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Evaluation of anti-B and anti-A,B monoclonal antibodies
Revue Frangaise Tome XXX. -
de Transfusion
N ° 5. -
et Immuno-h6matologie
1987
557
Evaluation of anti-B and anti-A,B monoclonal antibodies by L. Messeter UniversityHospital Blood Bank, Lurid, Sweden
Nineteen anti-B and twelve monoclonal anti-A,B were investigated. Specificity, potency and avidity in serological tests and binding to blood group substances in ELISA were determined. The antibodies were then evaluated for use as routine reagents for manual blood grouping according to the following requirements.
Anti-B The antibody must be completely specific. Non specific hemagglutination reactions must not occur at any temperature or after enzyme treatment of A or O red cells. Titer must be satisfactory, i.e. 128 or more with B, A1B and A2B red cells. Avidity must be high and comparable to the best polyclonal reagent, i.e. agglutination must start within a few seconds and be complete in about 30 seconds, also B and A1B cells. The antibody must give good agglutination reactions with cord cells and preferably also with weak variants.
Anti-A,B The antibody must be completely specific and may not agglutinate group O red cells at any temperature or after enzyme treatment. The titer must be satisfactory, and at least 128 with cells of all A and B groups. The avidity must be high and at least as good as that of a polyclonal reagent. Agglutination must start within a few seconds and be complete in about 30 seconds. Slightly lower avidity can be permitted for A2B red cells. The antibody must react well with cord cells and agglutinate the majority of A~ red cells.
558
MESSETER L
MATERIALS AND METHODS Manual hemagglutination tests were performed using 0.1 ml undiluted supernatant, or ascitic fluid diluted 1 : 10 with isotonic saline, and 3-5 % red cell suspensions in isotonic saline. The mixtures were incubated either in tubes for 1 h or on tiles for 20 rain at 22°C. Reactions were read against a well lit background, and positive reactions graded from 4+ (complete) to (+). Doubtful reactions were read microscopically. Freshly drawn samples from healthy blood donors, newborn infants (cord samples) and patients were used. Some few samples frozen in liquid nitrogen were thawed and washed before use. For titration tests, twofold serial dilutions with saline containing 1% BSA were performed. Tube technique with 1 h incubation at 22°C was used. Avidity testing was performed on slides, using a 40-50 % red cell suspension in homologous serum. The time (in seconds) when agglutination started and was complete / or at 2 min was recorded. Specificity testing was performed using a panel of extensively phenotyped group O and A red cells. Standard 2-step papain technique, and saline technique at 37, 22 and 4°C were used. Reactions were dead microscopically. Binding to blood group substances was investigated using a solid phase ELISA technique [1] in microfiter plates. These tests were performed at MonoCarb company, Lund, Sweden.
RESULTS All results have been compiled in Tables I-IV.
COMMENTS AND SUMMARY Anti-B Ten of the nineteen antibodies agglutinated papainized group A red cells, two of these ten and one further antibody also agglutinated papainized O cells. The latter (16 W2) contained a weak, papain-reactive anti-D, contamination ? One antibody (33 W 1) agglutinated A and O cells also in saline. All antibodies bound to B substance in ELISA except 11 W 2 and 33 W 1. These two antibodies did not bind to A or H blood group substance either. 11 W 2 clearly was a weak anti-B reacting with B red cells to a titer of 32, but also weakly agglutinated papainized A red cells. Binding to A, B and H glycolipids was not tested. 33 W 1 was not an anti-B, furthermore it did not bind in ELISA to any of the sugars used for characterizing the anti-glycoconjugate antibodies. All agglutination reactions were abolished upon neuraminidase treatment.
