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Evaluation of anti-A monoclonal antibodies
Revue Frangaise Tome XXX. -
de Transfusion
N°5. -
et I m m u n o - h 6 m a t o l o g i e 397
1987
Evaluation of anti-A monoclonal antibodies b y D . T i l l s * * a n d J. B u s h r o d * * South London Blood Transfusion Service and ** Department of Anthropology University of Cambridge United Kingdom
INTRODUCTION On receipt the culture supernatants were aliquoted in 1 ml. quantities and frozen at - 2 5 ° C. The ascitic fluids were diluted 1 : 10 in saline containing 6 % Bovine Albumin and frozen at - 25 ° C in 1 ml. quantities. Rare A variant cells were stored in glycerol at - 2 5 ° C and recovered by dialysis against saline overnight. S o m e of the cells stored in glycerol were also available fresh and f r o m liquid nitrogen. No difference in reactivity was found between fresh and stored cells. A~ and A2 cells were laid down in glycerol f r o m large samples and used as standard cells for inhibition, pH and temperature studies. Several culture supernatants which formed part of a previously reported study (Trots, 1986) of anti-A, anti-B and anti-A, B monoclonals were included as controls. S o m e of these were c o m m e r c i a l reagents and one was a recent h u m a n / h u m a n clone. This gave a total of 39 reagents in this survey, some of which had already been tested against a n u m b e r of the A variants which were also used in this study.
METHOD The testing m e t h o d used was that of WARLOW and TmLS (1978) using Ubottomed microtitre plates which had previously been rinsed in 0.2 % TWEEN 20 and dried. 50 t~1. of each antibody was placed in the microtitre plate wells, the plates were set up in batches and then frozen at - 25 ° C until use. 50 txl. of a 1% suspension of erythrocytes were added and the plates briefly shaken to mix. Incubation was p e r f o r m e d at 10 ° C for 10 minutes, this temperature was chosen as r o o m temperature was found to fluctuate between 10 and 30 ° C. Before reading the microtitre plates were centrifuged at 1,000 r p m for 30 seconds. Reading was effected by streaming at an angle of 80 ° and then reading over an illuminated concave mirror.
398
TILLS D, and coll.
The tests p e r f o r m e d during the study were. 1. Reactions with variants of the A antigen 2. Effect of temperature, 4 - 37 ° C 3. Effect of pH 4. Sugar inhibitions 5. Reactions with known A reactive B cells 6. Reactions with enzyme treated B cells 7. Reactions with enzyme treated O cells Unless otherwise stated culture supernatant 34 W 1 (In) reacted with the vast majority of cells by all techniques, including n o r m a l B and O cells which had been enzyme treated.
R e a c t i o n s with variants of the A antigen The m o n o c l o n a l antibodies were tested against the following A variants :
A~
19
Ab~,.~. Aend Am
13 3 4
A~t
1
A~I AFinn
1 1
Ah
3
A~B Weak A3
A3B BA Oriental Wk A Bombay TOTAL
4 8 3 2 12 1 78
Reactions with Ax cells Clone 34 W 1 reacted with most of the A variant cells and as it also reacts with most B's and some O's it has been omitted f r o m this analysis. Of the nineteen A~ cells included in the study only nine of them were detected by any of the anti-A clones. The reactions detected were as follows. 1 cell only reacted with 1 MAb, No 19 W 7 1 cell reacted with three MAb's 2 cells reacted with 5 MAb's
399
E VAL UA TION OF ANTI-A MONOCL ONAL ANTIBODIES
3 cells reacted with 6 MAb's 1 celt reacted with 10 MAb's 1 cell reacted with 14 MAb's Clone 19 W 7 reacted with nine cells. The most frequently reacting culture supernatants were 17 W 1, 9 W 1, 1 W 3, 19 W 10 and 19 W 8. The most frequently reacting ascitic fluid was 19 W 7. However, as previously found, TmLs 1986 the vast majority of clone are not capable of detecting Ax cells.
Reactions with
Abant u
The range of reactivity here was very considerable. Excluding 34 W 1 which reacted with all thirteen cells, the detection rate of the monoclonals for the cells ranged from 0 to 18. This covers the range f r o m A~ to A2B. Similar results with 37 monoclonals not included here were found in my previous study. It may be that the Ab~,u is either an heterogeneous group or that it's expression is affected by modifier genes.
