- Email: [email protected]
ISBT workshop on RBC monoclonal antibodies : report on group 2 (anti-B, -A,B) antibodies
Revue Francoaise d e T r a n s f u s i o n et I m m u n o - h t m a t o l o g i e T o m e XXX. -
on
N ° 5. -
503
1987
ISBT workshop RBC monoclonal antibodiesreport on group 2 (anti-B, -A,B) antibodies b y A . L u b e n k o , M . R e d m a n and M. Contreras North London Blood Transfusion Centre, Edgware, Middlesex, U.K.
INTRODUCTION The 19 anti-B and 12 anti-A,B monoclonal antibodies (MABs) of this groupe were assessed for their performance in blood grouping tests, and were hence investigated for (i) specificity, using our routine microtitre plate-based blood grouping technique; (ii) reactivity with a small selection of groupe B or A~B variants ; (iii) potency, in terms of titre and avidity and (iv), to a limited extent, for epitope specificity using saliva inhibitions and Synsorb absorptions.
MATERIALS A N D M E T H O D S Agglutination tests were done in 96 well, V-bottom microplates by incubating the MABs with an equal volume of 0.2 % rbc suspensions in PBS; scoring of agglutination strength was made using the <~method, after tilting the centrifuged V-well plates at 70°C to the horizontal for 5 to 10 min (at room temperature) following an approximative 1 hour incubation at room temperature. Titrations (and all dilutions) were made in 0.5 % foetal calf serum (FCS) in PBS containing 0.1% sodium azide as a bacteriostatic agent. Hence all ascitic fluids were employed at a working dilution of l x l 0 -3, with the exceptions of anti-B MABs 14W4 and 15 W2, which were used at a dilution of lxl0 -2. Avidity tests were carried out on a tile using equal volumes of 10 % rbc suspensions and undiluted MABs. Saliva inhibitions were performed using the antibodies diluted to give the same equivalent end points, and incubating equal volumes of the diluted MABs with serial two-fold dilutions of saliva for 30 minutes before adding indicator red cells, reincubating, and scoring"for agglutination as above. Synsorb absorptions were made by mixing 200 ~tl of undiluted MAB with 50 ~tg of Synsorb overnight on a rotary mixer. Cells of common ABO phenotype were from reasonably fresh
LUBENKO A. and coll.
504
samples taken into CPD or EDTA ; cells with variant phenotype had been stored in a preservative for periods u p to 6 weeks or m o r e before testing.
RESULTS AND DISCUSSION Specificity testing The 19 anti-B (including 6 ascitic fluids) a n d 12 anti-A,B (4 ascitic fluids) samples were tested against at least one example each of A1, A2, B a n d O cells to confirm their stated specificity. 16 out of 19 of t h e , anti-B ~ reagents were indeed group B specific. However, culture media 1 W 1 and 33 W 1 together with ascites 14 W 3 did not produce such clear cut results when used undiluted. 1 W 1 weakly agglutinated all of 6AI a n d 6A2 cells using saline microplate tests but not any group O cell tested; reactions with the group A I cells were only slightly stronger than those with A2 cells. We therefore classify this antibody's specificity as being anti-A,B with a weak affinity for A. MAB 33 W 1 a n d 14 W 3 agglutinated all 12 group O as well as 6A1 a n d 6Az cells tested a n d to similarly high titres. Similary, one group O ~ Bombay ~ sample also gave strongly positive results. Clearly, these antibodies were not ABO specific. Of the 12 anti-A,B MABs only one sample, antibody 11 W 6, did not yield o[3 specificity. Unlike the 11 other anti-A,B MABs, which agglutinated A 1, A2 a n d B cells only, 11 W 6 additionally agglutinated all of the 10 group O cells tested. In contrast to anti-B : 33 W 1 above, this n o n specific reactivity had a low titre (21 - 23 only) in comparison to the anti-A and -B activity of this antibody (see below). MAB 11 W 6 therefore did have ABO specificity, especially when used at a 1 in 8 dilution, b u t the n a t u r e of the reactivity with group O cells is u n c l e a r : this could be genuine cross-reactivity or simply contamination with another agglutinin.
