Synthetic membrane anchors

Abstract

Cells incorporating a synthetic molecule construct of the structure F—S.sub.1—S.sub.2-L where: F—S.sub.1 is an aminoalkylglycoside where F is a mono-, di-, tri- or oligo-saccharide and S.sub.1 is 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl; S.sub.2 is —CO(CH.sub.2).sub.2CO—, —CO(CH.sub.2).sub.3CO—, —CO(CH.sub.2).sub.4CO— or —CO(CH.sub.2).sub.5CO—; and L is phosphatidylethanolamine.

Claims

1. A method of preparing a synthetic molecule construct of the structure F—S.sub.1—S.sub.2-L, comprising the steps of: reacting a bis(succinimidyl)dicarboxylate with a phosphatidylethanolamine to provide a phosphatidylethanolamide derivative of the structure S.sub.2-L; and then reacting a primary aminoalkyl glycoside of the structure F—S.sub.1 with the phosphatidylethanolamide derivative to provide the construct, where F is a mono-, di-, tri- or oligo-saccharide; S.sub.1 is 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl; S.sub.2 is —CO(CH.sub.2).sub.2CO—, —CO(CH.sub.2).sub.3CO—, —CO(CH.sub.2).sub.4CO— or —CO(CH.sub.2).sub.5CO—; and L is the phosphatidylethanolamide.

2. The method of claim 1 where F is selected from the group consisting of: GalNAcα1-3(Fucα1-2)Galβ; Galα1-3Galβ; Galβ; Galα1-3(Fucα1-2)Galβ; NeuAcα2-3Galβ; NeuAcα2-6Galβ; Fucα1-2Galβ; Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ; Fucα1-2Galβ1-4GlcNAcβ1-6(Fucα1-2Galβ1-4GlcNAcβ1-3)Galβ; Fucα1-2Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ; NeuAcα2-3Galβ1-4GlcNAcβ1-6(NeuAcα2-3Galβ1-4GlcNAcβ1-3)Galβ; Galα1-4Galβ1-4Glc; GalNAcβ1-3Galα1-4Galβ1-4Glc; GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc; or GalNAcβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc.

3. The method of claim 1 where the primary aminopropyl glycoside is 3-aminopropyl glycoside.

4. The method of claim 1 where the phosphatidylethanolamine is 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE).

Description

(1) The invention will now be illustrated by reference to the following non-limiting Examples and Figures of the accompanying drawings in which:

(2) FIG. 1 shows Diamed results of Cellstab™ stored cells transformed by natural A glycolipid transformation solution at (L to R) 10 mg/mL, 5 mg/mL, 2 mg/mL, 2 mg/mL* and 1 mg/mL. Antisera used are Albaclone (top) and Bioclone (bottom). (*—transformation solution (containing glycolipids) was not washed out after the incubation, it was left in over night and washed out the next day (day 2).)

(3) FIG. 2 shows Diamed results of Cellstab™ stored cells transformed by natural B glycolipid transformation solution at (L to R) 10 mg/mL, 5 mg/mL, 2 mg/mL, 2 mg/mL* and 1 mg/mL. Antisera used are Albaclone (top) and Bioclone (bottom). (*—transformation solution (containing glycolipids) was not washed out after the incubation, it was left in over night and washed out the next day (day 2)).

(4) FIG. 3 shows FACS analysis following in vitro transformation of human Le(a-b-) red cells with natural Le.sup.b-6 glycolipid over time at three transformation temperatures, 37° C. (top), 22° C. (middle) and 4° C. (bottom).

(5) FIG. 4 shows Diamed results of cells transformed at 4° C. by A.sub.tri-sp-Ad-DOPE (I) transformation solution at (L to R): washed 0.08 mg/mL; unwashed 0.08 mg/mL; washed 0.05 mg/mL; unwashed 0.05 mg/mL; washed 0.03 mg/mL; and unwashed 0.03 mg/mL. The antisera used was Bioclone anti-A.

(6) FIG. 5 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by A.sub.tri-sp-Ad-DOPE (I) transformation solution at (L to R): 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL. The antisera used was Bioclone anti-A.

(7) FIG. 6 shows in the left column Diamed results of cells transformed at 4° C. by B.sub.tri-sp-Ad-DOPE (VI) transformation solution at (L to R): washed 0.6 mg/mL; unwashed 0.6 mg/mL; washed 0.3 mg/mL; unwashed 0.3 mg/mL; washed 0.15 mg/mL; and unwashed 0.15 mg/mL; and in the right column Diamed results of cells transformed at 4° C. by B.sub.tri-sp-Ad-DOPE (VI) transformation solution at (L to R): washed 0.08 mg/mL; unwashed 0.08 mg/mL; washed 0.05 mg/mL; unwashed 0.05 mg/mL; washed 0.03 mg/mL; and unwashed 0.03 mg/mL. The antisera used was Bioclone anti-B.

(8) FIG. 7 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by B.sub.tri-sp-Ad-DOPE (VI) transformation solution at (L to R): 0.6 mg/mL, 0.3 mg/mL and 0.15 mg/mL.

(9) FIG. 8 shows Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and Btn-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.07+B 0.3 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.07+B 0.3 mg/mL against anti-A and anti-B.

(10) FIG. 9 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.07+B 0.3 mg/mL against anti-A and anti-B.

(11) FIG. 10 shows Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.07+B 0.2 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.07+B 0.2 mg/mL against anti-A and anti-B.

(12) FIG. 11 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.07+B 0.2 mg/mL against anti-A and anti-B.

(13) FIG. 12 shows Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.06+B 0.3 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.06+B 0.3 mg/mL against anti-A and anti-B.

(14) FIG. 13 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.06+B 0.3 mg/mL against anti-A and anti-B.

(15) FIG. 14 shows Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.06+B 0.2 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.06+B 0.2 mg/mL against anti-A and anti-B.

(16) FIG. 15 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.06+B 0.2 mg/mL against anti-A and anti-B.

(17) FIG. 16 shows Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.05+B 0.3 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.05+B 0.3 mg/mL against anti-A and anti-B.

(18) FIG. 17 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.05+B 0.3 mg/mL against anti-A and anti-B.

(19) FIG. 18 shows Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain washed A 0.05+B 0.2 mg/mL against anti-A and anti-B. Wells 3 and 4 contain unwashed A 0.05+B 0.2 mg/mL against anti-A and anti-B.

(20) FIG. 19 shows cells that were no longer washed prior to testing. Diamed results of cells transformed at 4° C. by parallel transformation with A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI). Wells 1 and 2 (L to R) contain unwashed A 0.05+B 0.2 mg/mL against anti-A and anti-B.

COMPARATIVE EXAMPLES

(21) The Comparative Examples do not form part of the invention claimed. The Comparative Examples describe red blood cell transformation with natural glycolipids.

Comparative Example 1

Preparation of Natural Glycolipids

(22) Purification by HPLC

(23) In the first stage, columns were packed with dry silica (15-25 μm) before each run. Relatively dirty samples could be used in HPLC because the silica could be discarded along with the theoretically high level of irreversibly bound contaminants.

(24) Glycolipids were separated on silica gel with a mobile phase of increasing polarity. The program was a linear gradient beginning with 100% chloroform-methanol-water 80:20:1 (v/v) and ending with 100% chloroform-methanol-water 40:40:12 (v/v).

(25) The HPLC equipment used was a Shimadzu system capable of pumping and mixing four separate solvents at programmed ratios. As chloroform, methanol and water evaporate at different rates, a program was developed whereby the solvent components were not mixed prior to entering the HPLC.

(26) The Shimadzu HPLC mixes four different liquids by taking a “shot” from each of four bottles in turn. “Shots” of chloroform and water directly next to each other in the lines may cause miscibility problems. Methanol was sandwiched in between these two immiscible components. Additionally, the water was pre-mixed with methanol in a 1:1 ratio to further prevent problems with miscibility.

Comparative Example 2

Transformation of Red Blood Cell Transformation with Natural Glycolipids

(27) Agglutination

(28) Transformation of red blood cells was assessed by agglutination using the Diamed-ID Micro Typing System in addition to using conventional tube serology. Diamed ABO typing cards were not used. The cards used were NaCI, Enzyme test and cold agglutinin cards, which were not pre-loaded with any antisera or other reagents. This allowed the use of specific antisera with both methodologies.

(29) TABLE-US-00002 TABLE 1 Gel-cards. Manufacturer Catalogue ref Diamed NaCl, Enzyme test and cold agglutinin cards

(30) A comparative trial was carried out between tube serology and the Diamed system to establish the performance of the two systems. Cells were transformed at 25° C. for 4 hours. Seraclone and Alba-clone anti-A sera were used to gauge equivalency. The results are shown in Table 3 below.

(31) TABLE-US-00003 TABLE 2 Antisera used in comparison of tube serology with the Diamed system. Manufacturer Catalogue ref Lot Expiry Albaclone, SNBTS Anti-A. Z0010770 12.12.04 Seraclone, Biotest 801320100 1310401 12.04.03

(32) TABLE-US-00004 TABLE 3 Agglutination results comparing tube serology with the Diamed system. A glycolipid (mg/mL) 10 5 2 1 0 Tube Albaclone 3+ 2+ 0 0 0 Seraclone 3+ 2+ 0 0 0 Diamed Albaclone 2+ 2+ 0 0 0 Seraclone 3+ 2+   1+ w+ 0

(33) In this experiment, the Diamed system proved to be more sensitive to the weaker reactions than tube serology with the Seraclone anti-A, but not with Albaclone. These reagents are formulated differently, and are thus not expected to perform identically. However, the fact that the Seraclone anti-A tube serology combination did not detect positivity is probably due to operator interpretation. The weaker reactions are notoriously difficult to accurately score, and the difference between 1+and 0 can be difficult to discern in tubes.

(34) Optimisation

(35) The variables of glycolipid concentration, incubation temperature, incubation duration, diluent and storage solution were examined for their effect on cell health. Efficiency and stability of transformation was assessed by agglutination with the relevant antibody.

(36) TABLE-US-00005 TABLE 4 Tube serology agglutination of natural glycolipid A transformed cells over different times and temperatures. A 10 5 2 1 0.1 0.01 0.001 0.0001 0 Seraclone 3+ 2+ 0 0 0 (37° C. for 1.5 hours) Seraclone 4+ 3+   2+   1+ w+ 0 0 0 0 (25° C. for 4 hours)

(37) Glycolipid Concentration

(38) Initial transformation experiments were carried out with a highly purified (HPLC) Leb glycolipid sample and a less pure blood group A glycolipid sample. Transformation was performed at 37° C. for 1.5 hours

(39) The A glycolipid sample contained other lipid impurities and thus comparatively less blood group A molecules by weight than the Le.sup.b glycolipid sample of equivalent concentration (w/v). This seems to be borne out by the fact that higher concentrations of the A glycolipid than the Le.sup.b glycolipid were required to produce equivalent agglutination scores (see Table 6).

(40) The level of impurity in the A glycolipid sample may also have contributed to the lower stability over the 62 day period—the A-transformed cells ‘died’ at the highest concentration (having received the largest dose of impurity).

(41) TABLE-US-00006 TABLE 5 Anti-A and anti-Le.sup.b used in initial testing of natural glycolipid transformation. Manufacturer Catalogue ref Batch number Expiry Anti-A Seraclone, Biotest 801320100 1310401 12.04.03 Anti-Le.sup.b CSL 12801

(42) TABLE-US-00007 TABLE 6 Stability of RBCs transformed with natural A and Le.sup.b glycolipid as assessed by tube serology agglutination over the period of 62 days. Glycolipid Le.sup.b A (mg/mL) Day 1 Day 25 Day 62 Day 1 Day 25 Day 62 10 4+ 2-3+   3+ 2+ ? 5 4+ 2-3+   2+ 2+ w+ 2 3+ 1-2+ 0 1+ 0 1 4+   2+ 0 1+ 0 0.1 3+ 2+ 0 0 0.01 2+ 2+ 0 0 0.001 2+ 2+ 0 0 0.0001 2+ 0   0 0 0 0   0   0 0 0   0

(43) The above cells were also rated for haemolysis and these results are shown in Table 7 below.