Weak A2l's Early work on anti-A MAb's suggested that A2 and A2B cells showed wide variation in reaction strength. The four cells included here were found to give weak or negative reactions with a monoclonal anti-A widely used some years ago in the United Kingdom. The results obtained in this study are almost identical with those found in the earlier one. One cell did not react with 22 of the MAb's, another did not with 21 Mab's, a third showed no reaction with 17 Mab's and the last did not react with 13 Mab's. This illustrates the importance of testing all anti-A monoclonals with a panel of A2 and A2B cells known to be weak reactors.
Reactions of A 3 and AaB cells One of the eight A3 cells appears to have been missclassified as it only reacted with culture supernatant 34 W 1. Four A3B's were also tested. The reaction rate of t h e A 3 cells i.e. the n u m b e r of antibodies reacting with the cells ranged f r o m 9-20 with all but one being in the range of 13-20. The A3B's were in the mid range. Variation between A3 cells has been well reported in the same individual as well as between individuals. The earlier study of TmLs (1986) also showed considerable variation although with a superior detection rate where with the same cells 3 of the A3 samples were detected by 34 of the 37 MAb's.
Other variants of the A antigen The MAb's were also tested against 4 Am, 3 Aend, 3 A h, 1 Ain,, 1 Amn, 1 Apae and one cell only designated as A,B. OtheFthan 34 W 1 which reacted with all of the cells only 2 Ail, the A~nt and the AwB reacted with any of the MAb's. The Ai.t reacted with all of the clones except 15 W 3, one of the Ah cells was negative while of the others one reacted with 6 MAb's and the other with 14. The AwB cell reacted with 14 of the MAb's suggesting that it is either a very weak A2B or an A3B.
TILLS 12 and coll.
400
Oriental A variants Dr. Susan LEONGof the Hong Kong Blood Transfusion Service kindly supplied me with 12 A variant cells from Chinese subjects these comprised 4 A2's, 2 A2B's, 2 A~n,'S, 1 A~tB and 3 A3B's. All of these cells behaved as had the equivalent European cells.
1. Effects o[ temperature The monoclonal antibodies were tested against the standard A~ and A2 cells at 4, 10, 15 and 37 ° C. No marked differences in reaction strengths were found.
2. Effects o/ p H The buffers used included both phosphate and biological ones, both were used at pH 7.0 to see if any differences were caused by the different type of buffer. The range was pH 4.0 to 10.5. They were made up in normal physiological saline in 20 mM strength.
pH 4.0 5.5 6.0 6.5 7.0 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
Buffer Phosphate MES MES ADA BES Phosphate HEPES EPPS TAPPS Phosphate Phosphate CAPPS BIS TRIS PROPANE
The tests were set up with the 1% red cell suspension being made up in the appropriate buffer. Each buffer was tested against the standard A1 and A2 cells. As was expected the loss of reactivity was more easily detectable with A2 than with A1 dells. Most of the MAb's reacted well at median pH values between pH 5.5 and 8.5. Many of the reagents were sensitive to both high and low pH levels.
3. Sugar inhibitions The sugars used were
Lactose L-Fucose
Methyl Mannoside D-Fucose
EVALUATION OF ANTI-A MONOCLONAL ANTIBODIES
L-Mannitol Sucrose Melibiose D-Galactose D-Glucose D-Galactosamine
401
D-Mannitol Arabinose Raffinose N-Acetyl-Galactosamine D-Glucosamine
Some of the sugars used were specifically selected while others were included because they were being used for another project and were therefore available. 20 mM solutions were made up in normal physiological saline and added in 50 ~1. amounts to the monoclonal antibodies and left for 1 hour at 15° C. After this time the tests were carried out as previously described. A set of control tests with saline added rather than sugar were also set up. Two of the MAb's were inhibited by virtually all sugars, but as both of these had a very narrow pH range this inhibition may be more related to pH rather than to sugar inhibition. Again as would be expected inhibitions were more easily seen with A2 as the indicator cell. However both A~ and A2 cells were used. The sugars neutralising the most MAb's were L-Mannitol, D-Fucose and N-Acetyl-Galactosamine. L-Mannitol and D-Fucose in saline both have a pH of approximately 4.5 and we originally thought that the inhibitions were due to pH but as other low pH sugars do not cause inhibition this work will have to be repeated using buffered sugars. Two antibodies showed inhibition, numbers 31 W 1 (m) and 7 W 2 (m) with N-Ace@ Galactosamine with both A1and A2 cells at pH 6.0. The human/human clone was only inhibited by N-Acetyl Galactosamine. None of the commercial reagents were inhibited by any of the sugars.