Tests with ABO variants The 16 proven B specific MABs were tested against the only 4 group B variants available, i.e. 3 B 3 plus one sample designated as <~,as well as 3 group B and 1 group AB cord samples. Antibodies 14 W 3 and 33 W 1 were excluded from this analysis because of their panreactivity with all cells tested (including the B variants), while MAB 1 W 1 is discussed with the anti-A,B samples below. The 16 antibodies could be arranged into a hierarchy composed of 4 ranks (1 to 4, Table I) according to the n u m b e r B 3 samples they agglutinated, with the four ranks being 'split further into two divisions (designated (i) or (ii), Table II) on the basis of the MABs' ability to agglutinate the single By sample. Hence antibody 19 W 11 proved to be the highest r a n k i n g antibody because it agglutinated all four samples, and MABs 9 W 4, 9 W 5, 17 W 2, 14 W 5 a n d 15 W 2 were the lowest since they agglutinated n o n e of these samples. All 16 antibodies agglutinated all the cord samples tested ; agglutination was usually complete with the exception of MABs 9 W 4 and 9 W 5 which gave slightly weaker agglutinates (2+ strength only) with the single group AB a n d one of the three group B cord bloods. The 12 anti-A,B samples, using MAB 11 W 6 at a 1 in 16 dilution to ensure ABO specificity, together with ~
505
ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
I
TABLE REACTIVITY
OF
ANTI-B
NABs WITH
no.
GROUP
found positive MABs
Rank/Division
B3
1 i
ii 2
i
ii 3
i
ii
n
=
total
a b
= ascitic = culture
number fluid, medium
of s a m p l e s dild. dild.
B VARIANTS.
I
BV
(n=3)
(n=2)
3 3
1 0
19wl~ llw6-
2 2
1 0
23w4 15w7
1 1
1 0
25w5 a 5w3 9w3
0
0
9w4 9w5 17w2 14w4 a [plus 10anti-A,B]
[a~:4w2 a]
31w2
24w2 a
16w2
26w8
[a~:4wS a ] 15w2 a
tested.
ixl0 -2 (14w4 a n d 15w2) or ixl0 -3 (the rest) i in 16 to a v o i d n o n - s p e c i f i c a g g l u t i n a t i o n
bloods. Only two samples, ascitic fluids 4 W 2 and 4 W 3, reacted with any of the B variants : 4 W 2 gave complete agglutination of all four cells while 4 W 2 reacted weakly (1+ only) with the single By sample. With cord bloods, all antibodies except MABs 1 W 2, 7 W 1, 17 W 3, 23 W 7 and 26 W 4 gave complete agglutination with all cell samples tested ; the latter 5 antibodies failed to react with the group B cord bloods. The above antibodies were also tested against several group A variants, which included 4 A3, 2 Am and 11 A. samples as well as 8 group A cord bloods. The number of A~samples that were agglutinated by the different anti-A,B reagents was quite variable (Table II), ranging from 9 out of 10 for MABs 1 W 2 and 11 W 6 to only 1 out of 10 samples for MABs 23 W 6 and 23 W 7, with the last antibody exhibiting the weakest strength of agglutination that was observed with the A. cells i.e. 2+ strong only compared to 3+ or 4+ for all the other antibodies tested with practically all the other A~ samples. With cells of the A3phenotype, most MABs were capable of agglutinating 3 out of the 4 examples tested. One of the A3 cells failed to react with all the MABs except 26 W 4, whereas the other 3 examples reacted with respectively 12, 10 and 4 of the anti-A,B reagents. The two A~ samples reacted with one antibody only, i.e. MAB l 1 W 6 ; this occurred even with a 1 in 64 dilulion of this antibody and is hence unlikely to have been due to the weak non-specific activity of this reagent (at dilutions of less than 1 : 8) noted above.
506
LUBENKO A. and colt TABtE Reactivity
of
II
anti-A,B
No. A
MABs
found
with
positive A3
x
(n=10)
MAB
(n=4)
2 2 3 2 2 3 3
lw2 4w2 a 4w3 a 7wl . llw6 D 16w3 17w3 23w6 23w7 24w3 a 25w4 a 26w4
1 l(w) 3 2 3
(w) (lw)
n
=
total
no.
b
=
dild.
