Antibody-Dextran
Method C Preparation of 5X-Aminodextran
T-2M dextran (50 g, 0.308 mol, obtained from Sigma, St. Louis, Mo., or Pharmacia, Piscatawny, N.J.) was added to a 1-quart or 1-liter glass blender bowl containing 300 ml of distilled water. The mixture was blended at maximum speed until all the dextran dissolved, typically about 3-5 minutes. A solution of 26.75 g (0.125 mol) of NaIO4 in 300 ml distilled water was added to the dextran solution over about a 10 minute period using vigorous magnetic stirring. After the periodate addition was completed, the reaction mixture was stirred at room temperature for about an additional three hours. After the three hours, the 600 ml reaction volume had an initial specific conductivity of 9.7 mmho-cm-1 and an initial pH of 2.5. The reaction mixture was diluted to two liters with distilled water and desalted using the hollow fiber cartridge. Washing was done using 15-18 liter of distilled water to obtain 600 ml of washed, oxidized dextran solution having a specific conductance of 10 μmho-cm-1 and pH of 6.7.
The solution of oxidized dextran was cooled to about 8° C. using an ice bath and 23.2 ml (0.275 mol) of 1,3-diaminopropane was added over about 10 minutes to the oxidized dextran solution. The resulting reaction mixture was stirred and maintained at the ice bath temperature. The formation of the yellow Schiff's base was monitored ever 10-15 minutes by measuring the 335 nm near-UV absorbance of an extracted sample. In a typical experiment, the measurements at 335 nm using a 1 mm path length cell were:
TABLE 1______________________________________minutes absorbance values______________________________________ 0 0.100 5 2.06315 2.97530 3.69245 3.90160 4.10375 3.784______________________________________
After the absorbance had reached a plateau, 19.3 g (0.500 mol) of sodium borohydride in 19.3 ml of 1 mM aqueous potassium hydroxide were added to the reaction mixture over about 10 minutes at ambient room temperature using magnetic stirring. After the sodium borohydride addition was completed, the reaction mixture was stirred at ambient room temperature for about an additional two hours. After the stirring was completed, spectroscopic measurement at 335 nm using a 1 cm path length cell gave an absorbance value of 0.067 units which indicates that the Schiff's base compound had essentially disappeared. The reaction mixture, about 1000 ml volume, was then desalted using the hollow fiber cartridge. The initial specific conductance was 43 mmho-cm-1 and the initial pH was 11.0. About 18-20 liters of distilled water were used as wash liquid to produce about 300 ml of 5X-aminodextran solution having a specific conductance of about 4-6 μmho-cm-1 and a pH of 6.5-7.0. The 5X-aminodextran solution was filtered through a 0.2 μm cellulose nitrate filter and freeze-dried over 48 hours in a model TDS-00030-A, Dura-Dry® microprocessor-controlled freeze-dryer (FTS Systems, Inc.) to produce 24 g (48% yield) of flaky, pale yellow crystals. Elemental analysis: C=45.83%, H=7.00%, N=4.49%, O (by difference)=42.68%. Calculated analysis for C12 H22 O8.25 N: C=46.15%, H=7.10%, N=4.48%, 0=42.26%.
The empirical formula based on actual analysis is C12 H22 O8.3 N, which is very similar to the formula C12 H22 O8.25 N based on 6 units of glucose per one unit of fully diamine-substituted sugar ring (C12 H28 N4 O3). Therefore, the degree of diamine substitution in dextran was 1/7 in contrast to a theoretical value of 1/2.5 based on 100% periodate cleavage and diamine substitution.
Repeat experiments using an initial charge of 100 g and 300 g dextran produced a 5X-Amdex product having a similar degree of substitution. The preparation of 1X-Amdex was also scaled up to the 100 g and 300 g levels using Method C. At the 300 g scale the hollow fiber cartridge was changed to a polysulfone membrane of 8.5 ft.3 surface area, 1 mm diameter fibers having a 5,000 nominal molecular weight cut-off (model UFP-5-E-35, A/G Technology Corp.). The cartridge was used for ultrafiltration of both the oxidized dextran and the final aminodextran product to remove salts and low molecular weight reagents or by-products. At the 300 g scale, the yields of the 5X-Amdex and 1X-Amdex products were 135 g (45%) and 162.1 g (57%), respectively. The elemental analysis of the products were:
1X-Amdex, 300 g dextran scale.