(44) TABLE-US-00008 TABLE 7 Haemolysis as assessed visually. Day 1 - in the supernatant of the first wash after transformation; Days 25 and 62 - in the cell preservative solution before the cells are resuspended after storage. Scoring scale is analogous to the 4+ to 0 agglutination scale: hhhh—severely haemolysed, hhh—very haemolysed, hh—moderately haemolysed, h—mildly haemolysed, w—faintly haemolysed and 0—no haemolysis seen. Glycolipid Haemolysis concentration Le.sup.b A (mg/mL) Day 1 Day 25 Day 62 Day 1 Day 25 Day 62 10 h 0 h h h dead 5 hh 0 hhh w 0 hh 2 w 0 hhh w 0 hhhhh 1 w 0 hhh h 0 hhhh 0.1 h hhh 0.01 hh 0.001 h 0.0001 h Control h 0 h h h

(45) These results show that cell haemolysis can be shown to be associated with transformation with high concentrations of glycolipid. It is unclear whether the mechanism underlying this is disruption of the plasma membrane by large amounts of glycolipid being inserted, the rate of that insertion, or is possibly due to the quantity of associated impurity. However, the results for Le.sup.b at day 62 seem to support the first explanation.

(46) The Le.sup.b sample was highly purified—before being dissolved, it was a powder of pure white colour, and thus it is unlikely that the haemolysis was due to the deleterious effect of impurities. It is clear to see that at 62 days, the amount of haemolysis occurring diminishes in line with the decrease in the glycolipid concentration.

(47) Incubation Temperature

(48) Experiments were carried out to investigate other possible mechanisms for the reduction of haemolysis of RBCs during the insertion step. Previous experiments had shown that haemolysis was worse at higher glycolipid concentrations than at lower concentrations, and it is thought that haemolysis may also be related to the rate of glycolipid insertion. Since temperature is believed to affect the rate of insertion, experiments were conducted comparing transformation at 37° C. with transformation at room temperature (RT; 25° C.).

(49) Since the rate was expected to slow down as temperature decreased, the incubation period for the RT experiment was 4 hrs. Haemolysis was assessed visually and scored following insertion. Serology tests were also performed on the cells. The results are shown in Table 8.

(50) TABLE-US-00009 TABLE 8 The effect of incubation temperature on haemolysis and agglutination during insertion of glycolipids into RBC membranes. Haemolysis was scored visually at each of the three washes. Haemolysis Glycolipid RT 37° C. Serology (mg/mL) wash 1 wash 2 wash 3 wash 1 wash 2 wash 3 RT 37° C. 10 w 0 0 hh w 0 2+ 2+ 1 w 0 0 hh h vw 1+ w+

(51) Incubation Duration

(52) Incubation at 37° C. was carried out for 1 and 2 hours and its effect on cell health and transformation assessed by agglutination with the relevant antibody.

(53) TABLE-US-00010 TABLE 9 Antisera used in the duration of incubation trial. Batch Manufacturer Catalogue ref number Expiry date Albaclone, SNBTS Anti-A. Z0010770 12.12.04 Bioclone, OCD Anti-A, DEV01102 — experimental reagent Albaclone, SNBTS Anti-B Z0110670 01.07.05 Bioclone, OCD Anti-B, DEV01103 — experimental reagent

(54) TABLE-US-00011 TABLE 10 Effect of incubation time on agglutination of cells transformed with natural glycolipids. Concentration Albaclone BioClone Glycolipid (mg/mL) 1 hour 2 hours 1 hour 2 hours A 10 4+ 4+ 4+ 4+ 5 4+ 4+ 4+ 2+ 2 4+ 3+ 3+ 2+ 1 3+ 2+ 2+ 2+ 0.5 2+ 2+ 1+ w+ B 10 3+ 2+ 4+ 1+ 5 3+ 2+ 3+ 2+ 2 2+ 2+ 2+ 1+ 1 1+ w+ 1+ w+ 0.5 1+ w+ w+ w+

(55) These results indicate that increasing the duration of incubation during natural glycolipid insertion does not enhance agglutination. In fact, the agglutination scores are reduced after the two hour incubation. This may be due to the destabilisation of the membrane or exchange of the glycolipids back into solution.

(56) Diluent

(57) Experiments were also carried out to determine if changing the glycolipid diluent solution could reduce haemolysis. Working strength PBS was compared with 2× PBS and 2% Bovine Serum Albumin (BSA) in working strength PBS. Cells were incubated at 37° C. for 1.5 hours. The results are shown in Table 11.

(58) TABLE-US-00012 TABLE 11 Study on the effect on haemolysis of changing the glycolipid diluent solutions during insertion of glycolipids into RBC membranes. Glycolipid concentration Glycolipid Diluent Solution (mg/m L) PBS 2 × PBS 2% BSA in PBS 40 Hhh hhh hhh 30 Hhh hhh hhh 20 Hhh hhh hhh 10 Hhh hhh hhh 0 0 0 0

(59) Stability

(60) Once A and B blood group glycolipids had been HPLC purified to an acceptable level, an experiment to find the appropriate concentrations for stability trials was carried out.

(61) TABLE-US-00013 TABLE 12 Early stability trial of cells transformed with natural A glycolipid. A Expt Day 10 5 2 1 0.1 0.01 0.001 0.0001 0 1 7 4+ 3-4+ 1+ 0 0 0 0 0 0 2 43 3+ w+ 0 0 0 0 0 0 0 3 50 1+ 0 0 0 4 60 3+ 1+ 0 5 67 w+ vw vw 6 74 2+ 0 0 7 81 2+ 1+ 0

(62) TABLE-US-00014 TABLE 13 Antisera used in stability trials (Table 14 and Table 15). Batch Manufacturer Catalogue ref number Expiry date Albaclone, SNBTS Anti-A. Z0010770 12.12.04 Bioclone, OCD Anti-A, DEV01102 — experimental reagent Albaclone, SNBTS Anti-B Z0110670 01.07.05 Bioclone, OCD Anti-B, DEV01103 — experimental reagent

(63) TABLE-US-00015 TABLE 14 Tube serology of O RBCs transformed with A glycolipid in order to establish appropriate concentrations for stability trials. A glycolipid (mg/mL) Anti-A Expt 10 5 2 1 0.5 0.1 0.01 0.001 0 Alba 1 3+ 2+ 1+ 0 0 0 0 0 2 4+ 4+ 3+ 2+ w+ Bio 1 3+ 2+ 1+ 0 0 0 0 0 2 4+ 4+ 3+ 2+ w+ 1 & 2 Transformation at 25° C. for 4 hours

(64) TABLE-US-00016 TABLE 15 Tube serology of O RBCS transformed with B glycolipid in order to establish appropriate concentrations for stability trials. B glycolipid (mg/mL) Anti-B Expt 10 5 2 1 0.5 0.1 0.01 0.001 0 Alba 1 2+ 1+ w+ 0 0 0 0 0 2 1+ 1+ w+ 0 w+ Bio 1 3+ 2+ w+ 0 0 0 0 0 2 1+ 1+ w+ 0 w+ 1 & 2 Transformation at 25° C. for 4 hours

(65) Two sets of cells were transformed with different concentrations of natural A glycolipid. Transformation was performed at 25° C. One set of cells was tested long term, and one set of cells was tested weekly for agglutination. The agglutination results from tube serology and Diamed are shown in Table 16 below. All cells were stored in Cellstab™ in bottles with flat bases. The cells showed minimal to no haemolysis at any time.

(66) TABLE-US-00017 TABLE 16 Agglutination results for cells transformed with different concentrations of natural A glycolipid. Results were obtained using Albaclone anti-A. A glycolipid (mg/mL) 10 5 2 1 0.1 control Long term testing Day 1 Tube 4+ 3+   2+ 1+ +w 0 Diamed 3+ 3+ +w 0 0 0 Day 17 Tube 3+ 2+ 0 0 0 Diamed 3+ 2+   1+ 0 0 Weekly testing Day 1 Tube 3+   2+ 0 0 Diamed 3+ 0 0 0 Day 8 Tube 1+ 0 0 0 Diamed 3+ 0 0 0 Day 15 Tube 1+ 0 0 0 Diamed 3+   2+ 0 0 Day 22 Tube 3+ 0 0 0 Diamed 3+ 0 0 0 Day 29 Tube *+w *0  *0  *0 Diamed *3+  *0  *0  *0 Day 36 Tube * * * *0 Diamed *3+  *0  *0  *0 Day 43 Tube * * * *0 Diamed * * * *0 *Albaclone, while all others used Seraclone anti-A.

(67) Storage Solution

(68) Comparison of the two cell storage solutions, Celpresol™ (CSL) and Cellstab™ (Diamed) was carried out to test their relative abilities to support modified RBCs.

(69) The stability of RBCs transformed with blood group A and B antigen solutions of varying concentrations when stored in two different cell preservative solutions—Cellstab™ and Alsevers™—was trialed.

(70) A and B antisera from two different sources were used in serology testing.

(71) All cells were tested using the standard tube serology platform up to 42 days, at which time the cell agglutination reactions had become too difficult to score manually (see Table 17 for A results and Table 18 for B results).

(72) Diamed gel-card testing was carried out to day 56 for the Alsevers stored cells, and discontinued at day 63 due to fungal contamination (although still returning positive scores).

(73) The Cellstab™ stored cells continued to be tested up to day 70, and were still viable at this point (see FIG. 1 for A results and FIG. 2 for B results).

(74) The reagents used in the stability trial are shown in Table 13.

(75) TABLE-US-00018 TABLE 17 Tube serology results of stability trial of cells transformed with varying concentrations of A glycolipid and stored in either Cellstab ™ or Alsevers ™ Albaclone Anti-A Bioclone Anti-A (OCD - Cell (SNBTS) Developmental reagent) storage Transformation Solution (mg/mL) Day solution 10 5 2 2* 1 10 5 2 2* 1 2 Alsevers 4+ 3+ 2+ 1+ w+ 3+ 3+ 1+ 1+ 0 Cellstab ™ 4+ 4+ 3+ 1+ 1+ 3+ 3+ 2+ 1+ 0 8 Alsevers 4+ 4+ 2+ 1+ 1+ 2+ 2+ 1+ 1+ 0 Cellstab ™ 4+ 4+ 3+ 2+ 1+ 3+ 3+ 2+ w+ 0 14 Alsevers 4+ 3+ 2+ 2+ w+ 2+ 1+ w+ vw 0 Cellstab ™ 4+ 3+ 3+ 2+ w+ 3+ 2+ w+ vw 0 21 Alsevers 3+ 2+ 2+ 2+ 1+ 2+ 2+ 2+ 1+ 0 Cellstab ™ 3+ 3+ 2+ + .sup.‡ 2+ .sup.‡ .sup.‡ .sup.‡ 0 28 Alsevers 2+ 2+ 1+ 1+ 0 2+ 2+ 1+ 1+ 0 Cellstab ™ 2+.sup.‡ 2+.sup.‡ .sup.‡ .sup.‡ 0 1+ w+ 0 0 0 36 Alsevers 3+ 2+ 2+ 2+ 1+ 3+ 3+ 2+ 1+ 1+ Cellstab ™ 3+.sup.‡ 2+.sup.‡ .sup.‡ .sup.‡ .sup.‡ 3+.sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ 42 Alsevers 3+ 3+ 1+ w+ 0 2+ 2+ 2+ 1+ 1+ Cellstab ™ 4+.sup.‡ 4+.sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ 0 *transformation solution (containing glycolipids) was not washed out after the incubation, it was left in over night and washed out the next day. .sup.‡positive cell button, but cells fall off as negative (score assignment impossible).

(76) TABLE-US-00019 TABLE 18 Tube serology results of stability trial of cells transformed with varying concentrations of B glycolipid and stored in either Cellstab ™ or Alsevers ™. Albaclone Anti-B Bioclone Anti-B (OCD - Cell (SNBTS) Developmental reagent) storage Transformation Solution (mg/mL) Day solution 10 5 2 2* 1 10 5 2 2* 1 2 Alsevers 3+ 3+ 1+ 1+ 1+ 2+ 1+ 1+ 1+ 0 Cellstab ™ 3+ 3+ 2+ 2+ 1+ 2+ 2+ 2+ 1+ w+ 8 Alsevers 1+ 1+ w+ 0 0 0 0 0 0 0 Cellstab ™ 2+ 1+ w+ 0 1+ 1+ w+ 0 0 14 Alsevers 2+ 2+ 0 w+ 0 0 1+ 1+ 2+ 0 Cellstab ™ 1+ w+ 0 0 0 2+ 2+ w+ 1+ 1+ 21 Alsevers .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ 1 1 .sup.‡ .sup.‡ .sup.‡ Cellstab ™ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ + + + .sup.‡ .sup.‡ 28 Alsevers 2+ 1+ w+ 0 0 2+ 1+ 2+ 0 0 Cellstab ™ .sup.‡ .sup.‡ .sup.‡ 0 0 .sup.‡ 0 .sup.‡ .sup.‡ 0 36 Alsevers 2+ 2+ 2+ 1+ 1+ 2+ 2+ 2+ 1+ 1+ Cellstab ™ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ 42 Alsevers 2+ 2+ 2+ 2+ w+ 2+ 2+ 1+ w+ w+ Cellstab ™ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ .sup.‡ *transformation solution (containing glycolipids) was not washed out after the incubation, it was left in over night and washed out the next day. .sup.‡ positive cell button, but cells fall off as negative (score assignment impossible).