4. Reactions with A reactive B cells This subject has been well reviewed in the report of the Ortho Scientific Forum so we will not deal with it in any detail here. Merz-Dade in Switzerland very kindly sent us two examples they had detected of A reactive B cells. These both worked well without enzyme treatment and reacted with two of the reagents, 1 W 3 (m) and 19 W 7 (m) ascities.
5. Reactions with e n z y m e treated B cells A total of 50 B individuals from routine bleeds at the South London Blood Transfusion Centre were tested after enzyme treatment with Bromelin and Pronase. Mab 34 W 1 reacted with all of the B samples tested and sample 26 W 1 reacted with more than half of them. No differences in reaction was found between Bromelin or Pronase treated erythrocytes.
6. Reactions with e n z y m e treated 0 cells The O cells were again collected from the routine donor panel from South London BTS. Sample 34 W 1 reacted with 10 of the 50 O cells after they had been treated with enzyme. Sample 1 W 3 reacted very strongly with two O samples and is being further investigated for a possible cross-reacting antibody.
402
TILLS D. and colL
CONCLUSIONS Quality control The m a i n p r o b l e m we have in evaluating m o n o c l o n a l anti-A's is the selection of an appropriate reference cell which can be used for quality control. In the past w h e n we were using polyclonal reagents the cell of choice was an A2B with the anti-A c o m p o n e n t of the anti-A,B being expected to detect some if not all A~ cells. The use of MAb's raised our expectations and m a n y of us expected that MAb. anti-A's would b e c o m e available that would readily detect all N,'s. This would have enabled us to drop the anti-A,B f r o m routine testing so saving both time and money. This discovery of the A reacting B cell which reacted strongly with most of anti-A MAb's which also detected the A~ cell, even if this is technique dependent, has caused a rethink. Several workers now r e c o m m e n d using an anti-A which detects A2B's but not Ax'S and so gives no p r o b l e m with A reactive B cells. The anti-A, B is then retained to detect A~'s. This change in emphasis m a y explain why if we use the detection of the A~ cell as our criteria for a good m o n o c l o n a l anti-A then this batch of 30 clones is inferior to the 37 1 tested in the 1986 paper. In that study three MAb's detected 10 out of the fourteen A~ cells included.
Genetics S o m e workers, Lt~Eteao (1985) and GooI e t al. (1985) have suggested that the A antigen shows epitopes. However, on the evidence of this study and that of TILLS (1986), epitopes do not appear to be shown by agglutination tests. Both surveys suggest that the various sub-groups of A show overlapping reactivity and antigenicity. However, in m a r k e d contrast the evidence for the B antigen f r o m the survey carried out by TmLS (1986) (we were unable to obtain anti-B MAb's for this survey) suggests strongly the existence of epitopes. Acknowledgements Without the generous donations of reference cells by almost all of the Transfusion Centres in the UK we would not have been able to have done this work. In addition our gratitude is due to Dr. ImoNo and the staff of the Hong Kong Transfusion Center for the Oriental A variants and to Dr. Phyllis Moopms of the Natal Blood Transfusion Service for sending us the Abantu samples.
EVALUATIONOFANTI-A MONOCLONALANTIBODIES
403
REFERENCES WARLOWA., TILLS D. - Micromethods in blood group serology. Vox Sang., 1978, 35, 354-356. D. - ABH Monoclonals. Develop. biol. Standard., 1985, 67, 85-90. LOBENKOA. - Analysis of the heterogeneity of the Blood Group A2B using Monoclonal Typing Reagents. Abstract British Blood Transfusion Society Meeting. Oxford, 1985. GOOI H.C., HOUNSELLE.F., P~CARDJ.K,, LOWE A.D., VOAKD., LENNOX E.S., FEIZl T. - Differing reactions of monoclonal Anti-A antibodies with oligosaccharides related to blood group A. Journal of Biological Chemistry, 1985, 260, 13218-13224.