559
EVALUATIONOFAN'H-BANDANTI-A,B MONOCLONALANTIBODIES TABLE
I
SPECIFICITY:
ANTI-B
Clone n:o I WI 5 W3 9 W3 9 W4 9 W5 11 W2 14 W3 14 W4 15 WI 15 W2 16 W2 17 W2 19 W11 23 W4 24 W2 25 W5 26 W3 31 W2 33 WI ANTI-A,B: 1 W2 4 W2 4 W3 7 Wl II W6 16 W3 17 W3 23 W6 23 W7 24 W3 25 W4 26 W4
Hemagglutination Red c e l l s / p a p a i n i z e d B 0 A
Hemagg]utination Red c e l l s / s a l i n e A B 0
A
B
H
+
+
+
+
+
+
+
+
H-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
H-
4-
+
+
+
-r
+
+
+
H-
+
+
+
+
+
+
+
+
+
+
+
-
_
+
+
+
-
_
+
+
+
+
-
+
+ +
-
+
+ H4-
+
-
4-
4-
+ +
+
4-
-
+
-
+
+
+
+
+
+
+
+
-
(+)
(+)
+
4-
+
+
+
-
(+)
(+)
4-
H-
-
+
-
+
+
+
+
+
+
+
+
H-
4-
-
+
-
+
+
4-
+
-
+
-
+
+
H-
-
+
-
+
4-
+
-
+
4-
4-
-
+
H-
4-
--
+
-
+
4-
H-
--
+
-
+
ANTI-B: Clone n:o Agglutination AI AIB 3 5 W3 3 9 W3 2 15 WI 1 17 W2 2 Ortho
weak
+
+ +
+
(+)
TABLE IT
AVIDITY
ANII-A,B: 16 W3 17 W3 26 W4 Ortho
ELISA Blood group substance
starts, sec A2B B
3 2 2 0 1
A g g l u t i n a t i o n complete, sec AI AIB B A2B 12Q(4w) 120(4w)
120(4w) 120(aw) 30 20 22
120(4w) 15 30 8
40 15 15
120(4W) 60 15 12
560
MESSETER L.
TABLE
III
REACTIONS WITH CORD CELLS AND WEAK VARIANTS: ANTI-B CLONE N:O I Wl 5 W3 9 W3 9 W4 9 W5 11 W2 14 W3 14 W4 15 WI 15 W2 16 W2 17 W2 19 W11 23 W4 24 W2 25 W5 26 W3 31 W2 33 WI Poiyc]
ANTI-A,B: I W2 4 W2 4 W3 7 WI 11 W6 16 W3 17 W3 23 W6 23 W7 24 W3 25 W4 26 W4 Polycl
Cord cells A A A
A
A -
B
B
B
3
4W
3
3 3 3 2 3 4w Qw 4W 4w 4W 4W 3 4W
%W 3 4W 3W 3 3 3 4W 4w 4W 4W 4W 4W
3 3 3 2 3 4w 3 4W 4w 3 4W 3 4W
B
B
0
3
4W
-
3 3W 3 2 3W 4w 4w 4W 4w 3 4W 3 3
4W 3 3 2 3W 4w 3 4W 3 4W 4W 3 3
-
0
Weak v a r i a n t s Ax Bw Bw ABw lw 1
-
lw
1w
lw I
2w
3 Iw
lw -
2
3W
2
3W
3 3 2W 3
2
3
4W 3W 2W 4w
3W 3W 2W 3
3W 3 2 4w
3W 3 2 4
3W -
2 3
3 3 2 4W 3 3W 3 3W 3
3 3 2 3 4W 2
3 3
3 3 2 4W 4W 2
3W 3
I
3
3
3 3 2 4W 3 2
3W 3
2 I
I /4
2 /4 I
2w H--
3W 3W 2 3 3 2 3w
3w
-
IW 4W 3W 3 I 3 2W 3w
3 3 3 2W 3 2 3
3 3W
3 3W
3 3W
3 I
3 I
3 2W
H+
1W
3 4w
3w 3w
3
3
4 4 2
2w I
2w
2 3w
2W 3
2 3w
I lw 2w
I
I
/4 4 4 4
EVALUATION OF ANTI-B AND ANTI-A,B MONOCLONAL ANTIBODIES
561
TABLE IV POTENCY: ANTI-B: Clone N:o 1 W1 5 W3 9 W3 9 W4 9 W5 11 W2 14 W3 14 W4 15 WI 15 W2 16 W2 17 W2 19 W11
TiLer A1
PoLency cont.! A1
A2
A2
I AIB 128 128 128 256 16 32 >5800 >2560 512 I >5000 128 512 128
! l
W& W2 W5 W3 W2 WI
ANTI-A,8 I W2 4 W2 4 W3 7 W1 11 W6 16 W3 17 W3 23 W6 23 W7 24 W3 25 W4 26 W4
F
!
i I
}
l
AIB
i 23 24 25 26 31 33
1 i
A2B 128 128 256 512 64 64
512 256 512 128
I B
!o
128 I 256 128 l 512 64 32 >5000 >5000 i 512 >5000 256 512 128
J
I
[
0
A28 l
128 1280 >5000 64 512
I I
!
512
512
512
32 >5000 >5000 64 32 32 64 4 4 >5000 >5000 128
32 >5000 >5000 64 32 32 64 8 2 >5000 >5000 64
32 >5000 >5000 64 32 32 64 4 2
>5000 >5000 64
I I
!