(77) FACS Analysis of Glycolipid Insertion

(78) Transformation of human Le(a-b-) red cells with natural Le.sup.b-6 glycolipid over time at three transformation temperatures (37° C., 22° C. and 4° C.) was performed (FIG. 3). Natural Le.sup.b-6 glycolipid was dissolved in plasma and used to transform RBCs at a final concentration of 2 mg/mL and a final suspension of 10%.

(79) Reactivity was determined by FACS analysis using a Gamma anti-Leb. (The serological detection level is around 10.sup.2 molecules. The insertion of natural glycolipids at 4° C. for 8 hours was not detectable by agglutination with antibodies.) Projection of the rate of insertion curve from FACS analysis did not indicate that the rate of insertion at 4° C. would have reached agglutination detection levels within 24 hours.

(80) Low Incubation Temperature

(81) Transformation of RBCs with natural A or B glycolipid was perfomed at 37° C. for 1 hour and 2° C. for varying intervals. Cells were agglutinated with Bioclone anti-A or Bioclone anti-B. The results are provided in Tables 19 and 20.

(82) TABLE-US-00020 TABLE 19 Diamed results of comparison of natural A glycolipid transformation at 37° C. for 1 hour and 2° C. for varying intervals. Time Nat A (mg/mL) Temp (hours) 10 5 2 1 0 37° C. 1 3+ 3+ 2-3+ 2+ 0  2° C. 1 0 0 0 0 0 4 0 0 0 0 0 8 1-2+ 0 0 0 0 24 2-3+ 2+ 1-2+ 0 0 48 3+ 2-3+ 2-3+ 0 0 72 3-4+ 3+ 2+ 0 0

(83) TABLE-US-00021 TABLE 20 Diamed results of comparison of natural B glycolipid transformation at 37° C. for 1 hour and 2° C. for varying intervals. Time Nat B (mg/mL) Temp (hours) 10 5 2 1 0 37° C. 1 3+ 2-3+ 2+ 0 0  2° C. 1 0 0 0 0 0 4 0 0 0 0 0 8 0 0 0 0 0 24 1+ 0 0 0 0 48 2+ 1-2+ 0 0 0 72 2+ 1+ 0 0 0

(84) The rate of transformation is slow for both natural A glycolipid and natural B glycolipid as demonstrated by the negative agglutination scores after 1 hour at 2° C. Considerable insertion at 37° C. for this time interval has been demonstrated.

(85) Natural A glycolipid insertion at 2° C. required 48 hours to reach the same level of insertion obtainable by transformation at 37° C. After this time further insertion was not observed. Likewise, natural B glycolipid insertion at 2° C. was not as rapid as transformation at 37° C. The agglutination scores did not improve upon continued incubation and thus seemed to have reached maximal insertion at this time point for these concentrations.

EXAMPLES

(86) The Examples describe red blood cell transformation with the synthetic molecule constructs of the invention. In the context of these examples the term “synthetic glycolipids” is used to refer to these constructs.

Example 1

Preparation of Synthetic Glycolipids

(87) Materials and Methods

(88) TLC analysis was performed on silica gel 60 F.sub.254 plates (Merck), the compounds were detected by staining with 8% of phosphoric acid in water followed by heating at over 200° C. Column chromatography was carried out on silica gel 60 (0.2-0.063 mm, Merck) or Sephadex LH-20 (Amersham). .sup.1H NMR spectra were acquired on a Bruker DRX-500 spectrometer. Chemical shifts are given in ppm (δ) relative to CD.sub.3OD.

(89) Synthesis of activated 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and 1,2-O-distereoyl-sn-glycero-3-phosphatidylethanolamine (DSPE)(glycerophospholipids)

(90) To a solution of bis(N-hydroxysuccinimidyl) adipate (A) (70 mg, 205 μmol) in dry N,N-dimethylformamide (1.5 ml) were added DOPE or DSPE (L) (40 μmol) in chloroform (1.5 ml) followed by triethylamine (7 μl). The mixture was kept for 2 h at room temperature, then neutralized with acetic acid and partially concentrated in vacuo.

(91) Column chromatography (Sephadex LH-20, 1:1 chloroform-methanol, 0.2% acetic acid) of the residue yielded the activated lipid (A-L) (37 mg, 95%) as a colorless syrup; TLC (chloroform-methanol-water, 6:3:0.5): R.sub.f=0.5 (DOPE-A), R.sub.f=0.55 (DSPE-A).

(92) .sup.1H NMR (CDCl.sub.3/CD.sub.3OD, 2:1), δ:

(93) DOPE-A—5.5 (m, 4H, 2×(—CH═CH—), 5.39 (m, 1H, —OCH2—CHO—CH.sub.2O—), 4.58 (dd, 1 H, J=3.67, J=11.98, —CCOOHCH—CHO—CH.sub.2O—), 4.34 (dd, 1 H, J=6.61, J=11.98, —CCOOHCH—CHO—CH.sub.2O—), 4.26 (m, 2H, PO—CH.sub.2—CH.sub.2—NH.sub.2), 4.18 (m, 2H, —CH.sub.2—OP), 3,62 (m, 2H, PO—CH.sub.2—CH.sub.2—NH.sub.2), 3.00 (s, 4H, ONSuc), 2.8 (m, 2H, —CH.sub.2—CO (Ad), 2.50 (m, 4H, 2×(—CH.sub.2—CO), 2.42 (m, 2H, —CH.sub.2—CO (Ad), 2.17 (m, 8H, 2×(—CH.sub.2—CH═CH—CH.sub.2—), 1.93 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 1.78 (m, 4H, 2×(COCH.sub.2CH.sub.2—), 1,43, 1.47 (2 bs, 40H, 20 CH.sub.2), 1.04 (m, 6H, 2CH.sub.3).

(94) DSPE-A—5.39 (m, 1 H, —OCH.sub.2—CHO—CH.sub.2O—), 4.53 (dd, 1 H, J=3.42, J=11.98, —CCOOHCH—CHO—CH.sub.2O—), 4.33 (dd, 1 H, J=6.87, J=11.98, —CCOOHCH—CHO—CH.sub.2O—), 4.23 (m, 2H, PO—CH.sub.2—CH.sub.2—NH.sub.2), 4.15 (m, 2H, —CH.sub.2—OP), 3,61 (m, 2H, PO—CH.sub.2—CH.sub.2—NH.sub.2), 3.00 (s, 4H, ONSuc), 2.81 (m, 2H, —CH.sub.2—CO (Ad), 2.48 (m, 4H, 2×(—CH.sub.2—CO), 2.42 (m, 2H, —CH.sub.2—CO (Ad), 1.93 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 1.78 (m, 4H, 2×(COCH.sub.2CH.sub.2—), 1,43, 1.47 (2 bs, 40H, 20 CH.sub.2), 1.04 (m, 6H, 2 CH.sub.3).

(95) Condensing Activated DOPE (or DSPE) with Aminopropylglycoside.

(96) To a solution of activated DOPE (or DSPE) (A-L) (33 μmol) in N,N-dimethylformamide (1 ml) 30 μmol of Sug-S.sub.1—NH.sub.2 (F—S.sub.1—NH.sub.2) and 5 μl of triethylamine were added. For example, the Sug may be either the aminopropyl glycoside (F—S.sub.1—NH.sub.2) of either GalNAcα1-3(Fucα1-2)Galβ trisaccharide (A-glycotope) (F) or Galα1-3(Fucα1-2)Galβ trisaccharide (B-glycotope) (F).

(97) The mixture was stirred for 2 h at room temperature. Column chromatography (Sephadex LH-20 in 1:1 chloroform-methanol followed by silica gel in ethyl acetate-isopropanol-water, 4:3:1 (v/v/v) of the mixture typically yielded 85-90% of the synthetic molecule construct, for example, A.sub.tri-sp-Ad-DOPE (I) or B.sub.tri-sp-Ad-DOPE (VI).

(98) .sup.1H NMR (CDCl.sub.3/CD.sub.3OD, 1:1), δ:

(99) A.sub.tri-sp-Ad-DOPE (I)—5.5 (m, 4H, 2×(—CH═CH—), 5.43-5.37 (m, 2H, H-1 (GalNHAc) and —OCH.sub.2—CHO—CH.sub.2O—), 5.32 (d, 1 H, H-1, J=3.5 H-1 Fuc), 2.50 (m, 4H, 2×(—CH.sub.2—CO), 2.40 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.20 (m, 8H, 2×(—CH.sub.2—CH═CH—CH.sub.2—), 2.1 (s, 3H, NHAc), 1.92 (m, 2H, O—CH.sub.2CH.sub.2CH.sub.2—NH), 1.8 (m, 8H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and 2×(COCH.sub.2CH.sub.2—), 1,43, 1.47 (2 bs, 40H, 20 CH.sub.2), 1.40 (d, 3H, J=6.6, CH.sub.3 Fuc), 1.05 (m, 6H, 2 CH.sub.3).

(100) A.sub.tri-spsp.sub.1-Ad-DOPE (II)—5.5 (m, 4H, 2×(—CH═CH—), 5.43-5,37 [m, 2H, H-1 (GalNHAc) and —OCH.sub.2—CHO—CH.sub.2O—], 5.32 (d, 1 H, H-1, J=3.6 H-1 Fuc), 2.50 (m, 4H, 2×(—CH.sub.2—CO), 2.40- 2.32 (m, 6H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and COCH.sub.2— (sp.sub.1), 2.18 [m, 8H, 2×(—CH.sub.2—CH═CH—CH.sub.2—)], 2.1 (s, 3H, NHAc), 1.95(m, 2H, O—CH.sub.2CH.sub.2CH.sub.2—NH), 1.8 [m, 10H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO, 2×(COCH.sub.2CH.sub.2— . . . ), —COCH.sub.2CH.sub.2(CH.sub.2).sub.3NH—], 1.68 (m, 2H, CO(CH.sub.2).sub.3CH.sub.2CH.sub.2NH—), 1,43, 1.47 (2 bs, 42H, 22 CH.sub.2), 1.37 (d, 3H, J=5.6, CH.sub.3 Fuc), 1.05 (m, 6H, 2 CH.sub.3).

(101) A.sub.tri-sp-Ad-DSPE (III)—5.42-5.38 (m, 2H, H-1 (GalNHAc) and —OCH.sub.2—CHO—CH.sub.2O—), 5.31 (d, 1 H, H-1, J=3.5 H-1 Fuc), 2.48 [m, 4H, 2×(—CH.sub.2—CO)], 2.42 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.18 (s, 3H, NHAc), 1.95 (m, 2H, O—CH.sub.2CH.sub.2CH.sub.2—NH), 1.8 [m, 8H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and 2×(COCH.sub.2CH.sub.2—)], 1,43, 1.47 (2 bs, 56H, 28 CH.sub.2), 1.38 (d, 3H, J=6.6, CH.sub.3 Fuc), 1.05 (m, 6H, 2 CH.sub.3).

(102) B.sub.tri-sp-Ad-DOPE (VI)—5.5 (m, 4H, 2×(—CH═CH—), 5.42-5,38 [m, 2H, H-1 (Gal) and —OCH.sub.2—CHO—CH.sub.2O—], 5.31 (d, 1 H, H-1, J=3.7, H-1 Fuc), 2.48 [m, 4H, 2×(—CH.sub.2—CO)], 2.39 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.18 [m, 8H, 2×(—CH.sub.2—CH═CH—CH.sub.2—)], 1.93 (m, 2H, O—CH.sub.2CH.sub.2CH.sub.2—NH), 1.8 [m, 8H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and 2×(COCH.sub.2CH.sub.2—)], 1,43, 1.47 (2 bs, 40H, 20 CH.sub.2), 1.36 (d, 3H, J=6.6, CH.sub.3 Fuc), 1.05 (m, 6H, 2 CH.sub.3).

