was shown to persist in gut and fat body tissue of lectin-fed larvae chased with control diet for three days. Con A also shows more extensive binding to larval tissues in vitro than GNA, and these two factors are suggested to contribute to the higher levels of toxicity shown by Con A, relative to GNA, in previous long term bioassays.     

Lectins in fish skin: do they play a role in host-monogenean interactions?

Mucus samples from rainbow trout skin with or without infections by Gyrodactylus derjavini were tested for the presence of lectins reacting with mannose, galactose and lactose. The samples inhibited the binding of biotinylated lectins (from Canavalia ensiformis, Artocarpus integrifolia and Erythrina corallodendron, respectively) to microtitre plates with covalently bound carbohydrates (mannopyranoside, galactopyranoside and lactose, respectively). However, the inhibition of C. ensiformis and A. integrifolia lectins was slightly greater when mucus from infected (but recovering) fish was used, suggesting an increase of mannose and galactose binding lectins in fish skin exposed to parasites. As mannose, galactose and lactose are present on the glycocalyx of Gyrodactylus derjavini, it is suggested that lectins could play a dual role in interactions between fish hosts and their monogenean parasites. Thus, recognition between parasite and host and also host responses towards parasite infections could both, at least partly, involve carbohydrate-lectin binding.     

The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin A-peroxidase.

The plant lectin concanavalin A (Con A) possesses a remarkably specific capacity to bind primarily α-d-mannose or α-d-glucose sugar residues on macromolecules (cf. 1). The multivalent Con A will bind to carbohydrates on cell surfaces, and free binding sites on the attached Con A will bind to horseradish peroxidase which is a glycoprotein (2). Since peroxidase may be visualized by reaction with diaminobenzidine (3), it has been possible using this method to specifically “stain” carbohydrate residues on cell surface macromolecules (2, 4). The same principles for staining cell surfaces should apply to “staining” glycoproteins separated by polyacrylamide electrophoresis. In this paper, we examine the staining of glycoproteins in sciatic nerve by a Con A-peroxidase labeling technique. The method is more sensitive for mannose or glucose containing glycoproteins than the periodic acid-Schiff's (PAS) method commonly used.     

Lectin histochemistry of feline sphingomyelinosis.

The brain from a Siamese cat with sphingomyelinosis was examined with lectin histochemistry. Swollen neurons were stained with Canavalia ensiformis agglutinin (Con A). Some of them were also stained with Ricinus communis agglutinin-I (RCA-I) and Ulex europaeus agglutinin-I (UEA-I). A small number of axonal spheroids and glia cells were positive for Con A, RCA-I, UEA-I and wheat germ agglutinin. Control tissues were weakly stained with Con A, but not with any of the other lectins. These results indicate that affected neurons contain mannose and glucose residues in addition to sphingomyelin. This study points to the possibility that the characteristics of lectin histochemical study might be helpful for the diagnosis of sphingomyelinosis.     

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.     

Selective separation of human serum albumin with copper(II) chelated poly(hydroxyethyl methacrylate) based nanoparticles

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 300 nm in diameter and with a polydispersity index of 1.156 were produced by surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 996 m²/g. Metal-chelating ligand 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was covalently attached to the PHEMA nanoparticles. IMEO content was 0.97 mmol IEMO/g. The morphology and properties of these nanoparticles were characterized with scanning electron microscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The Cu²⁺-chelated PHEMA–IMEO nanoparticles were used in the adsorption-elution studies of human serum albumin (HSA) in a batch system. Maximum HSA adsorption amount of the Cu²⁺ chelated nanoparticles was 680 mg HSA/g. The PHEMA–IMEO–Cu²⁺ nanoparticles exhibited a quite high adsorption capacity and fast adsorption rate due to their high specific surface area and the absence of internal diffusion resistance.     

Catalase purification from rat liver with iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) cryogel discs

In this study, iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) (PHEMAGA/Fe³⁺) cryogel discs were prepared. The PHEMAGA/Fe³⁺ cryogel discs were characterized by elemental analysis, scanning electron microscopy, Fourier transform infrared spectroscopy, swelling tests, and surface area measurements. The PHEMAGA/Fe³⁺ cryogel discs had large pores ranging from 10 to 100?µm with a swelling degree of 9.36?g H₂O/g cryogel. Effects of pH, temperature, initial catalase concentration, and flow rate on adsorption capacity of the PHEMAGA/Fe³⁺ cryogel discs were investigated. Maximum catalase adsorption capacity (62.6?mg/g) was obtained at pH 7.0, 25°C, and 3?mg/ml initial catalase concentration. The PHEMAGA/Fe³⁺ cryogel discs were also tested for the purification of catalase from rat liver. After tissue homogenization, purification of catalase was performed using the PHEMAGA/Fe³⁺ cryogel discs and catalase was obtained with a yield of 54.34 and 16.67 purification fold.     