1:16
figure weak
A variants
dr.
samples
to
avoid
in p a r e n t h e s i s
(2+)
tested;
a
nonspecific
indicates
no.
of
= ascites;
agglutination. samples
giving
reactions.
Failures to react with the group A cord samples occurred with only 4 of the 12 anti-A,B MABs. Antibodies 4 W 2, 4 W 3 and 23 W 7 failed to react with the same 2 cord bloods while antibody 23 W 6 reacted weakly (2+ reaction) with one of the two but not at all with the other. Apart from MAB 4 W 3, these were the same anti-A,B reagents that performed most poorly with the A~ samples tested. Potency testing The potency of the anti-B reagents, especially with A1B cells which potentially could present quantitatively fewer anti-B binding epitopes, was first assessed by titration. We included a sample in these studies which we have provisionally described as AI(B): our previous work with MABs from other sources have indicated that this sample fails to react with most monoclonal anti-B but gives positive reactions with all polyclonal (human) reagents. Parallel titrations with two examples each of group B, A~B, A2B, and the AI(B) sample above revealed three patterns of reactivity (Table III) amongst the MAB, i.e.
ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
507
(1) : culture media 5 W 3, 9 W 3, 15 W 1 and 16 W 2 (and ascites 25 W 5) all gave titres in excess of 28 and equally high with the group B, A1B and A2B cells, but failed to react with the At(B) sample ; (2) : culture media 9 W 4, 9 W 5, 11 W 2 a n d 26 W 3 also failed to react with the AI(B) sample but, most significantly, gave m u c h reduced titres with cells of the AtB phenotype. (3) : the remaining 6 MABs gave high titres with all four target cells, with titres against the At(B) sample rivalling those exhibited against the other AB types ; antibody 23 W 4 was perhaps the exception since titres against At(B) cells were significantly lower than those against AIB cells. The potency of the anti-A,B MABs was first compared by parallel titration with group A2 vs group B cells (Table IV) ; also shown are figures for reactivity with A1B and A2B samples, as well as the range of titres exhibited against the weakest and strongest positively-reacting A~ cells (not all Ax samples gave positive results; see above and Table I1). Antibody 23 W 7 is omitted from this analysis because of its failure to react with group B samples when diluted. Comparison of the A2 vs the B titres revealed three reaction profiles amongst the MABs tested : (1) titre vs group A cells equal to that against group B cells (i.e. tA= % in table IV, 6 examples) ; (2) titre vs group A cells m u c h greater than that against group B cells-(i.e, tn > t B in table IV, 4 examples), and (3) titre vs group A cells m u c h less than those against the group B sample (i.e. tA < t8 in table IV, 2 examples). All MABs reacted equally well with the AtB a n d A2B target cells. Antibody 1 W 1 (so called < assays to compare the strength of agglutination produced by the various MABs at 10 and 60 seconds respectively (Table l/). Culture media were used undiluted whereas ascitic fluids were at their designated working dilution. The anti-B samples could be segregated into three groups according to their avidity for group B cells: antibodies 5 W 3, 4 W 4 etc. gave 3+ to 4+ strong agglutination within 10 seconds ; antibodies 19 W 11 a n d 26 W 3 in contrast gave macroscopically weak agglutinates (2+ only) only after 60 seconds incubation. Analysis of the avidity of the anti-A,B reagents was slightly more complicated by the need to account for their reactivity with cells of the A~ and A2 as well as of the B phenotype. Nevertheless, some antibodies (4 W 2, 4 W 3) agglutinated cells of all three phenotypes equally well ; other antibodies failed to react with Ax cells only (24 W 3, 25 W 4, 26 W 4) during the time course of the assay, whilst others performed better with the group B rather than the A2 cells (24 W 3, 25 W 4, 26 W 4), or vice versa (1 W 2, 7 W 1 and 17 W 3), while still failing to react with A~; one sample, 23 W 7, failed to react with any of the cells during the assay. Obviously the interpretation of this data is difficult : differences in the titre of the antibodies as well as differences in epitope specificity (i.e. in terms of which A~ sample is tested and whether A or B antigens are preferentially recognized) have to be accounted for as well as the differences in avidity of the various antibodies. Nevertheless, the performance of these antibodies was not necessarily related to titre : culture media were often m o r e potent than similarly titred ascitic fluids e.g. anti-B : 5 W 3, 9 W 4 etc vs MAB24 W 2. Neither was a broader specificity necessarily related to potency ; for example anti-B : 19 W 11 had poor avidity but the broadest specificity as demonstrated by its ability to agglutinate B 3 a n d A~(B) cells.