(103) H.sub.tri-sp-Ad-DOPE (VII)—5.5 [m, 4H, 2—(—CH═CH—)], 5.4 (m, 1 H, —OCH.sub.2—CHO—CH.sub.2O—), 5.35 (d, 1 H, H-1, J=3.2, H-1 Fuc), 4.65, 4.54 (2d, J=7.4, J=8.6, H-1 Gal, H-1 GlcNHAc), 4.46 (dd, 1 H J=3.18, J=12, —CCOOHCH—CHO—CH.sub.2O—), 4.38-4.28 (m, 2H, H-5 Fuc, CCOOHCH—CHO—CH.sub.2O—), 2.48 [m, 4H, 2×(—CH.sub.2—CO)], 2.40 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.18 [m, 8H, 2×(—CH.sub.2—CH═CH—CH.sub.2—)], 2.08 (s, 3H,NHAc), 1.92 (m, 2H, O—CH.sub.2CH.sub.2CH.sub.2—NH), 1.82-1.72 [m, 8H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and 2×(COCH.sub.2CH.sub.2—)], 1,48, 1.45 (2 bs, 40H, 20 CH.sub.2), 1.39 (d, 3H, J=6.5, CH.sub.3 Fuc), 1.05 (m, 6H, 2 CH.sub.3).

(104) H.sub.di-sp-Ad-DOPE (VIII)—5.49 (m, 4H, 2×(—CH═CH—), 5.37 (m, 1 H, —OCH.sub.2—CHO—CH.sub.2O—), 5.24 (d, 1 H, H-1, J=2.95, H-1 Fuc), 4.46 (d, J=7.34, H-1 Gal), 2.48 [m, 4H, 2×(—CH.sub.2—CO)], 2.42-2.35 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.17 [m, 8H, 2×(—CH.sub.2—CH═CH—CH.sub.2—)], 1.95 (m, 2H, O—CH.sub.2CH.sub.2CH.sub.2—NH), 1.81-1.74 [m, 8H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and 2×(COCH.sub.2CH.sub.2—)], 1,45, 1.41 (2 bs, 40H, 20 CH.sub.2), 1.39 (d, 3H, J=6.5, CH.sub.3 Fuc), 1.03 (m, 6H, 2 CH.sub.3).

(105) Galβ-sp-Ad-DOPE (IX)—5.51 [m, 4H, 2×(—CH═CH—)], 5.4 (m, 1 H, —OCH.sub.2—CHO—CH.sub.2O—), 4.61 (dd, 1 H J=3.18, J=12, —CCOOHCH—CHO—CH.sub.2O—), 4.41 (d, J=7.8, H-1 Gal), 4.37 (dd, 1 H, J=6.6, J=12, —CCOOHCH—CHO—CH.sub.2O—), 2.50 [m, 4H, 2×(—CH.sub.2—CO)], 2.40 (m, 4H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 2.20 [m, 8H, 2×(—CH.sub.2—CH═CH—CH.sub.2—)], 1.97 (m, 2H, O—CH.sub.2CH.sub.2CH.sub.2—NH), 1.82-1.72 [m, 8H, COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO and 2×(COCH.sub.2CH.sub.2—)], 1,48, 1.45 (2 bs, 40H, 20 CH.sub.2), 1.05 (m, 6H, 2 CH.sub.3).

Example 2

Solubility of Synthetic Glycolipids

(106) For use in the transformation of cells the first criterion that synthetic glycolipids must satisfy is that they are soluble in aqueous solvents, e.g. phosphate buffered saline. A number of techniques, including heat and/or sonication, were employed initially in order to maximise the solubility of the synthetic glycolipids tested (Table 21).

(107) The synthetic glycolipid must also be able to insert into the membrane and be recognisable to the appropriate antibody for transformation to be detected by agglutination. Initial tests on the molecules were to establish solubility and thus eliminate those molecules that were unsuitable for use in the transformation of cells.

(108) The results of these initial tests are provided in Table 22.

(109) TABLE-US-00022 TABLE 21 The range of synthetic glycolipid molecules tested. DOPE Lipid Tails: B.sub.tri-sp-Ad-DOPE (VI) A.sub.tri-sp-Ad-DOPE (I) Galβ-sp-Ad-DOPE (IX) H.sub.di-sp-Ad-DOPE (VIII) H.sub.tri-sp-Ad-DOPE (VII) A.sub.tri-spsp.sub.1-Ad-DOPE (II) B.sub.tri-PAA-DOPE (V) Different Lipid Tails: A.sub.tri-sp-lipid (IV) A.sub.tri-sp-Ad-DSPE (III)

(110) TABLE-US-00023 TABLE 22 Solubility of synthetic glycolipids in hot PBS and transformation ability. Detectable transformation Synthetic Water solubility ability A.sub.tri-sp-lipid (IV) No No B.sub.tri-PAA-DOPE (V) No No B.sub.tri-sp-Ad-DOPE (VI) Yes Yes A.sub.tri-sp-Ad-DOPE (I) Yes Yes Galβ-sp-Ad-DOPE (IX) Yes No H.sub.disp-Ad-DOPE (VIII) Yes No H.sub.tri-sp-Ad-DOPE (VII) Yes Yes A.sub.tri-spsp.sub.1-Ad-DOPE (II) Yes Yes A.sub.tri-sp-Ad-DSPE (III) Yes Yes

(111) The lack of detectable transformation for Galβ-sp-Ad-DOPE (IX) and H.sub.di-sp-Ad-DOPE (VIII) was thought to be due to the inability of the antibody to recognise the glycotope of these synthetic molecules. A.sub.tri-sp-lipid (IV) has a single rather than a diacyl tail and it was proposed that there was no insertion of this synthetic molecule into the membrane bilayer.

Example 3

Low Temperature Transformation of RBCs by Atri-sp-Ad-DOPE (I) and Btri-sp-Ad-DOPE (VI) Synthetic Glycolipids

(112) RBCs are healthier when stored at 4° C., and likewise are believed to be healthier when transformed at 4° C. It was not thought that a significant rate of insertion of the synthetic glycolipids would occur at 4° C. due to our previous studies (see Comparative Examples) and studies by others (Schwarzmann, 2000). These studies were performed with natural glycolipids. Surprisingly these studies did not predict the behaviour of the synthetic glycolipids of the invention.

(113) Whilst not wishing to be bound by theory, in the studies of Schwarzmann the low rate of insertion of the natural glycolipids may be due to the physicochemical properties of the natural glycolipid tail; a sphingolipid and a fatty acid.

(114) The diacyl tail of the glycolipid may be important in determining the rate of insertion. Certain diacyl tails may retain greater fluidity at lower temperatures. Alternatively, the domain of the plasma membrane into which the diacyl tail of these glycolipids inserts may retain this greater fluidity.

(115) It is known that the sphingolipid tails of natural glycolipids congregate in rigid domains and these domains may not allow further incorporation of glycolipid at low temperatures. Synthetic glycolipids with cis-desaturated diacyl tails may be favoured for use.

(116) Transformation of RBCs with synthetic glycolipids with different lipid tails was first evaluated (Tables 22 and 24).

(117) TABLE-US-00024 TABLE 23 Antisera used to obtain results presented in Tables 24 to 27. Batch Manufacturer Catalogue ref number Expiry date Anti-A Albaclone, SNBTS Z0010770 12.12.04 BioClone, OCD Experimental reagent 01102 — Anti-B Albaclone, SNBTS Z0110600 27.04.03 BioClone, OCD Experimental reagent 01103 —

(118) TABLE-US-00025 TABLE 24 Evaluation of insertion of different lipid tails by agglutination with the relevant antisera. Anti- Transformation solution (μg/mL) Molecule sera 1000 500 250 125 100 60 50 40 30 20 10 A.sub.tri-sp-Ad-DOPE (I) Alba w+ w+ 0 0 0 Bio 2+ 1+ w+ 0 0 Alba 4+ 3+ 2-3+ 2+ Bio 4+* 4+* 3+* 3+ DBA 0 B.sub.tri-sp-Ad-DOPE (VI) Alba 3+ Bio 3+ Alba 2+ 2+ 1+ 0 0 Bio 3+ 2+ 1+ 0 0 A.sub.tri-spsp.sub.1-Ad-DOPE (II) Alba 0 0 0 0 0 Bio 0 0 0 0 0 Alba 4+ 3+ 2+ 2+ Bio 4+* 3-4+* 3+* 2+ DBA 0 A.sub.tri-sp-lipid (IV) Alba 0 Bio 0 A.sub.tri-sp-Ad-DSPE (III) Alba 0 0 0 0 0 Bio 0 0 0 0 0 Alba 2-3+ 2-3+ 2+ 2+ Bio 3+ 2-3+ 2+ 2+ DBA 0 *splatter.

(119) Transformation of RBCs with synthetic glycolipids A.sub.tri-sp-Ad-DOPE (I) and B.sub.tri-sp-Ad-DOPE (VI) at 4° C. was then evaluated (Tables 25 to 28). These transformations were directed towards the preparation of cells expressing low levels of A, B or A and B glycotopes (“weak A, B and AB cells”).

(120) For the preparation of weak A and B cells transformation solutions (20 μL, A.sub.tri-sp-Ad-DOPE (I) at 0.08, 0.05 and 0.03 mg/mL, and B.sub.tri-sp-Ad-DOPE (VI) at 0.6, 0.3, 0.15, 0.08, 0.05 and 0.03 mg/mL) in 1× PBS were mixed with washed, packed group O RBCs (60 μL).

(121) For the preparation of weak AB cells transformation solutions (20 μL, A.sub.tri-sp-Ad-DOPE (I) at 0.07, 0.06 and 0.05 mg/mL, and B.sub.tri-sp-Ad-DOPE (VI) at 0.3, and 0.2 mg/mL) in 1× PBS were combined in block titre with washed, packed group O RBCs (60 μL). The combinations were: A.sub.tri-sp-Ad-DOPE (I) at 0.07 mg/mL+B.sub.tri-sp-Ad-DOPE (VI) at 0.3 mg/mL; A.sub.tri-sp-Ad-DOPE (I) at 0.07 mg/mL+B.sub.tri-sp-Ad-DOPE (VI) at 0.2 mg/mL; A.sub.tri-sp-Ad-DOPE (I) at 0.06 mg/mL+B.sub.tri-sp-Ad-DOPE (VI) at 0.3 mg/mL; A.sub.tri-sp-Ad-DOPE (I) at 0.06 mg/mL+B.sub.tri-sp-Ad-DOPE (VI) at 0.2 mg/mL; A.sub.tri-sp-Ad-DOPE (I) at 0.05 mg/mL+B.sub.tri-sp-Ad-DOPE (VI) at 0.3 mg/mL; and A.sub.tri-sp-Ad-DOPE (I) 0.05+B.sub.tri-sp-Ad-DOPE (VI) 0.2 mg/mL.

(122) Cells and transformation solutions were placed in a 4° C. fridge. Pipette mixing was performed at intervals. Cells were removed for testing at intervals against the relevant antisera and were tested in both washed and unwashed states (i.e. washed samples had the transformation solution removed).

(123) After 48 hours Celpresol™ was added to the cells so that the final cells:non-cells ratio was 3:5 (v/v). The cells continued to be tested at intervals. Testing was discontinued after 10 days because cells turned brown.

(124) This discolouration could be attributed to a number of factors including: cells were already 21 days old when transformed; 48 hour transformation was in PBS not Celpresol™ so cells stressed for this time; and cells may have been mishandled in transit between the transforming and testing laboratories. This may be mitigated by transformation of the cells in Celpresol™ as opposed to PBS.