de Transfusion
N°5. -
et I m m u n o - h 6 m a t o l o g i e 397
1987
Evaluation of anti-A monoclonal antibodies b y D . T i l l s * * a n d J. B u s h r o d * * South London Blood Transfusion Service and ** Department of Anthropology University of Cambridge United Kingdom
INTRODUCTION On receipt the culture supernatants were aliquoted in 1 ml. quantities and frozen at - 2 5 ° C. The ascitic fluids were diluted 1 : 10 in saline containing 6 % Bovine Albumin and frozen at - 25 ° C in 1 ml. quantities. Rare A variant cells were stored in glycerol at - 2 5 ° C and recovered by dialysis against saline overnight. S o m e of the cells stored in glycerol were also available fresh and f r o m liquid nitrogen. No difference in reactivity was found between fresh and stored cells. A~ and A2 cells were laid down in glycerol f r o m large samples and used as standard cells for inhibition, pH and temperature studies. Several culture supernatants which formed part of a previously reported study (Trots, 1986) of anti-A, anti-B and anti-A, B monoclonals were included as controls. S o m e of these were c o m m e r c i a l reagents and one was a recent h u m a n / h u m a n clone. This gave a total of 39 reagents in this survey, some of which had already been tested against a n u m b e r of the A variants which were also used in this study.
METHOD The testing m e t h o d used was that of WARLOW and TmLS (1978) using Ubottomed microtitre plates which had previously been rinsed in 0.2 % TWEEN 20 and dried. 50 t~1. of each antibody was placed in the microtitre plate wells, the plates were set up in batches and then frozen at - 25 ° C until use. 50 txl. of a 1% suspension of erythrocytes were added and the plates briefly shaken to mix. Incubation was p e r f o r m e d at 10 ° C for 10 minutes, this temperature was chosen as r o o m temperature was found to fluctuate between 10 and 30 ° C. Before reading the microtitre plates were centrifuged at 1,000 r p m for 30 seconds. Reading was effected by streaming at an angle of 80 ° and then reading over an illuminated concave mirror.
398
TILLS D, and coll.
The tests p e r f o r m e d during the study were. 1. Reactions with variants of the A antigen 2. Effect of temperature, 4 - 37 ° C 3. Effect of pH 4. Sugar inhibitions 5. Reactions with known A reactive B cells 6. Reactions with enzyme treated B cells 7. Reactions with enzyme treated O cells Unless otherwise stated culture supernatant 34 W 1 (In) reacted with the vast majority of cells by all techniques, including n o r m a l B and O cells which had been enzyme treated.
R e a c t i o n s with variants of the A antigen The m o n o c l o n a l antibodies were tested against the following A variants :
A~
19
Ab~,.~. Aend Am
13 3 4
A~t
1
A~I AFinn
1 1
Ah
3
A~B Weak A3
A3B BA Oriental Wk A Bombay TOTAL
4 8 3 2 12 1 78
Reactions with Ax cells Clone 34 W 1 reacted with most of the A variant cells and as it also reacts with most B's and some O's it has been omitted f r o m this analysis. Of the nineteen A~ cells included in the study only nine of them were detected by any of the anti-A clones. The reactions detected were as follows. 1 cell only reacted with 1 MAb, No 19 W 7 1 cell reacted with three MAb's 2 cells reacted with 5 MAb's
399
E VAL UA TION OF ANTI-A MONOCL ONAL ANTIBODIES
3 cells reacted with 6 MAb's 1 celt reacted with 10 MAb's 1 cell reacted with 14 MAb's Clone 19 W 7 reacted with nine cells. The most frequently reacting culture supernatants were 17 W 1, 9 W 1, 1 W 3, 19 W 10 and 19 W 8. The most frequently reacting ascitic fluid was 19 W 7. However, as previously found, TmLs 1986 the vast majority of clone are not capable of detecting Ax cells.
Reactions with
Abant u
The range of reactivity here was very considerable. Excluding 34 W 1 which reacted with all thirteen cells, the detection rate of the monoclonals for the cells ranged from 0 to 18. This covers the range f r o m A~ to A2B. Similar results with 37 monoclonals not included here were found in my previous study. It may be that the Ab~,u is either an heterogeneous group or that it's expression is affected by modifier genes.
