Allnrgy, 1991, 46, 520-528

1991
Analysis of allergenic components of Bermuda
grass pollen by monoclonal antibodies
Z. N. C H A N C , L . C . T.SAI, C . W . C m , M. C. WANC;, H . D . S H E N , D . T . LEE*
& S. H. HAN

* / • " • / " "

/ U H K)

I I ly M \l I I I I I I 11

II I I I I I II I 11 II I
I , 1 f 1 I M M 1 11
I I I I 1, I II M I M ^
II ^ II (, I I II ^ I lu I I II
1 I I I I ,11

related gras.ses (2, 22, 25, 28). In Taiwan, extract of BGP (2).
aboul 27% of the asthmatic jjaticnts showed
 ALLliRGENIC COMPONENT'S OF BGP DEFINED BY MOABS
Although major allergens of various grass-
pollen Ags, for example. Lolium peienne, have
been purified and well characterized (1, 5, 7, 8,
10, 13), progress in the study of allergens from
BGP has been limited. Some years ago, Oiren &
Dowdle (25) firsl reported ihat BGP-1, with a
molecular weioht of 30 kDa is the major allergen
of BGP. Later, Karlstam & Nilsson (16) found
that allergens oi BGP showed a high affinity for
C;on-A. Recently, the allergenic coin]3oncnt of
BGP has been examined in detail using immuno-
blotting tcchniqtie by Ford &Baldo (11) as well as
our group (27). A complex composition was
found in BGP extract There were al least 14 IgE
binding components wilh MW ranging from 8
kDa to 94 kDa. Among these, a'32-34 kDa
mpon
Mallhiesen el al. (21) u ed radio
Crude extrac'ts ofallcigens which are still being
used clinically for diagnosis and treatment may
genic components. In addition, the composition
may vary from lot to lol. Being a praclic al tool for
standardisatton and purification, MoAbs have
been tiscd to solve this problem and have been
applied successfully for the sttidy of pollen allergy
(1, 7, 9, 15, 24). To identify and purify the major
allergenic components ofBGP.anti-BGP MoAbs
were generated in the present study. Hie Ag
niolcculcs rccotrnizcd by these anti-BGP MoAbs
were analysed and the major allergenic compo-
nents of BGP identified. '
MATERIAL AND METHODS
Pollen extracts. Dried defatted pollen of BGP, an-
nx^A Hue •gvA^^ {Poaannua), corn {Zea mays), c&n-
•Ary gi-ARs {Phalaris minor) Mvl cane {Sorghum vulgare mice were immunized by intraperitoneal in-
var. saccharatum)
) were obtained from International
Biologicals, Inc. (Piedmont, OK, USA). Pollens
werc extracted with 0.15 M phosphate buffered
saline (PBS, pH 8.0) as previously described (27).
il
Sera were collected at the Allergy Glinic, Veterans
GrcncT'il l'losDit'il" J'lichnnti f l^ixichuntf Ttnwtin, I'lybudoifia. Icch^iic^uc. Spleen cells lroni Hyperini-
ROG)'from allergic- patients with po.s'itive skin munized BALB/c mice were fused with NS-1
521
1 BGP. c furthe
basis of strong positivit>' m the radioallergo-
sorbent test (RAST), Sera from individuals with
negative skin reaetions were used as negative
controls.
RAST was performed as described by Ekra-
moddoullah ct al.(8) with some modifications,
Briefly, the BGP extract was diluted in 50
mM bicarbonate buffer (pH 9.6) and coated
onto wells (1 ng/50 ^tl) of 96-well polyvinyl
plate (Gostar, Gambridge, MA, USA) at 4°G
overnight. The remaining binding sites were
blocked by incubation with PBS containing
5% bovine serum albumin (BSA) at room
temperature for at least 30 min. Serum
Thi
plate was washed with PBS containing 1%
BSA (PBS-BSA), followed by incubation with
' "'1-labelled anti-TgF MoAb (5 x 10' cpm/
well) at room temperature for 1 h. The anti-
described (3). The reaction was stopped by
washing with PBS-BSA at least five times and
the radioactivity in each well was determined
by a gamma counter (LKB, 1271 RIAGAM-
MA, Turku, Finland).
Iodination. One hundred |ag of proteins were
labelled with 1 mCi of Na''"'I (Amersham
Corp., Arlington HcightvS., IL, USA) by the
chloramine T method (12). Free "'I was re-
nioved by gel filtration through a Sephadcx G
Uppsal eden).
Produc of MoAbs
Iminmnzation. Eight-
Eight- to 12-week-old BALB/e
jection of 100 ng of the BGP extract emulsified
omplet Freund's adjuvant (Difco, Dc-
