Epitopes of HLA-A, B, C, DR, DQ, DP and MICA antigens.

Abstract

This chapter presents lists of HLA epitopes that have been defined to date. It also presents examples of reactions of mAb and eluted allosera with the class I, class II and MICAsingle antigen beads. To date, we have identified 110 class I epitopes, of which 47 were defined by mAbs and 63 by alloantibodies that were eluted from rHLA class I single antigen cell lines. We listed 34 epitopes shared by the HLA-A locus antigens, 44 epitopes shared by HLA-B locus antigens, 4 epitopes shared by HLA-C locus antigens, 20 inter-locus epitopes shared by HLA-A-B locus antigens, 5 inter-locus epitopes shared by HLA-B-C locus antigens and 3 inter-locus epitopes shared by HLA-A-B-C locus antigens. Sixty HLA-DR epitopes have been defined mostly by one amino acid (aa) residue on the HLA-DR beta chain. Eighteen HLA-DQ epitopes have been defined on the HLA-DQB chain and on the HLA-DQA chain of the HLA-DQ antigens. A few DQ epitopes were defined by one aa residue. However, most can be defined by several alternative combinations of aa residues. DQA and DP epitopes--few in number at this time--were identified. Only seven MICA epitopes have been defined to date. All epitopes can be defined by an exclusive amino acid.

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HLA EpitopEs
Clinical Transplants 2007, Terasaki Foundation Laboratory, Los Angeles, California
CHAptEr 17
Epitopes of the HLA-A, B, C, DR, DQ and MICA
Antigens
Nadim El-Awar1, Paul I. Terasaki2, Junchao Cai2, Chun-Tsan Deng1,
Mikki Ozawa1 and Anh Nguyen1
1One Lambda Inc., Canoga Park, CA
2Terasaki Foundation Laboratory, Los Angeles, CA
For the past 40 years in transplantation, we
have focused attention on the HLA antigen as the
target for anti-HLA antibodies. The common percep-
tion was that one HLA antibody recognizes one HLA
antigen. Monospecific sera (those that reacted with
a single HLA specificity) were sought after as sero-
logic reagents for HLA typing and although mono-
specific antibodies were found for many antigens
(1), their number was small relative to the much
larger number of sera that reacted with multiple HLA
specificities. Some cross-reacting groups (CREGs)
of HLA antigens that often reacted with sera contain-
ing HLA antibodies were identified in early studies of
HLA antisera (2) and their frequent occurrence sug-
gested that a single HLA antibody could react with
more than one HLA antigen.
In Clinical Transplants (1, 3-5) and other publi-
cations (6-9), we focused on defining HLA epitopes
(specific structures on the HLA molecules) that
could explain the cross-reactions between different
HLA antigens recognized by antibodies. Single-an-
tigen (SA) beads bearing individual recombinant
HLA antigens were used to demonstrate that a
monoclonal antibody or an alloantibody, eluted from
recombinant single antigen cell lines, reacted with
one or more HLA antigens when tested against an
SA bead panel. The reactive antigens were shown
to share one or a combination of amino acids that
defined the epitope recognized by that antibody. We
found that most of these epitopes were exposed on
the surface of the HLA antigen and that the amino
acids defining the epitopes were within the antibody
binding span (7). Since epitopes are the actual tar-
gets of antibodies, antibody specificities against
HLA antigens other than those that stimulated their
production - often found in the sera of transplant
patient - can now be understood as reactions to
epitopes shared with the immunizing antigens. The
importance of defining these epitopes is that they
may be the true transplantation antigens respon-
sible for antibody-mediated transplant rejection.
The main purpose of this chapter is to provide
lists of the HLA and MICA epitopes that we have
defined to date and their locations on the HLA anti-
gens. We also included several examples showing
the epitope-defining aa and their locations on the
various chains of the HLA and MICA molecules.
MAT ERIALS AND MET HODS
Monoclonal and Antibody Eluates
Monoclonal antibodies (mAbs), produced by
conventional methods, were tested as supernatants
of cloned hybridoma cell lines or aliquots of ascites
at dilutions of 1:10 to 1:500,000. Their specificities
have been previously characterized by both sero-
logical and immuno-binding assays by the manu-
facturer (One Lambda Inc, Canoga Park, CA, USA).
Since mAbs by definition are monospecific, adsorp-
tion experiments were generally not conducted. Anti-
HLA alloantibody samples were obtained from mul-
tiparous women, placentas, or from patients follow-
ing platelet transfusions or organ transplants. Sera
were adsorbed using recombinant HLA (rHLA) SA
cell lines derived from the LCL712.2 B host cell line
for HLA A, B, C and DR or homozygous cell lines for
HLA DQ (One Lambda Inc, Canoga Park, CA, USA).
All cell lines used for adsorption of the antibody were
selected based on the known serological specific-
ity of each sample obtained using SA microspheres
(LABScreen® beads: LS1A01, LS1A02, LS1A03,

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eL-AwAr, TerAsAkI, CAI eT AL
LS2A01 and LSMICA001 One Lambda Inc. Cano-
ga Park CA, USA). Forty μl of serum (diluted 1:3)
was mixed with 3 to 5 x106 cells and subsequently
incubated for 30 minutes at room temperature (RT).
The cells were then centrifuged and washed with
phosphate-buffered saline. Adsorbed antibody was
eluted by mixing 60 μl of ImmunoPure IgG elution
buffer (Pierce, Rockford, IL, USA Catalog #21004)
with the cells and incubating for 10 minutes at RT.
After incubation, the eluates were separated by cen-
trifugation, removed, and neutralized by 3 ul of 1M
TRIS-HCl pH 9.5. Most eluates were from a single
adsorption/elution step.
S ingle Antigen Beads Assays
Monoclonal antibodies, or antibody eluates
were examined using LabScreen® beads. The HLA
alleles represented in these products are listed in
Table 1. LABScreen® assays were performed ac-
cording to the manufacturer’s protocol. Briefly, 20
μl of test serum was added to 5 μl each of LS1A01,
LS1A02 SA, LS1A03, LS2A01 or LSMICA001 SA
beads, incubated in the dark for 30 minutes at RT,
and then washed with wash buffer. One hundred μl
of goat anti-human IgG or goat anti-mouse IgM or
IgG secondary antibody conjugated with R-Phyco-
erythrin (PE) was added to the beads, which were
incubated for 30 minutes in the dark at RT, then
washed and read on the LABScan™ 100 flow cy-
tometer (One Lambda Inc, Canoga Park, CA, USA).
Every assay included a negative control serum for
the allosera and a mouse IgM control (Sigma-Al-
drich, St. Louis, Mo, USA. #M5909 from tumor line
MOPC-104E) for the mAbs.
Data analysis
Data generated from the LABScanTM 100
were analyzed using computer software. Trimmed
mean fluorescence values for the SA beads reac-
tions were obtained from the output (.csv) file gen-
erated by the flow analyzer, and were adjusted for
background signal using the formula [(sample #N
bead – sample negative control bead) – (negative
control #N bead – negative control negative control
bead)]. For HLA class I, the adjusted reaction values
were then normalized by multiplying each value by
a corresponding normalization factor derived from
the results of the mAb W6/32 with the same beads.
Normalization factors were calculated by dividing
the average value of all mAb W6/32 reactions by the
adjusted fluorescence value for each bead. The data
were then graphed using either Excel spread sheets
(Microsoft, Redmond, WA USA) or HLA Visual® soft-
ware (One Lambda Inc. Canoga Park CA, USA). For
HLA class II and MICA, all values were adjusted for
background signals. All adjusted and/or normalized
reactions that were above zero were considered as
potential positive reactions for this study.
HLA amino ac id sequenc es,
epitopes, and distanc es between
residues
Amino acid (aa) sequences of the HLA antigens
or alleles were obtained from the Anthony Nolan
internet website (10). An epitope search program
was developed to identify distinguishing aa that
were exclusively shared by the positive antigens.
The program searched for one, 2, 3 or 4 common
unique aa positions. Among the many possibilities
generated, the results were further filtered by re-
stricting to positions that were exposed to the sur-
face of the molecule and that were within the anti-
body binding span estimated at 494Ǻ2 (19x26Ǻ) or
750Ǻ2 (11, 12). Approximate distances in angstroms
between 2 amino acids were calculated using the
Cn3D structure viewer software (13) and the 3D
structure of an HLA-A0201 molecule 1QEW (Orth,
et al., MMDB: Entrez’s 3D-structure database on
NCBI website) (14). Epitope ID numbers were as-
signed depending upon the number of unique aa
sites involved (1-200 for one aa, 201-400 for 2 aa,
or 401-500 for 3 or 4 aa positions).
RES ULT S
HLA Class I epitopes
Ninety-five Luminex beads each coated with dif-
ferent rHLA SA (Table 1) were used to identify a total
of 103 HLA-A, -B, and -C epitopes (Table 2), which
are recognized by a series of monoclonal antibodies
(40/103) and allosera (63/103). Of the 103 epitopes,
there were 31 A-, 43 B-, 4 C-, 17 A-B-, 5 B-C- and 3
A-B-C-locus antigen epitopes. Thirty-seven epitopes
can be defined by single aa at a single position and
other epitopes can be defined only by a combination
of 2-4 aa. Specifically, 49 epitopes were identified by

