Fluorescent Neoglycoproteins: Antibody-Aminodextran

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Bioconjugate Chem. 1999, 10, 1090−1106

Fluorescent Neoglycoproteins: Antibody-Aminodextran-Phycobiliprotein Conjugates Olavi Siiman,*,† Julie Wilkinson,† Alexander Burshteyn,‡ Patricia Roth,† and Stephen Ledis† Advanced Technology, and Reagents Applications & Development, Beckman Coulter, Inc., 11800 SW 147th Avenue, Miami, Florida 33196-2500. Received June 14, 1999; Revised Manuscript Received September 15, 1999

New, highly amino-substituted dextran or aminodextran (hereafter denoted Amdex) of various sizes between about 20 and 1000 kDa molecular mass and degrees of amino-substitution between 7 and 40% were prepared and characterized by elemental analyses and polyacrylamide gel electrophoresis. These aminodextrans together with others commercially available were shown by static light scattering, viscosity, and refractive index measurements to adopt a globular structure in aqueous salt solutions. Antibody and fluorescent protein dye, phycoerythrin, or its tandems with cyanin 5.1 and TEXAS RED, were covalently conjugated to the aminodextrans. The conjugates contained multiple dye molecules and were shown by dynamic light scattering and scanning electron microscopy to assume either globular structure or aggregates thereof. Streptavidin could be substituted for antibody to prepare streptavidin-aminodextran-PE conjugates, which were then used with biotinylated antibody to label subpopulations of white blood cells. The conjugates yielded up to 20-fold amplification of fluorescence intensity over direct antibody-dye conjugates in labeling white blood cells for flow cytometry.

INTRODUCTION

Water-soluble polysaccharides such as dextrans have enjoyed widespread use in the preparation of neoglycoproteins, the synthetic analogues of glycoproteins, as reviewed in ref 1. Dextrans and other polysaccharides are inert to most direct reactions with proteins, and must be chemically activated to allow conjugation to proteins. The use of antibody-dextran-type substances as carriers for drugs, toxins, photoprobes, etc. has been previously described for ternary conjugates of antibody-polyaldehyde dextran-methotrexate (2), antibody-aminodextran-chlorin (3), and antibody-dextran hydrazide-Sn(IV) chlorin (4). Other conjugated and dextran-crosslinked species have been described in several texts and review articles (5-9). One of the primary methods of preparing protein-dextran conjugates first involves preparation of “aminodextran”. Common practice for the introduction of amine groups at random positions in dextran has been to first cleave the sugar rings with periodate to form polyaldehyde-dextran. The second step is to react the cleaved rings with a diamine such as ethylenediamine or 1,3-diaminopropane to form a Schiffs’ base, which is then stabilized by reduction with sodium borohydride. The “aminodextran” compounds as described in the literature cited above were not well characterized. Typically, they were not isolated as solid compounds and, thus, were not characterized by elemental analyses and average molecular mass determinations. Further, the periodate oxidation-diamine method of preparing aminodextrans was restricted to a low percentage of amino groups per molecule. The percentage was less than 4-5%. Higher degrees of amine substitution were not possible under the usual conditions because high diamine concentrations caused extensive aminolysis * To whom correspondence should be addressed. E-mail: [email protected]. † Advanced Technology. ‡ Reagents Applications & Development.

of the glucosidic linkages between the sugar rings in dextran which resulted in very low molecular mass fragments. As a result, the yields were low and decreased drastically as higher degrees of amine substitution were pursued. An alternative method of producing aminodextrans without opening glucopyranose rings involves carboxymethylation of sugar residue hydroxyl groups by chloroacetic acid, followed by carbodiimide coupling of a diamine such as ethylenediamine. This method (10-11) has been used to produce an aminodextran having about one amine group per 67 glucose residues, which is then used to prepare anti-Ig antibody-aminodextran conjugates for use in inducing B cell activation and proliferation. Other methods of preparing protein-dextran conjugates include the direct reaction of protein with polyaldehyde-dextran (2), the reaction of an oxidized oligosaccharide moiety in a monoclonal antibody (MCA) with dextran hydrazide (4), the coupling with cyanogenbromide-activated dextran (12), the spontaneous Maillard reaction in controlled dry heating between the -amino groups in proteins and the reducing-end carbonyl groups in polysaccharides (13), and the selective reductive amination of the single terminal aldehyde group of dextran (14). Protein-dextran conjugates possess certain advantageous properties. It has been shown that enzymes possess enhanced thermal stability, when they have been immobilized by covalent attachment to polymers. Examples include glucoamylase on various periodate oxidized polysaccharides (15, 16) and trypsin on cyanogen-bromideactivated dextran (12). Other noted effects of protein conjugation to polysaccharides were improved emulsifying properties (17), improved solubility of insoluble gluten (18), enhanced antioxidant effect of ovalbumin (19), and broadened bactericidal effect of lysozyme (20). Our own work has been focused on the synthesis of new, highly substituted (7-40%) aminodextrans and their use as (1)

10.1021/bc990077g CCC: $18.00 © 1999 American Chemical Society Published on Web 10/26/1999

Antibody-Aminodextran-Phycobiliprotein Conjugates

conjugates with anti-CD3 monoclonal antibody (21) for the activation and proliferation of T cells; (2) a locus for nucleation and growth of magnetic metal oxide (22) particles and luminescent II-VI metal sulfide semiconductor1 nanoparticles; (3) a locus for nucleation and growth, and a reducing agent for preparation of colloidal metal particles (23-24); and (4) coatings (25, 26) for colloidal particles. Nevertheless, amplification of fluorescence by this method has not hithertofore been part of this success. Amplification of light signals for detection of species present in very low concentrations has been achieved in other ways, such as when enhanced light emission (27, 28) was observed by indirect fluorescence staining of cell receptor sites with multiple layers of phycoerythrinstreptavidin attached to biotinylated antibody at cell receptor sites. However, enhanced light-induced photochemistry (3) was used in excitation of chlorin e6 coupled through dextran to anti-T-cell monoclonal antibody to enhance singlet oxygen production. The common feature in these studies was the required increase in the number of probe molecules per targeted site. This method applied to fluorescence amplification in organic dyes of low molecular mass does not work well. In one attempt, ca. 1978, described in refs 29 and 30) at amplification of fluorescence signals, large numbers (several hundred) of fluorescein molecules were attached to a synthetic polymer, polyethylenimine, which was then conjugated with antibody. Since fluorescence emission from fluorescein molecules was quenched under these circumstances due to short nearest neighbor distances between fluorophores on the same polymer molecule, lack of control over fluorophore spatial orientation, and lack of control over the structure of the resultant conjugate, the method did not work. More than 100 fluorescent dextrans consisting of soluble dextrans (10-2000 kDa) conjugated with various fluorescent dyes such as fluorescein, dansyl, rhodamine, and TEXAS RED are commercially available (31). The degrees of substitution are 1-2 dyes/dextran of 10 kDa, 2-4 dyes/dextran of 40 kDa, 3-6 dyes/dextran of 70 kDa, ∼64 dyes/dextran of 500 kDa, and ∼134 dyes/dextran of 2000 kDa. Higher degrees of substitution than these may lead to quenching and nonspecific interactions. Most of the listed dextran conjugates are also available as so-called “lysine-fixable”, i.e., have incorporated lysine residues, which can be used for further reaction such as covalent attachment of antibody molecules. Fluorescein isothiocyanate (FITC) derivatives of dextran and poly-L-lysine are also commercially available from sources such as Sigma Chemical Co. with degrees of substitution ranging 0.003-0.020 mol of FITC/mol of glucose and 0.003-0.01 mol of FITC/mol of lysyl residue. For the largest molecular mass dextran of 2000 kDa that is listed, 33-222 FITC molecules/ molecule of dextran would be available. For a 70 kDa poly-L-lysine molecule, up to 5.5 FITCs are available. One molecule of phycoerythrin (PE) (32), however, contains 34 individual chromophoric groups, each of which exhibits about the same fluorescent intensity as one FITC unit. PE, isolated from phycobilisomes, the main light-harvesting pigment complexes in prokaryotes such as red algae and cyanobacteria, utilizes linear tetrapyrroles as chromophoric groups and is among the brightest fluorescent dyes currently available. On a molar basis, one molecule of PE has a fluorescence yield that is equivalent to at least 30 fluorescein or 100 rhodamine molecules at 1 Matijevic, E., Sondi, I., Koester, S., and Siiman, O., unpublished results.

Bioconjugate Chem., Vol. 10, No. 6, 1999 1091

comparable wavelengths. Thus, the largest molecular mass FITC-dextran conjugate with the greatest degree of FITC substitution has the potential to yield a fluorescence enhancement factor of 222/30 ) 7.4 over a single PE molecule. With so many FITC units per dextran carrier and a broad distribution in the molecular mass of each dextran, it would be difficult to synthetically mimic the monodisperse population of fluorescent groups that occurs naturally in molecules of PE. The spread in fluorescent intensities from one molecule of FITCdextran complex to another will offset the large enhancement factor that may be numerically anticipated. No enhancement factor greater than one, relative to PE fluorescence, has hithertofore been reported for the above FITC derivatives of dextran or poly-L-lysine, and no direct fluorescent stain which has a net fluorescence intensity greater than a single PE molecule is currently known. In phycoerythrin (PE) (32), there is a monodisperse population of fluorescent bilin groups naturally embedded in a protein through biosynthesis. PE exhibits maximal absorbance and fluorescence without susceptibility to either internal or external fluorescence quenching so that attachment of two or more PE molecules to a polymeric carrier should not quench PE fluorescence. The net fluorescence intensity from a PE-polymer complex should be the sum of fluorescence intensities from individual PE molecules. Only a single PE molecule (270 kDa) can normally be directly conjugated to an IgG antibody (160 kDa) and still retain antibody specificity (33, 34). Polymeric carrier molecules such as aminodextran of sufficiently large molecular weight can accommodate multiple antibody molecules. Our previous experience, described in ref 21 in conjugating anti-CD3 (T3) monoclonal antibody to aminodextran (1×-Amdex, ∼1000 kDa, 7% diamine substitution; 5×-Amdex, ∼350 kDa, 25% diamine substitution) under saturating conditions of antibody on dextran gave a CD3 antibody:aminodextran molar ratio in the conjugates of 37:1 for CD3-1×-Amdex and 20:1 for CD3-5×-Amdex, thus showing that aminodextran can be effectively loaded with many large protein molecules. Similar methods can be used to prepare protein-aminodextran conjugates with two or more protein molecules per aminodextran molecule, the protein being of one type or several different types. Herein, we describe the preparation of such conjugates for immunofluorescent labeling applications in which one protein would be PE and the other would be an antibody. Aminodextrans and the ternary conjugates were characterized by light scattering and scanning electron microscopy (SEM). Triple detector (light scattering, viscosity, and refractive index) measurements for all aminodextrans derived from linear, water-soluble dextran showed that the product aminodextrans were also linear, even though analytical evidence for branching through intermolecular cross-linking was suggested for some preparations of highly substituted aminodextran. Previous work in the high-resolution transmission electron microscopy (TEM) of antibodies, polysaccharides, and phycobilisomes and their component pigments has revealed the ultrastructure of antibody shape, the branching of polysaccharide chains in proteoglycans (35, 36), and the shape and size of R-phycoerythrin (32). Herein, moderate resolution SEM is used to determine the shape and size of ternary conjugates of MCA and PE with aminodextrans. Enhanced phycoerythrin fluorescence intensities per marker in labeling white blood cells were obtained by substituting the direct conjugates with aminodextrancross-linked conjugates, which results have been reported

