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Monoclonal antibodies specific to HLA class II DR4 and DR4-Dw13 subtypes and their application in studying the association of DR4 to rheumatoid arthritis
144
Abstracts Abstracts 98-103
Monoclonal antibodies
98
99
F O U R C Y T O T O X I C M O N O C L O N A L ANTIBODIES D E T E C T I N G T H E ANTIGENIC D E T E R MINANTS O F HLA-B21. S, Etessa.u, C.-T. Deng, M. LidS, S. Rojo, M Borns, D. Ngo, D Conners, N Conger. and J - H . Lee. One Lambda, Inc., 21001 Kittridge St., Canoga park, CA, 91303, U S A The H L A - B 2 I antigenic group a n d its splits, B49, B50 and B4005, have been studied during the 6th, 9th, 10th, and most recently the l l t h International Histocompatdiility Workshop. We are reporting for the first time the production of four new mono¢lonal antibodies which detect H L A B21 or its splits mAbs H384. H385, H390 a n d H302 have been tested agair~t a panel of 130 peripheral T-lymphocyies and B lymphoblastoid cell lines using microcytoloxicity testing. The reaction patterns of these four rnAbs are shown below:
TWO
HUMAN
ASSOCIATED
lgM
DISTINCT
A. W O l p l , K. S i e m o n e i t , Cross
Blood
ANTI-HLA-CLASS
WITH
H. Wintersinger,
Bank, Dept.
Transpl.
B60
1
Th. Eiermann,
Immunol.
and
NOT
SPECIFICITIES
SF. Goldmann.
Univ. UIm, Dept.
Red
Transt.
Med. UIm, Germany Beside
generating
human
anti-HLA
monoclonal
specificities equal to alloantisera, Epstein-Bar>Virus B-lymphoctes
from immunised
antibodies (EBV)
(mAb)
with
transformation
of
persons can provide insight into immunological
We have used EBV transformation 1349 B50 B4005 B57 B58 + + + + + + + + + + +
ANTIBODIES
relevant determinants.
specificity mAb H384 H385 H390 H302
1
HLA-CLASS
B48
B71 +
B75 +
class
1 specific
alloantibody.
mAb
Both
(UI-F21, mAb
to establish two human
UI-F24)
were
from
strongly
a woman
cytotoxic
IgM anti-HI.A-
with
a HLA-B38
demonstrating
a
panel
reactivity of 34% (20/59) and 62% 1188/301). No assogiation with a specific or +
+
supertypic
HLA-B4005 is reported as a low frequency antigen found in some Native American populations. It shares a core epitope with B49 and B50 To our knowledge, no monospecific antibody to B4005 has been reported H384 and H390 recogmze two separate epitopos shared by B49, B50 a n d B4I)05. whereas H385 recognizes a n epitope c o m m o n to only B50 and B 4 0 0 5 The fourth mAb. H302. reveals a cross-reactivity between B60, B48 and B4005 which has not been previously reported. By using these four mAbs, B4005 can be unambiguously distinguished from B50
seen.
HLA-class
The
lymphocytes random
I antigen or a combination
cytotoxic activity of the was
completely
abolished
platelets, suggesting HLA-class
demonstrated We
HLA-A,-B,-DR
agaist
by absorbing
peripheral
the
blood
antibodies
with
I association.
U I - F 2 4 r e a c t i v i t y w a s i d e n t i c a l in 13 H L A in u n r e l a t e d
of different antigens could be
two antibodies
identical sibling couples, whereas
identical patient
bone
marrow
donor
pairs 4/9
non identical reactivities.
conclude
that mAb
UI-F21
investigation of histocommpatibility
and
UI-F24
in u n r e l a t e d
may
be
a
useful
tool
li3r
transplantation.