128
256 512 512
128 640 >5000 256 512 512
32 >5000 >5000 128 32 128 32 8 1 >5000 >5000 128
I >5000 >5000 I 32 256 I 8 I >5000 >5000 64
i
]
512
i : 64 256
562
MESSETER L
Anti-A,B Four of the twelve antibodies agglutinated saline-suspended and/or papain treated group O red cells. Three antibodies were predominantly anti-A and gave barely perceptible agglutination with group B cells. One of these (7 W 1) bound only to A substance in ELISA, another (17 W 3) b o u n d to A and H, and the third (1 W 2) b o u n d to A, B and H. Two antibodies (4 W 2 and 4 W 3) b o u n d to B and H. Both of t h e m agglutinated group O cells to high titers. Eight antibodies had unacceptably low titer with both A and B cells, and one of these (26 W 4) b o u n d only to A in ELISA. Only five antibodies b o u n d to A and B but not H in ELISA. Thus, out of nineteen anti-B : s, only eight were undisputedly specific. Six of these had good titers and avidity but only few recognized the weak B variants. Of the other antibodies, one was probably not m o n o c l o n a l and one was no anti-B. This demonstrates clearly how difficult it is to produce an anti-B which is satisfactory as a blood grouping reagent. Among the twelve anti-A,B : s, only 24 W 3 gave strong titers with all ABO groups except O. It was specific in ELISA and the very weak reaction with papainized O cells disappeared at dilution 1 : 20 but this antibody did not react with A~ cells. All the other antibodies were either weak with group A or group B cells, or gave non specific reactions. The conclusion is that it is even m o r e difficult to produce a good monoclonal anti-A,B. If such a reagent really is considered necessary, it is probably better to prepare a blend of anti-A and anti-B. Antibodies which cross react with B and A red cells, respectively, are probably very useful in such a blend. ACKNOWLEDGEMENTS The skilful technical assistance of Mrs Karin LOw (MonoCarb Company, Lund, Sweden) who performed the ELISA tests and of Mrs Eva PERSSON (University Hospital Blood Bank, Lund, Sweden) who performed the serological tests, is gratefully acknowledged.
REFERENCES
[1]. MESSETERL., BRODINT., CHESTERM.A., KARLSSONK.A., ZOPFD., LUNDBLADA. - Immunochemical characterization of a monoclonal anti-Leb blood grouping reagent. Vox. Sang., 1984, 46, 66-74.
de Transfusion
N ° 5. -
et Immuno-h6matologie
1987
557
Evaluation of anti-B and anti-A,B monoclonal antibodies by L. Messeter UniversityHospital Blood Bank, Lurid, Sweden
Nineteen anti-B and twelve monoclonal anti-A,B were investigated. Specificity, potency and avidity in serological tests and binding to blood group substances in ELISA were determined. The antibodies were then evaluated for use as routine reagents for manual blood grouping according to the following requirements.
Anti-B The antibody must be completely specific. Non specific hemagglutination reactions must not occur at any temperature or after enzyme treatment of A or O red cells. Titer must be satisfactory, i.e. 128 or more with B, A1B and A2B red cells. Avidity must be high and comparable to the best polyclonal reagent, i.e. agglutination must start within a few seconds and be complete in about 30 seconds, also B and A1B cells. The antibody must give good agglutination reactions with cord cells and preferably also with weak variants.
Anti-A,B The antibody must be completely specific and may not agglutinate group O red cells at any temperature or after enzyme treatment. The titer must be satisfactory, and at least 128 with cells of all A and B groups. The avidity must be high and at least as good as that of a polyclonal reagent. Agglutination must start within a few seconds and be complete in about 30 seconds. Slightly lower avidity can be permitted for A2B red cells. The antibody must react well with cord cells and agglutinate the majority of A~ red cells.
558
MESSETER L
MATERIALS AND METHODS Manual hemagglutination tests were performed using 0.1 ml undiluted supernatant, or ascitic fluid diluted 1 : 10 with isotonic saline, and 3-5 % red cell suspensions in isotonic saline. The mixtures were incubated either in tubes for 1 h or on tiles for 20 rain at 22°C. Reactions were read against a well lit background, and positive reactions graded from 4+ (complete) to (+). Doubtful reactions were read microscopically. Freshly drawn samples from healthy blood donors, newborn infants (cord samples) and patients were used. Some few samples frozen in liquid nitrogen were thawed and washed before use. For titration tests, twofold serial dilutions with saline containing 1% BSA were performed. Tube technique with 1 h incubation at 22°C was used. Avidity testing was performed on slides, using a 40-50 % red cell suspension in homologous serum. The time (in seconds) when agglutination started and was complete / or at 2 min was recorded. Specificity testing was performed using a panel of extensively phenotyped group O and A red cells. Standard 2-step papain technique, and saline technique at 37, 22 and 4°C were used. Reactions were dead microscopically. Binding to blood group substances was investigated using a solid phase ELISA technique [1] in microfiter plates. These tests were performed at MonoCarb company, Lund, Sweden.