1092 Bioconjugate Chem., Vol. 10, No. 6, 1999

by us in meeting proceedings, and in issued and pending patents (37-39). The amplification of fluorescence was initially demonstrated for marked cells which usually showed dim fluorescence intensity (37, 38) when their receptors for monoclonal antibodies (BB27, IL12Rβ.44, and BY55) were saturated with direct antibody-PE conjugates and their fluorescence intensity was measured by flow cytometry. PE-tandem dye molecules that can be excited with the same laser line, 488.0 nm Ar+, as PE, were also incorporated into cross-linked conjugates to offer a set of six different markers using three colors, two intensity per color. One tandem dye is PC5 (PE-Cy5, phycoerythrin-cyanin 5.1) (40, 41), and a second tandem dye is ECD (phycoerythrin-TEXAS RED) (42, 43). This allowed multiplex labeling (39) of cells for analysis of mixed cell populations by flow cytometry. EXPERIMENTAL PROCEDURES

Materials. Dextran, T-2M, produced by Leuconostoc mesenteroides, strain B-512, was obtained from the Sigma Chemical Co., St. Louis, MO, as were other chemicals related to the preparation of aminodextrans. All inorganic chemicals were of reagent grade and were not further purified. Dextran, amino, nominally of 2 MDa Mr (molecular mass, was purchased from Molecular Probes, Inc., Eugene, OR, abbreviated as Amdex-M.P. Analytical data for lot 6551-3: 130 amines/mol. Measurement of the molecular mass of this lot of aminodextran by the Viscotek Corp. (Houston, TX) triple detection system (vide infra) gave ∼3 MDa. Therefore, there are (3 000 000 g/mol)/(162.1 g/mol glucose monomer) ) 18 507 glucose units/dextran molecule or 130/18 507 ) 0.007 02, i.e., ∼1/ 140 degree of substitution with single amine group per reacted glucose unit. The degree of substitution unless otherwise noted is defined as the total number of amino groups (primary and secondary) per monomeric glucose unit in the aminodextran molecule. R-PE (catalog no. PB31), extracted and purified from red algae, was obtained from Prozyme, Inc., San Leandro, CA. Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) was a Pierce product while 2-iminothiolane hydrochloride, L-cysteine, free base, and iodoacetamide were from Sigma. Column packing materials, G-25 or G-50 Sephadex and Bio-Gel A-5m and A-15m, were products of Pharmacia Biotech and Bio-Rad Laboratories, respectively. Phosphatebuffered saline, 1× PBS, pH 7.1-7.3, and conductivity, 13500-15500 µΩ-1 cm-1, was prepared by dilution with distilled water from a 20× PBS stock solution, which contains 26.9 g dm-3 of K2HPO4, 6.4 g dm-3 of KH2PO4, and 170.0 g dm-3 of NaCl. Desalting and purification of aminodextrans was carried out with hollow fiber (polysulfone, 9.9 ft2 membrane surface area, 1 mm diameter fibers) cartridges, A/G Technology Corp. model UFP-5-E-35 (5 kDa MWCO) or model UFP-30-E-35 (30 kDa MWCO), with tubing adaptor kit, KA12-3P. Lyophilization of the final washed aminodextrans was accomplished with an FTS Systems, Inc. model TDS-00030-A, Dura-Dry microprocessorcontrolled freeze-drier. The CD4 antibody [CD4 clone SFCI12T4D11 (IgG1)] and CD8β antibody [CD8β clone 2ST8.5H7(IgG2a)] are products of Coulter Corp., Miami, FL. Anti-CD4-FITC, -PE, and -ECD are products of Coulter Corp. Streptavidin (catalog no. 15532-039) was obtained from Gibco Life Technologies, Inc., Gaithersburg, MD. Avidin, NeutraLite, R-phycoerythrin conjugate (catalog no. A-2660) was obtained from Molecular Probes, Inc.

Siiman et al.

Preparation of Aminodextrans. Aminodextrans were obtained commercially or were prepared by the following procedures. In a standard preparation of 1×-Amdex, 8.56 g of NaIO4 in 100 mL of distilled water (DW) was added dropwise to 80 g of dextran (2 MDa) in 600 mL of DW over about 10 min with vigorous stirring, after which the mixture was stirred at room temperature for 3 h. The resulting viscous reaction mixture was then diluted to 2 L with DW and desalted using a hollow fiber cartridge and about 18-22 L of DW to obtain a solution having a final pH of 6.0-6.5. To the final, 800 mL volume of washed, oxidized dextran solution was slowly added 80 mL of colorless, liquid 1,3-diaminopropane (DAP) over about 10 min at room temperature. The resulting mixture was stirred for 3 h. Then, 3.2 g of NaBH4 in 40 mL of 1 mM aqueous NaOH was added to the aminodextran reaction mixture over about 5 min with stirring. The resulting mixture was stirred for 1 h and then desalted using a 5 kDa MW cutoff hollow fiber cartridge and about 20-25 L of DW to reduce the specific conductance to about 3-4 µΩ-1 cm-1 and the pH to 6.0-6.5. The final volume of aminodextran solution was 400 mL. This solution was passed through a 0.2 µm sterile cellulose acetate filter unit and then freeze-dried to obtain 48 g of flaky, pale yellow solid, a 52% yield. Elemental analysis observed: C, 42.53; H, 6.52; N, 1.01; O (by difference), 49.94. Elemental analysis calcd for C49H84NO40‚3H2O: C, 42.61; H, 6.57; N, 1.01; O, 49.81. The empirical formula C49H90NO43 is very similar to the formula based on 31 units of glucose, 1 unit of fully diamine substituted sugar ring, and three units of water. The degree of substitution in dextran by diamine was 1/32. Other runs varied between 1/28 and 1/45 degree of substitution. Similar results were obtained for aminodextrans prepared from dextrans having average Mr of 10 kDa to 2 MDa with 1×-5×-diaminopropane substitution. Modifications were made to periodate oxidation, diamine addition, and borohydride reduction reactions. The first modification was to use only a 10% excess of diamine over the stoichiometric 2:1 diamine:periodate molar ratio previously used. Second, the diamine addition reaction was conducted at a temperature in the range of 5-10 °C. Third, the diamine addition reaction was spectroscopically monitored in the near-UV region for Schiffs’ base formation, which was deemed completed when successive spectral analyses indicated a plateau was reached. These modifications reduced depolymerization and thus gave higher yield of product. Elemental analyses for 5×-Amdex, lot -11, which was prepared at a 500 g dextran scale and gave the best yield of conjugates were C , 44.45%; H, 7.20%; N, 3.79%, O (by difference), 44.56%. The empirical formula was C13.7H26.4O10.3N, which is similar to the formula C13H24O9N‚H2O based on 5 units of glucose/1 unit of 1.5 diaminopropane-substituted sugar ring (C9.5H21O2N3), or a degree of substitution in dextran of 1/6. Similar results were obtained for 5×-Amdex, lot -69 prepared at a 300 g dextran scale. High diamine concentrations caused depolymerization and thus gave significantly lower molecular weight aminodextrans than the starting dextran. β-Elimination and thereby depolymerization (44) has been previously noted in various oxidation reactions of cellulose and starch. The periodate oxidation of glucose units in dextran was shown (45, 46) to release one mole of formic acid per mole of reacted glucose unit by 95% of the anhydroglucose residues. Thus, the overall redox equation requires 2 mol of periodate/1 mol of glucose unit or 2 mol of aldehyde for complete reaction. However, in 5×-Amdex preparations, 2 mol of periodate/5 mol of glucose units were used so


that the theoretical degree of substitution is 1/5. Thus, the maximum degree of substitution of dextran with DAP is also 1/5. Partially cross-linked 5×-Amdex was prepared by a modified procedure. Dextran, T-2M, 500 g was blended in 4 L of DW to dissolve all solid. A solution of 267.5 g of sodium m-periodate in 2 L of DW was added to the dextran solution over a 5-15 min period using vigorous stirring. The reaction mixture was stirred for 4 h, over which 5-6 L of DW further was added to reduce the viscosity of the solution. The reaction mixture was then desalted using about 100 L of distilled water to obtain 6-9 L of washed, oxidized dextran solution having a specific conductance of about 6-20 µΩ-1 cm-1 and pH of 6.5-7.0. Then, the first portion of DAP, 70 mL of pure liquid, was added over about 5 min to the desalted, oxidized dextran solution. The resulting solution immediately began to show formation of a gel, which persisted for another 5-10 min before redissolving as a yellow solution. The reaction mixture was then put on an ice bath to maintain a reaction temperature of 8-10 °C and stirred vigorously before a second portion of 70 mL of DAP was added over a period of 5 min. After an additional 10 min of stirring, the third and final 70 mL portion of DAP was added to the reaction mixture. The total DAP addition and reaction time was 45 min. Then, 70 g of sodium borohydride in 700 mL of 1 mM aqueous KOH solution were added to the reaction mixture at 8-10 °C over about 10-15 min with overhead stirring. The reaction mixture was stirred for an additional 2 h until the yellow Schiffs’ base color had disappeared. The reaction mixture was then desalted using a 5 kDa MW cutoff hollow fiber cartridge and about 80 L of DW to produce about 1.6 L of aminodextran solution having a specific conductance of 10-20 µΩ-1 cm-1 and pH of 7.08.0. The aminodextran solution was filtered through a 1.6 µm glass filter and lyophilized for a minimum of 72 h to produce 75-90 g (15-18% yield) of flaky, white to pale yellow crystals. Analyses for 5×-Amdex, lot 1-5, which gave a good yield of conjugates were C ) 41.38%, H ) 7.81%, N ) 4.15%, O ) 45.64%, I ) 97 ppm, B ) 590 ppm. The empirical formula was C11.6H26.1O9.6N, which is similar to the formula C11.2H20.3O7.3N‚2H2O based on 4 units of glucose/one unit of 1.5 diaminopropane-substituted sugar ring. Thus, the degree of diamine substitution in dextran was 1/5. Similar procedures were used in the preparation of 5×-Amdex, lot 2-2, except 1/3 x DAP was added in two portions and a 30 kDa MW cutoff hollow fiber cartridge was used for desalting to give a product which analyzed as C, 40.44%; H, 7.75%; N, 3.48%; O, 48.48% with a degree of substitution of 1/8. Some 5×-Amdex lots require further description. The reaction mixture of the 5×-Amdex, lot 11-6, after addition of DAP and sodium borohydride, was adjusted to pH 8.5 with aqueous hydrochloric acid. It was then desalted, concentrated, and freeze-dried in the previously described manner. Elemental analyses for 5×-Amdex, lot 11-6, were C, 39.72%; H, 7.77%; N, 4.44%; Cl, 2.81%; O (by difference), 45.26%. The empirical formula based on actual analyses was C10.4H24.3O8.9NCl0.25. The chloride analysis showed that one of four total amine (primary and secondary) groups had a chloride counterion. As shown in the following section describing results of electrophoretic light-scattering experiments on suspensions of polystyrene latex beads coated with DAP or aminodextran, only primary amine groups are protonated near neutral pH to which the reaction mixture was desalted prior to lyophilization, this implies that 50% of the diaminopropane groups in aminodextran are bridging