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HLA EpitopEs
2 aa, 15 epitopes by 3 aa and 2 epitopes by 4 aa.
The combination aa positions were not contiguous,
but were within a conformational distance allowing
antibody binding (7).
For most epitopes, we found one or more alter-
natives of a single aa or aa combinations that might
define the epitope. It was beyond the scope of the
assays used in our studies to precisely determine
the aa that bind to the antibody.
Table 3 lists the number of epitopes defined for
each of the A-, B- or C-locus antigens. The number
of epitopes varied between 4 and 19 per antigen.
For example, HLA A1 antigen has 10 epitopes (1,
12, 13, 14, 15, 16, 208, 238, 241, and 242); HLA B7
has 19 epitopes (7, 20, 25,32, 33, 205, 216, 222,
223, 224, 229, 231, 233, 234, 235, 401, 402, 408,
and 410); and HLA Cw4 has 4 epitopes (32, 205,
232, and 244).
In general, there was no correlation between
the number of epitopes defined for the antigen and
the antigen’s population frequency. For example,
HLA A2, which is the most frequent antigen in the
population (30-54%), has 14 epitopes while A25,
with a frequency of 0-6%, has 16 epitopes.
Certain amino acid residue positions on the
HLA antigen were more frequently associated with
epitopes than others. Position 163 located in the α2
domain of the alpha chain is well exposed on the
surface of the molecule and was most frequently
implicated. In contrast, position 161, which is one
aa away from position 163 and is equally exposed,
defines only one epitope (Fig. 1). In addition, cer-
tain individual aa were observed more frequently
than others in defining epitopes. For example, the
amino acid threonine (T) was the most frequent
while cysteine (C) was the least frequent (Fig. 2).
The aa side chain length or structure, and whether
they were acidic, basic, hydrophobic, or hydrophilic
did not correlate with the frequency with which they
were found in epitopes.
Table 4 shows the number of epitopes defined
for each individual locus or the inter-locus epitopes
defined for the A-B-C loci. The number of aa that
define the epitopes are also shown. For example,
there were 23 epitopes shared exclusively among
B-locus antigens and each epitope was defined by
a combination of 2 aa.
Figures 3-13 are examples of HLA class I epi-
topes. All positive reactions in each example are
reactions of the SA beads with either a monoclo-
nal antibody, diluted sera or eluted alloantibodies.
aa shared exclusively among the positive antigens
define the epitope. The location of each epitope is
shown on the 3D structure of the HLA antigens.
table 1. single rHLA class i antigens coated on beads used for immuno-binding assay.
Antigen
A1
A11
A11
A2
A2
A2
A23
A24
A24
A25
A26
A29
A29
A3
A30
A31
A32
A33
A33
A34
Allele
A0101
A1101
A1102
A0201
A0203
A0206
A2301
A2402
A2403
A2501
A2601
A2901
A2902
A0301
A3001
A3101
A3201
A3301
A3303
A3401
Antigen
A36
A43
A66
A66
A68
A68
A69
A74
A80
Allele
A3601
A4301
A6601
A6602
A6801
A6802
A6901
A7401
A8001
Antigen
B41
B41
B42
B44
B44
B45
B46
B47
B48
B49
B50
B51
B51
B52
B53
B54
B55
B56
B57
B57
Allele
B4101
B4102
B4201
B4402
B4403
B4501
B4601
B4701
B4801
B4901
B5001
B5101
B5102
B5201
B5301
B5401
B5502
B5601
B5701
B5703
Antigen
B58
B59
B60
B61
B62
B63
B64
B65
B67
B7
B71
B72
B73
B75
B76
B77
B78
B8
B81
B82
Allele
B5801
B5901
B4001
B4002
B1501
B1516
B1401
B1402
B6701
B0702
B1510
B1503
B7301
B1502
B1512
B1513
B7801
B0801
B8101
B8201
Antigen
Cw1
Cw2
Cw4
Cw5
Cw6
Cw7
Cw8
Cw12
Cw14
Cw15
Cw16
Cw17
Cw18
Cw9
Cw10
Cw10
Allele
Cw0102
Cw0202
Cw0401
Cw0501
Cw0602
Cw0702
Cw0801
Cw1203
Cw1402
Cw1502
Cw1601
Cw1701
Cw1802
Cw0303
Cw0302
Cw0304
B13
B18
B27
B27
B35
B37
B38
B39
B39
B4005
B1301
B1801
B2705
B2708
B3501
B3701
B3801
B3901
B3905
B4005