Obsd.: C, 43.58; H, 6.50; N, 0.82; O (by difference), 49.10 Calculated for C62 H105 NO50 ·2H2 O: C, 43.79; H, 6.46; N, 0.82; O, 48.92
5X-Amdex, 300 g dextran scale.
Obsd.: C, 45.67; H, 6.90; N, 4.04 O (by difference), 43.39 Calculated for C13 H24 O9 N: C, 46.15; H, 7.15; N, 4.14; O, 42.56
The empirical formula obtained for 5X-Amdex, C19 H24 NO9, is very similar to the formula C13.2 H24 NO9.5 based on 68 units of glucose and one unit of fully diamine-substituted sugar ring. The degree of substitution of sugar residues in dextran was, therefore, about 1/8. The empirical formula C62 H110 NO52 obtained for the 1X-Amdex is similar to the formula C62 H105 NO50 ·2H2 O based on 39.3 units of glucose, one unit of fully diamine-substituted sugar ring and two units of water. The degree of substitution of sugar residues in the dextran was, therefore, about 1/40.
II. Preparation of Anti-CD3 Antibody-Aminodextran Conjugate
1. Activation of aminodextran with sulfo-SMCC.
25 mg of 1X-Amdex and 5X-Amdex were dissolved in 6.667 ml portions of 1X PBS in separate, sealable 15 ml tubes to give solutions having a concentration of 3.75 mg/ml. 1X PBS is made by dilution of a 20X PBS solution comprising 53.8 g K2 HPO4, 12.8 g KH2 PO4 and 340 g NaCl in 2 L distilled water as described in copending application Ser. No. 07/961,057. The 1X-Amdex was activated by the addition of 13.5 μL of 10 mg/ml sulfo-SMCC solution per milliliter 1X-Amdex solution (a total of 0,090 ml sulfo-SMCC solution). The 5X-Amdex was activated using five times the amount of sulfo-SMCC solution (0.450 ml). The sulfo-SMCC was pipetted into the respective tubes, vortexed to mix well and then roller mixed for about two hours. After the mixing was completed, each reaction mixture was chromatographed on a separate 100 ml G-50 Sephadex® column (2.5 cm×20 cm) equilibrated with 1X PBS. The samples were eluted using 1X PBS and collected in about 4 ml fractions. Fractions of the first band absorbing at 280 nm contain the high molecular weight activated aminodextran as was verified by Tyndall scatter with a focused light beam. These fractions were pooled to give about 10-11 ml total sulfo-SMCC activated aminodextran in each case. A second, larger band eluted from the column gave no Tyndall scatter. This second band was determined to contain excess low molecular weight sulfo-SMCC reagent.
2. Activation of Antibody.
T3 monoclonal antibody (an anti-CD3 antibody sold by Coulter Corporation) was activated by the addition of 1.423 ml of T3 concentrate (35.14 mg/ml) to a solution comprising 0.323 ml of 2 mg/ml iminothiolane in 1X PBS and 1.587 ml 1X PBS. The resulting solution which had an antibody concentration of 15 mg/ml and an iminothiolane molar concentration fifteen-fold larger was mixed at ambient temperature for about one hour. The entire reaction mixture was then applied to a 100 ml G-50 Sephadex® column equilibrated with 1X PBS and the sample was eluted using 1X PBS. First band peak fractions of about 5 ml volume were combined to give about 10.2 ml of 4.887 mg/ml antibody solution which contains a total of 49.847 mg iminothiolane-derivatized T3 antibody (abbreviated as IT-T3).
3. Conjugation of sulfo-SMCC-aminodextran and iminothiolane-T3 antibody.
5 ml of 4.887 mg/ml IT-T3 solution (about 24.435 ml antibody) were mixed with 10.4 ml of sulfo-SMCC-1X-Amdex (about 25 mg 1X-Amdex) solution for about two hours. In a like manner, 5 ml of 4.887 mg/ml IT-T3 solution were mixed with 11.2 ml of sulfo-SMCC-5X-Amdex (about 25 mg 5X-Amdex) solution. After the mixing was ended, the total volume of each mixture was determined and 0.120 times this volume of 5 mg/ml L-cysteine in 1X PBS was added to each conjugation mixture. The L-cysteine containing mixtures were then mixed for an additional 15 minutes to effect blocking of any unreacted sulfo-SMCC moleties. Lastly, 20 mg/ml iodoacetamide in 1X PBS solution in the amount of 0.120 times the total mixture volume and 1M 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 minutes to block any unreacted sulfhydryl groups.