or cross-linking groups between dextran chains. A portion of the lyophilized aminodextran hydrochloride was dissolved in DW and deionized in batchwise fashion with mixed bed (H+, OH- form), Bio-Rad AG 501-X8 resin, until the specific conductivity of the supernatant was minimized. The resin was removed by filtration of the suspension through rayon cloth, and the aminodextran (∼50 mg/mL) in the filtrate was fractionated by precipitation with acetone (0-43% cut). The solid precipitate was washed with acetone and dried in a vacuum desiccator under silica gel. Elemental analyses of this deionized and fractionated material gave C, 45.55%; H, 7.03%; N, 3.82%; Cl < 0.5%, O (by difference), 43.60%, showing that chloride ion had been removed. DELSA Results for DAP- and Amdex-Coated Polystyrene Latex Beads. 1,3-Diaminopropane (DAP) in aqueous solution is reported (47) to show acid dissociation constants, pKa1 ) 10.94 and pKa2 ) 9.03 at 10 °C. When either one of the primary amine groups of DAP is converted to a secondary amine in DAP derivatives, the pKa of the secondary amine is expected to drop significantly. This was borne out when protonation of primary and secondary amines of DAP derivatives was observed (48) in Coulter DELSA (Doppler Electrophoretic Light Scattering Analyzer) 440 experiments with DAP-PS and 1×-Amdex-polystyrene (PS) latex beads dispersed in aqueous media. Multiangle Doppler electrophoretic light scattering measurements have been reviewed (49). Mobility versus pH data were obtained for PS aldehyde/ sulfate beads, nominally of 2.0 µm diameter from Interfacial Dynamics Corp., and the same beads coated covalently with DAP or 1xAmdex at pH ∼10, followed by reduction of the Schiffs’ base with sodium borohydride, and washing of the beads by centrifugation at 30004000g, discarding the supernatant, and redispersion of the residue in an equal volume of distilled water. The chemistry that was used in converting aldehyde PS beads to DAP-PS beads was the same as that used in the conversion of oxidized dextran to aminodextran. Note that any unreacted aldehyde in the reaction with DAP is converted to alcohol by borohydride reduction. Bead suspensions, 4 or 1% w/v solids, were diluted about 1000fold with 0.01 M aqueous sodium nitrate solution, and pH was adjusted with either 0.1 M aqueous sodium hydroxide or 0.1 M aqueous sulfuric acid solutions. The mobility of DAP-PS beads shows a relatively flat, -0.275 to -0.92 cm2/V s, dependence from pH 7 to 9; but, a relatively steep rise from -0.275 to +4.87 cm2/V s between pH 7.0 and 5.6. Raw PS aldehyde/sulfate beads show a highly negative mobility of -6.6 to -6.0 cm2/V s between pH 9.9 and 5.6, and then a slight rise to -4.4 cm2/V s at pH 4.3. The data for DAP-PS beads indicate nearly complete compensation of the negative sulfate group charge by protonated primary amine groups in the pH 9 to 7 range, and further protonation of secondary amine at acidic pH less than 7. A similar trend is observed in electrophoretic mobilities of 1×-Amdex-PS beads. An almost flat mobility versus pH dependence of -1.161 to -1.083 cm2/V s from pH 7 to 9, but a rise from -1.161 to +0.567 cm2/V s was observed between pH 7.0 and 5.6. 1×-Amdex-coated PS beads show less compensation for the negative sulfate charge due to less than saturation coverage of the bead surface by amine groups of the polymeric diaminopropane-dextran derivative and correspondingly fewer secondary amine groups protonated at acidic pH less than 7. Preparation of Buffer-Exchanged Phycoerythrin (PE). In one run, four 2.5 mL portions of 20 mg/mL Prozyme PE concentrate (in 60% ammonium sulfate, 50


mM phosphate, and 5 mM sodium azide) were each pipetted into four 50 mL polypropylene centrifuge tubes. A total of 25 mL of PE storage buffer (50 mM phosphate and 2 mM EDTA, pH 7.0) was added to each of the four tubes. The tubes and contents were roller mixed for 30 min and then centrifuged for 30 min in a Beckman J6-B centrifuge at 3200 rpm. Residues were discarded. Supernatants were retained, and concentrated in CentriPrep-30 tubes (Amicon) by multiple centrifugations for about 20min each at 2000 rpm. The final 1-2 mL of PE concentrate was applied to the top of a 60 mL of G-50 Sephadex column, equilibrated, and eluted with PE storage buffer. The middle fraction, about 4 mL of total volume, was collected and retained. Absorbance readings for a 50-fold diluted sample at 565 nm gave a PE concentration in the concentrate of 35.050 mg/mL and a yield of 140.2 mg of PE. Further concentration of this fraction to 1-2 mL total volume gave a PE concentrate of 104.7 mg/mL. Preparation of Antibody-Phycobiliprotein Conjugates. Direct monoclonal antibody (MCA)-fluorochrome (PE, PC5, and ECD) conjugates are commercially available for most antibodies from Coulter Corporation or Immunotech, as prepared by established procedures of conjugation reactions of 2-iminothiolane (IT) activated PE (or PC5; ECD) with sulfo-SMCC activated antibody or sulfo-SMCC activated PE (or PC5; ECD) with DTT (dithiothreitol) reduced antibody. The molar ratio of MCA:fluorochrome in these conjugates is about 1:1. Conjugation of IgG monoclonal antibody to PC5 was accomplished by sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC) activation of PC5 and activation of antibody by reduction of disulfide bonds in the hinge region with DTT. As an example, a 100-fold dilution of the CD8β Antibody-PC5 conjugate pooled sample gave an A565.5 ) 0.4809, yielding 20.31 mg of total PC5 in the CD8β-PC5 conjugate, and an A280 ) 0.1513, giving 21.90 mg of total CD8β antibody in the CD8β-PC5 conjugate. The molar ratio of PC5/CD8β is, therefore, 0.618. A corrected F/P ratio based on the formula, MCA:PC5 ) [A280/A565.5 (conjugate) - A280/A565.5 (dye)] × 8.77, is 0.864. Similar methods were used to prepare an CD4-ECD conjugate having an F/P ratio of 0.96. Preparation of Antibody-Aminodextran-Phycobiliprotein Conjugates. The procedure was similar to the one previously (21) used to prepare conjugates of anti-CD3 antibody with aminodextran, either 1×-Amdex or 5×-Amdex. However, in the present work two different proteins, one, an iminothiolane-activated monoclonal antibody and the other, an iminothiolane-activated fluorescent protein, PE, PC5, or ECD, were conjugated simultaneously to the sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)-activated aminodextran. The IT to MCA and IT to PE (PC5 and ECD) molar ratios were maximized at 15 and 22.5, respectively, so as not to interfere with MCA activity (7) or with PE (PC5 and ECD) fluorescence intensity. Trials were typically done for new proteins at molar ratios of 7.5, 15, and 22.5. This is in accord with the exposure of CDR (complementary-determining or hypervariable region) and framework (or nonhypervariable region) residues in 34 human and murine antigen-binding regions of known three-dimensional structure (50). In 34 CDRs, of 78 total lys residues with an amine group which can react with IT, 60 are exposed to give an average of 2 exposed Lys/antibody, and of 3 total Cys residues, 0 are exposed, and 1 is partly buried. In 34 framework regions,

Siiman et al.