100
101
CYTOTOXIC MONOCLONAL ANTIBODIES FOR TYPING DRI. DRI03, AND DRI0, J LOOn1. C.-T Deng I , S Etessarnl I , A Manzo l. L Banh I, M. Chert I , S Denham l, G. Woo I . G. Semana2, J-H Lee I lone Lanthda. lnc. 21001 K~ttrldge St. Canoga park. CA., 91303. USA 2C.ILT S. Rue Pierre Jean Gineste. 350OO Refines. France The Eleverlth Inter'~atthnai Histocompatthility WorkShop reported that the DRBI*0101 is a predommmuly Cauc~ian allele, while "0102 is predominantly seen in North Amefrcan Bla~ks. Neither allele is serologically distinguishable although severed clusters of sera with "short" patterns for DRI were ohservefi. In contrast, the DRBr specificity wM ascrthod to the "0103 allele and officially recogutra~ as DRI03. The DRBr specificity wa~ seen as extra reactions in some DRI alloamisera Monospeclfic DRI0 reagents were said to remain unusual; most react with DR1 ceils. We have produced monoclonal antibodies recogmzing DRIi0101/0102). DR103 and DRI0. The antibodies HI26(DRl.103.10), HI24IDRl.103,10). H239(DR1,4,9,10,14), H395(DR103). HI51fDR10/, and HI751DRg,10) were tested against a panel of ld9 pefrpheral B lymphocytes (PBLL 51 chronic lymphocytic leuketma cells /CLLL and 16 B lymphoblastthd cell lines ILCL). many of which have been DNA typed. The serological reaction patterns are sumrmtnzefi below:
SEROLOGICAL HETEROGENEITY OF DR2 ELUCIDATED BY MONOCLONAL ANTIBODIES A Manzo' C -T DengI, R Pei I. / Loon 1. G g¢maga2. L 13ardi1, M Chcn ~. J Coblentz I S De.ham :. A Liu I G Woo ] J -H LeeI lOne Lambda. Inc, 2100i Klttndge St, Canoga park. CA. ')1303 UgA 2C R.T S Rue Pierre Jean Guleste, 35OOORetules. France Serological haterogeneltv of DR2 was recogdized in tile Tenth thternatlonul l-hstocompaubditv Workshop which defined Dal5 add OR16 Another s anmat called DR2LL~Mwas not omcially accepted bocause the nttmbor of cetls tested was deemed thsu/~¢ient The Eleventh Intemauondi Htstocerepauhthty Workshop recogmzod that the DRB5 gone mid its product. DIL5L are associated *~th the DRI31 genes for DR 15 and DR 16 It ls now known that the DR2LLrM(DRI501/haplotvpe lacks the DRB5 gone **bereasthe nR2ES haplorype contains a defective DRB5 gone Most DR2 anoanusera contain amthodies aganlsl both DRBI and DRB5 gone products, makang the distmcuon betv,~en DR 15 mid DR 16 extremely difiSctdt However. several sera among the Eleventh Workshop core sera ~ere useful in thtferenhaung a group of DR2 subtypes, including DR2LUM and DR2ES We describe here three groups of mouse monoclonul amthodies that can be used to dlsnngmsh DR2 from DR51 and to thffereodate DRI5 from DR16
DRI DRI03 DR10
# Cells 3-ested 23 ti 10
Moaoclonal Antibody ID HI25 H124 H239 H395 H151 + + + + + + + + +
Sboctficit)' of Monoclonul Annbedies H596 H174 H593 DR2{D~I) DR51(DRB5) DRI51DRB~ DRI501,1502. DR51 DR1601.1602, DRSl DRI50I (LLqVI) DRI501 (ES)
H175
+
The *0101 mad "0102 alleles are serologieally indistinguishable. However. both DR10 and DR103 can be unambiguously assigned. Heterozygous a~algnment of DR1, DRI03 or DR10 is not possible. The DRI03 antibodies react exclusively to DRI03 PBL and CLL, but also react positively to DRI102 and DR0402 LCL. At the molecular level, the primary ~tmmo acid sequence differences between DR1, DRI03 and DRI0 occur m the c~ helix around positions 67 to 71. It is interesting to speculate that these antibodies may bind to this region. Ualque amino acid differences for DRI0 occur th several other locations ~ well and may serve as possihle epitope sites. I/1 conclusion, these monocinnal antibodies are useful for serologically typing DRI, DRI03, and DR10 and are potentially useful in epitope mapping studies of these antigens.