LUBENKO A. and coll.
508
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ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
509
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ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
511
Similarly, anti-A,B : 11 W 6 and 1 W 2 were impotent with Ax cells in the avidity tests, but still agglutinated the greatest number of A~ samples in the microplate tests.
Inhibitions and Synsorb tests Simple attempts were made to analyse the epitope specificity of the MABs using saliva inhibitions and absorptions with Synsorb reagents. Amongst the anti-B reagents, two MABs were not inhibitable by group A1B or group B secretor saliva : anti-B : 9 W 3 and the non-specific 33 W 1. Many antibodies were inhibitable by both types of saliva (but not negative control A1secretor saliva) even at saliva dilutions greater than 1 in 1,000 (i.e. 21°). In contrast, 6 antibodies (9 W 4, 9 W 5, 15 W 2, 16 W 2, 23 W 4 and 31 W 2) were inhibitable only at high concentrations of saliva, i.e. dilutions of 1 in 26 or less. Significantly, even MAB 14 W 3 proved to be specifically inhibitable with group B (and AB) secretor saliva, even though this antibody has agglutinated group O and A cells in addition to group B cells during one specificity testing above. The anti-A,B reagents were also compared for their inhibitability with group A1 and group B secretor saliva, using both group A2 and group B red cells as indicator erythrocytes in the presence of each saliva. Two MABs (4 W 3 and 26 W 4) were not inhibitable by either saliva in agglutination tests on both target rbc ; three MABs (4 W 2, 23 W 7 and 25 W 4) were inhibitable by both salivas against both target cells, although antibody 4 W 2 was inhibited by higher concentrates of saliva than was antibody 23 W 7 (i.e. dilutions of 1 in 24 vs > 1 in 21° respectively), and the degree of inhibition of MAB 25 W 4 was greater when using A2 indicator cells. Two antibodies were inhibited by both salivas only when tested against the group A2 indicator cells, but their activity against group B cells was not affected at all by either saliva i.e. MABs 23 W 6 and 24 W 3. Lastly, four antibodies (1 W 2, 7 W 1, 11 W 6 and 16 W 3) were inhibited only by one saliva, i.e. group AI secretor but not at all by group B secretor saliva, when tested against both indicator cell. Antibody 17 W 3 could not be conclusively tested because of its poor reactivity with group B cells ; nevertheless group A secretor saliva did inhibit the activity of this antibody against both cell types. These results did not reflect either the avidity of the relevant MABs or their relative preference for A or B red cell antigens. For example, MABs 4 W 2 and 4 W 3 were equally avid but only the former was inhibitable. Similarly, sample 23 W 7 was the least avid yet one of the most easily inhibited; in comparison antibodies 11 W 6 and 16 W 3 had similar titres (but only slightly greater avidity for group B cells than A2cells) but were only inhibited by the group A saliva. Hence saliva inhibitions would seem to probe the epitope specificity of these antibodies, rather than the differences in their individual affinities. A more precise definition of the MABs epitope specificity was attempted using Synsorb reagents (Table VI). For the anti-B MABs, all samples except 9 W 3, 15 W 1, 17 W 2, 24 W 2 and 25 W 5 were completely exhausted (or nearly so) by Synsorb B treatment, but even for these exceptions the diminution of antibody titre was significant i.e.> 27 fall in titre. Even the non-specific antibodies 14 W 3 and 33 W 1 were partly neutralised by Synsorbs treatment, this diminution in activity having only marginal significance for;33 W 1. The anti-A,B MABs behaved similarly, with antibodies 4 W 2, 4 W 3, 24 W 3 and 26 W 4 being only weakly absorbed. For many antibodies, S~ynsorb A and B
512
LUBENKO A. and coll.