of 296 total Lys residues, 237 are exposed to give an average of 7 exposed Lys/antibody, and of 136 total Cys residues, 0 are exposed, all are buried. Thus, the tested range 7.5-22.5 in IT:MCA molar ratio covers the maximum of about 9 Lys residues that can react with IT. R-PE was further purified by centrifugation to remove high MW aggregates, followed by buffer exchange on G-50 Sephadex and concentration. Three protocols were used as follows. (i) For 1×-Amdex conjugates, the molar ratio of reactants that were used was 4:1:4 ) Dye:MCA:Amdex or the weight ratio was 25.713:4.287:10 ) Dye:MCA:Amdex at the 10 mg Amdex scale. (ii) For Amdex-Molecular Probes (hereafter denoted as Amdex or Amdex-M.P.), 50% less weight of dye, and MCA relative to Amdex was used so that molar ratio was 16:4:1 ) Dye:MCA:Amdex and weight ratio was 12.856: 2.143:10 ) Dye:MCA:Amdex at 10 mg Amdex scale. (iii) For 5×-Amdex, the same dye:MCA:Amdex weight ratios as previously listed and as used in 1×-Amdex trials were adopted, but the molar ratio was 4:1:15 ) Dye:MCA: Amdex. Described below is the preparative procedure for Amdex-M.P.-cross-linked CD8β-PC5. CD8β Antibody-Amdex-PC5 Conjugates. Conjugation was carried out with 50% less weight of dye and antibody relative to aminodextran so that molar ratio was 16:4:1 ) PC5:CD8β antibody:Amdex and weight ratio was 12.856:2.143:10 at the 10 mg Amdex scale. 1. Activation of Aminodextran with sulfo-SMCC. A total of 0.340 mL of a 29.412 mg/mL solution of Amdex in distilled water, to which 0.018 mL of 20× PBS buffer solution was added to make a 1× PBS solution, was activated with 0.180 mL of 10 mg/mL sulfo-SMCC solution in 1× PBS, to which was added 0.462 mL of 1× PBS solution to make a total Amdex concentration of 10 mg/mL. The mixture was roller mixed for about 1 h at room temperature. After the mixing was completed, the reaction mixture was immediately applied to the top of a 60 mL (1.7 cm × 28 cm) G-50 Sephadex column equilibrated with 1× PBS. The sample was eluted using 1× PBS and collected in about 2 mL fractions. Fractions of the first band absorbing at 280 nm contained the high molecular weight activated Amdex as was verified by Tyndall scatter with a focused visible light beam (model 650, Cambridge Instruments, Inc., Buffalo, NY). These fractions were pooled to give about 5.8 mL of total sulfoSMCC-activated Amdex. 2. Activation of Antibody. CD8β monoclonal antibody was activated by the addition of 0.033 mL of a 2 mg/mL solution of iminothiolane in 1× PBS and 0.219 mL of 1× PBS to 0.081 mL of CD8β concentrate (61.67 mg/mL). The resulting solution which had an antibody concentration of 15 mg/mL and an iminothiolane molar concentration 15-fold larger was mixed at ambient temperature for about 1 h. The reaction mixture was then chromatographed on a 60 mL (1.7 cm × 28 cm) G-50 Sephadex column equilibrated with 1× PBS, and the sample was eluted using 1× PBS. The first band peak fraction yielded about 4.2 mL of 1.113 mg/mL antibody solution, which contained a total of 4.67 mg of IT-CD8β antibody derivative. 3. Activation of PC5. PC5 was activated by the addition of 0.097 mL of a 2 mg/mL solution of iminothiolane in 1× PBS and 0.098 mL of 1× PBS to 0.179 mL of PC5 concentrate (83.625 mg/mL). The resulting solution which had a PC5 concentration of 40 mg/mL and an iminothiolane molar concentration 22.5-fold larger was mixed at room temperature for about 1 h. The reaction mixture


was then applied to the top of a 60 mL (1.7 cm × 28 cm) G-50 Sephadex column equilibrated with 1× PBS and the sample was eluted with 1× PBS. The first band peak fraction gave about 3.9 mL of 3.35 mg/mL PC5 at an A565/ A280 ratio of 6.2394, which contained a total of 13.065 mg of IT-PC5. 4. Conjugation of IT-CD8β and IT-PC5 to sulfo-SMCCAmdex. For a 15 mg total protein, 10 mg Amdex weight ratio, 1.926 mL of 1.113 mg/mL IT-CD8β solution (about 2.143 mg antibody) was first mixed with 3.839 mL of 3.35 mg/mL IT-PC5 solution (about 12.86 mg IT-PC5), to which was added 5.8 mL of sulfo-SMCC-Amdex solution (about 10 mg Amdex), and the entire mixture was roller mixed overnight for 16-24 h. After the mixing was completed, the total volume of the mixture was determined, and 0.120 times this volume of 5 mg/mL L-cysteine in 1× PBS was added to each conjugation mixture. The L-cysteine containing mixtures were then mixed for an additional 15 min to effect blocking of any unreacted sulfo-SMCC moieties. Last, 20 mg/mL iodoacetamide in 1× PBS in the amount of 0.120 times the total mixture volume and 1 M borate buffer solution, pH 9.8, in the amount of 0.020 times the total mixture volume were added to each mixture. The resulting mixtures were mixed for about 30 min to block any unreacted sulfhydryl groups. 5. Purification of CD8β-Amdex-PC5 conjugates. The total volume of conjugation mixture was reduced to about 2.5 mL by centrifuging an Amicon Centri-Prep 30 tube containing the sample for about 20 min at 2000 rpm using a refrigerated Beckman J-6B centrifuge. The sample was placed on the top of a Bio-Gel A-15m agarose column (2.5 cm × 48 cm) equilibrated with 1× PBS and chromatographed using 1× PBS as eluant. Eluant fractions of about 3.6 mL volume were collected using a Pharmacia LKB FRAC-100 collector operating in the drop collection mode. The fractions were monitored using a LKB 2138 Uvicord S monitor operating at 280 nm. The first narrow, intense band eluted from the column contained the CD8β-aminodextran-PC5 conjugate. A lower intensity shoulder of less than one-third the intensity of the first peak contained ∼1:1 PC5:aminodextran conjugate and excess PC5. A medium-to-low intensity well-separated third band was attributed to the low molecular weight excess blocking reagents. The fractions collected for the CD8β-Amdex-PC5 conjugate were analyzed spectrophotometrically at 565.5 and 280 nm using a 1 mm path-length cell. The concentration of PC5 in mg/mL in the conjugate was derived from the absorbance at 565.5 nm by using the formula A565.5/8.167. Data for fractions 19-24 under the first narrow peak gave 4.241 mg of total PC5 in the conjugate. Since 12.86 mg of IT-PC5 was used in conjugation, the yield was 33.32%. 6. Estimation of Molecular Masses of Antibody-Aminodextran-PE Conjugates. Blue dextran (Sigma, T-2M) was applied to an A-15m column that was used to purify the antibody-aminodextran-PE conjugates, eluted from the column with 1× PBS, monitored by A280, and collected at the same drop count of 120 drops/fraction. The first narrow peak (about fraction 20) in the elution profile of antibody-aminodextran-dye conjugates occurred in the same fraction as the first narrow peak in the elution profile of blue dextran. Therefore, we estimate the conjugates to have a similar molecular mass of about 2 000 000 Da. Trials with 5×-Amdex were run at a 4:1:15 molar ratio of Dye:MCA:Amdex or weight ratio of 25.713:4.287:10 at a 10 mg 5×-Amdex scale. Procedures for other amino-


Figure 1. A280 versus fraction number trace for 2 mL of concentrate of CD8β-5×-Amdex-PC5 conjugate on Bio-Gel A 15 m column of 2.5 × 48 cm size. Run conditions: 1 mm/min chart speed; ∼1.3 mL/min elution with 1× PBS; 0-1 absorbance unit scale. Positions of A280 maxima for either PC5 or CD8β antibody alone on the column are indicated with bars in fractions 74-76, and 92-94, respectively. Table 1. Fractionation Data for Trial 1, CD8β-5×-Amdex-PC5 Conjugate. fraction

A565.5

A280

A565.5/A280

PC5 (mg/mL)

CD8β (mg/mL)

36 37 38 39 40 41 42 43 44 45 46

0.0891 0.2661 0.7280 1.1681 1.1810 0.8906 0.5169 0.2720 0.1637 0.1160 0.0915

0.0344 0.0959 0.2450 0.3744 0.3646 0.2648 0.1452 0.0715 0.0397 0.0260 0.0191

2.59 2.77 2.97 3.12 3.24 3.36 3.56 3.81 4.13 4.46 4.79

0.109 0.326 0.891 1.430 1.446 1.090 0.633 0.333 0.200 0.142 0.112

0.193 0.507 1.212 1.758 1.639 1.134 0.573 0.252 0.118 0.063 0.035

A565.5 and A280 are absorbances measured at 565.5 and 280 nm with a 1 mm path length cell.

dextrans were similar to the ones described above for Amdex-M.P., i.e., activation ratios were the same but amounts were doubled to reflect the 2× greater amount of dye and antibody. In trial 1 for CD8β antibody5×-Amdex (lot -11)-PC5, purification of the conjugation mixture on Bio-Gel A-15m was carried out with collection of eluant fractions of 1.8 mL volume. A chromatogram showing the A280 versus fraction number recording is displayed in Figure 1. Data for fractions 36-46 under the first narrow peak containing the conjugate are listed in Table 1. The active CD8β antibody concentration was determined by an ELISA assay (51) for IgG1 antibody; however, assay results appeared to show interference from nonantibody parts of the conjugate. Thus, A280 values corrected for the PC5 contribution were used to obtain antibody concentrations. In another trial for CD8β antibody-5×-Amdex (lot 1-5)-PC5, the conjugation mixture gave an intense first band off the Bio-Gel A-15m column but the yield was low since the bulk of the bluepurple conjugate aggregated with the gel at the top of the column and could not be eluted. Fractions 20-25 collected at 3.6 mL/fraction gave 4.831 mg of conjugate, an 18.78% yield based on 25.724 mg of IT-PC5 used in the conjugation.


Siiman et al.

Table 2. Triple Detector System Data for Dextran and Various Aminodextrans. sample 1. 5X-Amdex, lot -11 2. 5X-Amdex, lot -11 3. 1X-Amdex, lot -75 4. 1X-Amdex, lot -75 5. Dextran, T-2M 6. 5X-Amdex, lot 1-5 7. 5X-Amdex, lot 1-5 8. Amdex-M.P. 9. 5X-Amdex, lot -69 10. 5X-Amdex, lot 11-6 11. 5X-Amdex, lot 2-2 Dextran (60) Dextran (63) Dextran (63) Dextran (63)

concentration (mg/mL)

dn/dc (mL/g)

Mw (kDa)