4. Purification of anti-CD3-aminodextran conjugates.
The total volume of each conjugation mixture was reduced to about 7.5 ml by centrifuging Amicon Centripep-30 tubes containing the samples for about 20 minutes at about 2500 rpm using a refrigerated Beckman J-6B centrifuge. After centrifuging, the reduced volume mixtures were placed on a Bio-Gel® A-5m agarose column (2.5 cm×48 cm) equilibrated with 1X PBS and chromatographed using 1X PBS as eluent. Eluent fractions of about 4 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 broad band eluted from the column contained the T3 antibody-aminodextran conjugate. A well-separated weaker band of less than half the intensity of the first band was attributed to excess IT-T3 since it showed no Tyndall effect and it had the same retention time on the column as the γ-globulin (Bio-Rad®) standard for gel filtration columns. A strong, well separated third band was assigned to low molecular weight excess blocking reagents. Comparing conjugates made with 1X-Amdex and 5X-Amdex, the T3 antibody-5X-Amdex conjugate had a narrower first band which showed a longer retention time and a very weak second band.
The fractions collected for each conjugate were analyzed at 280 nm using a 1 cm path length cell. Those fractions with an absorbance greater than 0.160 were pooled. The yields were 55 ml of T3-1X-Amdex conjugate having a T3 antibody concentration of 0.301 mg/ml and 54 ml of T3-5X-Amdex conjugate having a T3 antibody concentration 0.317 mg/ml. The results indicate that 66% and 68%, respectively, of the starting antibody was incorporated into the conjugate products. ELISA solid phase assays for T3 in the conjugates gave a T3 antibody concentration of 0.045 mg/ml in T3-1X-Amdex and 0.112 mg/ml in T3-5X-Amdex. The lower ELISA T3 values relative to the A280 values might indicate that either that there is some blocking of active T3 antibody sites by conjugation to aminodextran or that there is some interference with the A280 reading by an extra absorbance contribution arising from the sulfo-SMCC reagent.
The T3-1X-Amdex and T3-5X-Amdex conjugates were also analyzed by light scatter measurements (90°) using photon correlation spectroscopy. The samples were analyzed using a Coulter® N4MD sub-micron particle analyzer operating in the molecular weight and size distribution processor (SDP) weight analysis modes. Samples of dextran T-2M, 1X-Amdex and 5X-Amdex at a concentration of 10 mg/ml were analyzed. The average molecular weights were 3.1×106 daltons (92%, dextran T-2M), 1.0×106 daltons (100%, 1X-Amdex) and 3.5×105 daltons (100% 5X-Amdex). The T3 antibody-aminodextrans prepared above were concentrated to 10 ml total volume before light scatter measurements were made. The average molecular weights were 3.6×106 daltons for T3-1X-Amdex and 1.4×106 for T3-5X-Amdex. Using these results, the T3 antibody:1X-Amdex molar ratio was estimated as (3,600,000-1,000,000) ÷160,000=16:1 and the T3 antibody:5X-Amdex molar ratio was estimated as (1,400,000-350,000)÷160,000=6.6:1.
5. Saturation T3 Antibody Conjugation to Aminodextrans.
The chromatograms of the initial preparation of T3-1X-Amdex and T3-5X-Amdex conjugates showed little, if any, excess T3 antibody when conjugations were done using about 1:1 antibody:aminodextran weight ratios. Consequently, additional experiments were performed using 3:1 T3 antibody:aminodextran weight ratios and the same amount of each aminodextran. The activation, conjugation, blocking and chromatography procedures were the same as those described above. However, during chromatography, a narrow, intense band assigned to excess free IT-T3 trailed the broader T3 antibody-aminodextran band in each case. The IT-T3 band was sufficiently close to the T3-aminodextran bands so that only about half of the first band could be separated in each case. The T3-1X-Amdex preparation thus yielded 75 ml of 0.576 mg/ml T3 in the T3-1X-Amdex solution and 40 ml of 0.466 mg/ml T3 in the T3-5X-Amdex solution. ELISA solid phase assays gave 0.392 and 0.301 mg/ml T3 antibody, respectively. Light scatter results indicate average molecular weights of 6.9×106 daltons and 3.6×106 daltons, respectively. The T3 antibody:aminodextrap molar ratio in the conjugates were estimated as (6,900,000-1,000,000)÷160,000=37:1 for T3-1X-Amdex and (3,600,000-350,000)÷160,000=20:1 for T3-5X-Amdex.