. Ml, USA). They were boosted with 50
l" l^e BGP extract in incomplete Freund's
522 Z, N. GHANG ET AL.
myeloma c:ells as described by Rector et al.
(26). Hie fused c:ells were suspended in 200
ml RPMI with hypoxanthine-aminopterin-thy-
midine and 15% fetal calf serum (Flow Lab-
oratories, Svenska AB, Slockholm, Sweden).
One hundred nl aliquots of the cell-suspension
were added to 96-well PVG plates and ihcn
incubated at 37°G in 5% GO, for 7-10 days.
onies oVhybridoma were then harvested and
screened for anti-BGP antibody production by
radioimmunoassay (RIA)(3). Hybridomas
which secreted MoAb reaelive to BGP bul not
to ragweed poflen were chosen. While a few
cells from each positive well were cloned and
stibcloned, the rest ofthe c:ells were transferred
to a 48-wcll plale for furlher growth. 'Fhe
reaction pattern of the MoAb in culture super-
nalants against BGP was preliminarily ana-
ly.sed by iinmunobiot as previously described
(27), and further confirmed by RIP and gel
electrophoresis as described below. Only I'he
cloncs which showed different jjalterns were
saved for further study. After two rounds of
subcloning, one clone was selected from each
mother well to ensure that each clone was
from a different mother hybridoma. The
MoAbs secreted by these hybridomas were
designated by the original plate number-well
number when they were first .screened.
Hie ascites was produced by intraperitoneal
injcc:tion of hybridoma cells, and MoAbs were
purified with a protein A column (Pharmaeia)
(14).
Isolype eleterminalwn. The isotype of McjAbs was
determincd using a c;ommercial solid-phase en-
zyme immunoassay kit (Zymcd, San Francisco,
GA, USA) as described by the manufacturer.
other major grass pollens in Taiwan (canary,
'Fhe 96-well polyvinyl plate was coated at 4°C
ovc'rnighl with 1 |j.g pollen extract in 50 mNd
bicarbonate buffer (pFI 9.6). It was then salu-
rated with 5% BSA and followed by the addi-
tion of 50 |xl supernatant of the hybridoma or
NS-1 cells. After incubation at 37°G for 1 h
and washing 4 times with PBS-BSA, the bound
antibody was measured by incubation with '"'I-
labelled rabbit-anti-mouse Ig(G + M) (5 x lO'
cpm/well) (Kirkegaard and Perry Laboratories,
Gaithesburg, MD, USA).
^y,^^^^,^^,^^Va/,:o« „[ antigenic eomponents recognised by
^'"^^'^
RIP. Immunoprecipitation was performed by
the method of Ghorney et al. (4) with some
modifications. Briefly, the rabbit-anti-mousc Ig
(G + M) antibody was coupled to Sepharose 4B
beads (Pliarmacia) according to the description
ofthe manufacturer. Ten nl of a.scites fluid was
ihen added to 100 ^tl of rabbit-anti-mouse Ig
(G + M)-Sepharose 4B bead sus]3ension, and the
mixture shaken for 30 min at room tempera-
lure. Hic anlibody-bead complex was ihen
washed three times with 15 ml aliquots of ice-
cold PBS containing 0.5% noniclet P-40 (Sig-
ma). '•••'Flabelled BGP (5 x 10" cpm) was then
added to the bead pellet, and the mixture was
again shaken for 30 min at room temj^crature.
Reaction was stopped by washing the bound
immune complexes four limes with PBS con-
laining 0.5% nonidet P-40, and once wilh a
low-salt detergent buffer (2 mM Tris, 0.5%
nonidet P-40, pFl 7.4). Afier the final wash, the
beads were centrifuged through a cushion of
low-salt buffer containing 20% sucro.se, lol-
lowed by extraetion with 100 |il LaciniTili (17) or
O'Farrcll (23) sample buffer at 95°G for 3 min.
Gel eleetrophoresis. SDS-PAGE was performed
cording to the method of Laemmli (17) and IEF
by the method of O'Farrell (23) with some
modifications. The IEF gel (4.75% acrylamide,
8 M urea and 2% pharmalyte, pFI 3-10) was
radioimmunoprecipitates were isoeleetrically fo-
the control IFF gd was cut into 0.5 cm pieces
and immersed in degassed distilled water. Alter
extraction at rootn temperature for 1 h on a
.shaker, the pH of each extraet was determined,
For two-dimensional (2D) gel electrophoresis,
lhe IEF gel was equilibrated in a buffer contain-
