Note: Descriptions are shown in the official language in which they were submitted.
2~l0908~
~~'0 92~19970 PC~/US92/032~4
~THODS TO DETECT AND TREAT DISEASES
CAUSED BY BACTERIAL ALLERGENS
5 ~b~ -
Thi~ inventiorl is related to the f ield of
allergens, more specif ically to the identif icatio~ of
bacterial allergens, and to their use in di~ease
diagnosis aald treatment.
1~ Background of the Inverl~ion
A number of idiopathic recurrent diseases are
of unknown etiolsgy. Some of th~se d~iseases are thought
to be linked to in5Eection by a microorgarlism. However,
the ~::au~al relationship between the microorganism and the
15 disea~e is of~en not ~stabli~hecl. The twin diss:~rder~ of
~-hronic: gastritis and pept..c ulcer disease are within
this category.
Chronic gastritis and peptic ulcer diseas~ are
disease~ o~ ma~c:r signific:ance. Five to ten perc~nt of
2 ~ll indi~ridual . d~velop chronil: gastritiæ and or
gastroduodenal ulcers in their li~etime. Ulcer disea~;e
is a eommon cause o ~orbidity~. The annual preval@nce of
s~pto~atic p~ptic ulc~r di~s~ase in t~e Unit~d State~ o~
A~erica i~ apprc?xima~ely 18 per 1, 000 adults (or
4, 500, 000 p~ople~ proxi~a~aly 350, 000 new cas~ of
p~pkic ulc~2r dis~a~e ar~ diagn~ d zach y~ar.
r3~agnosi~; of th~se diseases is u~3ually
per~orD~d by ga~tro~uoderlal ~ndosl:opy, an invasive and
05~ 1y procedure. Treat~ent e3as:~0mpa~se~ oral medication,
3 dietary control~:, and surgery . Rarely is it def initive,
and t~e~e chrollic ::onditions ot~n have cycles of
i~prov~ell~ and r~lapse.
S~nc~ l:he r~port by ~r~hall (Lance~ 1983,
3) that the bacte~i~ }~Qk~ E2~ is
W0 9211~970 2 1 0 9 0 8 8 PCI/US92/03284 '` `
physically associated with the lesions of chronic
gastritis 0 a great deal of work has been done in an
ef f ort to elucidate a causal relationship be~ween the
5 organism and the chronic diseasen Early speculations
regarding localized pH c:hanges induced by H. E2~lori, the
release of toxins (Hupertz et al . ( 1988 ), Eux J . Clin
Microbiol Infect Dis 7: 576), and destructve enzymes
(Slomizlny et al. (l989~; Am J. Gastroenterol 84:1273),
10 and the di~feren~:es between different strains of the
bacteria (Eaton et al ~l989), Inect Immun (U.,S. )
~:1119) hav~ no~ result~d in firm conclusiorls concerning
the etiology of the diseas~ . Moreov@r, ~he sez~rch f or a
r~asonable explanation of cause and ef f ect has been
15 fur1~her co~a~licated by the recognition that a signif }cant
nu~her o~ clislic~lly well subj~cts al~o carry the
orgzlnîsm .
20 ~ : C~ska et al. (1972), Radioimmunosorbent Assay
of Allerg~n~ (J. ~A11~rgy and Clin. Immunol~
de~cribe~; a Radioa1lergo~o~bent ~ST) test to d~tec:t IgE
dir~acted~ to ~p8~ific allerg~n~
Nal~bu~ et ~ l979 ), Th~ S~ud~ of IgE in the
: 25 Diagno~æi23 o~ All~r~ic Di~ord~r~; in an Ol:olarygolos~y
:: Practic~ (Otolar~ngol H@~d Neck Surg. ~ 35~), describes
ied RAS~ ~:e~t. ~ :
,
La~rt:~t al~ (~978~, E)if:Eu~e Varioliform
: Ga~t~iti~ (Dlge~tion l7 :159~, report~dly prcvide ~udies
30 showing that the lesion~ o~; c~ronic g~$triti~ contain IgE
positiv~ ~;plzl~m~ cel1s, whieh the author in~erpret as
ugge5ting as~ rgic origin f or thi~; di~ea~e .
: ~dre et~al. (~983), Evidence or ~naphylactic
R~ctions in P~ptic:~Ulcer ancl V~riolifona Gast~ is
(Annals o Allergy ~ 3Z53. The aYerage number and
:
~ g2/19g70 2 1 0 9 1) ~ ~ Pcr/usg2/o3~
class distribution of IgE-containing cells in patients
with various types of g2stritis/ulcers as compared
healthy subjects reportedly were examined. The authors
interpret the r~sults as confirmatory for the theory that
mucosal anaphylaxis may be the cause of th~ gastric
lesions.
C~lenoff et al. (1983), Bacteria-Specific I~E
in Patients with Nasal Polyposis (Arch Otolaryn~ol lQ9:
372). The authors describe the use of a modified RAST
test to d~tact Ig~ speci~ic to bacterial antig~ns in
patients exhibiting chronic nasal polyposis.
Warren (1983), Unidentified Curved Bacilli on
Gastric Epithelium in Active Chronic Gastritis (Lancet,
127~). The author reports the observation that s~all
cu ~ ed and S shaped bacilli were
: obsexved in 135 gastric biopsy speclm~ns. The bacilli
~: were most ~re ~ ently correlated wi~h i~flammation, and
wer~ almo~t alway~ pre~nt in acute chronic gastritis.