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eL-AwAr, TerAsAkI, CAI eT AL
Epitope #
Assigned
1
2
3
4
5
6
Single antigena beads reactive with
monoclonal or eluted antibody
A1,36
A2,69
A23,24
A25,26,34,43,66
A29,43
A3
B7,8,13,18,27,35,37,38,39,4005,41,42,
44,45,46,47,48,49,50,51, 52,53,54,55,56,
59,60, 61,62,64, 65,67,71,72,73,75,76,77,
78,81,82
B13
B38,39,67
B46
Position and unique aa for
possible epitopeb Sites
44K/ 150V/ 158V/
107W
65G
(9Y)+149T/ (74D)+149T
62L
161D
Alloserum /
mAbs Tested
Z3945.OL
W6090.LO
Z1238.TO
Z8855.TO
X5518.TO
X8341.EO
rHLAa Cells
used for
adsorption
N/A (mAb)
N/A (mAb)
N/A (mAb)
N/A (mAb)
N/A (mAb)
N/A (mAb)
765Q
F1398-2EH1 N/A (mAb)
8
9
10
145L/ 41T+46A
158T
46A+66K [69R]c
(67F)+131R / (67F)+177D /
(67F)+180E [9D] c
163R
144k
166D/ 167G
76A
[90D]c
62G
142T/ 145H
127K
177D/180E
41T
Z0693.TO
Z7567.RO
Z5550.DM
N/A (mAb)
N/A (mAb)
N/A (mAb)
11B8
X7768.TON/A (mAb)
12
13
14
15
16
17
18
19
20
21
A1,11,25,26,43,6601
A1,2,3,11,24,36,68,69,80
A1,23,2402,80,B76
A1,26,29,36,43,80
A1,36,11,25,26,34,43,6601,80, B73
A2,B57,58
A2,68,69
A2,23,24,68,69
B7,8,41,42,48,60,81
B13,4005,41,44,45,47,49, 50,60,61
B8,13,18,35,37,38,39,3905,4005,41,
4102,44,4403,45,47,48,49,50, 51,52,
53,59,60,61,62,64,65,71, 72,75,76, 77,78
A23,24,25,32,B38,49,51,52,53,57,58,59,
63,77
AS264
W7252.AO
X9288.OO
X9288.OO
W6409
X4632
X4632
Z6303
ARC JA
W7248.BO
A2501
A0101
A2402
A2902
A8001
B5801
A6901
A6901
B0702
B4402
2269T Z7227.00B1301
23 80IRGF19B4901
24
A23,24,25,32,B13,2705,37,38, 44,47,49,
51,52,53,57,58,59,63,77
82L/ 83R
RGF19/
W8822.AO/
(mAb X8992.
H0)
B4403/ A3201
25
B7,8,18,2708,35,39,4005,41,42,
45, 46,48, 50,54,55,56,60,61,62,
64,65,67,71,72,73, 75,76,78,81, 82
A203,25,26,34,43,66
A23,24,80
[80N]c
X1779.BOB5501
27
28
149T
62E
56E / 62E+65R / 62E+76 /
144K+151R / 163E+166D /
163E+167G
S33S
Z5571.I0
nn
N/A (mAb)
29A80 2004-08361
nn
30A110219K
2002-05676 /
2002-00048
2002-01731
nn
nn
31A30,31
A2,3,11,2403,25,26,29,30,31,32, 33,
34,36,43, 66,68 ,69,74,B7,8, 13,18,
27,35,37,38,39,4005,41, 42,46,47,
48,49,50,51,52,53,54, 55,56,57,58,
59,60,61,62,63,64, 65,67,71,72,73,
75,77,78,81, Cw1,2,4,5,6,7,8, 9,10,
12,14,15, 16,17,18
A32,74,B7,8,13,18,27,35,37,38, 39,
4005,41,42,44,45,46,47,48, 49,50,51,
52,53,54,55,56,57,58, 59,60,61,62,
63,64,65,67,71,72, 73,75,76,77,78, 81,82
56R
32167WFS32-5D8B6N/A (mAb)
33[109L]c
F2164-4A7A3 N/A (mAb)
35 B18,35,37,51,52,53,78,5845T
FN3814-
3E1G4
2005-02583
A132
N/A (mAb)
nn
Cw0702
36
37
A30
Cw7
A2,A25,A26,A29, A31,A32, A33, A34,
A43,A66,A68,A69, A74,B73, Cw7,Cw17
17S / 56R+73T
194L
38253QA39 / A40 A2501 / A0201
table 2. HLA Class-i epitopes. The epitope #, HLA antigens exclusively sharing the epitope, amino acid or
the combination of amino acids, including alternatives that define the epitope, serum or monoclonal antibody,
and for allo sera the rHLA cell line used for absorption and elution of antibodies are shown from left to right.

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173
HLA EpitopEs
Epitope #
Assigned
Single antigena beads reactive with
monoclonal or eluted antibody
Position and unique aa for
possible epitopeb Sites
Alloserum /
mAbs Tested
Z7921.00 /
A113
A129
A61
rHLAa Cells
used for
adsorption
Cw0202 /
Cw0303(Cw9)
nn
Cw1701
39
40
41
Cw2,9,10,15 21H
Cw5,8
B73,Cw7,17
A2
177K
267Q
43Q+62G/ 62G+66K/
62G+76V/ 62G+79G
65G+151R/ 127K+144Q/
127K+151R
156Q+166D/ 156Q+167G
201 S8043 N/A (mAb)
202A23 Z1060.TON/A (mAb)
203A2402
A32,74,B8,18,37,38,39,41,42,54,55,59,
64,65,67
A32,74,B7,8,4005,41,42,48,60,
61, 73,81,Cw1,2,4,5,6,7,8, 9,10,
12,14,15,16,17,18
Z1022.HO N/A (mAb)
204[109L +163T] c
F760-5B5D8N/A (mAb)
205 109L+131RF1119-9F4E7N/A (mAb)
206A36
158V+163T/ 158V+166E/
158V+167W
43P+65R/ 65R+163L/
66N+131S/ 66N+163L
142I+144K/ 144K+145R
163R+166E/ 158A+163R/
163R+167W
76V+144K
[(152E+156W)] c
X7138.HON/A (mAb)
207B57,58,63
Z1203.TON/A (mAb)
208 A1,3,11,24,36,80X9174A8001
209A11,25,26,43,6601Z2076.@OA2501
210
211
A2,3,11,68,69
A203,25,26,34,43,66,B46,62,76
A23,24,32,B38,49,51,52,53,57,58,
59,63,77
A23,25,26,29,30,31,32,33,34,43,66, 74
A25,26,33,34,66,68,69
A33,34,68,69,B8,18,37,38,39,41,42,
54,55,59,64,65,67
B7,8,18,2708,35,39,4005,41,42,45,48,50,
54,55,56,60,61,62,64, 65,67,71,72,75,76,
78,81,82
SE896
RGF40
A1101
B76 (B1512)
212[80I+90A/ 80I+149A] c
Z8059.C0 B5701
213
214
138M+144Q
43Q+62R/ 62R+109F
Z6895.00
Z3044
A3101
A6901
215 62R+163T Z7227.00B5401/A6801
21676E+80N/ 76E+82R/ 76E+83G Z3038B4102
217B13,2705,37,44,47
76E+80T/ 79R+80T/ 80T+82L/
80T+83R
46A+76E
45T+62R/ 45T+65Q/ 45T+66I/
45T+69T/ 45T+71T
90A+(171H)
RGF41 /
Z6895.00
X9733
B4403 / B2705
218B13,57,62,63,75,76,77B76 (B1512)
219B18,35,37,51,52,53,78
A3301,B18,51,52,64,65,78
B35,4005,46,49,50,51,52,53,56, 57,58,
62,63,71,72,75,77,78
A6602,B7,13,27,47,48,60,61,73, 81,
Cw2,Cw17
B7,13,27,47,48,60,61,81
B7,27,42,54,55,56,57,58,63,67, 73, 81,82 43P+69A
B8,59
B8,18,37,38,39,41,42,54,55,59,64, 65,67 66I+163T
B8,18,35,39,4005,41,45,48,50,60, 61,62,
64,65,71,72,75,76,78
B18,37,38,39,54,55,59,64,65,67
B7,27,42,54,55,56,67,73,81,82
X6101B1801
220SE897
RGF19 (mAb:
FV3306-
1A1A3)
A4
A3301
221163L+167WB5601
222163E+166E/ 163E+167WCw0202
223
224
225
226
76E+163ERGF37
X0786.00
RGF38
Z6895.00
B0703
B5801/ B5502
B0801
B1801
(67F)+163T
22769T+80N/ 69T+ 82R/ 69T+83G X4715.D0B4501
228
229
131S+163T
65Q+69A
Z6331.00
Z6331.00
B3905
B8101
B5201 +
others
N/A
N/A (mAb)
230B38,49,51,52,53,59,7765Q+80I/ 69T+ 80I Z2980.BO
231
232
B7,48,81
B54,55,59,Cw1,4,5,6,7,8,12,14, 15,16,18 (103L)+163T
A25,32,B7,8,13,18,27,35,37,38, 39,4005,
41,42,44,45,46,47,48, 49, 50,51,52,53,
54,55,56,57,58, 59,60,61,62,63,64,65,
67,71,72, 73,75,76,77,78,81,82
B7,42,46,54,55,56,67,81,82
B27,47,61,7,48,60,73,81,13
41A+178K S35C
Z2611.R0
233[79R +127N] c
F352-10F9E1 N/A (mAb)
234
235
[43P+(70Q) / 65Q+(70Q)] c
138T+163E
AS627
X7021.E0
Z1173.
N0 / F667-
2E1E4/ 2005-
01035(Allo)
N/A
N/A (mAb)
236B57,58
43P+62G/ 41A+43P+62G
/ 17R+41A+43P+ 62G /
19E+41A+43P+ 62G
N/A (mAb)
table 2. (Cont’d)