 I LI RGl NIC COMPONINIS OF BGP DLFINLD B\ MO\BS
ing 125 mM Tris, 10% (v/v) glycerol, 4 % (w/v)
SDS, 10 niM dithiothreitol for 1 h at room teni-
]3erature, and then subjected lo electrophoresis
on 10% gel for 1000 voll-h. Low M W standards
(Bio-Rad, Richmond, GA USA) included rabbit
muscle phosphorylase b (97kDa), bovine serum
albumin (66kDa), ovalbumin (43kDa), carbonic
anhydrase (31kDa), soybean trypsin inhibitor
(2IkDa), and hen egg white lysozyme ( H k D a ) .
Following eleetrophoresis, the gels were stained
with Goomassie blue R-250, fixed, dried and
exposed to the X-ray film (Kodak X A R - 5 , Ko-
dak, Rochester, NY, USA) al -70°G. In the
2D gel electrophoresis was demonstrated by a
silver Slain kit (Bio-Rad) according to the manu-
Modified RAST The allergenicity of the anti-
genie components reactive lo the MoAbs was
by a modified RAS'F which wa.s .sirnphfied fi'om
523
kappa
the four-step sandwich RIA of Ley ct al. (18).
In brief, antigens were coated indirectly onto
polyvinyl plates by interaction with specific
MoAbs. Fifty [i] aliquots of the pre-determined
optimal concentration of MoAbs was coated to
each well and 50 nl aliquots of BGP (100 ng/ml)
was then added and tncubated at 37°G for 1 h.
After washing, sera (50 \xl) from BGP-allergic
patients or from normal persons was added and
further incubated at 37°G for 4 h. At the end of
incubation, the plate was washed again and
reacted with '^''I-labelled anli-human IgE
MoAbs (5 x 10'cpm/well) for 1 h and radioac-
^^,,^^^.^.5
Sixteen MoAbs reactive to BGP were selected
from two separate cell fusions. They arc
grouped into eight categories (Table 1) accoi-
ognized. In the first fusion, more then 50 hy-
524 Z, N, GHANG ET AL.
bridomas were generated and 10 were randomly
analysed. Il was found that seven hybridomas
secreted MoAbs belonging to (^.ategory I
MoAbs, anil ihrcc were ke]il for furlher sludy.
In lhe sc;cond fusion, even though the mice were
boosted with Gategory I MoAb-absorbed BGP,
two of the 10 MoAbs analysed still belonged to
G;itc:gory 1.
'Fhe live MoAbs in Category I formed mul-
tiplc' bands with BGP in RIP and cross-reacted
corn and annual blue) in RIA ('Fable 2). An
unexpected finding was ihal their light chains
were all of the rare lambda lypc. MoAbs ofthe
seven other categories (Categories II-VIIl), all
'Fhe MW and |)1 of these MoAb recognized
T!!!z!^z ^o:^onzz^J\.!C-
illustrates the representative 2D autoradio-
graph patterns of ihese anligenic componcnis.
Crude extract of BGP was also included for
comparison (Fig. la). As shown, Cyn d Bd67K
(Fig. 1 d) and Cyn d Bd58K (Fig. le) were
basic proteins. Cyn d Bd35K (Fig. Ig) c:on-
sislc-d of al leasl four isomeric proleins wilh ])I
ranging from 6.2 lo 7.2. Cyn d Bd68K, 48K,
3nK (Fig, 1 b) and Cyn d Bd20{)K (Fig. 1 c),
Cyn d Bd46K (Fig. 1 I), Cyn d Bd25K (Fig. 1
Table 2,
h) and Cyn d B(I12K (Fig. 1 i) were all acidic
proteins.
'I'able 4 shows the Igl'. binding capacily of
each anligenic coinponcnl of BGP using a
modified RAST. Sera from 11 BGP-allcrgic
patients were lested, bul none reacted with
Cyn d Bd200K. Among the oilier antigenic
c,oinponenls, three {Cyn d Bd35K , Cyn d
Bd58K and Cyn d Bd46K) were lound to react
wilh IgE antibodies in more than 50% of the
ihree, Cyn d Bd35K had the highest binding
capacity as well as reaction frequency (10 of
II), followed by Cyn d Bd58K (8 of 11) and
Cyn d Bd46K (7 of II). Olhers had less IgE
was < 50%. In comparison, five normal sera
i5'^^=ussioN
bodies to examine (he antigenic specificity of
BGP have found ihat BGP is unic|ue in that it
does nol share c:oinmon antigens with oilier
grass pollens (20, 21). However, in the present
sludy, in addition to specific anti-BGP
MoAbs, we have also found that MoAbs in
Category I not only recognized BGP but also
cross-reacted wilh pollens from four different
grasses. 'Fhis cross-rcactivc epitopr is only on
a minor allergen of BGP ('Fable 4). Moreover,
 ALLERGENIG COMPONENTS OF BGP DEFINED BY MOABS 525