Weak A2l's Early work on anti-A MAb's suggested that A2 and A2B cells showed wide variation in reaction strength. The four cells included here were found to give weak or negative reactions with a monoclonal anti-A widely used some years ago in the United Kingdom. The results obtained in this study are almost identical with those found in the earlier one. One cell did not react with 22 of the MAb's, another did not with 21 Mab's, a third showed no reaction with 17 Mab's and the last did not react with 13 Mab's. This illustrates the importance of testing all anti-A monoclonals with a panel of A2 and A2B cells known to be weak reactors.
Reactions of A 3 and AaB cells One of the eight A3 cells appears to have been missclassified as it only reacted with culture supernatant 34 W 1. Four A3B's were also tested. The reaction rate of t h e A 3 cells i.e. the n u m b e r of antibodies reacting with the cells ranged f r o m 9-20 with all but one being in the range of 13-20. The A3B's were in the mid range. Variation between A3 cells has been well reported in the same individual as well as between individuals. The earlier study of TmLs (1986) also showed considerable variation although with a superior detection rate where with the same cells 3 of the A3 samples were detected by 34 of the 37 MAb's.
Other variants of the A antigen The MAb's were also tested against 4 Am, 3 Aend, 3 A h, 1 Ain,, 1 Amn, 1 Apae and one cell only designated as A,B. OtheFthan 34 W 1 which reacted with all of the cells only 2 Ail, the A~nt and the AwB reacted with any of the MAb's. The Ai.t reacted with all of the clones except 15 W 3, one of the Ah cells was negative while of the others one reacted with 6 MAb's and the other with 14. The AwB cell reacted with 14 of the MAb's suggesting that it is either a very weak A2B or an A3B.
TILLS 12 and coll.
400
Oriental A variants Dr. Susan LEONGof the Hong Kong Blood Transfusion Service kindly supplied me with 12 A variant cells from Chinese subjects these comprised 4 A2's, 2 A2B's, 2 A~n,'S, 1 A~tB and 3 A3B's. All of these cells behaved as had the equivalent European cells.
1. Effects o[ temperature The monoclonal antibodies were tested against the standard A~ and A2 cells at 4, 10, 15 and 37 ° C. No marked differences in reaction strengths were found.
2. Effects o/ p H The buffers used included both phosphate and biological ones, both were used at pH 7.0 to see if any differences were caused by the different type of buffer. The range was pH 4.0 to 10.5. They were made up in normal physiological saline in 20 mM strength.
pH 4.0 5.5 6.0 6.5 7.0 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
Buffer Phosphate MES MES ADA BES Phosphate HEPES EPPS TAPPS Phosphate Phosphate CAPPS BIS TRIS PROPANE
The tests were set up with the 1% red cell suspension being made up in the appropriate buffer. Each buffer was tested against the standard A1 and A2 cells. As was expected the loss of reactivity was more easily detectable with A2 than with A1 dells. Most of the MAb's reacted well at median pH values between pH 5.5 and 8.5. Many of the reagents were sensitive to both high and low pH levels.
3. Sugar inhibitions The sugars used were
Lactose L-Fucose
Methyl Mannoside D-Fucose
EVALUATION OF ANTI-A MONOCLONAL ANTIBODIES
L-Mannitol Sucrose Melibiose D-Galactose D-Glucose D-Galactosamine
401
D-Mannitol Arabinose Raffinose N-Acetyl-Galactosamine D-Glucosamine
Some of the sugars used were specifically selected while others were included because they were being used for another project and were therefore available. 20 mM solutions were made up in normal physiological saline and added in 50 ~1. amounts to the monoclonal antibodies and left for 1 hour at 15° C. After this time the tests were carried out as previously described. A set of control tests with saline added rather than sugar were also set up. Two of the MAb's were inhibited by virtually all sugars, but as both of these had a very narrow pH range this inhibition may be more related to pH rather than to sugar inhibition. Again as would be expected inhibitions were more easily seen with A2 as the indicator cell. However both A~ and A2 cells were used. The sugars neutralising the most MAb's were L-Mannitol, D-Fucose and N-Acetyl-Galactosamine. L-Mannitol and D-Fucose in saline both have a pH of approximately 4.5 and we originally thought that the inhibitions were due to pH but as other low pH sugars do not cause inhibition this work will have to be repeated using buffered sugars. Two antibodies showed inhibition, numbers 31 W 1 (m) and 7 W 2 (m) with N-Ace@ Galactosamine with both A1and A2 cells at pH 6.0. The human/human clone was only inhibited by N-Acetyl Galactosamine. None of the commercial reagents were inhibited by any of the sugars.