m V
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V V V ~
v
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v
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V
ffi I11 ",~, 0
~D -
-p
4~0 0
11
ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
513
treatment caused similar falls in titre against A2 and B cells. MABs 1 W 2, 24 W 3 and 25 W 4 were exceptional in that Synsorb A exceeded Synsorb B in the degree of neutralisation that was effected, and for MABs 7 W 1, 16 W 3, 17 W 3 and 26 W 4 Synsorb B was pratically ineffective at reducing antibody titre. These effects did not mirror preference for A or B cells, since MAB 16 W 3 was equally reactive with group A and B cells but unreactive only with Synsorb B ; conversely antibody 23 W 6 was more reactive with B than A cells but equally reactive with B and A Synsorbs. Neither was absorbability related to avidity : three of the highest avidity antibodies (4 W 2, 4 W 3 and 24 W 3) were the most poorly absorbed. Similarly absorbability did not reflect inhibitability with saliva; MAB 4 W 3 was inhibitable but not absorbable, MAB 26 W 4 was (weakly) absorbable but not inhibitable and MAB 25 W 4 was equally inhibitable by A and B salivas but most strongly absorbable by Synsorb A. Once again, these differences probably reflect the differences of the fine specificity of the antibodies involved.
CONCLUSIONS Both groups of antibodies proved to be heterogenous in performance with the relevant cells and related variants. For the anti-B MABs, those samples that reacted avidily with group B cells did not necessarily recognize group B or A,B variants ; neither was a potent reactivity with group B cells paralleled by an ability to recognize group B saliva or a high affinity for group B trisaccharide epitopes presented on the Synsorb B reagent. For the anti-A,B reagents, the essential reactivity with A~cells did not necessarily reflect inhibitabilitywith group A and B salivas or affinity for either A or B Synsorb reagents. Both classes of MABs therefore probably recognize a variety of ABO epitopes and the dissection of this heterogenity in specificity probably awaits a definition of the relevant epitopes at the cellular and/or molecular level.
on
N ° 5. -
503
1987
ISBT workshop RBC monoclonal antibodiesreport on group 2 (anti-B, -A,B) antibodies b y A . L u b e n k o , M . R e d m a n and M. Contreras North London Blood Transfusion Centre, Edgware, Middlesex, U.K.
INTRODUCTION The 19 anti-B and 12 anti-A,B monoclonal antibodies (MABs) of this groupe were assessed for their performance in blood grouping tests, and were hence investigated for (i) specificity, using our routine microtitre plate-based blood grouping technique; (ii) reactivity with a small selection of groupe B or A~B variants ; (iii) potency, in terms of titre and avidity and (iv), to a limited extent, for epitope specificity using saliva inhibitions and Synsorb absorptions.
MATERIALS A N D M E T H O D S Agglutination tests were done in 96 well, V-bottom microplates by incubating the MABs with an equal volume of 0.2 % rbc suspensions in PBS; scoring of agglutination strength was made using the <
LUBENKO A. and coll.
504
samples taken into CPD or EDTA ; cells with variant phenotype had been stored in a preservative for periods u p to 6 weeks or m o r e before testing.
RESULTS AND DISCUSSION Specificity testing The 19 anti-B (including 6 ascitic fluids) a n d 12 anti-A,B (4 ascitic fluids) samples were tested against at least one example each of A1, A2, B a n d O cells to confirm their stated specificity. 16 out of 19 of t h e , anti-B ~ reagents were indeed group B specific. However, culture media 1 W 1 and 33 W 1 together with ascites 14 W 3 did not produce such clear cut results when used undiluted. 1 W 1 weakly agglutinated all of 6AI a n d 6A2 cells using saline microplate tests but not any group O cell tested; reactions with the group A I cells were only slightly stronger than those with A2 cells. We therefore classify this antibody's specificity as being anti-A,B with a weak affinity for A. MAB 33 W 1 a n d 14 W 3 agglutinated all 12 group O as well as 6A1 a n d 6Az cells tested a n d to similarly high titres. Similary, one group O ~ Bombay ~ sample also gave strongly positive results. Clearly, these antibodies were not ABO specific. Of the 12 anti-A,B MABs only one sample, antibody 11 W 6, did not yield o[3 specificity. Unlike the 11 other anti-A,B MABs, which agglutinated A 1, A2 a n d B cells only, 11 W 6 additionally agglutinated all of the 10 group O cells tested. In contrast to anti-B : 33 W 1 above, this n o n specific reactivity had a low titre (21 - 23 only) in comparison to the anti-A and -B activity of this antibody (see below). MAB 11 W 6 therefore did have ABO specificity, especially when used at a 1 in 8 dilution, b u t the n a t u r e of the reactivity with group O cells is u n c l e a r : this could be genuine cross-reactivity or simply contamination with another agglutinin.