These reagents were studied usulg a panel of 100 cells including lxnpherai blood B cells and chrome lymphocytal [eukenua cells. H174 tie.arty defines the DRSl speclfialty Tile reacnorts may be explained ff the epttope is the umqu¢ amino acid sequence 9QDKyI2 common to all DRB5 gone products H593 thsUngulshes DRI5 from DRI6 This anubodv rmght recograze an epnobo thvolwng 47F and 671LEQA71 U596 defines the broad DR2 specific ty and hthds to a DRBI coded epltobo The ttmque amino acid sequence common to the~ anugefts is 11P1Q~.13 In conclusthn, monoclonal anubodies have been produced slx~lficaay against the DPd~I encoded DtL2 products and the ass~latod DRB5 gcne product. OP.5] The DRI5 ~ l f i C l ~ ~ be urmmhlguoual~ disnngmsbed from DRI6 DRIS. DRI6, and DIL2LLrM/DR2EScan be assigned from the serologacal reaction paaerlts ldenttficanon of DR2ES and rela ed variants by serology ~s pamculanv useful, since DR51 would be fMsety asslgnod by PCR Accurate assignment using these mAbs should benefil the outcome of the transplant ot DR2 posnl~e panents
102
103
M O N O C L O N A L ANrHBODIES SPECIFIC TO HLA CLASS I1 DR4 AND DR4-DwI3 SUBTYPES AND THEIR APPLICATION IN STUDYING THE ASSOCIATION OF DR4 TO RHEUMATOID A R T H R I T I S C.-T Deng, J Loon. L. Banh, G Woo, S. Denham. J - H Lee. One Lambda, l n c , 21001 K i n n d g e St. Canoga Park, CA. 91303, USA HLA-DR4 has previously been described in association with rheumatoid arthritis IRA) in man,, populations worldwide. Serological studies from the Eleventh International Histocompatibilii3 Workshop have confirmed the DR4 association with RA in different population groups Furthermore, SSOP typing of RA patients showed a higher incidence of certain DR4 alleles, namely, DR0401, 0404 and 0 4 0 5 However, the results also indicate that the presence of the DR4D w l 3 (DR0403/07) alleles coincided with a reduced RA risk We introduce two cylotoxic monoclonal antibodies (mAbs) specific to DR4 or its subtypes M A b H453 was cytotoxic to all DR4 alleles tested H575 was cytoto×ic to: DR0403, 0406, 0407 and 0411; but negative to DR0401. 0402. 0404, 0405, and 0408. Both raAbs were negative to other D R B I specificities tested The specificities of the mAbs were detemuned by rmcrocytotoxicil3, testing against panels of DR4 cons already defined b) DNA A comprehensive panel of DR4 ulleles (missing only DR0409, 0410 and 0412) was achieved by using chrome lymphocytic leukemia cells and B lymphoblasmid ceil lines Both mAbs were also cytotoxic to peripheral B 13anpheeyltes. C o m p a n n g the amino acid sequences of all H L A class II DRB alleles revealed that the combined residues of 32Y and 33H are shared by all DR4 alleles. This region might be involved in the epitope recogmzed by mAb H 4 5 3 We also postulate that mAb H575 recognizes an epitupo related to residues 70 to 74 QRRAE, This sequence is in the helical regmn of the class II molecule and is shared by and unique to DR0403.0406, 0407 and 0411 alleles With these two mAbs. we hope that the rmcrocytotoxicity assa~ can further the studies of the association of DR4-Dw 13 subtypes with rheumatoid arthritis