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174
eL-AwAr, TerAsAkI, CAI eT AL
Epitope #
Assigned
237
Single antigena beads reactive with
monoclonal or eluted antibody
B57,63 (weak B58)
A1,2,3,11,25,26,29,32,33,34,36, 43,
66,68,69,74,B57,58,63
Position and unique aa for
possible epitopeb Sites
46A+65R / 41A+46A+65R
Alloserum /
mAbs Tested
X8442.E0
FG2755-
11A3D7
rHLAa Cells
used for
adsorption
N/A (mAb)
238 56G+65RN/A (mAb)
239B46,73
43P+76V/ 65Q+76V/ 76V+79R/
[76V+80N 41A+43P+76V/
41A+65Q+76V/ 73T+76V+79R/
73T+76V+80N] c
2002-00142
nn
240B76163L+166D/ 163L+167G
0-4 / 38991/
38995/ 38998/
39002
nn
241 A1,11,25,26,34,36,43,6601,80
65R+90D / 43Q+90D /
90D+138M
AS264 A3601
242
A1,2,3,1101,26,29,30,31,33, 34,36,
43,66,68,69,74,80
19E+79G0544HA N/A (mAb)
243
A25,26,A33,34,66,68,69,B63
62R+65R “B76 also positive (
62R+65Q)”
FC1043-
6B4D1B9
Eluted from
A2501 rHLA
cell line
A113
N/A (mAb)
244Cw2,4,5,6,15,17,18
B35,4005,46,49,50,51,52,53,56, 57, 58,
62,63,71,72, 75, 77,78, Cw9, Cw10
B46,73,Cw1,7,8,9,10,12,14,16
B7,27,42,54,55,56,57, 58,63,67, 81, 82
B7,42,54,55,56,67,81,82
B46,62,75,76,77
80KCw1701
B62 (B1501)
/ B35
245163L+167W A6
24676V+80N / 73T+76V+ 79R
Z9016.00 /
2002-00142
Z6328.00
Z9009.A0
Z6845.B0
Cw1802/ nn
401
402
403
43P+69A+76E
65Q+69A+ (70Q)
41A+46A+65Q
149A+150A+163R,149A+
158A+ 163R/149+163R+ 166E,
149A+163R+ 167W
65Q+69A+80T
65Q+69A+82L
65Q+69A+83R
127K+142I+ 144K/ 127K+142I+
151H/ 127K+144K+ 145R/
127K+145R+ 151H
(147W)+163E+ 177D
(147W)+163E+ 180E
43P+62R+65R
[41A+46E+67Y,43P+46E+67Y
43P+46E+ 70Q]c,43P+69A+
70Q, 43P+70Q+76E, [46E+
65Q+ 67Y, 46E+65Q+ 70Q] c
(63E)+69A+ 80N (70K)+76E+
80N (70K)+76E+ 82R
(70K)+76E+ 83G (70K)+80N+
131S
(9F)+142T+ 149A / (9F)+145H+
149A
62R+(63E)+80I
[ (63E)+(71A)+ 163L]c
[(9Y)+41A+ (63E)+ (95I)] c
(32Q)+62R+ (77N)+80T
(45M)+62R+ (77N)+80T
80I+90A+ 127N+(152E)/
80I+109L+ 131S+(152E)/
82L+90A+ 127N+(152E)/
83R+90A+ 127N+(152E)
69T+(74D)+ 158A+163T
(73T)+76V+ 80N+90A
B0702
B8201
B76 (B1512)
404
A112002-00323
nn
406B2705
X4221.DQ/
X3494.N0
N/A (mAb)
407A24
X8598.T0 /
S8036
N/A (mAb)
408B7 Z5754.R0 N/A (mAb)
409B63 Z6856.PKN/A (mAb)
410B7,42,54,55,56,67,81,82
2002-05252
nn
411B27082005-04739
nn
412 A0201
2002-00245 /
2004-08903
2002-01799
F633-3E8H4
F698-4F9F9
nn
nn
N/A (mAb)
N/A (mAb)
414
415
417
B49,52,63
B46,57,58,63
A11,B57,58
A26,B13418X6954.TON/A (mAb)
419 B49,51,52,63,77
FC2121-
5A4A1
N/A (mAb)
420
421
a Alleles are designated only when other alleles of the same antigen did not react with the eluate.
b Possible alternative epitopes are separated by “/”. Epitopes that are defined by more than a single position/aa are
separated by “+”. Amino acids that are not exposed at the surface of the HLA molecule are between parentheses.
c Epitope also shared by C-locus antigens based on aa sequences but not proven by antibody test with SA beads are
in square brackets.
B8,B64,B65
B46,Cw1,8,9,10,14,16
FS964-1A3B4 N/A (mAb)
A6Cw0102
table 2. (Cont’d)