Tests with ABO variants The 16 proven B specific MABs were tested against the only 4 group B variants available, i.e. 3 B 3 plus one sample designated as <~,as well as 3 group B and 1 group AB cord samples. Antibodies 14 W 3 and 33 W 1 were excluded from this analysis because of their panreactivity with all cells tested (including the B variants), while MAB 1 W 1 is discussed with the anti-A,B samples below. The 16 antibodies could be arranged into a hierarchy composed of 4 ranks (1 to 4, Table I) according to the n u m b e r B 3 samples they agglutinated, with the four ranks being 'split further into two divisions (designated (i) or (ii), Table II) on the basis of the MABs' ability to agglutinate the single By sample. Hence antibody 19 W 11 proved to be the highest r a n k i n g antibody because it agglutinated all four samples, and MABs 9 W 4, 9 W 5, 17 W 2, 14 W 5 a n d 15 W 2 were the lowest since they agglutinated n o n e of these samples. All 16 antibodies agglutinated all the cord samples tested ; agglutination was usually complete with the exception of MABs 9 W 4 and 9 W 5 which gave slightly weaker agglutinates (2+ strength only) with the single group AB a n d one of the three group B cord bloods. The 12 anti-A,B samples, using MAB 11 W 6 at a 1 in 16 dilution to ensure ABO specificity, together with ~
505
ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
I
TABLE REACTIVITY
OF
ANTI-B
NABs WITH
no.
GROUP
found positive MABs
Rank/Division
B3
1 i
ii 2
i
ii 3
i
ii
n
=
total
a b
= ascitic = culture
number fluid, medium
of s a m p l e s dild. dild.
B VARIANTS.
I
BV
(n=3)
(n=2)
3 3
1 0
19wl~ llw6-
2 2
1 0
23w4 15w7
1 1
1 0
25w5 a 5w3 9w3
0
0
9w4 9w5 17w2 14w4 a [plus 10anti-A,B]
[a~:4w2 a]
31w2
24w2 a
16w2
26w8
[a~:4wS a ] 15w2 a
tested.
ixl0 -2 (14w4 a n d 15w2) or ixl0 -3 (the rest) i in 16 to a v o i d n o n - s p e c i f i c a g g l u t i n a t i o n
bloods. Only two samples, ascitic fluids 4 W 2 and 4 W 3, reacted with any of the B variants : 4 W 2 gave complete agglutination of all four cells while 4 W 2 reacted weakly (1+ only) with the single By sample. With cord bloods, all antibodies except MABs 1 W 2, 7 W 1, 17 W 3, 23 W 7 and 26 W 4 gave complete agglutination with all cell samples tested ; the latter 5 antibodies failed to react with the group B cord bloods. The above antibodies were also tested against several group A variants, which included 4 A3, 2 Am and 11 A. samples as well as 8 group A cord bloods. The number of A~samples that were agglutinated by the different anti-A,B reagents was quite variable (Table II), ranging from 9 out of 10 for MABs 1 W 2 and 11 W 6 to only 1 out of 10 samples for MABs 23 W 6 and 23 W 7, with the last antibody exhibiting the weakest strength of agglutination that was observed with the A. cells i.e. 2+ strong only compared to 3+ or 4+ for all the other antibodies tested with practically all the other A~ samples. With cells of the A3phenotype, most MABs were capable of agglutinating 3 out of the 4 examples tested. One of the A3 cells failed to react with all the MABs except 26 W 4, whereas the other 3 examples reacted with respectively 12, 10 and 4 of the anti-A,B reagents. The two A~ samples reacted with one antibody only, i.e. MAB l 1 W 6 ; this occurred even with a 1 in 64 dilulion of this antibody and is hence unlikely to have been due to the weak non-specific activity of this reagent (at dilutions of less than 1 : 8) noted above.