RESULTS All results have been compiled in Tables I-IV.
COMMENTS AND SUMMARY Anti-B Ten of the nineteen antibodies agglutinated papainized group A red cells, two of these ten and one further antibody also agglutinated papainized O cells. The latter (16 W2) contained a weak, papain-reactive anti-D, contamination ? One antibody (33 W 1) agglutinated A and O cells also in saline. All antibodies bound to B substance in ELISA except 11 W 2 and 33 W 1. These two antibodies did not bind to A or H blood group substance either. 11 W 2 clearly was a weak anti-B reacting with B red cells to a titer of 32, but also weakly agglutinated papainized A red cells. Binding to A, B and H glycolipids was not tested. 33 W 1 was not an anti-B, furthermore it did not bind in ELISA to any of the sugars used for characterizing the anti-glycoconjugate antibodies. All agglutination reactions were abolished upon neuraminidase treatment.
559
EVALUATIONOFAN'H-BANDANTI-A,B MONOCLONALANTIBODIES TABLE
I
SPECIFICITY:
ANTI-B
Clone n:o I WI 5 W3 9 W3 9 W4 9 W5 11 W2 14 W3 14 W4 15 WI 15 W2 16 W2 17 W2 19 W11 23 W4 24 W2 25 W5 26 W3 31 W2 33 WI ANTI-A,B: 1 W2 4 W2 4 W3 7 Wl II W6 16 W3 17 W3 23 W6 23 W7 24 W3 25 W4 26 W4
Hemagglutination Red c e l l s / p a p a i n i z e d B 0 A
Hemagg]utination Red c e l l s / s a l i n e A B 0
A
B
H
+
+
+
+
+
+
+
+
H-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
H-
4-
+
+
+
-r
+
+
+
H-
+
+
+
+
+
+
+
+
+
+
+
-
_
+
+
+
-
_
+
+
+
+
-
+
+ +
-
+
+ H4-
+
-
4-
4-
+ +
+
4-
-
+
-
+
+
+
+
+
+
+
+
-
(+)
(+)
+
4-
+
+
+
-
(+)
(+)
4-
H-
-
+
-
+
+
+
+
+
+
+
+
H-
4-
-
+
-
+
+
4-
+
-
+
-
+
+
H-
-
+
-
+
4-
+
-
+
4-
4-
-
+
H-
4-
--
+
-
+
4-
H-
--
+
-
+
ANTI-B: Clone n:o Agglutination AI AIB 3 5 W3 3 9 W3 2 15 WI 1 17 W2 2 Ortho
weak
+
+ +
+
(+)
TABLE IT
AVIDITY
ANII-A,B: 16 W3 17 W3 26 W4 Ortho
ELISA Blood group substance
starts, sec A2B B
3 2 2 0 1
A g g l u t i n a t i o n complete, sec AI AIB B A2B 12Q(4w) 120(4w)
120(4w) 120(aw) 30 20 22
120(4w) 15 30 8
40 15 15
120(4W) 60 15 12
560
MESSETER L.