: 20: Peterson (1991), ~elicoka~ er ~Y~h~i and Peptic
Ulcer Disease ~New~England J. Med. ~?~:10~3)~ is a review
articl~ on the a~ociation between ~. ~YlQ~i ~formerly
called ~ D~ c~ and gastritis/ulcer diseas~.
:~ 25
; : $u~ary
Using a ~odified ~RAST te t, it wa~ discov~red
that thare was a high po itl~e correlation ~twe~n
: ga~triti /ulc~r dis~as@ and the pre~ence o~ Ig~ directed
ts ~pe~i~ic subfr~ctions of prot~in a}lergens of ~.
:~ Ç3~Lio Th~se re~ult~ indicate that a~ advers~ immune
~: reaction to t~e~ bact~ria is responsible for the
~: :
: '
.
~lOYU88
W~92/~70 PCT/U~92/~
pathological reaction, in particular, the existence of a
hypersensitivity reaction mediated by specific IgE~
In the modified RAST test purified protein
allergens were link~d to a solid support. Prior to
reaction with the protein allergens o ~O py_ori, the
ser~m to be tested was treated to remove IgA and IgG.
This "scrubbing" step was essential for the d~tection of
the allergen-specific IgE.
The identification of protein allerg~ns of H.
PYI~ i associated with ga triti~/ulcer disease allows for
a relatively non-invasive detection of the disease by a
modified RAST test. It also allows for treatment of the
disease by immunotherapy, using puri~ied protein
allergens.
Accord:ingly, one asp~ct Q~ the invention is
a ~ethod of ~asuring IgE which binds i~munologically to
:~ a bacterial ~llergen. Serum suspect~d o~ containing the
IgE is reacted with an extract o~ the bacteria Goupl~d to
a solid suppor~,~ followed by was~in~ and reacting with
lab~lled anti-IgE, a~d detac~in~ labeled anti-IgE bound
to~the 501id ~support, the improv ment compri8ing reacting
r th~ s~rum with a composition capable o~ removing IgA a~
: ~ Ig~ ro~ the~serum prior to the r~acti~g with the
25 : bact~rial extract~coupled to th~ solid ~upport, wherein
th~ a~ount c~compo8ition u~d i~ suffi~ient to remove
IgA~and IgG which interf~re. ~with the T~E binding ~o the
acterial all~ergen. ~: ;
Another a~pect of th in~ention IS a method of
~: 30 preparing purified prot2in all~rgen from bactexia
co~prising: (a~ treating ~acteria containing a protein
a}lergen with ac~tone~to remove lipid co~ponents; ~b~
: di~Fupting th~ acetone-treat~d b~cteria in a solution
;~ ~ : co~pri~ed of b~fer,~::salt, ~etal:~h~lator, protease
:35 inhibitor, and benazamidine; tc) separating a protein
:: :
.
- ')92/1~70 21 0 9 0 ~ ~ P~/US9~03284
containing fraction ~xom complex carbohydrates and
nucleic a~ids; ~d) colles:ting a composition compri;sed of
proteins which are of molecular weight at least about
5 1, 000; and (e) separating the proteins of the composition
of ( d ) by ion exchang~ chromatography .
Still another aspect of the invention an
i~unotherapeuk~ c method of treating an individual f or a
di ease resulting from an allergic r~action to a
10 baeterial in~e tion comprising in~rodu::ing into.. the
individual a compo~;ition consi~ting ~ssentially of
protein allerge!ns ~rom the b~cteri~, wh~rein the
condition~ f the introduction are 5uf f icient alleviate
the symptoms o~ the allergic: reaction.
Another aspect of the invention is a methc~d o~
d~t~rmining whether an indi~idual h~æ an allergic
respon~P to Helicobacter pylori, the method co~prising:
(a~ providing ~3erum ~rom an individual suspectsd of
eonta~inins~ I~E to H . pylori ~112rg~ns; ( b) providing a
20 com~o~;ition con~ ting e~;enti lly ~af ~I. py~lori protein
all@rg@n~; (c~ r~ac:ting lth~ e~ of (a) with the
::o~po~s;ition of (b) und~r c~ dition~ which allc:w
i3~su~010gic~1 binding b6~tw~aen IgE and an allerg~n to .
whic:h it i~ dir6~ t~d; and (d~ d~tec:~ing IgE-~llergerl
2~ c:o~apl~xes ~or~a~d, if any, ~b~tw@~ IgE in the s~rula o~ (a)
and a pr~@in all~rg@n :in th~ ao~osition o~ (b).
Still another a~p~c:t of the invention i8 a
~pcs~ition con~i ~ing aRs~nti.lly OE prot~in all~rg~n~E
o~ or~.
3 0 ~oth~r a~ ect of the inv~ntion i~ a protain
all~rg~n of H. wlori ce~upled to a fiol~d ~ub~trate~
Y~ anoth~r a~p~ct o~ the inttention is a m~thod
o 'cr~at~ng ~n individual ~o~ ~. pylori induc~d gastritis
6:~2~pra~ing ~ntr~sducisllg ~nto thæ individual a composition
cc3~pris~d o a polyp@pt~de whi~:h contains one or more
W~92/19970 2 1 0 ~ ~ ~ 8 PCT/VSg2~03~&
epitopes that are immunologically identifiable with
immunogenic epitopes of H. pylori, wherein the
polypeptide is in an amount su~ficient tv relieve an
allergic reaction to H. pylori in the individual, and
wherein the composition is further comprised of a
suitabl2 exGipient.