506
LUBENKO A. and colt TABtE Reactivity
of
II
anti-A,B
No. A
MABs
found
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(n=10)
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1:16
figure weak
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to
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a
nonspecific
indicates
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of
= ascites;
agglutination. samples
giving
reactions.
Failures to react with the group A cord samples occurred with only 4 of the 12 anti-A,B MABs. Antibodies 4 W 2, 4 W 3 and 23 W 7 failed to react with the same 2 cord bloods while antibody 23 W 6 reacted weakly (2+ reaction) with one of the two but not at all with the other. Apart from MAB 4 W 3, these were the same anti-A,B reagents that performed most poorly with the A~ samples tested. Potency testing The potency of the anti-B reagents, especially with A1B cells which potentially could present quantitatively fewer anti-B binding epitopes, was first assessed by titration. We included a sample in these studies which we have provisionally described as AI(B): our previous work with MABs from other sources have indicated that this sample fails to react with most monoclonal anti-B but gives positive reactions with all polyclonal (human) reagents. Parallel titrations with two examples each of group B, A~B, A2B, and the AI(B) sample above revealed three patterns of reactivity (Table III) amongst the MAB, i.e.
ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
507
(1) : culture media 5 W 3, 9 W 3, 15 W 1 and 16 W 2 (and ascites 25 W 5) all gave titres in excess of 28 and equally high with the group B, A1B and A2B cells, but failed to react with the At(B) sample ; (2) : culture media 9 W 4, 9 W 5, 11 W 2 a n d 26 W 3 also failed to react with the AI(B) sample but, most significantly, gave m u c h reduced titres with cells of the AtB phenotype. (3) : the remaining 6 MABs gave high titres with all four target cells, with titres against the At(B) sample rivalling those exhibited against the other AB types ; antibody 23 W 4 was perhaps the exception since titres against At(B) cells were significantly lower than those against AIB cells. The potency of the anti-A,B MABs was first compared by parallel titration with group A2 vs group B cells (Table IV) ; also shown are figures for reactivity with A1B and A2B samples, as well as the range of titres exhibited against the weakest and strongest positively-reacting A~ cells (not all Ax samples gave positive results; see above and Table I1). Antibody 23 W 7 is omitted from this analysis because of its failure to react with group B samples when diluted. Comparison of the A2 vs the B titres revealed three reaction profiles amongst the MABs tested : (1) titre vs group A cells equal to that against group B cells (i.e. tA= % in table IV, 6 examples) ; (2) titre vs group A cells m u c h greater than that against group B cells-(i.e, tn > t B in table IV, 4 examples), and (3) titre vs group A cells m u c h less than those against the group B sample (i.e. tA < t8 in table IV, 2 examples). All MABs reacted equally well with the AtB a n d A2B target cells. Antibody 1 W 1 (so called <
LUBENKO A. and coll.
508
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ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
509
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ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
511
Similarly, anti-A,B : 11 W 6 and 1 W 2 were impotent with Ax cells in the avidity tests, but still agglutinated the greatest number of A~ samples in the microplate tests.