0.860 1.940 0.940 3.480 0.800 0.800 3.010 0.770 2.400 2.610 2.390

0.155 0.132 0.155 0.132 0.147 0.190 0.132 0.155 0.132 0.132 0.132

414 25.6 1044 93.9 2,102 70.0 34.4 2999 69.95 44.5 168.4 54 600 65 20 500

CD4 Antibody-Amdex-ECD Conjugate. The procedures were the same as those outlined for the preparation of the anti-CD8β-Amdex-PC5 conjugate in Section II, except anti-CD4 antibody was activated with IT and used in the conjugation instead of CD8β antibody, and the tandem PE-Texas red or ECD fluorescent dye (41, 42) was used instead of PC5. In trial 2, IT-ECD (12.856 mg) and IT-CD4 (2.143 mg) were mixed with sulfoSMCC-Amdex (10 mg) at concentrations of 1.10, 0.184, and 0.857 mg/mL, respectively, during conjugation. The IT-ECD (A565.5/A280) ratio was 5.424. The conjugation mixture was concentrated to about 1.5 mL and applied to the top of an A-15m column. Data for fractions 2026, collected at about 3.6 mL/fraction under the first narrow peak in trial 2, gave 5.756 mg of total ECD in the conjugate. Since 12.858 mg of IT-ECD was used in conjugation, the yield was 44.77%. CD4 Antibody-Amdex-PE Conjugates. Again, the procedures were the same as those outlined for the CD8β-Amdex-PC5 conjugate in section 2, except CD4 antibody of the IgG class and PE were activated with IT and used in the conjugation instead of CD8β antibody and PC5. In trial 3, the reactants, 3.896 mL of 3.30 mg/ mL IT-PE (12.856 mg), were first mixed with 4.75 mL of sulfo-SMCC-Amdex (10 mg) solution, to which were then added 3.058 mL of 0.701 mg/mL IT-CD4 (2.143 mg). The IT-PE (A565/A280) ratio was 5.800. The conjugation mixture was concentrated to about 1.0 mL and applied to the top of an A-15m column. Data for fractions 18-24 collected at 3.6 mL/fraction under the first narrow peak in trial 3 gave 4.957 mg of total PE in the conjugate. Since 12.856 mg of IT-PE was used in conjugation, the yield was 38.56%. Streptavidin-Amdex-PE Conjugates. The procedures were similar to those already described for CD8βAmdex-PC5 and CD8β-5×-Amdex-PC5, except streptavidin was substituted for CD8β antibody and PE was substituted for PC5. To obtain concentrations of ITstreptavidin in milligrams per milliliter, the A280 values in two runs were divided by 2.5 [approximately the Mr ratio of IgG antibody/streptavidin ) 160000/60000 Da ) 2.67] in the 9-11 run and 3.0 in the 9-15 run. In both trials, 10 mg of Amdex (∼30 mg/mL in 1× PBS) was activated with 0.180 mL of 10 mg/mL sulfo-SMCC, and the three purified fractions showing the most Tyndall scatter were pooled to give about 5-9 mL of sulfoSMCC-Amdex solution. In run 9-11, 8.806 mg of IT-PE (8.30 mg/mL) and 2.143 mg IT-streptavidin (1.940 mg/mL) were mixed with 9 mL of sulfo-SMCC-AmdexM.P., and 17.613 mg of IT-PE and 4.287 mg of ITstreptavidin were mixed with 9 mL of sulfo-SMCC5×-Amdex (lot -11) overnight for 21 h. In run 9-15,

η (dL/g) 0.262 0.0602 0.442 0.205 0.609 0.167 0.100 0.674 0.141 0.0999 0.186

Rg (nm)

L (nm)

Rh (nm)

15.6 3.79 25.2 8.80 35.5 7.38 4.96 41.3 7.06 5.40 10.35 70.0

38.2 9.28 61.7 21.5 87.0 18.1 12.1 101.2 17.3 13.2 25.3

10.4 2.52 16.8 5.85 23.6 4.91 3.30 27.5 4.69 3.59 6.88 12.5 10.0 22.5

12.856 mg of IT-PE (8.85 mg/mL) and 2.143 mg of ITstreptavidin (1.202 mg/mL) were mixed with 8 mL of sulfo-SMCC-Amdex-M.P.; and 25.713 mg IT-PE and 4.287 mg IT-streptavidin were mixed with 5 mL of sulfoSMCC-5×-Amdex (lot -11) overnight for 20 h. Blocking with L-cysteine free base and iodoacetamide was done for each run in the same way as previous conjugations. The concentrated reaction mixtures of about 1 mL of total volume were each individually purified on a Bio-Gel A-15m column. Fractions (about 3.6 mL) of the first band, peaking in protein and PE concentrations at fraction 21 for the Amdex-M.P. conjugate and at fraction 22 for the 5×-Amdex conjugate, were retained. Triple Detector System Measurements. The Viscotek Corp. (Houston, TX) system consists of a model T-60 differential viscometer/light-scattering dual detector and a model LR40 differential laser refractometer with a 670 nm diode laser source. The principles of the method and analyses have been described (52). The responses of the three primary detectors are as follows: light scattering, M(dn/dc)2 × c; viscometer, ηc; refractometer, dn/dc × c. Right-angle light-scattering gives the molecular weight when combined with viscometry detection. The 90° LS is corrected for angular dissymmetry using the Debye equation, together with the molecular size information provided by the viscometer. Viscometry yields the molecular density, which is related to conformation and branching. The refractometer measures sample concentration, providing a direct determination of the refractive index increment, dn/dc, of the polymer sample. The hydrodynamic volume of a polymer molecule in solution, related to the cube of its radius of gyration, Rg, is directly proportional to the intrinsic viscosity (η) and the weight average molecular weight (Mw), divided by Avogadro’s number. The accuracy of Rg values determined by the triple detector system is claimed to be within 0.5 nm. RESULTS

Light Scattering, Viscosity, Refractive Index Measurements. Molecular data for 11 samples, one dextran and 10 aminodextrans, determined from duplicate batch analyses by the triple detector system are summarized in Table 2. The samples were run at a concentration of 0.7-3.5 mg/mL in 0.2 M aqueous sodium nitrate solution through a column consisting of a 50 ft coil of 0.01 in. i.d. stainless steel tubing without any column packing in batch mode. Some lots of solid aminodextran resisted dissolution and needed a more extensive protocol to afford complete dissolution. Two dissolution protocols were followed: (1) by minimum heating at ∼70-80 °C for 5-10 min for samples 1, 3, 5, 6, and 8; (2) by first soaking at


Figure 2. Triple chromatogram of Amdex-M.P. Run conditions: solvent, 0.05 M aqueous sodium sulfate; flow rate, 0.5 mL/min; injection volume, 50 µL; column temperature, ambient; concentration, 0.1 mg/mL. Traces of curves on RHS are for refractive index (RI), viscosity (Visc), and light scattering (LS) in order from left to right.

room temperature for 1-2 h and then heating at ∼7080 °C for 30 min for samples 2, 4, 7, 9, 10, and 11. The latter dissolution protocol gave reproducible results. Comparison of relative responses of viscosity and light scattering versus retention time within each sample showed that peak positions and band shapes were similar to each other as shown for Amdex-M.P. in Figure 2, implying little or no branching in the dextran and aminodextran chains. Lots of 5×-Amdex prepared by the same 500 g dextran scale, the same molar ratio of DAPto-sodium periodate, and the same mode of DAP addition in three equal portions consistently gave molecular mass figures in the 25-45 kDa range. SDS-PAGE Gel Electrophoreses. SDS-PAGE of aminodextrans was run at 400 V (65 V h) on the Pharmacia Phast system, using a 4 to 15 gel gradient for the 30-300 kDa molecular mass range and SDS buffer strips. The use of this system for aminodextrans is made possible by the protonation and, thus, charging of primary amine groups at pH 8.1 of SDS buffer strips and the selective electrostatic interaction between negatively charged sulfonate groups of the widely used Coomassie blue stain (53, 54) and protonated, primary amine groups of aminodextrans. Electrophoresis, applied to the study of synthetic polyelectrolytes, has been shown (55) to provide a more detailed molecular mass distribution than size-exclusion chromatography. We obtained an accurate measure of the size distribution in aminodextrans from the spread of bands in the electrophoretograms. Both dissolution protocols 1 and 2 were used to prepare 1 mg/mL Amdex solutions in 1× PBS buffer, each mixed 1:1 with SDS buffer, which were then run together with a high molecular mass (200, 116, 97, 66, and 45 kDa) standards sample. Gel electrophoresis bands stained with Coomassie blue for the two runs are shown in Figure 3. All the aminodextrans showed well-stained SDS-PAGE gels, indicating the presence of abundant primary amine groups. Molecular weight ranges for the aminodextrans were estimated from the range of the heaviest blue stain in each lane of the gel referenced to the relatively narrow band positions of five standards in the same gel. The 5×-Amdex lots 1-5, 2-2, -11, and 11-6 and 1×-Amdex lot -75 gave molecular mass ranges of about 30-75, 60190, 50-150, 35-250, and 150-250 kDa, respectively, for protocol 1 dissolution, and about 30-75, 55-175, 4090, 30-110, and 150-250 kDa, respectively, for protocol 2 dissolution. The 5×-Amdex lots showed the largest change to lower molecular mass fragments with the more extensive protocol 2 dissolution procedure. Also, the 5×Amdex lot 2-2, which was purified with a 30 kDa Mr


cutoff membrane, showed a significantly higher starting Mr range than other 5×-Amdex lots which were purified with a 5 kDa Mr cutoff hollow fiber cartridge. Thus, other higher MWCO (100, 300, and 500 kDa) membranes that are available can be used to obtain narrower and higher size distributions of aminodextrans where they are required. The average molecular masses obtained from the triple detector system measurements, and the molecular mass ranges obtained from electrophoresis for 1×and 5×-Amdex samples, both show the extensive depolymerization of dextran starting material (2-3 MDa) that occurred during the aminodextran preparation by the periodate oxidation-diamine addition route. Dynamic Light Scattering. Hydrodynamic radii, Rh, were obtained from photon correlation spectroscopy (PCS), also known as quasi-elastic light-scattering (QELS), measurements (56, 57) on the COULTER N4MD analyzer. The N4MD contains a correlator and microprocessor which converts autocorrelator results using Provencher’s program CONTIN to an intensity distribution. A second microprocessor then converts the intensity distribution by the exact Mie theory to give mass average molecular mass. Solutions of either PE, PC5, or ECD as well as their antibody-aminodextran-dye conjugates were measured at concentrations of 5 mg/mL or less in 1× PBS buffer, pH 7.2, with 632.8 nm He/Ne laser excitation. Measurements for freshly buffer-exchanged and purified PE at a concentration of 1.5 mg/mL in 50 mM phosphate, 2 mM EDTA buffer at pH 7.0 gave a mean diameter of 32.7 nm. The scattering angle was 22.8° instead of 90° to improve the intensity of scattered light by a factor of about 16, since PE is small and a poor scatterer which absorbs part of the incident laser light at 632.8 nm. PCS analyses of the peak chromatographic fraction for all three PE, PC5, and ECD conjugates with anti-CD4-Amdex-M.P. in 1× PBS gave the same mean diameter of 146 nm. Scanning Electron Microscopy. More direct evidence for the structure of the conjugates was obtained from scanning electron microscope (SEM) photos taken with a Philips model XL40-FEG instrument. Samples were prepared by filtering about 0.5 mL of conjugate at 0.4 mg/mL in 1× PBS, pH 7.2, through an 0.2 µm polycarbonate membrane filter (Poretics Corp., Livermore, CA) and drying the filter in air in a sterile Petri dish for 3-4 h to overnight. Portions of the sample on the filter were cut and attached to double-sided carbon tape on an aluminum stub. All samples were goldsputter-coated with a Cressington Model 108 auto coater at a 25 sec argon gas plasma cycle time and 7.5 cm platform-to-target distance for a thickness of 15-25 nm. Figure 4 shows images of four conjugate samples prepared in the above way for SEM. Note the globular shape of the conjugates, some of which are located inside the 0.2 µm pores of the membrane while others were retained on the surface of the filter. Hydrated samples, as shown in Figures 5 and 6 for CD4-Amdex-M.P.-PE (fraction 20) and CD8β-Amdex-M.P.-PC5 (fraction 19) conjugates, respectively, were measured within 1 day of sample preparation. This is necessary for retention of globular structure, especially for smaller conjugates with 5×- and 1×-Amdex; otherwise, the structures collapsed to varying degrees. The range of mean diameters from SEM photos is included in Table 3. Flow Cytometry. CD8β-PC5 and fractions of CD8β5×-Amdex-PC5 conjugates from trial 1 were titered with 3-0.5 µg of antibody in the conjugate per tube. Amounts


Siiman et al.