MONOCLONAL ANTIBODIES FOR SEROLOGICAL TYPING OF HLA CLASS I1 DRI401, DRI404 AND DR9 C E L L S C - T Deng. I. Loon, S Etessarm, L Banh. G Woo. S Denham, J - H L e e One Lambda, Inc.. 21001 Kitmdge St., Canoga park, CA. 91303, USA In the 19th Annual Meeting of ASHI, we reported a monocloanl antibody (mAb), H15, thai was cvtotoalc to DR9 DRI401, DR1404 and DRI405 cells. We also presented a possible epitopo of H I 5 Amino acid residues 70 to 74 RRRAE lie in the helical region of the class II molecule and are shared by and umque to DR0901, 1401. 1404. 1405, 1407, 1408. 1410. and 14I 1 alleles We now introduce two novel cytotoxic mAbs 14447 was specific to DRI401 and DRI404 cells, while H396 was specific to DR0901 cells Both mAbs were neganve to other DRBI specificines tested The spocificiides of the mAhs were detemuned by nucreeytotoyacits, tesUng agmnst panels of chrome tymphocync leukemia cells and B I)mlphohlastoid cell lines The DRBI alleles of our cell panels wore DNA typed by PCR-RFLP method. Both mAbs were also cytotoxic to panpberul B lymphocytes Comparing the amino acid sequences of all available HLA class II DRB alleles revealed that mAb H447 may reoogmze an epnope related to residues 57 to 60 AAEH. "Flus postulated epitope is also in the helical region o f t b e class II molecule and is shared by and umque to DRI401, 1404, 1407. and 1410 alleles Although we have DRI401 and DRI404 cells for cytotoyac tesOng, DRI407 and DRI410 cells are absent from our inventoD" C o m p a n n g the threeM]mensional structure of the HLA class II DRI molecule ( J H Brown et a l , Nature. 1993. 364:33) revealed that two armno acids at posnon 57 D and 60 Y of the DRI j3-cham differ from tile postulated epitope for rnAb H447 In particular, the charged residue 57 D in DR1 is substituted by the non-charged residue 57 A in DR]40I, 1404. 1407 and 1410 alleles This subsntu0on may affect the epitope cortformastun Because the DR0901 allele has many ttmque sequences, it is unclear which particular epitolx Js reeogmzed hy the DR9 monospeeific mAb. H396. Nevertheless. we are sure that H396 and H I 5 do not recogmze the same epitope Unlike H15, H396 was negative to hoth DRI401 and DR1404 celts These two mAhs, H447 and H396, pro'.ade addlttunal reagents for HLA class II typing of DRI401, DR1404 and DR9 cells.
Abstracts Abstracts 98-103
Monoclonal antibodies
98
99
F O U R C Y T O T O X I C M O N O C L O N A L ANTIBODIES D E T E C T I N G T H E ANTIGENIC D E T E R MINANTS O F HLA-B21. S, Etessa.u, C.-T. Deng, M. LidS, S. Rojo, M Borns, D. Ngo, D Conners, N Conger. and J - H . Lee. One Lambda, Inc., 21001 Kittridge St., Canoga park, CA, 91303, U S A The H L A - B 2 I antigenic group a n d its splits, B49, B50 and B4005, have been studied during the 6th, 9th, 10th, and most recently the l l t h International Histocompatdiility Workshop. We are reporting for the first time the production of four new mono¢lonal antibodies which detect H L A B21 or its splits mAbs H384. H385, H390 a n d H302 have been tested agair~t a panel of 130 peripheral T-lymphocyies and B lymphoblastoid cell lines using microcytoloxicity testing. The reaction patterns of these four rnAbs are shown below:
TWO
HUMAN
ASSOCIATED
lgM
DISTINCT
A. W O l p l , K. S i e m o n e i t , Cross
Blood
ANTI-HLA-CLASS
WITH
H. Wintersinger,
Bank, Dept.
Transpl.
B60
1
Th. Eiermann,
Immunol.
and
NOT
SPECIFICITIES
SF. Goldmann.
Univ. UIm, Dept.
Red
Transt.