Kahn & Marsh (16) recently reported thai A n o t h e r interesting point is thai light chains
anli-Lol p 1 M o A b cross-reacted wilh B G P to o f t h e five M o A b s in G a l e g o u 1 w c u ol l a m b d a
a cerftin deoree O u r results confirm this find- tyix-, which is rare I n BALB/c mice, the ratio
ing and, further, indic;ate that an cpilo] of kajjpa type immunoglobulins lo lambda type
epitopes oi' BC;P are shared by different immunoglobulins is about 20 1 Onl) loui anti-
pollens. gens have been reported to induce antibodies

4 B K , :iHK ri-coKiiizcd b y C a t c g c i r y I M o A b D - I J ; C) ( . > ti B d ^ O O K r c c d g i i i z o d b y C a t e g o r y II M o . \ b l l - H ; d ) Cyn d B d 6 7 K

irciurgl'.y'v'NloAb l'M(!rKU.> rf'Bd35K nloKuil-d by Cairsory \ ' l MoAb 4-:!7; I,) Cyn d \M25K .v.ognizod bv
526 Z, N, GlIANC; ET AL,
that conlain a high proportion of lambda
c hnins, namely, llie 4-hy<lroxy-3-mtiophenyl-
acc-lyl (NP) grou]), dcxtians having an allernat-
ing (1-3), (1-6) backbone, Ihe 2,4-dinilrophcnyl
(l)NP) group and ihc- 5-diinethylaminoaptha-
why all CJatc'gory I MoAbs were the rare lamb-
da lypc Is nol clear al present,
The c:omposition of BGP is complc:x, A large
nuinbcr of antigenic components wilh differenl
MW have been identified by immtmoblotting
(II, 27). In the modified RAS'F, antigenic com-
poncnts recognized by seven ofthe eight catcgo-
lies ol'MoAbs described reacted wilh Igl'. anii-
bodies in some allergic .sera. 'Fhese anligens are
presumably allergenic:. In this study, a'lolal of
nine allergens wilh differenl MW (12 - 68 kDa)
MW of these allergenic componcnis being rec-
ognizcxl were: all < 70 kDa, this is in agreenu:nt
with the restilts of other reports (11, 27).
Among the three allc:rgcnic c:c,inponcnts
whic:li reac tc:d wilh IgF antibodies in more ihan
50% of the sera lestcxl, Cyn d Bcl35K showed
the sironuesi reaction and the highest binding
lrcc|uency in the modificxl RAS'F. In an earlier
report (27), we found ihal a 32 kDa component
of BGP was the major allergen which reacted
willi Igl-, antibodies in sera from 75% BGP-
allergic palients in an inimunoblolling sludy.
other investigators. Orren & Dowdle: (25) isola-
led a 30 kDa allergen from BGP ihat elicited
positive skin lesl in 100% of BCJP-allergic ])a-
tients. Ford & Baldo (11) reporled a 34 kDa