III. Activation of Peripheral Blood T cells With Anti-CD3-Aminodextran Conjugates A. Isolation of Peripheral Blood Mononuclear Cells (PBMC)
The procedure used herein was performed at room temperature. Normal whole blood was collected into tubes containing EDTA (ethylenediaminetetraacetic acid). The tubes were centrifuged at 500 g for 10 minutes. Using sterile techniques, the buffy coat was collected, diluted 1:2 (cell:solvent) with 1X PBS and layered over Ficoll-Hypaque®. The resulting sample was then centrifuged at 400 g for 30 minutes, the PBMC interface was collected and diluted with 1X PBS. The diluted sample was centrifuged at 300 g for 10 minutes and then washed twice with 1X PBS by resuspending the pellet, diluting with 1X PBS and centrifuging at 300 g for 10 minutes. After a final resuspension in 1X PBS, the cells were counted and cell viability was ascertained. The cells were divided into two parts, centrifuged as above and resuspended in the appropriate media for the following steps.
B. Culture and Activation of T Cells with Anti-CD3-Aminodextran
Non-activated cells for use as the control were cultured in Nutricyte® medium containing 10% (v/v) CPSR-2 serum replacement (obtained from Sigma Chemical). The initial cell concentration was 2.5×106 cells/mi. Cultures destined for T cell activation were established in the same medium supplemented with 2-4 ng/ml phorbol 12-myristate 13-acetate (PMA) and 0.125-0.5 μg/ml of anti-CD3-aminodextran. Activated cultures were established using both T3-1X-Amdex and T3-5X-Amdex. The cell suspensions were incubated in T-150 flasks at 37° C., 5% CO2 for 72 hours.
C. Harvesting of Control and Activated Cells
The control cells and the T3-1X-Amdex and T3-5X-Amdex activated cells were harvested by scraping the bottom of the tissue culture flasks with a disposable scraper, collecting the cells with a pipette and placing them in a centrifuge tube. The cells were counted, centrifuged at 300 g for 10 minutes and washed twice with 1X PBS as has been described.
D. Immunofluorescent Staining and Flow Cytometric Analyses of T Cell Activation
The degree of T cell activation was determined by immunofluorescent staining of the cells, followed by flow cytometric analysis of the stained cells for the expression of both activation-associated antigens (CD71, transferrin receptor and CD25, IL2 receptor) and proliferation-associated events (DNA and proliferating cell nuclear antigen, PCNA). Aliquots containing 1×106 cells of each culture were placed in six labelled sample tubes (3 sets of six tubes). One tube in each culture set was stained with the following reagents which are sold by Coulter Corporation.
Tube 1: IgG2a-FITC isotype control
Tube 2: IL-2R1-FITC (IgG2a, CD25)
Tube 3: IgM-FITC isotype control
Tube 4: T9-FITC (IgM, CD71)
Tube 5: IgG1, isotype control and propidium iodide (to label DNA)
Tube 6: PCNA (IgG1) and propidium iodide.
For each set of cells (control, T3-1X-Amdex activated and T3-5X-Amdex activated), tubes 1-4 were processed for cell surface staining by incubating the cells with the appropriate FITC conjugated monoclonal antibody for 15 minutes at room temperature. The incubated cells were then diluted with 1X PBS, centrifuged at 300G for 10 minutes, washed with 1X PBS by suspension and centrifugation and finally resuspended in 1 ml of 1X PBS.
The cells in tubes 5 and 6 were processed for the staining of intracellular antigens by suspending the control cell or activated cell pellets in 1 ml of a solution containing 20 μg/ml lysophosphatidyl choline in 1% paraformaldehyde and incubating the resulting suspensions for 2 minutes at room temperature. After incubation, the cells were resuspended in 1 ml of about -10° C. absolute methanol, incubated on ice for about 10 minutes and centrifuged. The resulting pellet was then incubated in 1 ml of 0.1% NP-40 (available from Sigma Chemical) for about 5 minutes at about 0° C., centrifuged and then incubated again for about 15 minutes at room temperature with IgG1, (tube 5) or PCNA (tube 6) monoclonal antibody. The cells were washed in 1X PBS and incubated for an additional 15 minutes at room temperature with goat anti-mouse immunoglobulin conjugated to FITC. After washing in 1X PBS, 1 ml of propidium iodide was added to each tube.