Med. UIm, Germany Beside
generating
human
anti-HLA
monoclonal
specificities equal to alloantisera, Epstein-Bar>Virus B-lymphoctes
from immunised
antibodies (EBV)
(mAb)
with
transformation
of
persons can provide insight into immunological
We have used EBV transformation 1349 B50 B4005 B57 B58 + + + + + + + + + + +
ANTIBODIES
relevant determinants.
specificity mAb H384 H385 H390 H302
1
HLA-CLASS
B48
B71 +
B75 +
class
1 specific
alloantibody.
mAb
Both
(UI-F21, mAb
to establish two human
UI-F24)
were
from
strongly
a woman
cytotoxic
IgM anti-HI.A-
with
a HLA-B38
demonstrating
a
panel
reactivity of 34% (20/59) and 62% 1188/301). No assogiation with a specific or +
+
supertypic
HLA-B4005 is reported as a low frequency antigen found in some Native American populations. It shares a core epitope with B49 and B50 To our knowledge, no monospecific antibody to B4005 has been reported H384 and H390 recogmze two separate epitopos shared by B49, B50 a n d B4I)05. whereas H385 recognizes a n epitope c o m m o n to only B50 and B 4 0 0 5 The fourth mAb. H302. reveals a cross-reactivity between B60, B48 and B4005 which has not been previously reported. By using these four mAbs, B4005 can be unambiguously distinguished from B50
seen.
HLA-class
The
lymphocytes random
I antigen or a combination
cytotoxic activity of the was
completely
abolished
platelets, suggesting HLA-class
demonstrated We
HLA-A,-B,-DR
agaist
by absorbing
peripheral
the
blood
antibodies
with
I association.
U I - F 2 4 r e a c t i v i t y w a s i d e n t i c a l in 13 H L A in u n r e l a t e d
of different antigens could be
two antibodies
identical sibling couples, whereas
identical patient
bone
marrow
donor
pairs 4/9
non identical reactivities.
conclude
that mAb
UI-F21
investigation of histocommpatibility
and
UI-F24
in u n r e l a t e d
may
be
a
useful
tool
li3r
transplantation.
100
101
CYTOTOXIC MONOCLONAL ANTIBODIES FOR TYPING DRI. DRI03, AND DRI0, J LOOn1. C.-T Deng I , S Etessarnl I , A Manzo l. L Banh I, M. Chert I , S Denham l, G. Woo I . G. Semana2, J-H Lee I lone Lanthda. lnc. 21001 K~ttrldge St. Canoga park. CA., 91303. USA 2C.ILT S. Rue Pierre Jean Gineste. 350OO Refines. France The Eleverlth Inter'~atthnai Histocompatthility WorkShop reported that the DRBI*0101 is a predommmuly Cauc~ian allele, while "0102 is predominantly seen in North Amefrcan Bla~ks. Neither allele is serologically distinguishable although severed clusters of sera with "short" patterns for DRI were ohservefi. In contrast, the DRBr specificity wM ascrthod to the "0103 allele and officially recogutra~ as DRI03. The DRBr specificity wa~ seen as extra reactions in some DRI alloamisera Monospeclfic DRI0 reagents were said to remain unusual; most react with DR1 ceils. We have produced monoclonal antibodies recogmzing DRIi0101/0102). DR103 and DRI0. The antibodies HI26(DRl.103.10), HI24IDRl.103,10). H239(DR1,4,9,10,14), H395(DR103). HI51fDR10/, and HI751DRg,10) were tested against a panel of ld9 pefrpheral B lymphocytes (PBLL 51 chronic lymphocytic leuketma cells /CLLL and 16 B lymphoblastthd cell lines ILCL). many of which have been DNA typed. The serological reaction patterns are sumrmtnzefi below:
SEROLOGICAL HETEROGENEITY OF DR2 ELUCIDATED BY MONOCLONAL ANTIBODIES A Manzo' C -T DengI, R Pei I. / Loon 1. G g¢maga2. L 13ardi1, M Chcn ~. J Coblentz I S De.ham :. A Liu I G Woo ] J -H LeeI lOne Lambda. Inc, 2100i Klttndge St, Canoga park. CA. ')1303 UgA 2C R.T S Rue Pierre Jean Guleste, 35OOORetules. France Serological haterogeneltv of DR2 was recogdized in tile Tenth thternatlonul l-hstocompaubditv Workshop which defined Dal5 add OR16 Another s anmat called DR2LL~Mwas not omcially accepted bocause the nttmbor of cetls tested was deemed thsu/~¢ient The Eleventh Intemauondi Htstocerepauhthty Workshop recogmzod that the DRB5 gone mid its product. DIL5L are associated *~th the DRI31 genes for DR 15 and DR 16 It ls now known that the DR2LLrM(DRI501/haplotvpe lacks the DRB5 gone **bereasthe nR2ES haplorype contains a defective DRB5 gone Most DR2 anoanusera contain amthodies aganlsl both DRBI and DRB5 gone products, makang the distmcuon betv,~en DR 15 mid DR 16 extremely difiSctdt However. several sera among the Eleventh Workshop core sera ~ere useful in thtferenhaung a group of DR2 subtypes, including DR2LUM and DR2ES We describe here three groups of mouse monoclonul amthodies that can be used to dlsnngmsh DR2 from DR51 and to thffereodate DRI5 from DR16
DRI DRI03 DR10
# Cells 3-ested 23 ti 10
Moaoclonal Antibody ID HI25 H124 H239 H395 H151 + + + + + + + + +
Sboctficit)' of Monoclonul Annbedies H596 H174 H593 DR2{D~I) DR51(DRB5) DRI51DRB~ DRI501,1502. DR51 DR1601.1602, DRSl DRI50I (LLqVI) DRI501 (ES)
H175
+
The *0101 mad "0102 alleles are serologieally indistinguishable. However. both DR10 and DR103 can be unambiguously assigned. Heterozygous a~algnment of DR1, DRI03 or DR10 is not possible. The DRI03 antibodies react exclusively to DRI03 PBL and CLL, but also react positively to DRI102 and DR0402 LCL. At the molecular level, the primary ~tmmo acid sequence differences between DR1, DRI03 and DRI0 occur m the c~ helix around positions 67 to 71. It is interesting to speculate that these antibodies may bind to this region. Ualque amino acid differences for DRI0 occur th several other locations ~ well and may serve as possihle epitope sites. I/1 conclusion, these monocinnal antibodies are useful for serologically typing DRI, DRI03, and DR10 and are potentially useful in epitope mapping studies of these antigens.
These reagents were studied usulg a panel of 100 cells including lxnpherai blood B cells and chrome lymphocytal [eukenua cells. H174 tie.arty defines the DRSl speclfialty Tile reacnorts may be explained ff the epttope is the umqu¢ amino acid sequence 9QDKyI2 common to all DRB5 gone products H593 thsUngulshes DRI5 from DRI6 This anubodv rmght recograze an epnobo thvolwng 47F and 671LEQA71 U596 defines the broad DR2 specific ty and hthds to a DRBI coded epltobo The ttmque amino acid sequence common to the~ anugefts is 11P1Q~.13 In conclusthn, monoclonal anubodies have been produced slx~lficaay against the DPd~I encoded DtL2 products and the ass~latod DRB5 gcne product. OP.5] The DRI5 ~ l f i C l ~ ~ be urmmhlguoual~ disnngmsbed from DRI6 DRIS. DRI6, and DIL2LLrM/DR2EScan be assigned from the serologacal reaction paaerlts ldenttficanon of DR2ES and rela ed variants by serology ~s pamculanv useful, since DR51 would be fMsety asslgnod by PCR Accurate assignment using these mAbs should benefil the outcome of the transplant ot DR2 posnl~e panents
102
103
M O N O C L O N A L ANrHBODIES SPECIFIC TO HLA CLASS I1 DR4 AND DR4-DwI3 SUBTYPES AND THEIR APPLICATION IN STUDYING THE ASSOCIATION OF DR4 TO RHEUMATOID A R T H R I T I S C.-T Deng, J Loon. L. Banh, G Woo, S. Denham. J - H Lee. One Lambda, l n c , 21001 K i n n d g e St. Canoga Park, CA. 91303, USA HLA-DR4 has previously been described in association with rheumatoid arthritis IRA) in man,, populations worldwide. Serological studies from the Eleventh International Histocompatibilii3 Workshop have confirmed the DR4 association with RA in different population groups Furthermore, SSOP typing of RA patients showed a higher incidence of certain DR4 alleles, namely, DR0401, 0404 and 0 4 0 5 However, the results also indicate that the presence of the DR4D w l 3 (DR0403/07) alleles coincided with a reduced RA risk We introduce two cylotoxic monoclonal antibodies (mAbs) specific to DR4 or its subtypes M A b H453 was cytotoxic to all DR4 alleles tested H575 was cytoto×ic to: DR0403, 0406, 0407 and 0411; but negative to DR0401. 