(125) TABLE-US-00026 TABLE 25 Diamed results of weak A RBCs transformed at 4° C. against anti-A. A.sub.tri-sp-Ad-DOPE (I) (mg/mL) Washed unwashed Time 0.08 0.05 0.03 0.08 0.05 0.03 2 hrs 0 0 0 0 0 0 4 hrs 1+ 0 0 2+ 0 0 6 hrs 2+ 0 0 2+ 0 0 8 hrs 2+ 0 0 2-3+ 0 0 12 hrs 2-3+ 0 0 3+ 1+ 0 24 hrs 3-4+ 1+ 0 3-4+ 2+ 0 30.5 hr 3-4+ 1+ 0 3-4+ 2+ 0 48 hrs 4+ 2+ 0 4+ 2+ 0 72 hrs 4+ 2+ 0 4+ 2-3+ 0 96 hrs 4+ 2-3+ 0 4+ 2-3+ 0 Day 7  3-4+ 2+ 0 Day 10 3-4+ 2+ 0

(126) TABLE-US-00027 TABLE 26 Diamed results of weak B RBCs transformed at 4° C. against anti-B. B.sub.tri-sp-Ad-DOPE (VI) (mg/mL) washed unwashed Time 0.6 0.3 0.15 0.6 0.3 0.15 2 hrs 0 0 0 0 0 0 4 hrs 0 0 0 1+ 0 0 6 hrs w+ 0 0 1+ 0 0 8 hrs 2+ 0 0 2+ w+ 0 12 hrs 2+ w+ 0 2-3+ 2+ 0 24 hrs 4+ 3+ 2+ 4+ 3+ 2+ 30.5 hr 4+ 2-3+ 0 4+ 2-3+ w+ 48 hrs 4+ 3+ 1+ 4+ 3+ 2+ 72 hrs 4+ 4+ 2+ 4+ 4+ 2+ 96 hrs 4+ 3-4+ 2-3+ 4+ 3-4+ 2-3+ Day 7  4+ 2-3+ 0 Day 10 4+ 2+ 0

(127) TABLE-US-00028 TABLE 27 Diamed results of weak AB RBCs transformed at 4° C. in block titre against anti-A. B.sub.tri-sp-Ad- A.sub.tri-sp-Ad-DOPE (I) (mg/mL) DOPE (VI) washed unwashed Day (mg/mL) 0.07 0.06 0.05 0.07 0.06 0.05 1 0.3 2+ 1-2+ w+ 2-3+ 2+ 1+ 0.2 2+ 1-2+ 0 2-3+ 2+ 1+ 5 0.3 2+ 1-2+ 1+ 2-3+ 2+ 1-2+ 0.2 2+ 1-2+ w+ 2-3+ 2+ 1-2+ 8 0.3 2-3+ 2+ 2+ 0.2 2-3+ 2+ 1-2+

(128) TABLE-US-00029 TABLE 28 Diamed results of weak AB RBCs transformed at 4° C. in block titre against anti-B. B.sub.tri-sp-Ad- A.sub.tri-sp-Ad-DOPE (I) (mg/mL) DOPE (VI) washed unwashed Day (mg/mL) 0.07 0.06 0.05 0.07 0.06 0.05 1 0.3 3+ 3+ 2+ 3+ 3+ 2-3+ 0.2 1+ 1-2+ 0 2+ 2+ 1-2+ 5 0.3 2+ 2+ 1+ 2+ 2+ 2+ 0.2 0 w+ vw 1+ w+ vw 8 0.3 2+ 2+ 2+ 0.2 1+ 1+ 0

Example 4

Insertion Efficiency of Transformation of RBCs by Atri-sp-Ad-DOPE (I) and Btri-sp-Ad-DOPE (VI) Synthetic Glycolipids

(129) The post-transformation supernatant solutions (from A.sub.tri-sp-Ad-DOPE (I) at 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL, and B.sub.tri-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added neat and in a 1:2 dilution to washed, packed RBCs (60 μL). The tubes were incubated in a 37° C. waterbath for one hour, with mixing taking place every 15 minutes.

(130) The transformed RBCs were washed 3× with PBS and then suspended in Cellstab™ at the appropriate concentration for serology testing.

(131) TABLE-US-00030 TABLE 29 Tube serology Pre-trans conc (mg/mL) Score A.sub.tri-sp-Ad-DOPE (I) at 0.08 0 1:2 of A.sub.tri-sp-Ad-DOPE (I) 0 at 0.08 A.sub.tri-sp-Ad-DOPE (I) at 0.05 0 1:2 of A.sub.tri-sp-Ad-DOPE (I) 0 at 0.05 A.sub.tri-sp-Ad-DOPE (I) at 0.03 0 1:2 of A.sub.tri-sp-Ad-DOPE (I) 0 at 0.03 B.sub.tri-sp-Ad-DOPE (VI) at vw+ 0.60 1:2 of B.sub.tri-sp-Ad-DOPE 0 (VI) at 0.60

(132) The score given by the post-transformation supernatant solution (from the 0.08 mg/mL pre-transformation solution) is not even that of the 0.03 mg/mL transformation solution in the first pass (w+). These results indicate that >75% of the molecules are inserted into the RBC membrane on the first pass.

(133) In addition, the post-transformation solutions were concentrated 20× and compared in parallel with the transformation solutions of known concentration. Only the post-transformation solutions derived from the 0.08 mg/mL A.sub.tri-sp-Ad-DOPE (I) and 0.6 mg/mL B.sub.tri-sp-Ad-DOPE (VI) solutions were tested.

(134) Post-transformation solutions (20 μL) were dialysed (pore size 500 Da) against de-ionised water for 2 days. The samples were left to dry in a fumehood for 10 days. At the end of this time they were transferred into a rotavapor flask and set on the rotavapor to rotate under vacuum with no heat overnight.

(135) Samples were dried in a water bath at 40° C. and washed over into smaller vessels with chloroform-methanol 2:1 leaving significant amounts of dried cellular material. The chloroform-methanol 2:1 washings were dried down, washed over again into test-tubes with chloroform-methanol 2:1 and dried down. These samples were redissolved in 1 mL of 1× PBS and used for transformation experiments. The cellular material in the bottom of the flasks was washed out with water into another set of tubes.

(136) The post-transformation solutions (from A.sub.tri-sp-Ad-DOPE (I) at 0.08 mg/mL and B.sub.tri-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added to washed, packed RBCs (60 μL). In parallel, the transformation solutions (A.sub.tri-sp-Ad-DOPE (I) at 0.08 mg/mL, 0.05 mg/mL and 0.03 mg/mL, and B.sub.tri-sp-Ad-DOPE (VI) at 0.6 mg/mL, 20 μL) were added to washed, packed RBCs (60 μL).

(137) The tubes were incubated in a 37° C. waterbath for one hour, with mixing taking place every 15 minutes. The transformed RBCs were washed 3× with PBS and then suspended in Cellstab™ at the appropriate concentration for serology testing.

(138) TABLE-US-00031 TABLE 30 Diamed serology conc (mg/mL) Score A.sub.tri-sp-Ad-DOPE (I) at 3+ 0.08 A.sub.tri-sp-Ad-DOPE (I) at 2+ 0.05 A.sub.tri-sp-Ad-DOPE (I) at 1+ 0.03 From A.sub.tri-sp-Ad- 0  DOPE (I) at 0.08 B.sub.tri-sp-Ad-DOPE (VI) 4+ at 0.60 From B.sub.tri-sp-Ad- 0  DOPE (VI) at 0.60

(139) These results suggest that there are not enough molecules in the post-transformation solution, even when concentrated 20×, to be detected by serology.

Example 5

Transformation of Murine RBCs by Htri-sp-Ad-DOPE (VII) Synthetic Glycolipid

(140) Mouse cells were transformed at 37° C. for 1 hour.

(141) TABLE-US-00032 TABLE 31 Anti-H reagents used for results in Tables 32 and 33. Antisera Manufacturer Batch Anti-H IgM Japanese Red Cross HIRO-75 UEA Lorne Laboratories 11549E D.O.E. 06.2004 Bio-UEA EY Labs 201105-2

(142) TABLE-US-00033 TABLE 32 Tube Serology. H Antisera UEA Cells IgM T = 0 T = 20 Bio-UEA Mouse RBCs (− control) 0  0  0 Mouse RBCs + 0.01 mg/mL 0  H.sub.tri-sp-Ad-DOPE (VII) Mouse RBCs + 0.05 mg/mL 1+ H.sub.tri-sp-Ad-DOPE (VII) Mouse RBCs + 0.1 mg/mL 3+ H.sub.tri-sp-Ad-DOPE (VII) Mouse RBCs + 0.25 mg/mL 4+ 1+ H.sub.tri-sp-Ad-DOPE (VII) Mouse RBCs + 1 mg/mL 2+ 2+ H.sub.tri-sp-Ad-DOPE (VII) Human O RBCs (+ control) 4+ 1+ 2/3+ 4+

(143) TABLE-US-00034 TABLE 33 Diamed Cells Score Mouse RBCs + 0.01 mg/mL H.sub.tri-sp-Ad-DOPE 0  (VII) Mouse RBCs + 0.05 mg/mL H.sub.tri-sp-Ad-DOPE 0  (VII) Mouse RBCs + 0.1 mg/mL H.sub.tri-sp-Ad-DOPE (VII) 2+ Mouse RBCs + 0.25 mg/mL H.sub.tri-sp-Ad-DOPE 3+ (VII)

Example 6

Transformation of RBCs by Filtered Atri-sp-Ad-DOPE (I) Synthetic Glycolipid

(144) Some Atn-sp-Ad-DOPE (I) had been sterile-filtered through a 0.2 μm filter. To investigate whether transformation would be the same with this product a comparative trial was done.

(145) TABLE-US-00035 TABLE 34 Anti-A used for results presented in Table 35. Manufacturer Catalogue ref Batch number Expiry date BioClone, OCD Experimental reagent 01102 —

(146) TABLE-US-00036 TABLE 35 Column agglutination of A RBCs transformed with varying concentrations of sterile-filtered vs unfiltered A.sub.tri-sp-Ad-DOPE (I). Concentration Sterile-filtered (mg/mL) A.sub.tri-sp-Ad-DOPE (I) Unfiltered A.sub.tri-sp-Ad-DOPE (I) 0.2   4+ 4+ 0.1   4+ 3-4+ 0.05 2-3+ 2-3+ 0.01 0 0  Control 37° C. 0 Control 25° C. 0

(147) These results show no significant difference between the two preparations of A.sub.tri-sp-Ad-DOPE (I) and suggests that filtration through a 0.2 μM filter did not remove molecules or change the composition or properties of the fluid to the point that transformation was affected.

Example 7

Storage of Transformed Cells

(148) To investigate whether storage at 4° C. or 37° C. changed the agglutination results of A.sub.tri-sp-Ad-DOPE (I) and natural A glycolipid transformed O RBCs, identified as “Syn-A” and “Nat-A” cells respectively, were divided in two and suspended to 5% in Cellstab™.

(149) One set of cells was stored at 4° C. and the other set of cells was stored at 37° C. in a waterbath. Agglutination of the stored transformed cells was assessed (Table 36).

(150) TABLE-US-00037 TABLE 36 Syn-A A.sub.tri-sp-Ad- Nat-A Time Temp DOPE (I) at At 1 At (hours) Platform (° C.) 0.1 mg/mL mg/mL 10 mg/mL Control 0 Tube 3+ 0 1-2+ 0 20 Column 4 4+ 0 3+ 0 37 4+ 0 3+ 0 44 Column 4 4+ 3+ 0 37 4+ 3+ 0

Example 8

RBC Transformation with A- and B-antigen Synthetic Glycolipids with Different Non-carbohydrate Structures

(151) The water soluble synthetic glycolipids designated A.sub.tri-sp-Ad-DOPE (I), A.sub.tri-sp.sub.1sp.sub.2-Ad-DOPE (II), A.sub.tri-sp-Ad-DSPE (III), and B.sub.tri-sp-Ad-DOPE (VI) were prepared according to the method described in Example 1 with necessary modifications.

(152) Washed packed group O red blood cells (RBCs) (3 parts by volume) and the synthetic glycolipid solution (1 part by volume, varying concentrations) were added to an eppendorf tube. The tube was incubated in a 37° C. waterbath for one hour, mixing every 15 minutes. The transformed RBCs were washed 3× with PBS and then suspended in Cellstab™ at the appropriate concentration for serology testing.

(153) Tube serology and Diamed gel-card results for RBCs transformed with the different synthetic molecule constructs are provided in Table 38. Results for the stability of the RBCs transformed with the different synthetic glycolipids at different concentrations are provided in Tables 39 to 44.