Still another aspect of the invention is a
diagnostic kit comprised of a polypeptide containing at
least one epitope which is immunologically identîfiable
with an H. pylori epitope, packaged in a suitable
container, and a means for detecting immunological
complexes formed between the polypeptide and IgE in the
biological sample, if any.
lS Yet another aspec~ of the invention is a
composition comp~i~ed of a stru~tur~l analog of an
epitope of an N. pylori allergen, wherein the ~tructural
analog binds to an IgE~parat~pe~
other a~pect of the invention i~ a
compo i~ion comprised of a purified polyclonal antibody
; directed to 2 polypeptide allergen of H. p~lo~i.
Yet another a~ect o~ the invention is a
: : composition comprised of a monoclonal antibody dir~cted~
~; to a polypeptide allergen of H. pylori.
; : 25:
: Figure: 1 ~ i8 ~ graph;showing the e~ect o~
~crubb~ng ~ru~ wi~h Protéin A on the d~tection of anti-
~ ~YlÇ~i Igl3 in a ~odified RAST te~t
3 0 Figure 2A i~ a graph sbowing ~e serum IgE
levels of IgE: directQd to subfral::tior1~ 0~ ~I- }2Y~i.
protein aller~ns in healthy indi~iduals ~controls).
Figu~e 2B i a graph showing th~ serum IgE
lev~ls o Ig~ dir~cted t~ sub~raetions of
~ 3~ protein all~rgens in gastritis patient~.
:
21090~8
V-'092~9g70 ' PCT/US92~03
Figure 3 is a plot of the net total IgE
immunological r~activity of serum from control and
gastritis patients using all available H. ~ylori protein
fractions isolated from an ~PLC DEAE column.
Figure 4 is a plot of the net total IgE
immunological reactivity of serum from control and
gastritis pati~nts with the proteins in ~ractions 59,
64, 66, 68, 72 and 74 of the HPLC DEAE column.
Mod~s for Carryinq Out the Invention
The present invention stems from th~ discovery
that individuals with chronic gastritis or gastroduodenal
ul ers have serum IgE specific for protein all~rgens of
~o py~o~i, implicating hypersensitivity to this
microorganism in the etiology of the di~ea~e~.
. ~Q~i. i5 most likely an innocuous colonizer
of the gastric mucosa.~ It dwells just beneath the
protective m~cous layer and probably feed~ ~rom it
wi~out much harm to th~ hoct or to the hos~'~ protective
d~f~nses against the ga~tric acid~ The in~lam~atory
proc~ss recognized in chronic gastrtitis results in those
individuals who pos~e~s:th~ g~ne~ic proclivity toward~
all~r~y ~n~ ~h~n ~have the n~c~ssary MHC II antigen
: ~ ~ 25 fr~work for pres~nting the Ho ~ all~xg~nic
prot~3ins as ;~allerg-n~. A qualitativQ a~ad/or quan~ita~ive
r~duction in t:he ~s~c:r~tion of protective ~ucll~ by the
gQblet c:ell probably~ occurs thus making th~a underlying
D~ucosa vuln~rable. In ad~tion, a likely increase in
loc~l hista~i:n~ productlon may tak~ plac~ in respons~ to
l:he allergic r~action and is absorbed into the vascular
plexus o~ the sto~D~ch ~ thu~ l~ading to an increase in
gastric as:id pro~ction. ~ ~hese two phenomena may
ogeth~r ~r~ult in inereased irritation o~ the early
gastric lesion~ and, along w~th ~he constant alleryic
.
WO 92/19g70 21 U 9 0 8 8 PCT/US92/0328'
r~action to H. py?,ori, lead to lasion enlar~ement and
chronicity .
Based upon th~ discovery discussed above, it is
possible to design immulloassays to detect an H pvlori
induced allexgic reaction in individuals. In one aspect,
these immunoassays utiliz~ puri~i~d protein allergens,
and are preferable to endoscopy since they may be
performed in vi~ and are relatively non-invasive. In
10 addition, the disco~ery allow~ for a novel trea'cment of
the~e cliseases; i.e,, immunotherapy with compositions
compris2d of at leas~ one purif ied protein allergen of H .
~ , and/or wi~h an allergoid o~ a protein allergen of
H. vlori.
The practice of the pres~nt invention will
employ, unless otherwise indica~ed, conventional
techniques o~ proteir~ puri~ication, microbiology,
molecular ~biology, and i~munology, which are within the
skill o~ tlle art. Such technique ~r~ explained fully in
20 'ch~ literature.
~ s uæed herein, the term "all~rgen'~ refer~ to
an a~tigen that g ives ris~ to all~rgi~: sensitization by
: Ig~ antibodies . ,.~,
~ h~term '~all~rgoid" re~rs to a chemically
~: 25 modified all~rge~ ~at giYes ri~ to antibody o$ ~he IgG
: ~ bu~ not I~E cl~ her~by raducing ~llargic ~ymptoms.
T~ er~ N~ndividual~O a~ u~ed herein, ref~rs
~:a:~rtebrate,~; particularly ~e~b~r~ of th~ ~amm~lian
~p~ie~ and includ~ but:i~ not li~ited to domeetic
' 30 anim~l~, sports anim~l~, and prima e~, including humans.
:: T~e te~ "allergyi', a~ u~d h~rein, denote~ an
:al~ered s~ate of i~mune reaativity, usually d~noting
hypersensitivity.
A us~d herein, nI~unologically identi~iable
wi~h/as" refers to the presence:o~ ~pi~ope~s~ and
'
:: .