allergen reactive wilh Igl- in all BGP-allergic
|)atients in immunoblotting analysis. Matthic-
sen ct al. (21) also found that Igf: in seni fiom
97% BCiP-allergic |5alienls reacied wilh a 32
kDa allergen in BGP. 'Flic size of lhe major
allergen oV BGP reporled by different laborato-
ries is similar lo Cyn d Bcl35K described in ihis
the major allergen remains to be solvc,:d. 'Fhe pi
of BGP-1 from Orren & Dowdle (25) was 5.4.
In conlrasl, Matlhiescn el al. (21) reported ihal
pi of the major allergenic component of BGP
was distributed from 6.0 to 6.8. Using RIP, lhe
present study demonstrated clearly that Cyn d
Bcl35K had at least four i.someric compoiienls.
 ALLLRGI^.N1C" (X)MPONENTS OF BGP DEFINED BY MOABS .527

and the pis lan^eel h o m b 2 to 7 2 I h

R E l ERENC ES
eonesponds well with the lesult of Matlhiese
et al Moiecnei it seems that lhe ma]C)i allei
n /.„//, I (Rxc
lound b^ othe

hcd RAS I

Malthicscn lound ihat tlu !2 kDa maioi BCP

alkl^cn has piccipitatcs and Ii;F btndmu; chai

^(ns (2f) In this stud\, all foui Catcqon \'f

McjAbs ^^hlch u a c t c d with C yti d Bd TiK n o
the
(d:
shemn) I lus su^^ests tl
d BdTiK IS ume|ue an
pe)llens tested

ma\ be the same as a bO kDa allel^en lound

m immunoble)t anahsts h\ Fold 6. Balelo (11)

li;l m ??% ol B(xPallel^le se , a m out pieM [I,',','.',''|,'*,,7

otts lepoit (27) loiel 6. Baldo 11 also lound a ,, , ,^, ,,,,,,,|a,,,
16 k D r alU.^enR eomponenl m B(rP whieh ,,t .Kcntuc

BCP alle
Due leehnual ost ol the
alleii;ens e)l BCP ha\e not been puitlied noi
well chaiacteii/ed With anti BCP MoAbs
axailable we hope that this pie)blem wtll soon
be solved In addition the loui uiti BCP
MoAbs m ( ate^oi) VI whieh aie agamsl the
ma|oi alleiqen i yu d BelTiK will be uselul
te5ols foi studies of epitope speeilieitN and I[?E
binding e a p a n u ol the e p,topc(s) em the ma|o,
alle^^en of BCP

ACKNOWLEDCl M F N l S
.528 Z. N. CHANC; KT AL.

17. l.aenm.li UK. Olcavagc of slrucUiral i>rolcin.s ilming 24. Olson JR, Klai)|K'r DG. 'rwo major luimai
by

18. Ley y , C;orl)i AL, Sanclu'/.-Madrid F. C:arrcira JC:. A 2115, I98(i.

dactylo,, (Bermuda ^rass) |,oll<:n. J Allergy Clin Imnumol 972, I9fi2.

22. Meyers RL, Bcriis-ma.son AW, Thayer KH, I'eldnian Address:

23. C)'Farr,-ll PH. High re.sok.tion lwo-din,ensional rice- Taipei, 'raiwan 11217 (Republic of China)