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175
HLA EpitopEs
table 3. Epitopes on HLA class i antigens.
Antigen
# of
Epitopes
10
13
14
9
12
16
16
7
7
5
5
11
9
13
10
14
15
11
12
8
9
14
15
16
12
13
14
13
12
14
17
7
8
15
11
14
14
11
15
16
13
17
17
14
18
Epitopes
A1
A11
A0201
A23
A24
A25
A26
A29
A3
A30
A31
A32
A33
A34
A36
A43
A66
A68
A69
A74
A80
B13
B18
B27
B35
B37
B38
B39
B4005
B41
B42
B44
B45
B46
B47
B48
B49
B50
B51
B52
B53
B54
B55
B56
B57
1
12
2
3
3
4
4
5
6
31
31
23
32
4
1
4
4
13
2
32
13
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
17
12
13
13
14
13
12
12
15
13
32
32
24
38 213 214 215 220 238 242 243
16273238 211 213 214 215 238 241 242 243
13151632 206 208 238 241 242
51215162732
1216 273238 209 211 213 214 222 238 241 242 243
18193238 210 214 215 238 242 243
1318193238 210 214 215 238 242 243
3338 204 205 213 238 242
1415 16 2829 208 241 242
82122 243233 217 218 222 223 233 235 418
2225323335 204 215 216 219 220 226 227 228 233
242532 33 216 217 222 223 224 229 233 235 401 406 411
2225323335 216 219 221 227 233 245
2224 323335 204 215 217 219 226 228 233
9222324 3233 204 212 215 226 228 230 233
9222532 33 204 215 216 226 227 228 233
21 2225 3233 205 216 221 227 233 245
20 2122 2532 33 204 205 215 216 226 227 233
202532 33 204 205 215 216 224 226 229 233 234 401 402 410
21222433 217 233
21222533 216 227 233
10 253233 211 221 233 234 239 245 246 403 415 421
21222432 33 217 222 223 233 235
20 22253233 205 216 222 223 227 231 233 235
212223243233 212 221 230 233 245 414 419
21222532 33 216 221 227 233 245
22 2324 3233 35 212 219 220 221 230 233 245 419
2223 2432 33 35 212 219 220 221 230 233 245 414 419
22 23 2432 3335 212 219 221 230 233 245
2532 33 204 215 216 224 226 228 229 232 233 234 401 402 410
253233 204 215 216 224 226 228 229 232 233 234 401 402 410
253233 216 221 224 229 233 234 245 401 402 410
23243233 207 212 218 221 224 233 236 237 238 245 401 415 417
13
16
17
19
14
16
15
32
32 208 210 238 242
36 213 242
38 213 242
3233 38 204 205 212 213 233 238
14
30
18
23
19
23
16
38 213 238 242
15
32 208 209 210 238 241 242 404 417
19 27 3238 201 210 211 238 242 412
2428 202 212 213
23 24 2832 203 208 212 407
24273238 209 211 213 214 233 238 243 241
273238 209 211 213 214 238 241 242 418 243
16 208 238 241 242
38 209 211 213 238 241 242

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eL-AwAr, TerAsAkI, CAI eT AL
table 3. Epitopes on HLA class i antigens.
Antigen
# of
Epitopes
17
15
14
13
13
19
14
14
17
19
10
10
15
12
12
14
13
16
18
11
5
5
4
5
4
7
6
6
6
4
5
5
5
6
4
Epitopes
B58
B59
B60
B61
B62
B63
B64
B65
B67
B7
B71
B72
B73
B75
B76
B77
B78
B8
B81
B82
Cw1
CW2
CW4
CW5
CW6
CW7
CW8
CW9
CW10
CW12
CW14
CW15
CW16
CW17
CW18
17
7
7
7
7
23
7
7
7
7
7
7
7
7
7
7
7
7
7
7
32 205 232 246 421
32 39 205 222 244
32 205 232 244
32
40 205 232 244
32 205 232 244
323738
32
40 205 232 246 421
3239 205 245 246 421
3239 205 245 246 421
32 205 232 246
32 205 232 246 421
32 39 205 232 244
32 205 232 246 421
323841 205 222 244
32 205 232 244
23
22
20
21
22
24
22
22
9
20
22
22
16
22
14
22
22
11
20
25
24
23
21
22
25
32
25
25
25
25
25
25
25
25
22
23
25
20
25
33 216 224 229 233 234 401 402 410
32
24
22
25
32
33 207 212 218 221 224 233 237 238 243 245 401 409 414 415 419
3233 204 215 216 220 226 227 228 233 420
3233 204 215 216 220 226 227 228 233 420
3233 204 215 216 224 226 228 229 233 234 401 402 410
3233 205 216 222 223 224 229 231 233 234 235 401 402 408 410
32 33 216 221 227 233 245
3233 216 221 227 233 245
32 3338 41 205 222 224 229 233 235 239 246
3233 216 218 221 227 233 245 403
2533 211 216 218 227 233 240 403
243233 212 218 221 230 233 245 403 419
323335 216 219 220 221 227 233 245
22 2532 33 204 205 215 216 225 226 227 233 420
3233 205 216 222 223 224 229 231 233 234 235 401 402 410
33
32
25
32
33 211 216 218 221 227 233 245 403
35 207 212 221 224 233 236 238 245 401 415 417
33 204 212 215 225 226 228 230 232 233
3233 205 216 222 223 227 233 235
33 205 216 222 223 227 233 235
41 205 232 246
table 4. Distribution of 103 HLA class i epitopes among the ABC loci. Also shown the number of
amino acids that define the epitopes.
Number of
Epitopes
23 1712 10886333222211
HLA - Locus
B
AA
BB ABAB
C
BC
A
BC ABCABBABC
C
Number of aa
defining epitope
2123121123123
4
12