~o g2/~9970 2 1 0 9 0 8 8 Pcr/usg2/o3284
polypeptides~s) which are also present in the designated
polypeptide(s). Immunological identity may be determined
by antibody binding and/or competiti~n in binding; these
techniques are ~nown to those of average skill in the
art, and are also illustrat~d infra.
As used herein, "epitope" refers to an
antigenic determinant of a polypeptide. An spitope could
comprise 3 amino acids in a ~patial conformation which is
unique to the epitope. Generally an epitope consists of
at least 5 such amino acids, and more usually, consists
of at l~ast ~-10 such a~ino acids. Methods of
determining the spatial con~ormation of amino acids are
known in the art, and include, for example, x-ray
lS crystallography and 2-dimensional nuclear magnetic
: resonance.
:: A pol ~ eptide i~ '~i~mu~oreactive" when it is
munoloqically reactive" with an antibody, i.e., when
it binds to an antibody due to antibody r~cognition of a
z~ specific epitope contained within the polypeptide.
unological reac~ivity may be determinad by antibody
bind~ng, more partieularly by the kinetics of antibody
binding, and/or by co~petition in binding using as ~.
co~pet~tor~s):~ known polypept~de(~ containing an
25 epitop~ aga1nsk which th~ ~ntibody i~ dir~c~d. The
t~chJiique~ ~or d~e~inins wh~ther ~ polypeptid~ i~
i~munologically reactive with an antibody ar~ known in
~: th~ art. ~n "~unore~ iv~" ~pol~peptid~ m~y ~lso be
~l~unogenic~ A~ u3ed h~r~in, the t~rm "immunogenic
30 polyp~ptid~ a polypeptid~ tha~ it~ a cellular
and/or hu~aoral i~un- r~ pon~, whethe2 alon~ or linked
to a ca`rrier in the presencçç or ab~nce o~ an adjuvant.
` :
A~ used herein, th~ ~er~ '~an~ibody" ref ~rs ~o a
ps:~lypeptide or group of polyp~ptide~ which are comp~ised
35 of at least one antibody combining eite. An 'antibody
. 2l0snss
WO92/19970 P~T/US92/032P~'~
--10--
combining site" or ~'binding domain", is formed from the
folding of variable domains of an antibody molecule5s~ to
form thr~e-dimensional binding spaces with an internal
surface shape and charge distribution complementary to
the featur~s of an epitope of an antigen, which allows an
immunological reaction with the antigen. An antibody
combinlng site may be Porm~d from a heavy and/or a light
ch~in domain (VN and VL, respectively), which form
hypervariable loops which contribute to ant7gen binding.
: A "paratope" is an antibody combining site for an
epitope, the simplest orm of an antigenic determinant.
The term "antibody" includes, for example, vertebrate
antibodies, hybrid antibodies, chimeric antibodies,
alter~d antibodies, univalen~ antibodies, the Fab
~:: proteins, and single domain antibodies.
~ he term ~pQlypeptîdel~ re~ers to a polymer of
;~ amiDo acids and does:not re~r to a specific length of
the product; thu , pept~des, oli~op~ptides, and prot~ins
; 20 ar~ included within the definition o~ polypeptide. This
t~rm also doe~ not reXer to or ex~Iude pQst-expression
: m ~ ification~ of the:polypeptide, fox example,
glyco~ylations~ a~etyl~tions, phosphorylations and th~-
~lik~. ~ncluded wiShi~ th~ definition ~r~, for example,
2~ ~olypeptide~containing one or ~ore ~nalogs of an am~no
a~id ~in~lud~n~, for exampl~, unnatural amino a~ids,
etc~), polypeptldes:with substituted linkages, a well as
her modiricat~ion~ kno~n in th~ art, both naturally
o~curring and non-na~urally occurring. ~he term
~' 30 ~polypeptid~N doe~ not conn~te th~ m~thod by which th~
: ~ ~olecul~ was~fflada, and thu~ in¢lud~s naturally occurring
: molecules, a~:w~ $~ ~m~l~culQ~ made by chemical or
recombinant sy~thesis.
A~ used herein, a "biolo~ical ~a~ple" ref~rs to
a s~mple of ti~sue or fluid iso~ated from an individual,
:
2109088
92/19970 PCT/US~2/03~
including but not limited to, for example, plasma, ser~m,
spinal fluid, lymph f~uid, the external sections of the
skin, respiratory, intestinal, and genitourinary tracts,
tears, sali~a, milk, blood cells, tumors, organs, and
also sampl~ of ~a vitXo cell culture constituents.
The term 'tcoupled" as us~d herein refers ts
attachment by covalent bonds or by strong non-covalent
interaction~ (e.g., hydrophobic interaction~, hydrogen
bonds, etc.~. Covalent ~onds ~ay be, for example, ester,
ether, phosphoester, amide, peptide, imide, carbon~sulfur
bonds, carbon-phosphorus bonds, and the like.
~ The term "support'~ refers to any solid or semi-
solid sur~ace to which a desired polypeptide. Suitable
su~port~ include gl~8~, plastic, metal, polymer gels, and
the like, and ~ay t~k~ the ~or~ of b~ds, well~,
dipsticks, m~mbranes, and th~ lik~.
The t~r~ "label" as used her~in re~ers to any
~ ato~ or moiety which can b~ used to provide a d~tec~able
: ~ : 20 (pref~rably gu~nti~iable) signal, ~nd which can be
attached to a polynu¢leotide sr pol ~ eptide.