TABLE
III
REACTIONS WITH CORD CELLS AND WEAK VARIANTS: ANTI-B CLONE N:O I Wl 5 W3 9 W3 9 W4 9 W5 11 W2 14 W3 14 W4 15 WI 15 W2 16 W2 17 W2 19 W11 23 W4 24 W2 25 W5 26 W3 31 W2 33 WI Poiyc]
ANTI-A,B: I W2 4 W2 4 W3 7 WI 11 W6 16 W3 17 W3 23 W6 23 W7 24 W3 25 W4 26 W4 Polycl
Cord cells A A A
A
A -
B
B
B
3
4W
3
3 3 3 2 3 4w Qw 4W 4w 4W 4W 3 4W
%W 3 4W 3W 3 3 3 4W 4w 4W 4W 4W 4W
3 3 3 2 3 4w 3 4W 4w 3 4W 3 4W
B
B
0
3
4W
-
3 3W 3 2 3W 4w 4w 4W 4w 3 4W 3 3
4W 3 3 2 3W 4w 3 4W 3 4W 4W 3 3
-
0
Weak v a r i a n t s Ax Bw Bw ABw lw 1
-
lw
1w
lw I
2w
3 Iw
lw -
2
3W
2
3W
3 3 2W 3
2
3
4W 3W 2W 4w
3W 3W 2W 3
3W 3 2 4w
3W 3 2 4
3W -
2 3
3 3 2 4W 3 3W 3 3W 3
3 3 2 3 4W 2
3 3
3 3 2 4W 4W 2
3W 3
I
3
3
3 3 2 4W 3 2
3W 3
2 I
I /4
2 /4 I
2w H--
3W 3W 2 3 3 2 3w
3w
-
IW 4W 3W 3 I 3 2W 3w
3 3 3 2W 3 2 3
3 3W
3 3W
3 3W
3 I
3 I
3 2W
H+
1W
3 4w
3w 3w
3
3
4 4 2
2w I
2w
2 3w
2W 3
2 3w
I lw 2w
I
I
/4 4 4 4
EVALUATION OF ANTI-B AND ANTI-A,B MONOCLONAL ANTIBODIES
561
TABLE IV POTENCY: ANTI-B: Clone N:o 1 W1 5 W3 9 W3 9 W4 9 W5 11 W2 14 W3 14 W4 15 WI 15 W2 16 W2 17 W2 19 W11
TiLer A1
PoLency cont.! A1
A2
A2
I AIB 128 128 128 256 16 32 >5800 >2560 512 I >5000 128 512 128
! l
W& W2 W5 W3 W2 WI
ANTI-A,8 I W2 4 W2 4 W3 7 W1 11 W6 16 W3 17 W3 23 W6 23 W7 24 W3 25 W4 26 W4
F
!
i I
}
l
AIB
i 23 24 25 26 31 33
1 i
A2B 128 128 256 512 64 64
512 256 512 128
I B
!o
128 I 256 128 l 512 64 32 >5000 >5000 i 512 >5000 256 512 128
J
I
[
0
A28 l
128 1280 >5000 64 512
I I
!
512
512
512
32 >5000 >5000 64 32 32 64 4 4 >5000 >5000 128
32 >5000 >5000 64 32 32 64 8 2 >5000 >5000 64
32 >5000 >5000 64 32 32 64 4 2
>5000 >5000 64
I I
!
128
256 512 512
128 640 >5000 256 512 512
32 >5000 >5000 128 32 128 32 8 1 >5000 >5000 128
I >5000 >5000 I 32 256 I 8 I >5000 >5000 64
i
]
512
i : 64 256
562
MESSETER L
Anti-A,B Four of the twelve antibodies agglutinated saline-suspended and/or papain treated group O red cells. Three antibodies were predominantly anti-A and gave barely perceptible agglutination with group B cells. One of these (7 W 1) bound only to A substance in ELISA, another (17 W 3) b o u n d to A and H, and the third (1 W 2) b o u n d to A, B and H. Two antibodies (4 W 2 and 4 W 3) b o u n d to B and H. Both of t h e m agglutinated group O cells to high titers. Eight antibodies had unacceptably low titer with both A and B cells, and one of these (26 W 4) b o u n d only to A in ELISA. Only five antibodies b o u n d to A and B but not H in ELISA. Thus, out of nineteen anti-B : s, only eight were undisputedly specific. Six of these had good titers and avidity but only few recognized the weak B variants. Of the other antibodies, one was probably not m o n o c l o n a l and one was no anti-B. This demonstrates clearly how difficult it is to produce an anti-B which is satisfactory as a blood grouping reagent. Among the twelve anti-A,B : s, only 24 W 3 gave strong titers with all ABO groups except O. It was specific in ELISA and the very weak reaction with papainized O cells disappeared at dilution 1 : 20 but this antibody did not react with A~ cells. All the other antibodies were either weak with group A or group B cells, or gave non specific reactions. The conclusion is that it is even m o r e difficult to produce a good monoclonal anti-A,B. If such a reagent really is considered necessary, it is probably better to prepare a blend of anti-A and anti-B. Antibodies which cross react with B and A red cells, respectively, are probably very useful in such a blend. ACKNOWLEDGEMENTS The skilful technical assistance of Mrs Karin LOw (MonoCarb Company, Lund, Sweden) who performed the ELISA tests and of Mrs Eva PERSSON (University Hospital Blood Bank, Lund, Sweden) who performed the serological tests, is gratefully acknowledged.
REFERENCES
[1]. MESSETERL., BRODINT., CHESTERM.A., KARLSSONK.A., ZOPFD., LUNDBLADA. - Immunochemical characterization of a monoclonal anti-Leb blood grouping reagent. Vox. Sang., 1984, 46, 66-74.