B. Preparation of Antibody-Aminodextran-Phycobiliprotein Conjugates
[0100]
The procedure was similar to the described in U.S. Pat. No. 5,791,741, incorporated by reference herein, to prepare antibody-Aminodextran-PE conjugates. Herein, an iminothiolane-activated streptavidin or dithiothreitol(DTT) (Sigma)-activated monoclonal antibody and, then, an iminothiolane-activated fluorescent protein, PE, PC5, or ECD, were conjugated stepwise to the sulfo-SMCC-activated aminodextran. The IT to SA, DTT to MCA, and IT to PE (PC5, ECD) molar ratios were maximized at 15, 50, and 22.5, respectively, so as not to interfere with SA or MCA activity, or with PE (PC5, ECD) fluorescence intensity.
[0101]
C. CD8β Antibody-Aminodextran-PC5 Conjugate
[0102]
Conjugation was carried out with molar ratio of 43:4.3:1=PC5:CD8β antibody:purified aminodextran-MP8 of Example 1. The weight ratio was 12.856:0.858:10 at a 10 mg aminodextran scale.
[0103]
Aminodextran was activated with sulfo-SMCC as follows: 1.58 mL of a 6.63 mg/mL solution of Aminodextran in 1×PBS were activated with 0.180 mL of 10 mg/mL sulfo-SMCC solution in 1×PBS. The mixture was roller mixed for about one hour 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 aminodextran as was verified by Tyndall scatter with a focused visible light beam (Model 650, Cambridge Instruments, Inc., Buffalo, N.Y.). These fractions were pooled to give about 3.3 mL total sulfo-SMCC-activated aminodextran.
[0104]
CD8β monoclonal antibody was activated by the addition of 0.048 mL of a 5 mg/mL solution of DTT in 1×PBS, 2 mM EDTA and 0.871 mL 1×PBS, 2 mM EDTA to 0.081 mL of CD8β concentrate (61.67 mg/mL). The resulting solution, which had an antibody concentration of 5 mg/mL and a DTT molar concentration fifty-fold larger, was mixed at ambient temperature for about one hour. The reaction mixture was then chromatographed on a 60 mL (1.7 cm×28 cm) G-50 Sephadex column equilibrated with 1×PBS, 2 mM EDTA and the sample was eluted using 1×PBS, 2 mM EDTA. The first band peak fraction yielded about 1.0 mL of 3.722 mg/mL antibody solution, which contained a total of 3.72 mg DTT-activated CD8β antibody derivative.
[0105]
Conjugation of DTT- CD8β to sulfo-SMCC-aminodextran occurred by reacting 0.858 mg MCA, a 10 mg aminodextran weight ratio, and adding 0.231 mL of 3.72 mg/mL DTT-CD8β solution were added into a 3.3 mL of sulfo-SMCC-aminodextran solution. The mixture was roller mixed for 2 hours.
[0106]
PC5 was activated by the addition of 0.116 mL of a 2 mg/mL solution of iminothiolane in 1×PBS and 0.032 mL 1×PBS to 0.302 mL of PC5 concentrate (59.7 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 one hour. 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 4.14 mL of 4.005 mg/mL PC5 at an A565/A280 ratio of 5.623, which contained a total of 16.581 mg IT-PC5.
[0107]
Conjugation of IT-PC5 to the DTT- CD8β+sulfo-SMCC-aminodextran reaction mixture occurred as follows: After mixing DTT- CD8β and sulfo-SMCC-Aminodextran for 2 hours, 3.210 mL of 4.005 mg/mL IT-PC5 representing 12.856 mg IT-PC5 was added to the mixture, which was roller mixed overnight for 16-24 hours. 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 the conjugation mixture. The L-cysteine containing mixtures were then mixed for an additional 15 minutes to effect blocking of any unreacted sulfo-SMCC moieties. Lastly, 20 mg/mL iodoacetamide in 1×PBS in the amount of 0.120 times the total mixture volume and 1M 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 minutes to block any unreacted sulfhydryl groups.
[0108]
Purification of CD8β-Aminodextran-PC5 conjugate was accomplished as follows: The total volume of conjugation mixture was reduced to about 2.0 mL by centrifuging an Amicon Centri-Prep 30 tube containing the sample for about 30 minutes at 2000 rpm using an IEC Centra-8 centrifuge. The sample was placed on the top of a Bio-Gel A-15 m agarose column (BioRad Laboratories, Inc., 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.