Page 9

177
HLA EpitopEs
Epitope
#
1001
1002
DR antigens sharing epitope
Position/
amino acida
4Q
9W
DR7,DR9,DR53
DR1,DR7,DR15,DR16,DR103
DR1,DR4,DR7,DR9,DR15,DR16,
DR51,DR53,DR103
DR4,DR10
DR1,DR9,DR10
DR11,DR14,DR15,DR17,DR18,
DR52
DR7,DR51
DR7
DR4,DR8,DR11,DR13,DR14,
DR15,DR16,DR17
DR1,DR9,DR51,DR53
DR7,DR14
DR12
DR1,DR14,DR15,DR103
DR10,DR53
DR11,DR12,DR13,DR15,DR17
DR4,DR8,DR13
DR11
DR7,DR8.DR11,DR12,DR13,
DR16,DR51,DR103
DR9,DR10,DR14,DR53
DR15
DR4,DR13,DR17,DR18,DR52
DR7,DR17,DR18,DR52
DR9,DR14,DR53
DR8
DR7,DR52
DR17,DR18
DR17,DR18,DR52
DR1,DR4,DR7,DR9,DR10,DR11,
DR12,DR13,DR14,DR15,DR16,
DR51,DR53,DR103
DR7,DR9
DR1,DR12
DR1,DR51,DR103
100310Q
1004
1005
11V
13F
100613S
1007
1008
13Y
25Q
100928D
1010
1011
1012
1013
1014
1015
1016
1017
31I
37F
37L
37S
40Y
47F
57S
58E
101870D
1019
1020
1021
1022
1023
1024
1025
1026
1027
70R
71A
71K
73G
74E
74L
74Q
74R
77N
102877T
1029
1030
1031
78V
85A
96E
Epitope
#
DR antigens sharing epitope
Position/
amino acida
1032
DR7,DR8,DR9,DR10,DR11,DR12,
DR13,DR14,DR17, DR18, DR52
DR10,DR15,DR16,DR53
DR4
DR4,DR7,DR9
DR52
DR4,DR7,DR9,DR51,DR52
DR4,DR10,DR51,DR53
DR1,DR7,DR9,DR15,DR16,DR51,
DR52,DR53,DR103
DR4,DR8,DR10,DR11,DR12,
DR13,DR14,DR17,DR18
DR8,DR11,DR12,DR13,DR14,
DR17,DR18
DR4
DR7,DR9,DR10
DR9
DR51
DR1,DR7,DR15,DR16,DR103
DR9,DR51
DR7
DR4
DR8,DR12
DR10,DR53
DR13
DR7,DR9,DR12
DR4,DR7,DR9
DR8,DR11,DR12,DR13,DR14,
DR17,DR18
96H
1033
1034
1035
1036
1037
1038
96Q
96Y
98E
98Q
104A
120N
1039140A
1040140T
1041149H
1042
1043
1401
1402
1403
1404
1405
1406
1407
1408
1409
1410
1411
180L
181M
9K,30G
9Q,108T
9W,10Q
11D,28H
11G,14K
11V,13H
13G,16Y
38A,40Y
57S,71K
57V,60S
98E,104A
9E,10Y,11S,
12T,13S
11G,14K,
25Q, 30L
11P,13R,
133L,142M
11S,12T,13S
13H,33H,
96Y,180L
57A,60H,
112Y
1601
1602DR7
1603DR15,DR16
1604DR11,DR13,DR14,DR17,DR18
1605DR4
1606DR14
a Possible alternative epitopes are separated by commas.
HLA Class II epitopes
table 5. HLA Dr Epitopes. the epitope #, HLA antigens exclusively sharing the epitope, and
amino acid or the combination of amino acids, including alternatives that define the epitope are
shown from left to right. Most amino acid positions listed in this table are not exposed on the
surface of the molecule and therefore may not be the amino acids that bind to the antibody.
Figures 14 – 16 show all epitopes that have
been identified for the HLA antigens A1, B8, or
Cw4. Most epitopes are well exposed on the sur-
face of the HLA molecule and therefore can bind to
the antibody.
Sixty HLA-DR epitopes and 18 HLA-DQ epit-
opes have been defined to date (Tables 5,6). Most
HLA Class II epitopes (3, 5, 9) were defined by one
aa residue on the HLA-DR, -DQB or -DQA chains.

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178
eL-AwAr, TerAsAkI, CAI eT AL
However, as is the case with Class I epitopes, some
DR epitopes can also be defined by more than one
alternative residue (epitopes 1401 – 1606). On the
other hand, almost half of the DQB and DQA epit-
opes can be defined by combination of two aa resi-
dues (Table 6).
Table 7 shows the number of epitopes defined
for each HLA-DR antigen. The number varied from
8 to 21. For example, DR7 has 21 epitopes (1001,
1002, 1003, 1007, 1008, 1011, 1018, 1022, 1025,
1028, 1029, 1032, 1035, 1037, 1039, 1043, 1403,
1405, 1410, 1411, and 1602). Similarly, all DQ anti-
gens have more than one epitope, most of them on
the beta chain. Epitopes that have been identified
on the alpha chain of the DQ antigen are limited in
number at this time but may not be rare.
Figures 17-20 show one example of a DR epit-
ope, 2 DQB epitopes, and one DQA epitope.
MICA epitopes
Seven MICA epitopes have been defined to
date (Table 8). All the MICA epitopes can be de-
fined by a single amino acid. For example, the epi-
tope shared by the MICA*001, 002, 004, 007, 009,
012, 018, and 027 antigens is defined by the amino
acid, glutamine, (Q) at position 91 of the MICA mol-
ecule (Fig. 21). In 2 cases, the sera were absorbed
by rMICA cell lines and the eluted antibodies re-
acted exclusively with certain MICA antigens. One
antibody eluted from MICA*018 reacted exclusively
with MICA*001, 012, and 018 antigens on the MICA
SA beads. The 3 MICA antigens exclusively share
threonine (T) at position 24, which defines this epi-
tope. Another antibody eluted from a MICA*004
rMICA cell line reacted with MICA*027, 004 and
009 antigens which share tyrosine (Y), valine (V)
or glutamic acid (E) at positions 36, 129, and 173,
respectively. Either one of the amino acids define
this epitope.
table 6. HLA DQB and DQA Epitopes. the epitope #, HLA antigens exclusively sharing the
epitope, and amino acid or the combination of amino acids, including alternatives that define the
epitope are shown from left to right.
Epitope # a
DQ antigens sharing epitope
2001DQ2
2002DQ4
2003DQ4,DQ5,DQ6,DQ7,DQ8,DQ9
2004DQ5,DQ6
2005DQ7
2006DQ7,DQ8,DQ9
2007DQ4,DQ5,DQ6
2008DQ2,DQ5,DQ7,DQ8,DQ9
2009 DQ2,DQ4,DQ5,DQ6,DQ8,DQ9
2010DQ4,DQ5,DQ6,DQ8,DQ9
2011DQ5,DQ0601
2012 DQ8,DQ9
2013DQ2,DQ4,DQ7,DQ8,DQ9
2014DQ4,DQ7,DQ8,DQ9
2015DQ5
2017DQA1*0201
2018 DQA1*04/DQA1*05/DQA1*06
2019DQA1*03
Position/ amino acid b
28S/30S/37I/52L/55L
56L
28T/46V/52P
84E/85V/86A/89G/90I/221Q
45E
55P
52P+55R
(9Y+11F)
34R+45G
45G+46V
38V+46V
45G+55P
84Q/85L/86E/87L/89T/220H/221H
77T+84Q/77T+85L/77T+86E/77T+87L/182N
70G+71A/116I/125S
47K/52H/54L
40G/47C
26S/47Q/56R/187T
a 2008 defined by mAb. b Possible alternative epitopes are separated by “/”. Epitopes that are defined by more than
a single position/aa are separated by “+”. Amino acids that are not exposed at the surface of the HLA molecule are
between parentheses.