Inhibitions and Synsorb tests Simple attempts were made to analyse the epitope specificity of the MABs using saliva inhibitions and absorptions with Synsorb reagents. Amongst the anti-B reagents, two MABs were not inhibitable by group A1B or group B secretor saliva : anti-B : 9 W 3 and the non-specific 33 W 1. Many antibodies were inhibitable by both types of saliva (but not negative control A1secretor saliva) even at saliva dilutions greater than 1 in 1,000 (i.e. 21°). In contrast, 6 antibodies (9 W 4, 9 W 5, 15 W 2, 16 W 2, 23 W 4 and 31 W 2) were inhibitable only at high concentrations of saliva, i.e. dilutions of 1 in 26 or less. Significantly, even MAB 14 W 3 proved to be specifically inhibitable with group B (and AB) secretor saliva, even though this antibody has agglutinated group O and A cells in addition to group B cells during one specificity testing above. The anti-A,B reagents were also compared for their inhibitability with group A1 and group B secretor saliva, using both group A2 and group B red cells as indicator erythrocytes in the presence of each saliva. Two MABs (4 W 3 and 26 W 4) were not inhibitable by either saliva in agglutination tests on both target rbc ; three MABs (4 W 2, 23 W 7 and 25 W 4) were inhibitable by both salivas against both target cells, although antibody 4 W 2 was inhibited by higher concentrates of saliva than was antibody 23 W 7 (i.e. dilutions of 1 in 24 vs > 1 in 21° respectively), and the degree of inhibition of MAB 25 W 4 was greater when using A2 indicator cells. Two antibodies were inhibited by both salivas only when tested against the group A2 indicator cells, but their activity against group B cells was not affected at all by either saliva i.e. MABs 23 W 6 and 24 W 3. Lastly, four antibodies (1 W 2, 7 W 1, 11 W 6 and 16 W 3) were inhibited only by one saliva, i.e. group AI secretor but not at all by group B secretor saliva, when tested against both indicator cell. Antibody 17 W 3 could not be conclusively tested because of its poor reactivity with group B cells ; nevertheless group A secretor saliva did inhibit the activity of this antibody against both cell types. These results did not reflect either the avidity of the relevant MABs or their relative preference for A or B red cell antigens. For example, MABs 4 W 2 and 4 W 3 were equally avid but only the former was inhibitable. Similarly, sample 23 W 7 was the least avid yet one of the most easily inhibited; in comparison antibodies 11 W 6 and 16 W 3 had similar titres (but only slightly greater avidity for group B cells than A2cells) but were only inhibited by the group A saliva. Hence saliva inhibitions would seem to probe the epitope specificity of these antibodies, rather than the differences in their individual affinities. A more precise definition of the MABs epitope specificity was attempted using Synsorb reagents (Table VI). For the anti-B MABs, all samples except 9 W 3, 15 W 1, 17 W 2, 24 W 2 and 25 W 5 were completely exhausted (or nearly so) by Synsorb B treatment, but even for these exceptions the diminution of antibody titre was significant i.e.> 27 fall in titre. Even the non-specific antibodies 14 W 3 and 33 W 1 were partly neutralised by Synsorbs treatment, this diminution in activity having only marginal significance for;33 W 1. The anti-A,B MABs behaved similarly, with antibodies 4 W 2, 4 W 3, 24 W 3 and 26 W 4 being only weakly absorbed. For many antibodies, S~ynsorb A and B
512
LUBENKO A. and coll.
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ISBT WORKSHOP ON RBC MONOCLONAL ANTIBODIES
513
treatment caused similar falls in titre against A2 and B cells. MABs 1 W 2, 24 W 3 and 25 W 4 were exceptional in that Synsorb A exceeded Synsorb B in the degree of neutralisation that was effected, and for MABs 7 W 1, 16 W 3, 17 W 3 and 26 W 4 Synsorb B was pratically ineffective at reducing antibody titre. These effects did not mirror preference for A or B cells, since MAB 16 W 3 was equally reactive with group A and B cells but unreactive only with Synsorb B ; conversely antibody 23 W 6 was more reactive with B than A cells but equally reactive with B and A Synsorbs. Neither was absorbability related to avidity : three of the highest avidity antibodies (4 W 2, 4 W 3 and 24 W 3) were the most poorly absorbed. Similarly absorbability did not reflect inhibitability with saliva; MAB 4 W 3 was inhibitable but not absorbable, MAB 26 W 4 was (weakly) absorbable but not inhibitable and MAB 25 W 4 was equally inhibitable by A and B salivas but most strongly absorbable by Synsorb A. Once again, these differences probably reflect the differences of the fine specificity of the antibodies involved.
CONCLUSIONS Both groups of antibodies proved to be heterogenous in performance with the relevant cells and related variants. For the anti-B MABs, those samples that reacted avidily with group B cells did not necessarily recognize group B or A,B variants ; neither was a potent reactivity with group B cells paralleled by an ability to recognize group B saliva or a high affinity for group B trisaccharide epitopes presented on the Synsorb B reagent. For the anti-A,B reagents, the essential reactivity with A~cells did not necessarily reflect inhibitabilitywith group A and B salivas or affinity for either A or B Synsorb reagents. Both classes of MABs therefore probably recognize a variety of ABO epitopes and the dissection of this heterogenity in specificity probably awaits a definition of the relevant epitopes at the cellular and/or molecular level.