The ~er~ ~treat~ent~ as us~d herein, refer~ to
prophylaxi~ and/or th~rapy. ~
Th~ ter~ " iDunog~nic~ rer~r~ to an agent used
to stimul~ta th~ i D une sys~e~ o~ a li~ing organi~m, so
that one o~ ~or~:~unctions of the immune ~y~e~ are
in~r~a~d and direetad tow~rd~ ~he immunogenic ag~nt.
: _ In one efflbodim-~t of the invention, an
i~dividu~les all~rgic~s~n~itiv~ty ~o ~. E~lQ~ is
dst~r~ n~d ~y deteat~ng IgE sp~cific to ~. ~}~cL
allergen~. Any ~Q~h~d ~ detecting IgÆ spee~ic for an
allergen known in the art may be used.
For exa~ple, in one m~thod, on~ or more
polypeptid~s co~prised of epi~op@s i~unologically
id~ntifiable with epitopes of allergen~ (a te~m which
,
WO92/19970 2 1 0 9 0 8 8 PC~/US92/03~ `
-12-
includes allergen polypeptides) are coupled to a solid
substrate. A biological sample su~pected of containing
IgE speci~ic for allergens from the material is reacted
with the allergen-substrate complex, and IgE that reacted
immunologically with the allergen of the complex is
detected. An example af this kind of assay is the
Radioallergosorbent (RAST) test.
Generallyt in the RAST test an allergen extract
i~ coupled to cellulose particles or paper discs.
Patient's s~r~m containing IgE antibody or a standard
seru~ is reacted with the allergen-coupled immunosorbent~
After thorough washing, labeled anti-IgE is reacted with
the immunosorbent. ~fter furth~r wa~hing, the label on
the s~parated sorbent is d~termined and i~ a measur~ of
th~ a~iount o~ p~c~fic serum Ig~ antibodies to that
a}lergen. In a preferred mode~ the ~AST te~t i5 modified
to increase its sensitivity by re~ioving IgG and/or Ig~
antibodies which~ay interfere with IgE binding to the
allergen. This ~is particularly critical when measuring
serum~IgE specific to H. ~lQ~i allergenis~ Reac~ants
~;~; capable of re~i~oving IgG and/or Ig~ ar~ known in the art,
and include, :for exa~ple, Prot@in G, anti human IgGjand~
anti-h~an IgRt a~ wall a Protein A. For convenience,
the e r~ ant~ y~b~ ~a~ix~d~ to a solid sub~trate~
inalud~ng, fox exampl~, Sepharos~ ~hQ amount of the
`~ ro ctants u~ed:is:suficient~to r~mo~ing interfering IgG
: and IgA, bu~ no~ the IgE which i8 to be det~ct~d. The
d~termination of ~h~ d~ired a~oun~ is by metho~ known
to t~os~ of ~kill in ~h~ art.
A m~th~d of removing interfering ~gG and/~r IgA
antibodies by incu~ation o~ the ~eru~ with Protein ~ is
di~cussed in th~Exa~ples, in~ra. G~nerally, the amount
of Protein A which is us~d is suf~icient to prevent the
~: ~5
~lo 9 ~ 88 ~3s 92 ~ O 3 2 8 4
- 13 -
blocking antibodies from competing with the IgE having
the same specificity.
The modi~ied RAST test may also include the use
of purif~ed protein allergens. Methods of purifying
proteins are known in the art, and include, for example,
differential extraction, salt fractionation,
chromatography on ion exchange resins, af f inity
chromatography, centrifugation, and the like~ See, for
~0 example, Methods in E~zymolo~y for a variety o~ mathods
for purifying proteins. An example o~ a purification
procedure which separate~ protein allergens o~ H pYlori
~rom carbohydrates, lipids, and nuc~eic acids i5
present~d in the Examples. Further separa~ion of the
protein allergens by HPLC chromatography on DEAE
identified subfractions o~ protein allerg~ns from Ho
pylori that bind to IgE from individuals with chronic
gastritis and/or gastroduodenal ulcers; IgEs speci~ic for
these allergens were essentially absent in normal control
individuals. Allergens Xrom these fractions would be
especially useful in immunoassays.
For conveni~nce, po7ypeptides comprised of
one or more epi~opes which ar~ immunologically
identi~iable with epitop~s of H. pvlori allergens may be
25 packaged in diagnostic kits. Diagnost~c kits include the
polypeptides in suitable containers and a means ~or
det~cting immunological compl~xes formed between the
pol~peptide and IgE in the biological sample, if any.
In ome cases~ the polypeptides may be affixed to a solid
~ubstrate. The kit may also contain other ~uitably
pac~aged rPagents and materials needed for the particular
diaynostic protocol, for example, stand~rd~/ buffers, as
well as instructions for conducting the test using the
kit ingredientsO
SUBSTITUTE SHEET
IPEAIUS
~lU~U88
~ ,.. .. .. . " .. .. .
WO g2/lg970 . ' . PCr/VS9~/~328~V`
-14-
In another embodiment of th~ invention,
individuals suspect~d of having a propensity for, or
su~fering from H. ~y1QE~ induced gastric disease are
treated with substances which reduce the allergic
r~sponse to the microorgani~m. Treatment may be with,
Xor example, a composition containing puri~ied protein
allergens, or with recombinant polypeptides or anti-
: idiotype antibodi~s which are im~unologically
id~nti~iable with th~ protein allerg~n by vir.tue of one
or ~ore im~unogenic epi~opes which are immunolo~ically
cross-reactive with those on H. ~YlQEi protein ~ller~en.
On~ or more allergens contained within DEAE frartions 59,
64, 66, 6~, 72 and 74, the pr@paration of which is
d~ecribed in Example 1, may be particularly suilable.