4. Reactions with A reactive B cells This subject has been well reviewed in the report of the Ortho Scientific Forum so we will not deal with it in any detail here. Merz-Dade in Switzerland very kindly sent us two examples they had detected of A reactive B cells. These both worked well without enzyme treatment and reacted with two of the reagents, 1 W 3 (m) and 19 W 7 (m) ascities.
5. Reactions with e n z y m e treated B cells A total of 50 B individuals from routine bleeds at the South London Blood Transfusion Centre were tested after enzyme treatment with Bromelin and Pronase. Mab 34 W 1 reacted with all of the B samples tested and sample 26 W 1 reacted with more than half of them. No differences in reaction was found between Bromelin or Pronase treated erythrocytes.
6. Reactions with e n z y m e treated 0 cells The O cells were again collected from the routine donor panel from South London BTS. Sample 34 W 1 reacted with 10 of the 50 O cells after they had been treated with enzyme. Sample 1 W 3 reacted very strongly with two O samples and is being further investigated for a possible cross-reacting antibody.
402
TILLS D. and colL
CONCLUSIONS Quality control The m a i n p r o b l e m we have in evaluating m o n o c l o n a l anti-A's is the selection of an appropriate reference cell which can be used for quality control. In the past w h e n we were using polyclonal reagents the cell of choice was an A2B with the anti-A c o m p o n e n t of the anti-A,B being expected to detect some if not all A~ cells. The use of MAb's raised our expectations and m a n y of us expected that MAb. anti-A's would b e c o m e available that would readily detect all N,'s. This would have enabled us to drop the anti-A,B f r o m routine testing so saving both time and money. This discovery of the A reacting B cell which reacted strongly with most of anti-A MAb's which also detected the A~ cell, even if this is technique dependent, has caused a rethink. Several workers now r e c o m m e n d using an anti-A which detects A2B's but not Ax'S and so gives no p r o b l e m with A reactive B cells. The anti-A, B is then retained to detect A~'s. This change in emphasis m a y explain why if we use the detection of the A~ cell as our criteria for a good m o n o c l o n a l anti-A then this batch of 30 clones is inferior to the 37 1 tested in the 1986 paper. In that study three MAb's detected 10 out of the fourteen A~ cells included.
Genetics S o m e workers, Lt~Eteao (1985) and GooI e t al. (1985) have suggested that the A antigen shows epitopes. However, on the evidence of this study and that of TILLS (1986), epitopes do not appear to be shown by agglutination tests. Both surveys suggest that the various sub-groups of A show overlapping reactivity and antigenicity. However, in m a r k e d contrast the evidence for the B antigen f r o m the survey carried out by TmLS (1986) (we were unable to obtain anti-B MAb's for this survey) suggests strongly the existence of epitopes. Acknowledgements Without the generous donations of reference cells by almost all of the Transfusion Centres in the UK we would not have been able to have done this work. In addition our gratitude is due to Dr. ImoNo and the staff of the Hong Kong Transfusion Center for the Oriental A variants and to Dr. Phyllis Moopms of the Natal Blood Transfusion Service for sending us the Abantu samples.
EVALUATIONOFANTI-A MONOCLONALANTIBODIES
403
REFERENCES WARLOWA., TILLS D. - Micromethods in blood group serology. Vox Sang., 1978, 35, 354-356. D. - ABH Monoclonals. Develop. biol. Standard., 1985, 67, 85-90. LOBENKOA. - Analysis of the heterogeneity of the Blood Group A2B using Monoclonal Typing Reagents. Abstract British Blood Transfusion Society Meeting. Oxford, 1985. GOOI H.C., HOUNSELLE.F., P~CARDJ.K,, LOWE A.D., VOAKD., LENNOX E.S., FEIZl T. - Differing reactions of monoclonal Anti-A antibodies with oligosaccharides related to blood group A. Journal of Biological Chemistry, 1985, 260, 13218-13224.