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179
HLA EpitopEs
table 7. Epitopes on HLA class ii antigens.
Antigen
# of epitopes
DR110
DR1011
DR1038
DR1111
DR1211
DR1312
DR14 13
DR1512
DR169
DR1712
DR18 10
DR4 15
Epitopes
1002 1003 1005 1010 1013 1028 1030 1031 1039 1403
1004 1005 1014 1019 1028 1032 1033 1038 1040 1043 1408
1002 1003 1013 1018 1028 1031 1039 1403
1006 1009 1015 1017 1018 1028 1032 1040 1041 1601 1604
1012 1015 1018 1028 1030 1032 1040 1041 1407 1410 1601
1009 1015 1016 1018 1021 1028 1032 1040 1041 1409 1601 1604
1006 1009 1011 1013 1019 1023 1028 1032 1040 1041 1601 1604 1606
1002 1003 1006 1009 1013 1015 1020 1028 1033 1039 1403 1603
1002 1003 1009 1018 1028 1033 1039 1403 1603
1006 1009 1015 1021 1022 1026 1027 1032 1040 1041 1601 1604
1006 1021 1022 1026 1027 1032 1040 1041 1601 1604
1003 1004 1009 1016 1021 1028 1034 1035 1037 1038 1040
1042 1406 1411 1605
1003 1007 1010 1018 1028 1031 1037 1038 1039 1402 1404
1006 1021 1022 1025 1027 1032 1036 1037 1039
1001 1003 1010 1014 1019 1023 1028 1033 1038 1039 1408
1001 1002 1003 1007 1008 1011 1018 1022 1025 1028 1029
1032 1035 1037 1039 1043 1403 1405 1410 1411 1602
1009 1016 1018 1024 1032 1040 1041 1407 1601
1001 1003 1005 1010 1019 1023 1028 1029 1032 1035 1037
1039 1043 1401 1404 1410 1411
2017
2019
2018
2001 2008 2009 2013
2002 2003 2007 2009 2010 2013 2014
2003 2004 2007 2008 2009 2010 2011 2015
2003 2004 2007 2009 2010 2011
2003 2005 2006 2008 2013 2014
2003 2006 2008 2009 2010 2012 2013 2014
2003 2006 2008 2009 2010 2012 2013 2014
DR51
DR52
DR53
DR7
11
9
11
21
DR8
DR9
9
17
DQA1*0201
DQA1*03
DQA1*04/DQ
DQ2
DQ4
DQ5
DQ0601
DQ7
DQ8
DQ9
1
1
1
4
7
8
6
6
8
8
table 8. MiCA Epitopes. the epitope #, MiCA antigens exclusively sharing the epitope, amino
acid or the combination of amino acids, including alternatives, that define the epitope, serum or
monoclonal antibody, and for x allo sera the recombinant MiCA cell line used for absorption and
elution of antibodies are shown from left to right.
Single antigen beads reactive with eluted
antibodypossible epitopea Sites
MICA*001, 012, 018(24T)
MICA*001, 002, 004, 007, 009, 012, 018, 027 b
91Q
MICA*004, 009122V
MICA*027, 004, 009 b
36Y/129V/173E
MICA*01791R
MICA*004
181R
MICA*027 b
213I / 251R
ND = Not done. a Possible alternative epitopes are separated by “/”. Epitopes that are defined by more than a
single position/aa are separated by “+”. Amino acids that are not exposed at the surface of the HLA molecule are
between parentheses. b MICA*019 also share the amino acid(s) shown. Due to higher than acceptable background
fluorescence with the MICA*019 beads, it is not listed here.
Position and unique aa for Alloserum /
mAbs Tested
URUALB77
ND
ND
USAKAM 056
ND
ND
ND
rMICA Cells used
for adsorption
MICA*018
ND
ND
MICA*004
ND
ND
ND

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180
eL-AwAr, TerAsAkI, CAI eT AL
Figure 1. The frequencies at which poly-
morphic aa positions appear in the definition
of the HLA class I epitopes are shown in this
Figure. For example, position 163 (the most
frequent) defined more than 30 epitopes while
position 161, only one aa away, defined only
one epitope.
Figure 2. Amino acids were found at differ-
ent frequencies in defining the HLA class I an-
tigens epitopes. Threonine (T) was associated
with 373 of all the epitope alternative sites for
the 103 class I epitopes identified. On the other
hand, Cysteine (C) was found only once.
Figure 3. Antibody eluted from the A2501 rHLA
SA cell line only reacts strongly with A11, A25, A26,
A43, and A6601 SA beads. These antigens have
the unique combination of arginine (R) at position
163 and glutamic acid (E) at position 166 on the
HLA molecule (epitope #209, Table 2).
Z2076.@0: Eluted from A2501 rHLA cell line
A1101
A1102
A2501 A2601
A4301
A6601
B4201
A3401
A6602
Hundreds
25
Reaction strength
Single Antigen Beads specificities
5
10
15
20
Antigen
A01011
A1101
A1102
163
R
R
R
166
D
E
E
A2501
A2601
A4301
A6601
B4201
A3401
A6602
R
R
R
R
T
T
E
E
E
E
E
E
E
E
aa position
RE
Polymorphic amino acid positions on HLA class I antigens:
Number of epitopes defined by each position
35
Amino acid polymorphic position in HLA class I alpha chain
0
5
10
15
20
25
30
9
9
19
19
32
32
43
43
45
45
56
56
63
63
66
66
69
69
71
71
74
74
77
77
80
80
83
83
95
95
107
103
127138
131
144
142
147
145
150
151
152
156
158
161
163
161
167
166
177
171
180
178
253
194
Number of epitopes
17 21
21
41
41
44
44
46
46
62
62
65
65
67
67
73
73
76
76
79
79
82
82
90
90
109145
149
267
70
70
107
103
127138
131
144
142
147
150
151
152
156
158 163
167
166
177
171
180
178
253
194
109
149
267
376 376
337 333 337 333
259259
220212220212
188188
153137
111988481716552503428
11
00
5050
100100
150150
200200
250250
300300
350350
400400
Count
T E R Q LT E R Q LAA N W G
Amino acids Amino acids
IIPPD V KD V KS H YS H YF M CF M C
Frequency of each amino acid found
In HLA class I epitopesIn HLA class I epitopes
Letter
A
C
D
E
FF
Name
Alanine
Cysteine
Aspartic Acid
Glutamic Acid
Phenyalanine Phenyalanine
Letter
M
N
P
Q
RR
Name
Methionine
Asparagine
Proline
Glutamine
Arginine Arginine
Letter
G
H
I
K
LL
Name
Glycine
Histidine
Isoleucine
Lysine
LeucineLeucine
Letter
S
T
V
W
YY
Name
Serine
Threonine
Valine
Tryptophan
TyrosineTyrosine
153137
111988481716552503428
Count
N W G
Frequency of each amino acid found
Letter
A
C
D
E
Name
Alanine
Cysteine
Aspartic Acid
Glutamic Acid
Letter
M
N
P
Q
Name
Methionine
Asparagine
Proline
Glutamine
Letter
G
H
I
K
Name
Glycine
Histidine
Isoleucine
Lysine
Letter
S
T
V
W
Name
Serine
Threonine
Valine
Tryptophan