Figure 3. SDS-PAGE results for various aminodextran lots dissolved in 1× PBS buffer solution: (A) protocol 1 dissolution; (B) protocol 2 dissolution.

of antibody for the titers were determined from corrected A280 values for the conjugates. Dilutions were added to 100 µL of whole blood and incubated for 60 min at room temperature. For analysis on a flow cytometer (COULTER EPICS 4-color XL-MCL), the blood mixtures were lysed and quenched using the COULTER ImmunoPrep reagent system on the COULTER Q-Prep workstation, washed once with 1× PBS (addition of 2 mL of 1× PBS, centrifugation at 500 g for 5min, discarding supernatant), and the cell pellet was resuspended in 1 mL of 1× PBS. The lymphocyte cell population was distinguished with forward light scatter vs 90° light scatter, electronically gated, and displayed on single parameter histograms [y-axis ) number of fluorescent events (count); x-axis ) fluorescence intensity]. Flow cytometer instrument settings were adjusted to display the nonstaining negative lymphocyte subpopulation only within the first decade on the 4-decade log scale for fluorescence intensity. Figure 7 shows the ability of CD8β-5×-Amdex-PC5 conjugate as a fluorescent marker containing more than two PC5 molecules per dextran molecule, to enhance the mean channel PC5 fluorescence intensity (MFI) of the positive cell population over that obtained with the direct CD8β-PC5 conjugate which contains only 0.86 PC5/ antibody. The histograms show that the cross-linked conjugate has 3.5-fold higher fluorescence intensity on CD8β+ lymphocytes. In another run, it was found that the mean channel fluorescence intensity of labeled T cells could be enhanced up to 8-fold by examining various fractions of trial 1, CD8β-5×-Amdex-PC5 conjugate as the fluorescent marker. Titers of the control, CD8β-PC5,

and the sample, CD8β-5×-Amdex-PC5, with the same instrument settings were run and the results, mean channel PC5 fluorescence intensities and MFI ratios, sample fraction-to-control at same titers, are presented in Table 4 for another donor. MFI ratios corrected for the F/P ratio, 0.860, of the control, direct CD8β-PC5 conjugate, were about 16-17% higher than values listed in Table 4. CD4-ECD and a single pooled sample of CD4-5×Amdex-ECD conjugates were titered with 0.5-0.015 µg (based on the corrected A280 value for each conjugate) per tube in the same way as in the previous example. The results are shown in Figure 8 for the positive population mean channel ECD fluorescence intensities (MFI) for the CD4+ lymphocytes with the direct and cross-linked antibody-ECD conjugates. The MFI ratios, maximizing at 9.7 were calculated from the mean channel positions of each 5×-Amdex sample compared to the saturating bottle product dose of CD4-ECD (0.5 µg). Using the F/P ratio of 0.96 for the CD4-ECD conjugate, the corresponding maximum MFI ratio is 10.1. CD4 antibody conjugates were prepared to obtain an enhanced fluorescence standard with a narrow distribution of fluorescence intensities for positive lymphocytes. To 100 µL of whole blood from a single normal blood donor, 0.5 µg each of the CD4-PE control conjugate and the CD4-5×-Amdex-PE fraction samples were added and mixed for 30 min at room temperature. Concentrations of CD4 antibody in the conjugates were based on corrected A280 values. The samples were prepared for flow cytometric analyses (FCM) as before. The amplification



Figure 4. Scanning Electron Micrographs of CD4-Amdex, M.P.-PE, top, left; CD4-5×-Amdex(lot 1-5)-PE, bottom, left; CD4-1×Amdex(lot-75), top, right; and CD8β-5×-Amdex(lot-11)-PC5 conjugates. The instrument was operated with the secondary emission detector and 3-5 kV electron beam intensity. Magnification was 13697×, 54913×, 88829×, and 14479×, respectively, on original photo, but should otherwise be determined from bar length.

Figure 5. Scanning electron micrograph of CD4-Amdex, M.P.PE conjugate, pooled and concentrated fractions 19 and 20.

Figure 6. Scanning electron micrograph of CD8β-Amdex, M.P.-PC5 conjugate, fraction 19.

of the mean channel PE fluorescence intensities relative to the control, obtained at the same instrument settings, is shown in Table 5. The maximum positive amplification ratio was 4.7; however, the negative population of MFI was also amplified, although not to the same extent. The signal-to-noise (S/N) amplification, which encompasses both the positive and negative amplification ratios, was approximately 2.0. Both the amplified and nonamplified CD4 antibodies detected similar percentages of CD4+ and CD4- lymphocytes. Maximum amplification ratios of 8.5, 4.2, and 2.2 occurred in the CD4,PE; CD4,ECD; and CD8β,PC5 conjugates with Amdex-M.P. (fractions 19, 21, and 19, respectively) as shown in Tables 6-8. These fractions coincided with the lower fraction side (higher molecular mass) of the first intense band observed in chromatog-

raphy of conjugation mixtures. The CD4-Amdex-PE conjugate demonstrated the highest amplification ratio ever obtained for a CD4, PE conjugate with any lot of aminodextran. Also, this ECD conjugate is the first one to show excellent yield in the first band as well as the highest amplification ratio of any ECD conjugate. Also notable in Tables 6-8 is that the Amdex M.P. gives low negative amplification ratio values, and thus higher S/N amplification ratios, as compared to the 5×-Amdex values in Table 5. A degree of substitution of ∼1/140 with a single amino group per reacted glucose unit was shown to be sufficient to obtain MCA-Amdex-PE (PC5, ECD) conjugates in good yields. For CD4-Amdex-PE conjugates, AmdexM.P. has provided the highest amplification of fluorescence intensity over that obtained with direct CD4-PE


Siiman et al.

Table 3. Mean Diameters of PE and Its Conjugates and Molar Ratios of PE:Aminodextran mean diameter by SEM diameter MW mole fraction PE/Amdex PE/Amdex PE/Amdex QELS (nm) range (nm) (kDa) [PE/(PE + Ab)] by packing by MW by flow cytometry

species PE CD4-1X-Amdex-PE CD4-Amdex-PE CD4-5X-Amdex (lot -11)-PE CD4-5X-Amdex (lot 1-5) -PE CD8β-5X-Amdex(lot -11)-PC5

27.7 90.2 146 116 57.4 153

36-71 69-172 34-69 67-200

270 2556 6697 4227 1035

0.553 0.488 0.401 0.560

1.2 23 0.16 0.39

6.3 8.4 7.8 2.6

2.4 8.2 4.2 4.4

Figure 7. Flow cytometric histograms of CD8β-PC5 and CD8β-5×-Amdex-PC5 conjugates mixed with whole blood. 100 µL of whole blood from a normal donor was stained with 10 µL (2.5 µg) of CD8β monoclonal antibody either as (A) a CD8βPC5 or (B) a CD8β-5×-Amdex-PC5 conjugate. The fluorescence intensity of a light scatter-gated lymphocyte sub-population is displayed as histograms where the y-axis represents the number of fluorescent events (count), and the x-axis shows fluorescence intensity. The nonstaining negative cell population is seen in the left quarter of the histogram. Table 4. Flow Cytometric Data for CD8β-5×-Amdex-PC5 versus Control, CD8β-PC5 CD8β in sample/ control control(µg) CD8β-PC5

sample fraction no. CD8β-5X-Amdex-PC5 36 37 38 39 40 41 42 43

Positive Mean Channel PC5 Fluorescence Intensities (MFI) 3 53 265 325 379 419 376 377 381 2.5 53 282 303 352 334 335 357 368 2 55 236 266 302 293 293 319 320 1.5 51 194 226 252 240 240 213 292 336 1 50 174 196 178 183 183 166 216 262 0.5 51 93 130 134 132 132 155 117 177 Positive MFI Ratios (Fraction/Control) 3 1.0 5.0 6.1 7.1 7.9 7.1 7.1 7.1 7.1 2.5 1.0 5.3 5.7 6.6 6.3 6.3 6.7 6.9 2 1.0 4.3 4.8 5.5 5.3 5.7 5.8 5.8 1.5 1.0 3.8 4.4 4.9 4.7 4.2 5.2 5.7 6.6 1 1.0 3.5 3.9 3.6 3.7 3.3 4.0 4.3 5.3 0.5 1.0 1.8 2.5 2.6 2.6 3.0 2.2 2.3 3.5

conjugate in labeling CD4+ cells in whole blood. Flow cytometry histograms of the number of fluorescence events versus mean channel fluorescence intensity are shown in Figure 9 for CD4-PE versus various CD4Amdex-PE-labeled lymphocytes of the same blood donor. Good separation between the direct conjugate and each Amdex-cross-linked conjugate was obtained. Also, no interference from CD4 negative populations ever was observed. Similar histograms for CD4-ECD versus CD4Amdex-ECD are shown in Figure 10. A graph comparing CD4,PE with carriers of various molecular masses is shown in Figure 11, and shows the direct, linear proportionality between amplified fluorescence intensity and molecular mass of carrier only for the direct conjugate and conjugates with 1×-Amdex and Amdex-M.P. Both cross-linked, 5×-Amdex conjugates with CD4 antibody and PE show much higher amplification than predicted from the linear plot in Figure 11 for linear chain aminodextrans of low degree of amino substitution