(154) TABLE-US-00038 TABLE 37 Antisera used for results presented in Tables 38 to 44. Antisera Manufacturer Batch Albaclone anti-A SNBTS Z0010770 - D.O.E 12.12.04 Bioclone anti-A Ortho Diagnostics 01102 - D.O.M 16.05.02 Albaclone anti-B SNBTS Z0110670 - D.O.E 12.12.04 Bioclone anti-B Ortho Diagnostics 01103 - D.O.M 16.05.02

(155) TABLE-US-00039 TABLE 38 Comparison of transformation of RBCs using A-antigen synthetic glycolipids at different concentrations. A Antisera Albaclone Bioclone Conc anti-A anti-A Synthetic mg/mL Tube Diamed Tube Diamed A.sub.tri-sp-Ad-DOPE (I) 0.25 n.d. 4+ n.d. 4+ 0.1 n.d. 4+/3+ n.d. 4+/3+ 0.05 w+ 2+ 2+ 2+ 0.04 w+ n.d. 1+ n.d. 0.03 0 n.d. w+ n.d. 0.02 0 n.d. 0 n.d. 0.01 0 0 0 0 A.sub.tri-sp-Ad-DSPE (III) 0.25 n.d. 0 n.d. 0 0.1 n.d. 0 n.d. 0 0.05 0 0 0 0 0.04 0 n.d. 0 n.d. 0.03 0 n.d. 0 n.d. 0.02 0 n.d. 0 n.d. 0.01 0 0 0 0 A.sub.tri-sp.sub.1sp.sub.2-Ad-DOPE 0.25 n.d. 4+ n.d. 4+ (II) 0.1 n.d. 4+ n.d. 4+/3+ 0.05 0 3+ 0 3+ 0.04 0 n.d. 0 n.d. 0.03 0 n.d. 0 n.d. 0.02 0 n.d. 0 n.d. 0.01 0 0 0 0 Incubated control — 0 n.d. 0 n.d. Bench control — 0 n.d. 0 n.d. Abbreviations: n.d. Not determined

(156) TABLE-US-00040 TABLE 39 Stability trial of RBCs transformed with A.sub.tri-sp-Ad-DOPE (I) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL). Agglutination by manual tube serology. Cell Albaclone anti-A Bioclone anti-A storage Concentration of Transformation Solution (mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 4+ 4+° 4+° 4+° Cellstab ™ 4+ 4+ 3+ 4+° 4+° 4+° 10 Alsevers 3+ 2+ 2+ 4+° 4+° 3+ Cellstab ™ 4+° 3+° 2+ 4+° 4+° 4+° 17 Alsevers 4+ 4+ 4+ 4+° 4+° 4+° Cellstab ™ 4+ 4+ 4+ 4+° 4+° 4+° 24 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+° 4+ 4+ Abbreviations: °splatter

(157) TABLE-US-00041 TABLE 40 Stability trial of RBCs transformed with A.sub.tri-sp-Ad-DOPE (I) at low concentrations (0.1 mg/mL, 0.05 mg/mL and 0.025 mg/mL). Agglutination by manual tube serology. Cell Albaclone anti-A Bioclone anti-A storage Concentration of Transformation Solution (mg/mL) Day solution 0.1 0.05 0.025 0.1 0.05 0.025 2 Alsevers 3+/2+ 1+ 1+/w+ 2+ 2+/1+ 1+ Cellstab ™ 3+/2+ 2+ 1+ 3+/2+ 3+/2+ 2+ 8 Alsevers 2+ 1+ w+ 3+/2+ 2+ 2+ Cellstab ™ 2+ 1+/w+ vw 3+° 2+ 1+ 15 Alsevers 2+ 1+ 0 3+ 2+ Vw Cellstab ™ 4+ w+ 0 4+ 4+ 1+ 22 Alsevers 2+ 2+ 0 3+ 2+ w+ Cellstab ™ 4+ 4+ 1+ 4+ 4+ 1+ 44 Alsevers n.d. n.d. n.d. n.d. n.d. n.d. Cellstab ™ 4+ 2+ w+ 4+ 2+ w+ Abbreviations: n.d. Not determined °splatter

(158) TABLE-US-00042 TABLE 41 Stability trial of RBCs transformed with A.sub.tri-sp-Ad-DOPE (I) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL). Agglutination in Diamed gel-cards. Albaclone anti-A Bioclone anti-A Cell Concentration of storage Transformation Solution (mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 10 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 17 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 24 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 45 Alsevers 4+ 4+ 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 59 Alsevers 4+ 4+ 4+ 4+ Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 73 Alsevers Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ 88 Alsevers Cellstab ™ 4+ 4+ 4+ 4+ 4+ 4+ Where there were insufficient cells for testing, blank spaces have been left.

(159) TABLE-US-00043 TABLE 42 Stability trial of RBCs transformed with A.sub.tri-sp-Ad-DOPE (I) at low concentrations (0.1 mg/mL, 0.05 mg/mL and 0.025 mg/mL). Agglutination in Diamed gel-cards. Cell Albaclone anti-A Bioclone anti-A storage Concentration of Transformation Solution (mg/mL) Day solution 0.1 0.05 0.025 0.1 0.05 0.025 2 Alsevers 4+ 2+ 0 4+ 3+ 1+ Cellstab ™ 4+ 2+ 0 4+ 3+ 1+ 8 Alsevers 4+ 3+ 0 4+ 4+ 1+ Cellstab ™ 4+ 3+ 0 4+ 4+ 1+ 15 Alsevers 4+ 2+ 0 4+ 3+/2+ 1+ Cellstab ™ 4+ 4+ 0 4+ 4+ 1+ 22 Alsevers 4+ 3+/2+ 0 4+ 3+ w+ Cellstab ™ 4+ 4+ 0 4+ 4+ 1+ 29 Alsevers 4+ 2+ 0 4+ 3+ w+ Cellstab ™ 4+ 3+ 0 4+ 4+ 2+ 43 Alsevers 4+ 3+ w+ 4+ 4+ 2+ Cellstab ™ 4+ 4+/3+ 0 4+ 4+ 1+ 50 Alsevers 4+ 3+ w+ 4+ 4+ 2+ Cellstab ™ 4+ 3+ 0 4+ 4+ 1+ 57 Alsevers 4+ 3+/2+ 4+ 4+ Cellstab ™ 4+ 3+ 0 4+ 3+ w+ 63 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 0 71 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 0 86 Alsevers Cellstab ™ 4+/3+ 2+ 0 4+ 3+ 0 Where there were insufficient cells for testing, blank spaces have been left.

(160) TABLE-US-00044 TABLE 43 Stability trial of RBCs transformed with B.sub.tri-sp-Ad-DOPE (VI) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL). Agglutination by manual tube serology. Cell Albaclone anti-B Bioclone anti-B storage Concentration of Transformation Solution (mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 3+ 3+ 2+ 2+ 1+ 1+ Cellstab ™ 3+ 2+ 2+ 2+ 2+ 1+ 9 Alsevers 4+ 4+ 2+ 4+ 3+ 2+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 2+ 16 Alsevers 4+ 4+ 3+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 23 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 30 Alsevers 3+ 3+ 2+ 2+ 2+ 2+ Cellstab ™ 4+ 3+ 2+ 3+° 3+° 2+ 37 Alsevers 3+ 2+ 1+ 3+ 2+ 1+ Cellstab ™ 3+ 3+ 2+/1+ 4+° 3+ 1+ 44 Alsevers 4+ 3+ 1+ 3+ 3+ w+ Cellstab ™ 4+ 4+ n.d. 4+ 4+ .sup.‡ 51 Alsevers 3+ 3+ 2+ 4+ 3+ 2+ Cellstab ™ 4+ 4+ n.d. 4+ 4+ 2+ Abbreviations: °splatter

(161) TABLE-US-00045 TABLE 44 Stability trial of RBCs transformed with B.sub.tri-sp-Ad-DOPE (VI) at high concentrations (1 mg/mL, 0.5 mg/mL and 0.25 mg/mL). Agglutination in Diamed gel-cards. Cell Albaclone anti-B Bioclone anti-B storage Concentration of Transformation Solution (mg/mL) Day solution 1 0.5 0.25 1 0.5 0.25 2 Alsevers 4+ 4+ 2+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 9 Alsevers 4+ 4+ 2+ 4+ 4+ 2+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 16 Alsevers 4+ 4+ 2+ 4+ 4+ 1+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 23 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 30 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 37 Alsevers 4+ 4+ 3+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 44 Alsevers 4+ 4+ 2+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 4+/3+ 51 Alsevers 4+ 4+ 2+ 4+ 4+ 3+ Cellstab ™ 4+ 4+ 3+ 4+ 4+ 3+ 58 Alsevers 4+ 1+ 4+ 2+ Cellstab ™ 4+ 4+ 2+ 4+ 4+ 2+ 72 Alsevers 4+ 2+ 4+ 3+ Cellstab ™ 4+ 4+ 3+/2+ 4+ 4+ 3+ 87 Alsevers Cellstab ™ 4+ 4+/3+ 1+ 4+ 4+/3+ 2+/1+ 116 Alsevers Cellstab ™ 4+ 3+ 0 4+ 4+/3+ 1+ Where there were insufficient cells for testing, blank spaces have been left.

Example 9

Red Blood Cell Transformation with H-antigen Synthetic Glycolipids

(162) The water soluble synthetic glycolipids designated H.sub.tri-sp-Ad-DOPE (VII), H.sub.di-sp-Ad-DOPE (VIII) and Galβ-sp-Ad-DOPE (IX) were prepared according to the method described in Example 1 with necessary modifications.

(163) Washed packed mouse RBCs (3 parts by volume) and the synthetic glycolipid solutions (1 part by volume of varying concentrations) were added to an eppendorf tube. The tube was incubated in a 37° C. waterbath for one hour, mixing every 15 minutes. The transformed RBCs were washed 3× with PBS and then suspended in Cellstab™ at the appropriate concentration for serology testing.

(164) Tube serology and Diamed gel-card results for RBCs transformed with the different synthetic glycolipids are presented in Table 46. The results show that three sugars (H.sub.tri) are required for detection by anti-H IgM, at least by the reagent used.

(165) TABLE-US-00046 TABLE 45 Antisera used for results presented in Table 46. Antisera Manufacturer Batch Anti-H IgM Japanese Red Cross HIRO-75 UEA Lorne Laboratories 11549E D.O.E. 06.2004 Bio-UEA EY Labs 201105-2

(166) TABLE-US-00047 TABLE 46 Comparison of transformation of RBCs using H-antigen synthetic glycolipids with different glycotopes made to different concentrations. Conc H Antisera mg/ IgM UEA Bio-UEA Synthetic mL Tube Diamed Tube T0 Tube T20 Tube H.sub.tri-sp-Ad- 1 n.d. n.d. 2+ n.d. 2+ DOPE (VII) 0.25  4+  3+ n.d. n.d. 1+ 0.1  3+  2+ n.d. n.d. n.d. 0.05  1+ 0 n.d. n.d. n.d. 0.01 0 0 n.d. n.d. n.d. H.sub.di-sp-Ad- 0.25 0 n.d. n.d. n.d. n.d. DOPE (VIII) 0.1 0 n.d. n.d. n.d. n.d. 0.05 0 n.d. n.d. n.d. n.d. 0.01 0 n.d. n.d. n.d. n.d. Galβ-sp-Ad- 0.25 0 n.d. n.d. n.d. n.d. DOPE (IX) 0.1 0 n.d. n.d. n.d. n.d. 0.05 0 n.d. n.d. n.d. n.d. 0.01 0 n.d. n.d. n.d. n.d. Human O —  4+ n.d. 1+ 2/3+ 4+ cells Incubated — 0 n.d. 0 0 n.d. control Bench — 0 n.d. n.d. n.d. n.d. control Abbreviations: n.d. Not determined

Example 10

Insertion of Hdi-sp-Ad-DOPE (VIII) and Galβ-sp-Ad-DOPE (IX) Synthetic Glycolipids into Murine Red Blood Cells

(167) The water soluble synthetic glycolipids designated H.sub.di-sp-Ad-DOPE (VIII) and Galβ-sp-Ad-DOPE (IX) were prepared according to the method described in Example 1 with necessary modifications.

(168) Murine RBCs were washed 3× in 1× PBS. 30 μl of packed RBCs were combined with 30 μl of H.sub.di-sp-Ad-DOPE (VIII), and 30 μl of packed RBCs were combined with 30 μl Galβ-sp-Ad-DOPE (IX), respectively. Both synthetic molecule constructs were at a concentration of 1.0 mg/ml. 30 μl of 1× PBS was added to 30 μl of packed RBCs to act as the control group. Cells were incubated for 90 minutes in a 37° C. shaking water-bath. RBCs were washed 3× in 1× PBS.