[0109]
The first well-separated, narrow, intense band eluted from the column in fractions #19 to #23 contained the CD8β-aminodextran-PC5 conjugate. The broad hump usually overlapping and following this narrow first band was absent due to the removal of almost all of the low molecular weight contaminant of aminodextran, which had a broad size distribution. A second intense band peaking at about fraction #47 contained excess PC5, and a medium-to-low intensity well-separated third band peaking at about fraction #62 was attributed to the low molecular weight excess blocking reagents. The fractions collected for the CD8β-Aminodextran-PC5 conjugate were analyzed spectrophotometrically at wavelengths of 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 to 23 under the first narrow peak gave 6.264 mg total PC5 in the conjugate. Since 12.856 mg IT-PC5 was used in conjugation, the yield was 48.7%.
[0110]
D. CD4Antibody-Aminodextran-ECD Conjugate
[0111]
The procedures were similar to those outlined for the preparation of the anti-CD8β-Aminodextran-PC5 conjugate, except anti-CD4 antibody was activated with DTT and used in the conjugation instead of CD8β antibody, and the tandem PE-Texas red or ECD fluorescent dye was used instead of PC5. In trial 2, DTT-CD4 (0.858 mg) was mixed with sulfo-SMCC-Aminodextran (10 mg) at concentrations of 0.141 and 1.647 mg/mL, respectively, during the first 2 hour step in conjugation. Then, 5.102 mL of IT-ECD (12.856 mg) were added to the mixture, which was further roller mixed overnight for 16-24 hours. The IT-ECD (A565.5/A280) ratio was 4.968. The conjugation mixture was concentrated to about 1.0 mL and applied to the top of a Sepharose 4B column. Data for fractions 19-23, collected at about 3.6 mL per fraction under the first narrow peak in trial 2, gave 6.098 mg total ECD in the conjugate. Since 12.856 mg IT-ECD was used in conjugation, the yield was 47.4%.
[0112]
An example of the effect of aminodextran-MP8 purification on the chromatograms of CD4 antibody-aminodextran-PE conjugates applied to a Sepharose 4B column, 2.5 cm×46 cm, equilibrated and eluted with 1×PBS is shown in FIG. 6B.
[0113]
In the above experiments, because of the very large amounts of phycobiliprotein in the conjugates, the usual calculation of antibody concentration from absorbance measurements at 280 and 565 nm could not be reliably performed. However, the concentration of reacting (conjugate) units of antibody-Aminodextran-PE was obtained by dividing the PE concentration, determined from the absorbance at 565 nm, by the normalized molar ratio of PE to Aminodextran. First, the mol PE was calculated by summing the concentration of PE in the fractions under the first narrow conjugate band, and multiplying by the volume per fraction, and dividing by the MW of PE. Second, all of the aminodextran was assumed to react and be retained in the first band. The mol Aminodextran was calculated by dividing the mass of Aminodextran used by the MW of 8.0 MDa determined.
Example 6 Flow Cytometric Analyses Using Improved Amdex A. Use of the Conjugates of Example 5
[0114]
CD8β-PC5 (commercially available as Item 6607109, in the 2003 Beckman Coulter, Inc., Cell Analysis Catalog) and fractions of CD8β-Aminodextran-MP8, both standard MP8 and improved (or prepurified) MP-8,-PC5 conjugates were titered with 2.5, 1.25, and 0.625 μg of antibody in the conjugate per tube. Amounts of antibody for the titers were determined from corrected A280 values for the direct conjugate, and by an indirect method described below for the aminodextran-crosslinked conjugates. Dilutions were added to 100 μL of whole blood and incubated for 10 minutes at room temperature.
[0115]
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 5 min, 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° C. 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 non-staining negative lymphocyte sub-population only within the 1st decade on the 4-decade log scale for fluorescence intensity.
[0116]
FIG. 10 shows the ability of CD8β-Aminodextran-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 1 PC5 per antibody and no aminodextran. This is the control against which fluorescence amplification is measured. Again, the data demonstrate the significant improvement in fluorescent intensity using the amplified CD8β reagent as opposed to standard bottled product.
[0117]
FIGS. 7A and 7B are histograms showing that the crosslinked conjugates with standard Aminodextran-MP8 and purified Aminodextran-MP8 have up to 3.5- and 10-fold, respectively, higher fluorescence intensity on CD8β+lymphocytes. Titers of the control, CD8β-PC5, and the samples, CD8β-Aminodextran-MP8 (standard and purified)-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 Tables 3 and 4.