Figure 8. Flow cytometric results in tabulated and graphical form: positive population mean fluorescence intensity (MFI) data for direct CD4-ECD and CD4-5×-Amdex-ECD conjugates mixed with 100 µL whole blood at various titers of 0.015-0.5 µg of CD4.

and for the direct conjugate. Possibly, a more globular structure and higher degree of amino substitution in 5×-Amdex, albeit cross-linked, allows formation of multiply cross-linked complexes of the type -Amdex-MCA, PE-Amdex-, which, as a single unit in binding to antigenic sites on biological cells can yield greater amplification of fluorescence intensity. The broad range of yields of MCA-5×-Amdex-PE, PC5, or ECD conjugates and their much greater sensitivity to the nature of the antibody that was used in the conjugations, point to the formation of multimeric complexes. Furthermore, several trials with aminodextran-crosslinked conjugates, streptavidin-Amdex-PE and streptavidin-5×-Amdex-PE, were used as indirect secondary reagents with biotinylated CD4 primary antibody to label and analyze for CD4+ cells in whole blood by flow cytometry. Titers between 0.25 and 5.0 µg of protein in the peak fraction 21 of lot 9-15, streptavidin-AmdexPE conjugate with 100 µL of whole blood (previously stained with biotinylated CD4 antibody and washed once), were lysed, quenched, washed, and analyzed. These are shown in the overlaid histograms of Figure 12A. A plateau indicating saturation of CD4+ receptors in the sample was reached in mean channel fluorescence intensity at a titer of 2.5 µg. The Figure 12 histograms for different fractions of conjugates containing either (B) Amdex-M.P. or (C) 5×-Amdex, lot -11, showed that all fractions were equally good at providing fluorescence intensity, i.e., within each series of fractions the number of PE molecules per conjugate molecule was about the same. When compared against trials with 2.5 µg amounts



Table 5. Flow Cytometric Amplification Data for CD4-5×-Amdex-PE Conjugate Compared to Direct CD4-PE Conjugatea % fraction

neg. pop. MFI

pos. pop. MFI

S/N

S/N ampl. ratio

neg. ampl. ratio

pos. ampl. ratio

neg

pos

CD4-5X-Amdex-PE 21 22 23 24 25 26 direct conjugate CD4-PE

0.42 0.39 0.42 0.47 0.60 0.33 0.19

271 301 334 332 343 290 73

651 769 794 715 569 870 376

1.7 2.0 2.1 1.9 1.5 2.3 1.0

2.2 2.0 2.2 2.4 3.1 1.7 1.0

3.7 4.1 4.6 4.6 4.7 4.0 1.0

66 68 64 65 63 64 64

34 32 35 35 37 36 36

a MFI ) mean fluorescence intensity; positive (pos.) amplification (ampl.) ratio ) pos. population (pop.) MFI Amdex conjugate:pos. pop. MFI direct conjugate; negative (neg.) amplification ratio ) neg. pop. MFI Amdex conjugate:neg. pop. MFI direct conjugate; S/N ) MFI pos. pop./MFI neg. pop.; S/N ampl. ratio ) S/N Amdex conjugate:S/N direct conjugate; % neg. ) percentage of negative nonstaining cells displayed on histogram; % pos. ) percentage of positive cells displayed on histogram.

Table 6. Flow Cytometric Data for CD4-Amdex-PE Conjugate Compared to Direct CD4-PE Conjugatea % fraction

neg. pop. MFI

pos. pop. MFI

S/N

S/N ampl. ratio

neg. ampl. ratio

pos. ampl. ratio

neg

pos

CD4-Amdex-PE 18 19 21 22 23 24 CD4-PE

0.47 0.51 0.44 0.37 0.42 0.43 0.24

501 624 508 426 351 319 73

1072 1226 1168 1157 837 738 300

3.6 4.1 3.9 3.9 2.8 2.5 1.0

1.9 2.1 1.8 1.5 1.7 1.8 1.0

6.9 8.5 7.0 5.8 4.8 4.4 1.0

46 45 45 55 45 51 46

54 55 55 45 55 49 54

a

For headings, see footnote to Table 5.

Table 7. Flow Cytometric Data for CD4-Amdex-ECD Conjugate Compared to Direct CD4-ECD Conjugatea % fraction

neg. pop. MFI

pos. pop. MFI

S/N

S/N ampl. ratio

neg. ampl. ratio

pos. ampl. ratio

neg

pos

CD4-Amdex-ECD 19 20 21 22 23 24 25 26 27 28 CD4-ECD

0.21 0.23 0.24 0.24 0.24 0.26 0.26 0.22 0.22 0.21 0.22

129 240 243 228 200 191 189 150 146 133 58

624 1032 1016 936 823 733 722 690 674 624 272

2 4 4 3 3 3 3 3 2 2 1

1.0 1.1 1.1 1.1 1.1 1.2 1.2 1.0 1.0 1.0 1.0

2.2 4.1 4.2 3.9 3.4 3.3 3.2 2.6 2.5 2.3 1.0

63 63 62 63 63 64 62 63 63 63 62

37 37 38 37 37 36 38 37 37 37 38

a


Table 8. Flow Cytometric Data for CD8β-Amdex-PC5 Conjugate Compared to Direct CD8β-PC5 Conjugatea % fraction

neg. pop. MFI

pos. pop. MFI

S/N

S/N ampl. ratio

neg. ampl. ratio

pos. ampl. ratio

neg

pos

CD8β-Amdex-PC5 18 19 20 21 22 23 CD8β-PC5

0.18 0.20 0.19 0.20 0.21 0.26 0.15

52 102 97 95 92 82 47

280 519 498 480 436 313 307

0.9 1.7 1.6 1.6 1.4 1.0 1.0

1.2 1.3 1.3 1.3 1.4 1.7 1.0

1.1 2.2 2.1 2.0 2.0 1.8 1.0

82 82 81 82 82 82 83

18 18 19 18 18 19 17

a


of avidin in the direct conjugate, avidin, NeutraLite -PE, the streptavidin-Amdex-PE conjugates gave about 8-11fold greater fluorescence intensity from labeled CD4+ lymphocytes as shown in Figure 13 for four runs. DISCUSSION

Data from Table 2 were further used to construct a Mark-Houwink plot of log(η) versus log(Mw), shown in Figure 14. Linear regression analysis for all data gave a correlation coefficient of 0.976 and a slope of 0.451. The

slope corresponds to the exponent in the equation, [η] ) KMa , where K is the proportionality constant. In the above samples, the value of the exponent is very similar to the a ) 0.50 btained (58) for linear dextran (20-100 kDa) in water at 25° C. This value of the exponent has been established for linear, flexible polymers under “theta” temperature or solvent conditions, whereas the branched fraction of dextran of 800 kDa in water at 25° C gave a ) 0.20. A similar plot of log(Rg) versus log(Mw) for all data gave a correlation coefficient of 0.998 and a


Siiman et al.

Figure 9. Comparison of flow cytometric histograms of direct CD4-PE and various CD4-Amdex-PE conjugates, 1X ) 1×-Amdex, Amdex M.P., and two lots of 5×-Amdex, mixed with 100 µL of whole blood. The fluorescence intensity of a light scatter-gated lymphocyte subpopulation is displayed as histograms where the y-axis represents the number of fluorescent events (count), and the x-axis shows fluorescence intensity. The nonstaining negative cell population is seen in the left quarter of the histogram.

Figure 10. Comparison of flow cytometric histograms of direct CD4-ECD and CD4-Amdex, M.P.-ECD conjugates mixed with 100 µL of whole blood. The fluorescence intensity of a light scatter-gated lymphocyte subpopulation is displayed as histograms where the y-axis represents the number of fluorescent events (count), and the x-axis shows fluorescence intensity. The nonstaining negative cell population is seen in the left quarter of the histogram.

slope of 0.481. We conclude, therefore, that the aminodextrans behave in aqueous sodium nitrate solution as

Figure 11. Graph of the positive amplification ratio [MFI (CD4-Amdex-PE)/MFI (CD4-PE)] for CD4 conjugates labeling CD4 positive lymphocytes in whole blood versus molecular mass for various aminodextrans.

flexible, linear chains arranged in a more relaxed, globular structure than the compact globular structures assumed by typical proteins. The positive charges of primary amino groups in 5×-Amdex are not sufficient to


Figure 12. Flow cytometric histograms of streptavidinAmdex-PE conjugates mixed with 100 µL of whole blood pretreated with biotinylated CD4 antibody. (A) Amdex, M.P. conjugate, lot 9-15, fraction 21 mixed in titers of 0.25, 0.5, 2.5, and 5 µg of protein. (B) Amdex, M.P. conjugate, lot 9-11, fractions 19, 20, 21, 22, and 23, all mixed individually at 2.5 µg of streptavidin. (C) 5×-Amdex conjugate, lot 9-15, fractions 21, 22, 23, 24, and 25, all mixed individually at 2.5 µg of streptavidin. The fluorescence intensity of a light scatter-gated lymphocyte subpopulation is displayed as histograms where the y-axis represents the number of fluorescent events (count), and the x-axis shows fluorescence intensity. The nonstaining negative cell population is seen in the left quarter of the histogram.

force aminodextrans to assume an extended, straight chain structure. The radius of gyration for a randomly coiled, linear, polymer molecule has been derived from random walk statistics as Rg2 ) 1/6 nl2, where n is the number of statistical segments in the polymer chain and l is the length of each statistical segment. Also, the mean square end-to-end distance, L2, in a random coil is given by L2 ) nl2, (59). Thus, the Rg and mass average molecular mass values obtained from light scatter and viscosity, and elemental analyses (empirical formulas and degree of substitution) can be used to calculate L and other parameters. Values of Rh, the hydrodynamic radius, were calculated from Rg values by using the relationship, Rh ) 0.665Rg, derived for random polymer coils. Statistical segment lengths were 7.5-9.0 Å for the aminodextrans, compared to the length of a single unit of 1,6-linked glucose reported (60) as about 8 Å long. Free rotation about the methylene group between residues of 1,6glucosyl units in aminodextran also confers extra flexibility on the polymer chain in contrast to the rigidity of


Figure 13. Comparison of flow cytometric histograms for avidin, NeutraLite-PE conjugate (vertical dotted line) and streptavidin-Amdex-PE conjugates, both individually mixed with 100 µL of whole blood pretreated with biotinylated CD4 antibody. (A) Amdex, M.P. conjugate, lot 9-15, fraction 21; (B) Amdex, M.P. conjugate, lot 9-11, fraction 21; (C) 5×-Amdex conjugate, lot 9-11, fraction 25; (D) 5×-Amdex conjugate, lot 9-15, fraction 26. Each sample contained 2.5 µg of avidin or streptavidin. The fluorescence intensity of a light scatter-gated lymphocyte sub-population is displayed as histograms where the y-axis represents the number of fluorescent events (count), and the x-axis shows fluorescence intensity. The nonstaining negative cell population is seen in the left quarter of the histogram.

the chain of 1,4-glucosyl units in cellulose nitrate in which glucose residues are joined only by an ether linkage. Light-scattering results and a Zimm plot for cellulose nitrate in acetone (61, 62) gave Mw ) 400 kDa, a statistical segment length of 35 Å or about seven 1,4glucosyl units, and a root-mean-square end-to-end distance, L, of 1500 Å. The longer statistical segment length and high L value confirm that cellulose nitrate has an unusually stiff and extended structure. The above data for randomly coiled polymer, dextran and aminodextrans, have shown them to assume much more tightly coiled and compact structures. Previous studies of dextran of molecular mass greater than 2000 Da also indicated a


Siiman et al.