0402. 0404, 0405, and 0408. Both raAbs were negative to other D R B I specificities tested The specificities of the mAbs were detemuned by rmcrocytotoxicil3, testing against panels of DR4 cons already defined b) DNA A comprehensive panel of DR4 ulleles (missing only DR0409, 0410 and 0412) was achieved by using chrome lymphocytic leukemia cells and B lymphoblasmid ceil lines Both mAbs were also cytotoxic to peripheral B 13anpheeyltes. C o m p a n n g the amino acid sequences of all H L A class II DRB alleles revealed that the combined residues of 32Y and 33H are shared by all DR4 alleles. This region might be involved in the epitope recogmzed by mAb H 4 5 3 We also postulate that mAb H575 recognizes an epitupo related to residues 70 to 74 QRRAE, This sequence is in the helical regmn of the class II molecule and is shared by and unique to DR0403.0406, 0407 and 0411 alleles With these two mAbs. we hope that the rmcrocytotoxicity assa~ can further the studies of the association of DR4-Dw 13 subtypes with rheumatoid arthritis
MONOCLONAL ANTIBODIES FOR SEROLOGICAL TYPING OF HLA CLASS I1 DRI401, DRI404 AND DR9 C E L L S C - T Deng. I. Loon, S Etessarm, L Banh. G Woo. S Denham, J - H L e e One Lambda, Inc.. 21001 Kitmdge St., Canoga park, CA. 91303, USA In the 19th Annual Meeting of ASHI, we reported a monocloanl antibody (mAb), H15, thai was cvtotoalc to DR9 DRI401, DR1404 and DRI405 cells. We also presented a possible epitopo of H I 5 Amino acid residues 70 to 74 RRRAE lie in the helical region of the class II molecule and are shared by and umque to DR0901, 1401. 1404. 1405, 1407, 1408. 1410. and 14I 1 alleles We now introduce two novel cytotoxic mAbs 14447 was specific to DRI401 and DRI404 cells, while H396 was specific to DR0901 cells Both mAbs were neganve to other DRBI specificines tested The spocificiides of the mAhs were detemuned by nucreeytotoyacits, tesUng agmnst panels of chrome tymphocync leukemia cells and B I)mlphohlastoid cell lines The DRBI alleles of our cell panels wore DNA typed by PCR-RFLP method. Both mAbs were also cytotoxic to panpberul B lymphocytes Comparing the amino acid sequences of all available HLA class II DRB alleles revealed that mAb H447 may reoogmze an epnope related to residues 57 to 60 AAEH. "Flus postulated epitope is also in the helical region o f t b e class II molecule and is shared by and umque to DRI401, 1404, 1407. and 1410 alleles Although we have DRI401 and DRI404 cells for cytotoyac tesOng, DRI407 and DRI410 cells are absent from our inventoD" C o m p a n n g the threeM]mensional structure of the HLA class II DRI molecule ( J H Brown et a l , Nature. 1993. 364:33) revealed that two armno acids at posnon 57 D and 60 Y of the DRI j3-cham differ from tile postulated epitope for rnAb H447 In particular, the charged residue 57 D in DR1 is substituted by the non-charged residue 57 A in DR]40I, 1404. 1407 and 1410 alleles This subsntu0on may affect the epitope cortformastun Because the DR0901 allele has many ttmque sequences, it is unclear which particular epitolx Js reeogmzed hy the DR9 monospeeific mAb. H396. Nevertheless. we are sure that H396 and H I 5 do not recogmze the same epitope Unlike H15, H396 was negative to hoth DRI401 and DR1404 celts These two mAhs, H447 and H396, pro'.ade addlttunal reagents for HLA class II typing of DRI401, DR1404 and DR9 cells.