Page 13

181
HLA EpitopEs
Figure 4. Example of a conformational epi-
tope dependent on threonine (T) at position 45
and arginine (R) at position 62. Eluate from the
B1801 rHLA SA cell line recognized only B18,
B35, B37, B51, B52, B53, and B78 SA beads.
These are the only HLA molecules with this ep-
itope. Although they are separated in sequence
by 17 aa residues, the 2 positions are 9 Ǻ apart
and within the antibody’s binding span (epitope
#219, Table 2).
Figure 5. Epitope 21 (Table 2). SA beads
B13, B4005, B41, B44, B45, B47, B49, B50, B60,
and B61 were positive with alloantibody eluted
from a B4402 rHLA SA cell line. The positive an-
tigens all share the aa threonine (T) at position
41 on a side loop of the HLA molecule.
W7248.B0: Elution from B4402 rHLA cell line
2
4
6
8
10
12
14
16
B1301
B4005
B4101B4102 B4402B4403B4501B4701B4901B5001B4001B4002
B4801B8101
Hundreds
Single Antigen Beads specificities
Reaction strength
41
A
T
T
T
T
B07021
B1301
B4005
B4101
B4102
B4402
B4403
T
T
B4501
B4701
B4901
B5001
B4001
B4002
B4801
B8101
T
T
T
T
T
T
A
A
aa & position
62
45
Antigen
B07021
B1801
B3501
B3701
B5101
B5201
B5301
B7801
A8001
B4402B4402
62
R
R
R
R
R
R
R
R
E
RR
45
E
T
T
T
T
T
T
T
E
KK
aa position
45
E
T
T
T
T
T
T
T
E
Antigen
B07021
B1801
B3501
B3701
B5101
B5201
B5301
B7801
A8001
62
R
R
R
R
R
R
R
R
E
aa position
X6101: Elution from B1801 rHLA cell line
2
4
6
8
10
12
14
B1801B3501
B3701B5101B5201
B5301
B7801A8001
B4402
Hundreds
Single Antigen Beads specificities
Reaction strength

Page 14

182
eL-AwAr, TerAsAkI, CAI eT AL
Figure 6. Epitope 401 (Table 2). SA
beads B54, B5701, B55, B5703, B56, B63,
B58, B82, B7, B67, B81, B2708, B42, and
B2705 were positive with antibody eluted
from B0702. All 14 antigens share proline
(P), alanine (A), and glutamic acid (E) at
positions 43, 69, and 76, respectively. The
aa combination is exclusive to this group
of antigens, and within the binding span
(19x26Ǻ) of a single antibody. The farthest
residues, 43P and 76E, are separated by
~18.9 Ǻ.
aa& positions
43 69 76 43 69 76
B07021 P A E
B5401
B07021
P A E
P A E
B8201
P A E
B5701
P A E
B6701
P A E
B5502
P A E
B8101
P A E
B5703
P A E
B2708
P A E
B5601
P A E
B4201
P A E
B1516
P A E
B2705
P A E
P T EB5801
P A E
B5901
aa& positions
69 69
7676
4343
Z6328.00: Eluted from B0702 rHLA cell line
20
40
60
80
100
120
140
B5401 B5701B5502
Single antigen beads specificities
B5703B5601B1516 B5801B8201
B0702
B6701B8101B2708B4201
B2705
B5901
Hundreds
Reaction strength
Figure 7. Epitope 205 (Table 2). SA
beads A32, A74, B4005, B4101, B4102,
B42, B48, B60, B61, B7, B73, B8, and B81
exhibited strong binding with mAb F1119-
9F4E7, and share leucine (L) at position
109 and arginine (R) at position 131.The
combination is exclusive to this antigen
group. The 2 positions are approximately
15 Ǻ apart and are located on the side
loops of the HLA molecule.
mAb: F1119-9F4E7
50
100
150
200
250
A3201
A7401
B4005
Single Antigen Beads Specificities
aa position
B4101
B4102
B4201
B4801
B4001
B4002
B0702B7301 B0801
B8101
B4402B8201
Hundreds
Reaction strength
109 131109 131
F A01011 F R A01011R
B07021 L
A3201
A7401
B4005
R
R
R
R
R
R
R
R
B07021
B4002
B0702
L
L
L
L
L
R
R
R
R
R
B4801
L
L
L
L
L
L
L
B4101
B4102
B4001
B8101
B4201
B7301
B8201
L
L
L
R
S
S
B4402
B0801
LR
aa position
LR
LR

Page 15

183
HLA EpitopEs
Figure 10. Epitope 39 (Table 2). Allo-
antibody eluted from the Cw0202 rHLA SA
cell line reacted with single antigens Cw10
(0302), Cw9, Cw10 (0304), Cw15 and
Cw2. The positive antigens share histidine
(H) at position 21, which is located in the
alpha 1 domain.
Z7921.00: Eluted from Cw0202.L rHLA cell line
Hundreds
2
4
6
8
10
12
14
16
Cw*0302
Cw10 Cw9 Cw10 Cw15 Cw2
Cw*0303
Cw*0304
Cw*1502Cw*0202
B4601
Cw*0602
Cw6 Cw17
Cw*1701
B46
Single Antigen Beads Specificities
Reaction Strength
2121
19
E
E
E
E
E
E
E
E
E
E
E
21
R
R
R
H
H
H
H
H
R
R
R
24
A
S
S
A
A
A
A
A
A
S
A
A*01011
B*07021
Cw*010201
Cw*0302
Cw*0303
Cw*0304
Cw*1502
Cw*0202
B4601
Cw*0602
Cw*1701
aa & positions
Figure 8. Epitope 24 (Table 2). All SA
beads with Bw4-associated antigens were
positive with alloantibody from RGF19 se-
rum adsorbed and eluted from a B4403
rHLA SA cell line. Only the reactive anti-
gens have leucine (L) at position 82 and
arginine (R) at position 83 in the alpha 1
domain helix.
Figure 9. Epitope 37 (Table 2). Mono-
specific antibody eluted from the rHLA an-
tigen Cw0702 reacted exclusively with the
Cw0702 SA bead. Cw0702 is the only anti-
gen with leucine (L) at position 194, which
is located on the alpha chain.
AlphaAlpha
Beta-2Beta-2
194L194L
A132: Eluted from Cw0702 rHLA cell line
5
10
15
20
25
30
Cw*0702
A4301
A1102
Hundreds
Single Antigen Beads Specificities
Reaction Strength
193
P
P
P
194
I
I
V
199
A
A
A
A*01011
B*07021
Cw*010201
Cw*0702
A*1102
P
P
P
L
I
A
A
AVA
A*3401
aa & positions
P
RGF19: Eluted from B4403 rHLA cell line
20
40
60
80
100
120
140
160
180
A3201
B4901
B5301B5101
A2501B5801
B5201
B3801
A2402A2403B5703
B5701
B1513B1516B3701B4403B5901B2705
A2301
B4701B1301B4402B7801B3501
Hundreds
Single Antigen Beads Specificities
Reaction Strength
83
82
82
R
83
G
82
R
83
G
B07021
A2301
A2402
A2403
A2501
A3201
B1301
B2705
B3701
B3801
B4402
B4403
B4701
R
L
L
L
L
L
L
L
L
L
L
L
L
G
R
R
R
R
R
R
R
R
R
R
R
R
B7R
L
L
L
L
L
L
L
L
L
L
R
R
G
R
R
R
R
R
R
R
R
R
R
G
G
B4901
B5101
B5201
B5301
B5701
B5703
B5801
B5901
B1516
B1513
B7801
B3501
aa & positionsaa & positions
A01011A1