(169) Three groups of packed RBCs were incubated with an equal volume of lectin UEA-1 for 30 minutes at room temperature. The lectin was prepared in 1× PBS at a concentration of 0.1 mg/ml. 50 μl of a 3% cell suspension was spun for 15 seconds in an Immunofuge at low speed. Results were read by tube serology. The results are presented in Table 48. The results show that neither anti-H IgM nor UEA-1 detects two sugars (H.sub.di).

(170) TABLE-US-00048 TABLE 47 Antisera used for results presented in Table 48. Antisera Manufacturer Batch Biotest anti-H Biotest AG UEA EY Labs 201105-2

(171) TABLE-US-00049 TABLE 48 Murine RBCs transformed with Galβ-sp-Ad-DOPE or H.sub.di-sp-Ad-DOPE, assessed by agglutination. Cell Type Inserted Molecule UEA-1 Mouse IgM.sup.H Murine RBC Galβ (1 mg/ml) 0 n.d. Murine RBC H.sub.di (1 mg/ml) 0 0 Murine RBC Control (PBS) 0 0 Human RBC Control (PBS)  4+  3+ Abbreviations: n.d. Not determined

Example 11

Preparation of Sensitivity Controls

(172) The synthetic glycolipids of the invention may be used in the preparation of “sensitivity controls” (also referred to as “quality control cells”, “serology controls”, or “process controls”) as described in the specification accompanying international application no. PCT/NZ02/00214 (WO 03/034074). The synthetic glycolipids provide the advantage that the transformation of the RBCs may be achieved at reduced temperatures.

(173) RBC Transformation Solutions

(174) Two stock solutions are used: Solution 1: 1 mg/mL A.sub.tri-sp-Ad-DOPE (I) suspended in Celpresol™ solution. Solution 2: 5mg/mL B.sub.tri-sp-Ad-DOPE (VI) suspended in Celpresol™ solution.

(175) Glycolipids are manufactured in a white dry powder. Glycolipids in this form (enclosed in a sealed container under a controlled temperature) are stable for an indefinite period of time. The glycolipids are suspended in solution (e.g. Celpresol™) by weight in order to formulate the transformation solutions.

(176) Once the transformation solutions are received at CSL, they are filtered (through a MILLEX®-GV 0.22μ filter unit) under aseptic conditions.

(177) Processing of RBCs

(178) RBC donations are processed using a continuous flow centrifuge washer under aseptic conditions. RBC donations are washed in buffered saline followed by Celpresol™ solution. The PCV of the RBC donations is measured on a Beckman Coulter AcT Diff analyser. The donations are then adjusted to a packed cell volume (PCV) of 50% with the addition of Celpresol™.

(179) Transformation of RBCs to Provide “Weak AB Cells”

(180) RBCs are washed in buffered saline and Celpresol™. The cells are suspended in Celpresol™ solution to a PCV of >50%. The PCV of red cells is measured using a Beckman Coulter AcT Diff. The mass of the red cell solution is weighed.

(181) The amount of A.sub.tri-sp-Ad-DOPE (I), B.sub.tri-sp-Ad-DOPE (VI) and Celpresol™ for transformation is calculated using the following equations:

(182) $a=\frac{P⨯F}{S}$$b=\frac{P⨯F}{S}$$c=P-\left(1-P\right)-a-b$
where a=amount of A.sub.tri-sp-Ad-DOPE (I) to be added per 1 mL of red cells (mL) b=amount of B.sub.tri-sp-Ad-DOPE (VI) to be added per 1 mL of red cells (mL) c=amount of Celpresol™ to be added per 1 mL of red cells (mL) to dilute cells to 50% PCV P=PCV of red cell solution F=Final desired concentration of glycolipid S=Concentration of stock glycolipid solution

(183) To determine the amount of glycolipid and Celpresol™ to add to a bulk sample of red cells, multiply each of a, b and c by the red cell volume. Add A.sub.tri-sp-Ad-DOPE (I), B.sub.tri-sp-Ad-DOPE (VI) and Celpresol™ to the red cell bulk sample aseptically.

(184) Incubate the sample for 3 hours at 20° C. under controlled temperature conditions and constant gentle agitation. At the end of the 3 hour period, aseptically remove a sample of red cells and test the sample to confirm transformation of the RBCs. Perform blood grouping using tube, tile and column agglutination technology (CAT) techniques.

(185) Incubate the red cell sample for 3 hours at 2-8° C. under controlled temperature conditions and constant gentle agitation for 18 hours. At the end of the 3 hour period, aseptically remove a sample of red cells and test the sample to confirm transformation of the red cells. Perform blood grouping using tube, tile and CAT techniques.

(186) Wash the transformed red cells using a continuous flow centrifuge method, under aseptic conditions using Celpresol™ solution. Measure the PCV of the washed red cells and adjust to 50% PCV by the addition of Celpresol™ solution.

(187) Formulation and Dispensing

(188) Aseptically combine a volume of the transformed RBCs with a volume of simulated plasma diluent (SPD). The plasma may contain monoclonal and polyclonal antibodies. Antibodies are selected according to the desired characteristics of the sensitivity controls. The plasma may additionally contain tartrazine and bovine serum albumin.

(189) Blood grouping and antibody screening is performed on the bulk samples using tube, tile and CAT techniques. The transformed RBC-SPD blend is then aseptically dispensed into BD Vacutainer tubes and the tubes labelled accordingly.

(190) Validation Testing

(191) Weak AB cells produced by the use of synthetic glycolipids (designated A.sub.wB.sub.w in Tables 51 to 53) were used to validate a range of testing platforms in parallel with naturally occurring weak A, weak B and weak AB cells.

(192) TABLE-US-00050 TABLE 49 Reagents and cards used in validation testing. Method Reagent Tube Epiclone Tile Epiclone Ref Manufacturer and type Batch Expiry CAT 1 OCD BioVue ABD/Rev ABR528A 16.06.05 CAT 2 OCD BioVue ABD/Rev ABR521A 06.05.06 CAT 3 OCD BioVue ABD/ABD ACC255A 24.05.05 CAT 4 Diamed ID-MTS 50092.10.02 Apr-05 CAT 5 Diamed ID-MTS Donor typing 51051.05.04 Mar-05 CAT 6 Diamed ID-MTS Recipient typing 50053.07.02 Apr-05 CAT 7 Diamed ID-MTS Cord typing 50961.08.03 Jul-05

(193) TABLE-US-00051 TABLE 50 Testing platform methodology for validation testing. Tile 1 drop 3% cells, 2 drops reagent, 15 min @ RT in moist chamber. Tube 2 drops @ RT, 10 min. ID-MTS As per manufacturers instructions using Dil-2. BioVue As per manufacturers instructions using 0.8% RCD.

(194) TABLE-US-00052 TABLE 51 Validation results across all methods against anti-A. Testing platform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1 A.sub.x w+ 0 2+ 1+ 0 0 0 0 2 A.sub.x w+ 0 2+ 2+ 0 0 0 0 3 A.sub.1B 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4 A.sub.x w+ 0 2+ 2+ 0 0 0 0 5 A.sub.2B 3+ 3+ 4+ 3+ 3+ 1+ 2+ 3+ 6 A.sub.x w+ 0 2+ 2+ 0 0 0 0 7 A.sub.x 1+ 0 2+ 2+ 0 0 0 0 8 A.sub.x w+ 0 2+ 2+ 0 0 0 0 9 A.sub.x 0 0 1+ 1+ 0 0 0 0 10 A.sub.x w+ 0 2+ 2+ 0 0 0 0 11 A.sub.3 4+ 4+ 4+ 3+ 3+ 1+ 1+ 3+ 12 A.sub.3B 3+ 3+ 3+ 3+ 2+ w+ w+ 2+ 13 B.sub.3 0 0 0 0 0 0 0 0 0 14 B.sub.3 0 0 0 0 0 0 0 0 0 15 A.sub.wB.sub.w 2+ 2+ 2+ 2+ 2+ 0 0 0 0

(195) TABLE-US-00053 TABLE 52 Validation results across all methods against anti-B. Testing platform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1 A.sub.x 0 0 0 0 0 0 0 0 2 A.sub.x 0 0 0 0 0 0 0 0 3 A.sub.1B 4+ 4+ 4+ 4+ 4+ 4+ 3+ 3+ 4+ 4 A.sub.x 0 0 0 0 0 0 0 0 5 A.sub.2B 4+ 4+ 4+ 4+ 4+ 3+ 3+ 4+ 6 A.sub.x 0 0 0 0 0 0 0 0 7 A.sub.x 0 0 0 0 0 0 0 0 8 A.sub.x 0 0 0 0 0 0 0 0 9 A.sub.x 0 0 0 0 0 0 0 0 10 A.sub.x 0 0 0 0 0 0 0 0 11 A.sub.3 0 0 0 0 0 0 0 0 12 A.sub.3B 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 13 B.sub.3 2+ 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+ 14 B.sub.3 2+ 2+ 2+ 2+ 2+ 2+ 1+ 1+ 2+ 15 A.sub.wB.sub.w 3+ 3+ 1+ 1+ 1+ 0 0 0 0

(196) TABLE-US-00054 TABLE 53 Validation results across all methods against anti-AB. Testing platform Cell Type Tube Tile CAT 1 CAT 2 CAT 3 CAT 4 CAT 5 CAT 6 CAT 7 1 A.sub.x 3+ 2+ 2+ 2 A.sub.x 4+ 2+ 3+ 3 A.sub.1B 4+ 4+ 4+ 4 A.sub.x 3+ 2+ 3+ 5 A.sub.2B 4+ 4+ 4+ 6 A.sub.x 4+ 4+ 3+ 7 A.sub.x 4+ 4+ 3+ 8 A.sub.x 3+ 4+ 3+ 9 A.sub.x 4+ 2+ 2+ 10 A.sub.x 3+ 4+ 3+ 11 A.sub.3 4+ 4+ 4+ 12 A.sub.3B 4+ 4+ 4+ 13 B.sub.3 2+ 2+ 2+ 14 B.sub.3 2+ 2+ 2+ 15 A.sub.wB.sub.w 3+ 3+ 3+

Example 12

Attachment of Modified Embryos to Transformed Endometrial Cells

(197) The ability to effect qualitative and quantitative differences in the cell surface antigens expressed by cell types other than RBCs was investigated. The ability to enhance the adhesion of embryos to endometrial cells was adopted as a model system.

(198) The synthetic molecules may be used as synthetic membrane anchors and/or synthetic molecule constructs. Therefore, they may also be employed in the method of enhancing embryo implantation as described in international patent application no PCT/NZ2003/000059 (published as WO 03/087346) which is incorporated by reference.

(199) Endometrial Cell Transformation

(200) Insertion of Water Soluble Synthetic Molecule Construct

(201) A single cell suspension of endometrial epithelial cells was prepared. The endometrial cells were washed 3× by resuspending in CMF HBSS and centrifuging at 2000 rpm for 3 minutes.

(202) The washed cell preparation was resuspended in 50 μl of M2.

(203) Micro-centrifuge tubes each containing a 50 μl solution of 5 M/ml endometrial cells were prepared. To separate tubes of endometrial cells 50 μl of synthetic glycolipids A.sub.tri-sp-Ad-DOPE (I) or B.sub.tri-sp-Ad-DOPE A (VI), or 50 μl M2 were added to the control cells. The cells were incubated for 90 minutes at 37° C. on a mixer. The endometrial cells were washed 3× by resuspending in CMF HBSS media and centrifuging at 2000 rpm for 3 minutes. The washed cell preparation was resuspended in 50 μl of M2.

(204) Test For Insertion Using Fluorescent Probe:

(205) 50 μl of corresponding primary murine monoclonal antibody was added to each tube. Each tube was incubated at room temperature for 10 minutes. Cells were washed 3× in M2 media. 10 μl of mouse anti-IgG FITC was added to each tube. Tubes were incubated at room temperature in dark conditions for 10 minutes. Endometrial cells were mounted on glass slides and viewed under a fluorescence microscope.

(206) Test for Direct Agglutination:

(207) 5 μl of each group of cells was placed onto separate microscope slides. To each 5 μl drop of cells 5 μl of a corresponding antibody was added. The cells were gently mixed on the slide for 2 minutes. Agglutination was visualised under the microscope. The results are presented in Table 55.