Treatment may also b~ wit~,.for example,
allergoids o~ YlQ~i protein allerge~. Methods of
pr~paring all~rgoid~ ~rom antigens are known in the art~
Typically~ mild formali~ or glutaral~ehyde treatment of
th~ antig~n reduce~ the allerg~nicity (I~E formation)
w~thout affecting the a~tigenic~ty (IgG '~blocking"
: antibody fo ~ ~tion~
Tr~ t~Qnt ~ay ~lso b~ with, ~or ~ample, ~.~
c~po~itio~ cont~inI~g ~t l~a~t one ~tructural analog of
an:~pitops o~ a p~ot~in all~rgQn, whiah binds to the
corra~po~d~ng IgE p~ratope. Structural analog~ ar~
organio 2~le~ul~ which ara capablQ o~ a~u~ing the
appropriat~ ~harg~:distributio~ and
~y~rophobic/hydrophili~ characterist~cs ~o allow bi~ding
to tX~ paratope in a faæhion which ~ic~ th~ im~unologic
bind~ng of th~ epi~ope.
Wh~n the ~oal is all~viation of ~he all~rgic
r~action by~im~unothsrapy in th~ for~ o~
hypo~n i~ization, the trQ~ted individ~l receives
: 35 in~ection~ o~ a aomposition co~pri~ed o~ one or more
uVo g2/19g70 2 1 0 9 0 ~ g PCI/USg2/03284
--15--
rele~ant allerqens continuously. Treatment is begun at a
dosag~ w enough to avoid any loc:al or systemic
reactions~ and frequent injections, usually once or twi~e
5 a weeJc are administered at increasing dosages until the
highest dose the patient can tolerate without excessive
local or systemiC reactions is reached. This is a
maintenance dos2, which is then continu~d at less
frequent intervals, u~ually every 2 6 week5 d~pending
lO upon the individua1 ' s re~ponse. However, th~ ~ctual
dcs~:age and treatment regimen will d~pend upon l:he
individual treated, and will be determined by the person
administering the treatmPnt.
In another embodiment o~ the invention, the
15 immunoreaGtive pslypeptides ( including al~ergens) or
structur~l analogs o~ epitopes, are prepared into
vaccines . Vacc:ines may b~ prepared f ro~ orle or more
immunogenic polypeptides. If r~combinant, ~hese
poIypeptid~ may b~ express~d in a variety o~ host cells
20: (~ g ~ bactexia, y~a~t, insact, or mammalian cells), or
~;: alternativ~aly may ~e isolated from th~ bacterial
~; . preparationsO
~: ~ . me preparat~on of vaccines whieh c:orl~ain a,~,,
im~o~enic: polypeptide~s) or structural ~nalogs of
25 e~sitop~ c:tiv~ ingxed:ient, i~ knowrl to one slcilled
artO Typic~lly, such vaccine~ are prepared as
inj~ct~ble~, ~ither ~ liquid ~olution~ or suspénsions;
~: aQlid form~ ~uitabl~ ~or solution in, or su~pension in,
liquid prior ~to~ in~ction may 21180 be pr~pared. Tlle
3 0 prgparation ~ay also b@ emulsi~ied, or ~he prot~in
. encapsulat~d ~in lipo~o~es. The activ~ i~unogenic
di~nts ar~ often ~ixed with excipients which ar~
Esh~ c~utic lly acceptable and colapatible with the
activ~ ingredi~nt.: Suitabl~ excipient~ are, for exampl~,
35 wa~:er~ saline, dextrose, glyce~ol~ ethanol, or ~h~ like
WO92/1~70 PCT/US92/03~
21 ~8~
and combina~ions thereof~ I~ addition, if desired, the
vaccine may contain ~inor amounts of auxiliary sub~tances
such as w~tting or emulsifying agents, pH buffering
agents, and/or adjuvants which enhance the e~fectiYeness
of th~ vaccine. Examples o adjuvants which may be
~ffectiv~ include but are not limited to: aluminum
hydroxide, N acetyl-muramyl-
L-threonyl-DDisoglutamine (thr-MDP), N-acetyl~nor-
muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to
as nor-MDP~, N-acetylmuramyl~L-alanyl-D-isoglutaminyl-
~-alanine-2~ 2~-dipalmitoyl-sn-glycero-3 hydroxyphosph
oryloxy)-ethylamine ~(CGP 19835~, referred to as ~TP-PE),
and RIBI, which con~ains three compon~nts extracted from
bac~eria, monophosphoryl lipid ~, ~rehalose dimycolate
and cell wall sk~leton (~PL~TDM+CW5~ in a 2%
squal~ne/Tween 80:e~mulsion. The e~ectivene~s of an
~djuvant m~y be deter~ined by measuring the amount of
a~tibodies directed aga~n t an i~munogenic polypeptide
containing an ~. ~ immunoreactive ~equ~nce resulting
fro~ administration oP thi~ polypeptide in vaccin~s which
~ are al80 comprised of the variou ~djuvants.