外源激素对番茄质膜结合植物凝集素的影响

番茄愈伤组织培养于附加不同激素的 MS 培养基上。经过3次继代培养后,分别从这些愈伤组织分离原生质体,并使之与植物凝集素 ConA 结合。再从原生质体制备质膜。比较在相同膜蛋白含量条件下,质膜上结合~(125)I-ConA。结果表明:在含有2,4-D 的培养基上番茄培养细胞的质膜结合 ConA 的能力保持在很高的水平上。其它生长物质和激动素对细胞表面特性的影响各不相同。     

Sugar–lectin interactions investigated through affinity capillary electrophoresis

The affinity interactions of Concanavalin A (Con A) with various saccharide oligomers (dextrins, dextrans, and selected N-linked glycans from various glycoproteins) have been investigated through a capillary electrophoresis approach. Con A has shown a notable binding discrimination between the α-1,6-linked dextran and α-1,4-linked dextrin oligomers. Both the binding capacity and binding discrimination appear to decrease with an increase in sugar chainlength. While the core structure of N-linked glycans is deemed to be responsible for the overall binding of various glycans to Con A, the presence of mannose units at the non-reducing ends was found to be very beneficial to the affinity interaction with Con A. Finally, a connection between the glycan–lectin interaction and glycoprotein–lectin interaction has also been suggested.     

Con A conjugated to Europium(III) cryptate as a new histological tool for prostate cancer investigation using confocal microscopy

Lectins are carbohydrate recognition proteins that can be used as probes to reveal the glycosylation state of cells. They frequently have been used for diagnostic and prognostic cancer studies. For fluorescence based analysis, lectins commonly are conjugated to fluorescein isothiocyanate (Con A-FITC); however, this molecule loses its fluorescence quickly. We conjugated Europium cryptate to Con A (Con A-cryp-Eu) for use as a histochemical luminescent probe to recognize glucose/mannose residues in benign prostatic hyperplasia and prostatic carcinoma tissues, and used confocal microscopy instead of commercial Con A-FITC. Tissues were treated with Evans blue to suppress intrinsic tissue fluorescence before incubation with Con A-cryp-Eu or Con A-FITC. Con A-cryp-Eu exhibited hemagglutinating activity. Con A-cryp-Eu showed the same binding pattern as Con A-FITC in prostate stroma and gland cells. Staining was strong in benign prostate hyperplasia and prostate carcinoma tissues. Con A-cryp-Eu probe stained glucose/mannose residues in prostatic carcinoma more intensely than Con A-FITC. Furthermore, staining with Con A-cryp-Eu showed greater fluorescence intensity than Con A-FITC and the emission of Con A-cryp-Eu was more stable than the Con A-FITC for seven days under the same storage conditions. Maintenance of the luminescent properties and the binding pattern of Con A-cryp-Eu favor its use as an auxiliary histochemistry probe for prostatic tissue studies.     

Surface carbohydrate changes on Onchocerca lienalis larvae as they develop from microfilariae to the infective third-stage in Simulium ornatum

Use was made of seven FITC labelled lectins as tools to investigate the surface of Onchocerca lienalis larvae as they develop through to the infective third-stage in a natural vector, Simulium ornatum. The lectins were derived from Canavalia ensiformis (Con A), Lens culinaris (lentil), Triticum vulgaris (wheat germ), Arachis hypogaea (peanut), Helix pomatia, Phaseolus vulgaris (kidney bean) and Tetragonolobus purpureus (asparagus pea). Between 70 and 100 living parasites were examined for each developmental stage; i.e. skin microfilariae, late first-stages, second-stages, preinfective third-stages and infective third-stages isolated from the mouth parts of the flies. None of the lectins used bound to the surface of the microfilariae. However, progressive binding to the cuticle of the first- and second-stages was observed using Con. A, lentil lectin and wheat germ agglutinin (WGA). Following moulting to the third-stage, binding of these three lectins declined. Furthermore, as these lectins decreased, peanut and Helix pomatia lectins progressively increased in their binding, despite the fact that they showed little or no binding to the first- and second-stages; stages at which Con A, lentil and WGA were at their maximum. Asparagus pea and kidney bean lectins failed completely to bind to any of the larvae examined. Carbohydrate inhibition tests showed that the lectin was indeed binding specifically to glycoconjugates on the parasite surface. WGA binding was not inhibited by prior incubation with N-acetyl-D-glucosamine, even at high concentrations, but neuraminic acid did completely inhibit its binding. Judging from the patterns of binding on the nematodes themselves, the carbohydrates may not be vector in origin, but derive from the worms. The lectin specificities indicate that initially mannose/glucose type derivatives are present on the surface. Following moulting to the third-stage these are progressively replaced, or overlaid with galactosamine type derivatives, also present on the infective third-stage as it enters the bovine host. The availability of these surface glycoconjugates to attack mediated by natural insect lectins may be of importance in the parasite regulatory mechanisms of the blackfly. Variability in these surface carbohydrates, and in the response to them could well be a contributing factor in the cytospecific variation in S. damnosum susceptibility to geographical variants of O. volvulus.     