(208) TABLE-US-00055 TABLE 54 Antisera used for results presented in Table 55. Antisera Manufacturer Bioclone anti-A Ortho Diagnostics 01102 D.O.M. 16.05.02 Bioclone anti-B Ortho Diagnostics Developmental reagent

(209) TABLE-US-00056 TABLE 55 Endometrial cells transformed with A.sub.tri-sp-Ad-DOPE (I) or B.sub.tri-sp-Ad-DOPE A (VI), as visualised using fluorescence. Agglutination Fluorescence score reaction by after incubation with microscopic Cell Type Inserted Antigen 1° antibody IgFITC Probe visualisation Endometrial A.sub.tri-sp-Ad-DOPE Anti-A Bioclone 4+ 4+ cells (I) (1 mg/ml) Endometrial B.sub.tri-sp-Ad-DOPE Anti-B Bioclone 1+ 3+ cells (VI) (1 mg/ml) Endometrial Control (M2 Anti-A Bioclone 0  0  cells media)

(210) Embryo Modification

(211) Insertion of Water Soluble Synthetic Molecule Construct:

(212) The embryo zona pellucida was removed by treating embryos with 0.5% pronase in a 37° C. oven for 6 minutes or until all zonas were removed. Micro-drops were prepared by adding 5 μl of synthetic glycolipid A.sub.tri-sp-Ad-DOPE (I) or B.sub.tri-sp-Ad-DOPE (VI), at a concentration of 1 mg/mL to a 45 μl drop of M2 media overlaid with mineral oil. All embryo groups were incubated in the 50 μl micro-drops for 1 hour at 37° C. Embryos from experimental and control groups were washed 3× with M2 media.

(213) Test for Insertion:

(214) Embryos from experimental and control groups were placed into a micro-drop of corresponding antibody and incubated for 30 min at 37° C. Embryos from experimental and control groups were washed 3× with M2 media.

(215) Embryos from all experimental and control groups were placed into micro-drops of anti-mouse Ig FITC (1:50 dilution anti-mouse Ig FITC in M2) and incubated for 30 min at 37° C. Embryos from experimental and control groups were washed 3× with M2 media. Embryos were mounted on microscope slides in a 5 μl drop of M2 and the drops overlaid with oil.

(216) The slides were viewed under a fluorescence microscope. Results are presented in Tables 56 and 57. The negative result for transformation with B.sub.tri-sp-Ad-DOPE (VI) is attributed to a lack of 1° antibody sensitivity.

(217) TABLE-US-00057 TABLE 56 Embryos transformed with A.sub.tri-sp-Ad-DOPE (I) as visualised using fluorescence. Fluorescence score Embryo after incubation with Morphology 24 hr Cell Type Inserted Antigen 1° antibody IgFITC Probe post insertion Embryos A.sub.tri-sp-Ad-DOPE Anti-A Bioclone 2+/1+ Appeared viable (I) Embryos Control Anti-A Bioclone 0 Appeared viable

(218) TABLE-US-00058 TABLE 57 Embryos transformed with A.sub.tri-sp-Ad-DOPE (I) or B.sub.tri-sp-Ad-DOPE (VI), as visualised using fluorescence. Fluorescence score Embryo after incubation with Morphology 24 hr Cell Type Inserted Antigen 1° antibody IgFITC Probe post insertion Embryos A.sub.tri-sp-Ad-DOPE Anti-A Bioclone  2+ n.d. (I) Embryos B.sub.tri-sp-Ad-DOPE Anti-B Bioclone 0 n.d. (VI) Embryos Control (M2 Anti-A Bioclone 0 n.d. media) Abbreviations: n.d. Not determined

(219) Enhanced Attachment Transformed Endometrial Cells to Modified Embryos

(220) Modified embryos (BioG-Avidin-BiolgG.sup.B and BioG-Avidin-BioIgM.sup.A) were prepared in accordance with the methods described in the specification accompanying the international application no. PCT/NZ03/00059 (published as WO03/087346).

(221) Two concave glass slides were prepared, one with two wells of synthetic glycolipid A.sub.tri-sp-Ad-DOPE (I) inserted endometrial cells and the other with two wells of synthetic glycolipid B.sub.tri-sp-Ad-DOPE (VI) inserted endometrial cells.

(222) The two groups of embryos were transferred to each of the concave glass slides: Slide 1 A.sub.tri/IgG.sup.B embryos A.sub.tri/IgM.sup.A embryos Slide 2 B.sub.tri/IgG.sup.B embryos B.sub.tri/IgM.sup.A embryos

(223) The embryos were surrounded with endometrial cells. The wells were covered with mineral oil and incubated for 15 minutes at 37° C. Using a wide bore handling pipette each group of embryos were carefully transferred to a fresh drop of M2 media. The embryos were gently washed. The embryos were gently transferred into 2 μL of M2 media on a marked microscope slide. Each drop was overlaid with mineral oil

(224) Under a central plane of focus on an Olympus microscope the number of endometrial cells adhered to the embryos in each group was assessed. The number of cells adhered to each embryo was recorded. Results are presented in Table 58.

(225) TABLE-US-00059 TABLE 58 Endometrial cells transformed with A.sub.tri-sp-Ad-DOPE (I) or B.sub.tri-sp-Ad-DOPE (VI), and embryos modified with BioG-Avidin-BioIgG.sup.B or BioG-Avidin-BioIgM.sup.A. Assessment by attachment of endometrial cells to embryos. Average number of endometrial cells Transformed attached endometrial to modified Cell Type cells Modified embryos embryos Endometrial A.sub.tri-sp-Ad- BioG-Avidin-BioIgG.sup.B 2.3 cells DOPE (I) BioG-Avidin-BioIgM.sup.A 7.25 Endometrial cells B.sub.tri-sp-Ad- BioG-Avidin-BioIgG.sup.B 6.7 DOPE (VI) BioG-Avidin-BioIgM.sup.A 3.4

(226) Where in the foregoing description reference has been made to integers or components having known equivalents then such equivalents are herein incorporated as if individually set forth.

(227) Although the invention has been described by way of example and with reference to possible embodiments thereof it is to be appreciated that improvements and/or modification may be made thereto without departing from the scope or spirit of the invention.

REFERENCES

(228) Abe K, McKibbin J M & Hakomori S I. (1983) The monoclonal antibody directed to difucosylated type 2 chain (Fucα1.fwdarw.2Galβ1.fwdarw.4[Fucα1.fwdarw.3]GlcNAc; Y determinant). J. Biol. Chem. 258: 11793-11797.

(229) Adamany A M, Blumenfeld O O, Sabo B & McCreary J. (1983) A carbohydrate structural variant of MM glycoprotein (glycophorin A). J. Biol. Chem. 258: 11537-11545.

(230) Blanchard D, Cartron J P, Fournet B, Mountreuil J, van Halbeek H & Vliegenthart J F G. (1983) Primary structure of the oligosaccharide determinant of blood group Cad specificity. J. Biol. Chem. 258: 7691-7695.

(231) Fukuda M, Dell A & Fukuda M. (1984a) Structure of fetal lactosaminoglycan. The carbohydrate moiety of band 3 isolated from human umbilical cord erythrocytes. J. Biol. Chem. 259: 4782-4791.

(232) Fukuda M, Dell A, Oates J E & Fukuda M. (1984b) Structure of branched lactosaminoglycan, the carbohydrate moiety of band 3 isolated from adult human erythrocytes. J. Biol. Chem. 259: 8260-8273.

(233) Fukuda M, Lauffenberger M, Sasaki H, Rogers M E & Dell A. (1987) Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins. J. Biol. Chem. 262: 11952-11957.

(234) Fukuda M N, Dell A, Oates J E, Wu P, Klock J C & Fukuda M. (1985) Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells. J. Biol. Chem. 260: 1067-1082.

(235) Gillard B K, Blanchard D, Bouhours J F, Cartron J P, van Kuik J A, Kamerling J P, Vliegenthart J F G & Marcus D M. (1988) Structure of a ganglioside with Cad blood group antigen activity. Biochemistry. 27: 4601-4604.

(236) Hakomori S I, Nudelman E, Levery S B & Kannagi R. (1984) Novel fucolipids accumulating in human adenocarcinoma. I. Glycilipids with di- or trifucosylated type 2 chain. J. Biol. Chem. 259: 4672-4680

(237) Hanfland P. (1975) Characterisation of B and H blood group active glycosphingolipids from human B erythrocyte membranes. Chem. Phys. Lipids. 15: 105-124.

(238) Hanfland P, Kordowicz M, Niermann H, Egge H, Dabrowski U, Peter-Katalinic J & Dabrowski J. (1984) Purification and structures of branched blood-group-B-active glycosphingolipids from human erythrocyte membranes. Eur. J. Biochem. 145: 531-542.

(239) Hanfland P, Kordowicz M, Peter-Katalinic J, Pfannschmidt G, Crawford R J, Graham H A & Egge H. (1986) Immunochemistry of the Lewis blood-group system: isolation and structures of Lewis-c active and related glycosphingolipids from the plasma of blood-group O Le(a-b-) nonsecretors. Arch. Biochem. Biophys. 246: 655-672.

(240) Hiraiwa N, Tsuyuoka K, Li Y T, Tanaka M, Seno T, Okubo Y, Fukuda Y, Imura H & Kannagi R. (1990) Gangliosides and sialoglycoproteins carrying a rare blood group antigen determinant, Cad, associated with human cancers as detected by specific monoclonal antibodies. Cancer Res. 50: 5497-5503.

(241) Kannagi R , Nudelman E, Levery S B, & Hakomori S I. (1982) A series of human erythrocytes glycosphingolipids reacting to the monoclonal antibody directed to a developmentally regulated antigen, SSEA-1. J. Biol. Chem. 257: 14865-14874.

(242) Kewitz S, Groβ H J, Kosa R & Roelcke D. (1995) Anti=Pr cold agglutinins recognise immunodominant α2,3- or α2,6-sialylgroups on glycophorins. Glycocon. J. 12: 714-720.

(243) Koscielak J, Miller-Podraza H, Krauze R & Piasek A. (1976) Isolation and characterisation of poly(glycosyl)ceramides (megaloglycolipids) with A, H, and I blood group activities. Eur. J. Biochem. 71: 9-18.

(244) Laine R A. (1994) Invited commentary. Glycobiol 4: 759-767

(245) Lidowska E, Duk M & Dahr W. (1980) Comparison of alkali-labile oligosaccharide chains of M and N blood-group glycopeptides from human erythrocyte membrane. Carbohydr. Res. 79: 103-113.

(246) Lloyd K O & Kabat E A. (1968) Immunochemical studies on blood groups. XLI. Proposed structures for the carbohydrate portions of blood group A, B, H, Lewis.sup.a, and Lewis.sup.b substances. Proc. Natl. Acad. Sci USA. 61: 1470-1477.

(247) Lundblad A. (1977) Urinary glycoproteins, glycopeptides, and oligosaccharides. In: The Glycoconjugates Eds Horowitz M I & Pigman W. Vol 1: 441-458.

(248) Magnani J L, Nilsson B, Brockhaus M, Zopf D, Steplewski Z, Koprowski H & Ginsburg V. (1986) A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-N-fucopentaose II. J. Biol. Chem. 257: 14365-14369.

(249) Nudelman E, Fukushi Y, Levery S B, Higuchi T & Hakomori S I. (1986) Novel fucolipids of human adenocarcinoma: disialoyl Le.sup.a antigen (III.sup.4FucIII.sup.6NeuAcIV.sup.3NeuAcLc.sub.4) of human colonic adenocarcinoma and the monoclonal antibody (FH7) defining this structure. J. Biol. Chem. 261: 5487-5495.

(250) Slomiany A, Zdebska E & Slomiany B L. (1984) Structures of the neutral oligosaccharides isolated from A-active human gastric mucin. J. Biol. Chem. 259: 14743-14749.

(251) Takasaki S, Yamashita K & Kobata A. (1978) The sugar chain structures of ABO blood group active glycoproteins obtained from human erythrocyte membrane. J. Biol. Chem. 253: 6086-6091.

(252) Tanaka M, Dube V E & Anderson B. (1984) Structures of oligosaccharides cleaved by base-borohydride from an I, H, and Lea active ovarian cyst glycoprotein. Biochim. Biophys. Acta. 798: 283-290.

(253) Thomas D B & Winzler R J. (1969) Structural studies on human erythrocytes glycoprotein. Alkali-labile oligosaccharides. J. Biol. Chem. 244: 5943-5946.

(254) Watkins W M. (1966) Blood group substances. Science. 152: 172-181.

(255) Yoshima H, Furthmayr H & Kobata A. (1980) Structures of the asparagine-linked sugar chains of glycophorin A. J. Biol. Chem. 255: 9713-9718.