:~ The vaccines ~re conv~ntionally administ~r~d
parenterally, by injection, for~ex~mple, ei~her
~;~ 2~5 ~ubcutaneou ly or intramusGul~rly. Aadition~l
fornulation~ which ~re ~uitabl~ ~or other modes of
~d~inistrat~Gn:include suppo~ito~ies ~nd, i~ ~ome cases,
r~l form~lation~. For suppo~itorie~, ~raditional
bind~rs and arri~rs ~ay inelude, ~or exa~ple,
polyalXylene ~lycols or triglycerides; such supposi~ories
~ay ~ formed fr~ ~ixtuxe~ con~aining the active
: ingr~di~nt in ~he range o~ 0.5%~to 10~, preferably ~%-2%.
ormul2tion~ include s~ch nor~ally Qmployed
~xcipie~t~ a~, for ~xa~pl~9 pharmaceu~ical grad~s of
~-nnitol~ lactose, ætar~h, ~a~nesiu~ stear~te,.sodium
~Og2/19970 210 9 0 8 8 PC~/US92103~
-17-
saccharine, cellulose, magnesium carbonate, and the like.
These compositions take th~ ~orm of solutions, suspen-
sions, tablet~, pills, capsul~s~ sustained release
formulations or powders and contain 10%-95% of active
ingredient, preferably 25%-70%.
The proteîns may b~ for~ulated into the vaccine
as neutral or salt forms. Pharmaceutically acceptable
sal~s include the acid addition salts (formed with free
amino groups o~ th~ peptide) and which are ~ormed with
inorganic acids such as, for example, hydrochloric or
pho~phoric acids, or such organic acids such as acekic,
oxalic, tar~aric, m~l~ic, and the like. Salts ~ormed
with the free car~oxyl groups m~y also be derived from
inorganic bases such ~s, for example, sodium, potassium,
a~oniu~, calcium, or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, 2-~thylamino
: ~ ethanol~ histidin~ procaine, and the like,
Th~ Yaccines are administ~r~d in a manner
compatibl~ with the:dosag~ for~ulation, and in such
amoune as wiIl be prophylactically a~d/or th~rapeutically
:eff~ctiYe. The;quantity to b~ administered, which is
gen~rally in the range of about 5 micrograms to about ~50
~icro~ra~s o~ an~igen:p~r do e, depend~ on the subject to
~25 be tr~ated, ~ap~Gity o~ th~ ~u~e~t~ Lmmune sy~em to
syDthesiza antibodies,~nd t~de~ree o~ protection
d~si~ad.~: Pre~is~:a~ounts o~ active in~redi~nt requir~d
~:: g~ b- ad~inistsr~d may depand on th~ ~ud~m~nt og t~e
pract~tion~r and ~y be peculi~r to ~ach subjeet.
vaccin~ may be g~ ven in a single dose
schedule, or pre~erably in a multipl~ dose schedule. A
multiple dose schedule is on~ in which a prim~ry eourse
~: of:vaccination may ~e with l-10 s~p~rat~ do~es, followed
~y ot~er dos~s giv~n a~ SUb8Q~U~nt ti~ i~tervals
r~uired to m~intain and or reenforce the i~une
,
210!~088
W092/1g~70 PCT/USg~/03
-18-
response, for example, at 1-4 months for a s~cond dose,
and if needed, a subsequent dose(s) after several months.
The dosage regimen will also, at least in part, be
determined by the ne~d of the individual and be dependent
upon the judgment of the practitioner.
In another embodiment of the in~ention, a
polypeptide containing one or more epitopes
immunologically identifiable with epitopes of an H.
Dylori allergen are used to prepare antibodies to ~.
py~ori epitopes, using the polypeptlde as an immunizing
a~ent, and methods known to those o~ skill in th~ art.
The antibodies prepared may be purified polyclonal
antibodies, single-chain antibodies, monoclonal
antibodies, antibody fragments, and th~ like. These
: antibo~ies may;be used, for example, ~or purification by
affinity chromatography po1ypeptides of interest. More
specifically, they~can be used to purify polypeptides
containing epitope~ immunologically identi~iable with
;; zO épitopes of ~ pylori àllerqens, including the allergens
themselves.~
In:t~rn~ ant~ibodi-s to H. EYlQ~ Bpitopes may
:be used:for the preparation of:anti-idiotype antibodies.
hese~anti-idiotype~antibodies:are comprised of a region
; 25 which~mi~io8~the~epitop-~of the alle~g~n. Anti-idiotype
: ~ay~ synthe8ized u~ing mèthods known in the art, and
will u~ually~use ~ntibodieg~directed to ~. pylori
epitop-~ 8~ ~n :im~uniz~ing~:agent.
Anti-id~iotyp~antibodies may bQ useful in
im~unotherapy of~individualg sen5itive to H- EYlQ~i
allergen~,~a~ well~a~:for~ e:p~rification of and/or
detection of~antibodiè~directed to ~ PYLQ~i antig~ns
containing:~pitopeg~which~:immunologically cross-react
with the anti-idiotype~antibodies.
:
~vo 92/~9970 2 1 0 9 0 8 8 PCr/US92/03~84
--19--
The immunogeni~ polypeptides prepared as
described above are used to produce antibodiee, in~luding
pGlys:lonal and monoclonal. If polyclonal antibodies are
5 desired , a selected mammal ~ e . g ., mouse ~ rabbit , goat ,
horse, e~c:.) i5 immunized with an immunog~nic polypeptide
bearing an H. Dylo~i epitope (s) . Serum from the im-
munized animal is ~ollected ar~d treated according to
lulown procedures. If serum cont~ining polyclonal
10 antibodie to an ~. pylori epi~ope contains antibodies to
other antigens, the polyclonal antibodies can be puri~i~d
by immunoaf f inity chro~atography . Techniques f or
producing and proc~ssing polyclonal antisera are known in
the art, see for example, Mayer and Walker (1987)
15 I~OC~EMICAL METHODS IN CEI,L AND MOLECUhAR BIOLOGY
(Aaademic Press, ~ don) . ~l~ernatively ~ polyclonal
antibodies may be isolated from an individual previously
infected with ~O ~Q~,~ and purified by the methods
di~cuss~ad abo~
~noclonal antibodies direc~ed against
epitop~s ~¢an al80 b~ r~adily produced }:y one
skilled: in the~ar~ h~ gen~ral methodology for ~nakirlg
monoclonal antib~odie~ ~y hybridoma~ is we}l kn~wn. Im,~
mortal antibody-producing:c~ll lin~ can be created by
25 ~ ~ll fusion,~and al~o:by~ot~er techniques such a~ direc~
transror~atlon;~of~B~ly~pho~yt~ with onGogenic DN~, or
trans~tion~with Ep~tsin-~arr viru a ~a~, ~.g~, M.