Immobilization of catalase via adsorption onto metal-chelated affinity cryogels

A poly (acrylamide-allylglycidyl ether) [p(AAm-AGE)] cryogel was prepared by radical polymerization of acrylamide (AAm) and allylglycidyl ether (AGE). Cibacron Blue F3GA (CB) was covalently attached to the p(AAm-AGE) cryogel via the reaction between the chloride groups of the reactive dyes and the epoxide groups of the AGE. The CB-attached p(AAm-AGE) cryogel was chelated with Fe³⁺ ions. This immobilized metal ion affinity chromatography (IMAC) cryogel carrying 25.8 ± 2.0 μmol Fe³⁺ ions was used in adsorption studies to interrogate the effects of pH, protein initial concentration, flow rate, temperature and ionic strength on enzyme activity. Maximum adsorption capacities were found to be 75.7 ± 1.2 mg/g for p(AAm-AGE)-CB-Fe³⁺ cryogels and 60.6 ± 1.0 mg/g for p(AAm-AGE)-CB cryogels, respectively. The adsorbed amounts of catalase per unit mass of cryogel reached a plateau value at about 1.5 mg/mL at pH 6.0. The K_m values were found to be 0.73 ± 0.02 g/L for the free catalase and 0.18 ± 0.02 g/L for the immobilized catalase. The V_max value of free catalase (2.0 × 10³ U/mg enzyme) was found to be lower than that of the immobilized catalase (2.5 × 10³ U/mg enzyme). It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the p(AAm-AGE)-CB-Fe³⁺ cryogel.     

Molecularly imprinted cryogel for L-glutamic acid separation

A molecular recognition based L-glutamic acid (L-GLU) imprinted cryogel was prepared for L-GLU separation via chromatographic applications. The novel functional monomer N-methacryloyl-(L)-glutamic acid-Fe(3+) (MAGA-Fe(3+) ) was synthesized to be complex with L-GLU. The L-GLU imprinted cryogel was prepared by free radical polymerization under semifrozen conditions in the presence of a monomer-template complex MAGA-Fe(3+) -L-GLU. The binding mechanism of MAGA-Fe(3+) and L-GLU was characterized by Fourier transform infrared (FTIR) spectroscopy in detail. FTIR analyses on the synthesized MAGA-Fe(3+) -GLU complex reveals bridging bidentate and monodentate binding modes of Fe(3+) in complex with the carboxylate groups of the glutamate residues. The template L-GLU could be reversibly detached from the cryogel to form the template cavities using a 100 mM solution of HNO(3) . The amount of adsorbed L-GLU was detected using the phenyl isothiocyanate method. The L-GLU adsorption capacity of the cryogel decreased drastically from 11.3 to 6.4 μmol g(-1) as the flow rate increased from 0.5 to 4.0 mL min(-1) . The adsorption onto the L-GLU imprinted cryogel was highly pH dependent due to electrostatic interaction between the L-GLU and MAGA-Fe(3+) . The PHEMAGA-Fe(3+) -GLU cryogel exhibited high selectivity to the corresponding guest amino acids (i.e., D-GLU, L-ASN, L-GLN, L-, and D-ASP). Finally, the L-GLU imprinted cryogel was recovered and reused many times, with no significant decrease in their adsorption capacities.     

Purification of a lectin from Canavalia ensiformis using PEG-citrate aqueous two-phase system

A PEG/citrate aqueous two-phase system was tested in the partition of commercial Concanavalin A (Con A) and subsequently applied to the extraction and purification of Con A from the crude extract of Canavalia ensiformis seeds. Con A was successfully extracted to the bottom phase of a system composed of 22% (w/w) PEG8000 and 12% (w/w) citrate at pH 6.0. The obtained purification factor was 11.5 without any loss in the hemagglutinating activity. The purity of extracted lectin was confirmed by SDS-PAGE analysis.