Figure 14. Mark-Houwink plot of log[intrinsic viscosity, η] versus log[molecular weight, Mw] for various aminodextrans and dextran as compiled in Table 2.

globular structure (60, 63) in contrast to the open, extended structures of fiber and extracellular matrix polysaccharides such as cellulose (61, 62) and proteoglycans (35, 36). Dextran prepared by freeze fixation and detected by electron microscopy (63) showed dextran globules. Direct observation (60) of the polymerization reaction to form dextran in the cell of a light-scattering instrument followed by construction of Zimm plots for dextran solutions showed uniform spheres of 75-85 nm radii of gyration for molecular masses of 64-105 MDa; however, the process of purification of these high molecular mass dextrans invariably gave products insoluble in aqueous media. These results have made it easier to structurally visualize the formation of protein, antibody, and phycobiliprotein, conjugates of aminodextrans by constructing a working model of a central, loose, globular aminodextran core surrounded by a shell of protein molecules. A direct proportionality between the total accessible surface area and molecular mass has been proposed (64) for large protein molecules. If this relationship, 4πRh2 ∝ M, is applied to the dextran and aminodextran data of Table 2 using the surface area of a sphere of radius Rh in angstroms, we find that 4πRh2 ) 0.366 M on the average for 11 measurements. This suggests that the total accessible surface area, 4πRh2/0.366, of aminodextrans in random coil configurations is about 2.7 times greater than the surface area of a sphere of the same size. This figure is higher than the one for proteins for which the accessible surface area is quoted (64) as being nearly two times greater than that expected for a sphere of the same size. In most 5×-Amdex samples, the average molecular mass determined from the triple detector system measurements fell in the middle of the range shown by SDSPAGE results, even though the position of species in electrophoretograms depends strictly on their charge-tosize ratio. Two factors may have contributed to the high, apparent 150-250 kDa molecular range for 1×-Amdex. First, the molecular mass standards were globular proteins, which are more compact than aminodextrans of the same molecular mass, having a relatively open chain, random coil structure. The gel will be more effective in separating aminodextrans of somewhat lower molecular mass as compared with proteins. Second, aminodextrans of higher positive charge such as 5×Amdex will travel more quickly through the gel compared to aminodextrans of low positive charge such as 1×Amdex. Several mechanisms for depolymerization of dextran dialdehyde by strongly alkaline media may be proposed. The mechanisms involve either of two acidic hydrogen atoms in the periodate cleaved, dialdehyde units of dextran dialdehyde polymer as shown in Figure 15 for each of the acidic hydrogen sites. Mechanism 1, β-car-

Figure 15. Schematic of the depolymerization mechanisms for dextran dialdehyde.

bonyl elimination, appears to be the more favored (65, 66) one because of the presence of a carbonyl in β-position to a C-atom involved in the glycosidic linkage, which yields a conjugated CdO, CdC structure in the product. Mean hydrodynamic diameters of PE and conjugates were measured by PCS. Molar ratios, PE/Amdex, were obtained in three different ways (two, by model calculations, and one, from flow cytometry measurements): (1) by calculating the number of hard spheres of PE that can be arranged in a closest packed array around a hard sphere of aminodextran, i.e., surface area of sphere of aminodextran, 4πRh2, divided by d2(PE) × x3/2, and then multiplying by the mole fraction of PE; (2) by subtracting the molecular mass of one aminodextran molecule from the molecular mass ()4πRh2) of the conjugate, then, dividing by the average of the molecular masses of IgG antibody (160 kDa) and PE (270 kDa), and finally multiplying by the mole fraction of PE; and (3) by taking the ratio of mean channel fluorescence intensity of the CD4-aminodextran-PE conjugate to the mean channel fluorescence intensity of the direct CD4-PE conjugate, both measured by flow cytometry at saturation of the receptor sites on the targeted CD4+ lymphocytes in whole blood. Molecular masses (M) of the corresponding PE conjugates listed in Table 3 were calculated from hydrodynamic diameters determined from PCS measurements by assuming the relationship, 4πRh2 ) M with radius Rh in angstroms, for large protein molecules (64). Estimates of the PE/Amdex ratios by packing and by molecular mass differ most widely from each other for 5×-Amdex conjugates. When the PE/Amdex ratio was less than 1 or close to 1 as with 5×-Amdex and 1×-Amdex conjugates, the size of the Amdex molecule was smaller than the size of either PE or antibody molecule. In these cases, the smaller Amdex (25-94 kDa) molecules will tend to pack around a larger, central PE (270 kDa) or antibody (160 kDa) molecule. Further, amplification factors exceed packing limits around a single, unassociated 5×-Amdex molecule for the CD4-5×-Amdex-PE conjugates and, thus, suggest that more than one 5×-Amdex molecule is part of the conjugate. This allows a network of 5×-Amdex-MCA, PE-5×-Amdex to form and contain additional PE molecules. The most consistent amplification ratios were obtained in conjugates with Amdex-M.P., which has a low degree of amine substitution and very large size. The main criteria for success in obtaining good yields of antibody, fluorescent dye conjugates with aminodex-


tran, that produce large enhancements in fluorescence intensity of targeted cells appear to be the following: (1) use of the highest molecular mass possible for the aminodextran carrier while still retaining solubility in aqueous media; (2) a low degree of amine substitution (1/140) in dextran of 3 MDa still allows conjugation of antibody and phycobiliproteins; however, the percentage of saturation coverage of the aminodextran carrier may be low; (3) more highly substituted 5×-Amdex (1/5 to 1/8) allows a greater packing density of protein, and also the possibility of network structures containing even more aminodextran and phycobiliprotein. With the advances now being made in microfluidics (67-69), it will soon be possible to use these large conjugates to perform fluorescence-activated sorting of targeted molecules instead of just micron size objects such as biological cells and particles. Development of new detection and sequencing methods for nucleic acids could arise from using polymeric carriers such as aminodextrans with conjugated oligo and fluorescent dye. LITERATURE CITED (1) Lee, Y. C., and Lee, R. T. (1992) Synthetic Glycoconjugates. In Glycoconjugates. Composition, Structure and Function (H. J. Allen and E. C. Kisailus, Eds.) pp 121-165, Marcel Dekker, Inc., New York. (2) Manabe, Y., Tsubota, T., Haruta, Y., Kataoka, K., Okazaki, M., Haisa, S., Nakamura, K., and Kimura, I. (1984) Production of a Monoclonal Antibody-Methotrexate Conjugate Utilizing Dextran T-40 and Its Biologic Activity. J. Lab. Clin. Med. 104, 445-454. (3) Oseroff, A. R., Ohuoha, D., Hasan, T., Bommer, J. C., and Yarmush, M. L. (1986) Antibody-Targeted Photolysis: Selective Photodestruction of Human T-Cell Leukemia Cells Using Monoclonal Antibody-Chlorin e6 Conjugates. Proc. Natl. Acad. Sci. U.S.A. 83, 8744-8748. (4) Rakestraw, S. L., Tompkins, R. G., and Yarmush, M. L. (1990) Antibody-Targeted Photolysis: In Vitro Studies with Sn(IV) Chlorin e6 Covalently Bound to Monoclonal Antibodies Using a Modified Dextran Carrier. Proc. Natl. Acad. Sci. U.S.A. 87, 4217-4221. (5) Wong, S. S. (1991) Chemistry of Protein Conjugation and Cross-Linking, p 279, CRC Press, Inc., Boca Raton, FL. (6) Hermanson, G. T. (1996) Bioconjugate Techniques, pp 618629, Academic Press, San Diego, CA. (7) Aslam, M., and Dent, A. (1998) Bioconjugation-Protein Coupling Techniques for the Biomedical Sciences, Chap. 8, Grove’s Dictionaries Inc., New York. (8) Maeda, H., Seymour, L. W., and Miyamoto, Y. (1992) Conjugates of Anticancer Agents and Polymers: Advantages of Macromolecular Therapeutics in Vivo. Bioconjugate Chem. 3, 351-362. (9) Brinkley, M. (1992) A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Cross-Linking Reagents. Bioconjugate Chem. 3, 2-13. (10) Brunswick, M., Finkelman, F. D., Highet, P. F., Inman, J. K., Dintzis, H. M., and Mond, J. J. (1988) Picogram Quantities of Anti-Ig Antibodies Coupled to Dextran Induce B Cell Proliferation. J. Immunol. 140, 3364-3372. (11) Mongini, P. K. A., Blessinger, C. A., Highet, P. F., and Inman, J. K. (1992) Membrane IgM-Mediated Signaling of Human B Cells. J. Immunol. 148, 3892-3901. (12) Marshall, J. J., and Rabinowitz, M. L. (1976) Preparation and Characterization of a Dextran-Trypsin Conjugate. J. Biol. Chem. 251, 1081-1087. (13) Kato, A. (1996) Preparation and Functional Properties of Protein-Polysaccharide Conjugates. In Surface Activity of Proteins: Chemical and Physicochemical Modifications (S. Magdassi, Ed.) Chap. 5, pp 115-129, Marcel Dekker, Inc., New York. (14) Zhao, Q., Gottschalk, I., Carlsson, J., Arvidsson, L.-E., Oscarsson, S., Medin, A., Ersson, B., and Janson, J.-C. (1997)

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