2i~r~et al.~(l980):: HYBRIDOM~Tg~NIQUES PRINCIP~ES
~: AND ~RACT~ OE,:~E~OND EDITION (Springer-Verlag, N.Y.)~;
` H~merling et al. (1~81)~0~0C ~ AL ANT~ODIE~ AND T-CELL
RIDO~AS~; Kennett~et a1. (1980) ~ON~C~0NAL
ANT}BODIES; ~ç~ Q,~:U.S. Pat~nt~Nos. 4,341,761;
4,39~,121; 4,427,783; 4,444,887:; ~,46~,gl7; 4~472,500;
4,491,63~; and~40493,~90. :P~nels o~ ~onoclo~al
antibodi~s produced~ga~ins~ X~~ epi~op~s.can be
: ~ :
2~ 0~0~8
WO 92/19g70 PC~/U~2~328
--20--
screen~d for various properties; i.~., for isotype,
epitope af f inity, etc .
Antibodies, both monoclonal and polyrlonal,
5 whic:h are directed against H. ylGxi epitopQs are
particularly use~ul in diagnosis, and those which are
n~utralizing are useful in passive immunotherapy.
Monoc:lonal antibodies, in particular, may b2 used to
rais~ an~i-idiotype antibodies.
Anti-icliotype antibodi~s are im~unoglQbuli~s
which carry an i'internal image" of ~he antigen of the
infectious agent again5t which prot~ction is desired.
See, fo~ example, Nisonoff , P.., et al. (1981), Clin.
Im~aunol. Immunopathc~ 397-406; and Dreesman et a}.
~1985), J. Tn~ect. Disease ~:761. Techniques for
rai~ing anti idioty~ae antibodies are known in the art.
Sea, for example, G~ch (1985~, Nature ~:74; ~acNam~ra
et al. (1984~, Sci~nc~ 1325; and Uy1:dehaag ~t al.
( 198S), J. ID~nol . ;L~: 1225. These anti-idlotype
2 0 antibodi~ may also be u~ful ~or i:re~t~ent, vaccinat:ion
andtor diaçlno~i~ of ~. ~ induced ga~t:ritis andJor
g~stroduodenzll ulcer~, a~ well a~ for an elucidation of
~he im~unog~nic regiQns of ~0 ~Q~ antigens
_.
D~scribed ~lc~w ar~ ~xamplec o~ the pr~ent
~: in~antion which ar@~ pro~ided for illustrat:ive pu~oses,
a~d not to li~ the ~cope of the pre ent invention. In
light of th~ pr~senk di~clo~ure, r~w~arous embodim~nts
: ' 3 0 wi~in thQ aope o~ thQ claims will b~ apparen~ ~o those
of ordinary skill :in tha art~
:
~u~ 92/19g70 210 9 0 8 8 PCI/US~2/03284
Processing o~ H. pylori
Four grams, wet weight, o~ Il. pylori (PTCC
5 strain 4 3 504; ATCC , Bethesda , MD , USA) were cultured
~ssentially by th~ method of S~ibert. Smibert, Ann. Rev~
~icrobio~. 1978 32:67. More specifically, H. pylo~
obtained from the Am~rican Type Culture Collection, ATCC
No. 43504, wa~ removed aspectically from its vial,
suspended in 1 ml sterile Difco Brucella ~roth, and
trans~srr2d by an in inoculating loop to 3 separate
Bruaella Agar plates (Anaerobe syste~s, San Jose, CA).
The plates were incubated at 35 deg C for 5 days in a
microae~ophilic atmosphere o~ 85% N2, 10~ C02, and 5% 2
After incubation the plate~ were removed and examined.
Tiny grayish white:colonies were obs~rved. Microscopic
~: : exa~ination~of a Gram-stained smear showed large oxbow-
: shaped and loopc of Gram-negative rods (5 ~icrons), which
are typical O~ EYL9~i-
; 20 ~. ~ in colonies fro~ the 5 day plates
w~re trans~erred to:a fresh set of Brucella plates, and
the plates were:in~ubated microaerophilically at 35 deg C
for 3 ~o 5 day$.: APter 3 days a~ ~orQ luxuriant growth.~f
cQloni~s:occurr~d. Thase colonie~ w~re used as
:the inoc~lu~ for a~broth;~ed cultuse.
A;broth ~ed cult~ e w~ pr~pared by
tran~ferring:~to several 10 ml scr~w-capped tubes 5 ml
rile ~ruc~la broth with 5% horse ~ru~ (GI~C0 BRL),
,~
and colonies coll~`cted by swab ~ro~ th~ plate3~ All
tu~e~ were in~ubated at 35 d~g C under ~ ~icroaerophilic
: at~o~phere for ;3 ~to;5~day~ heavy degre~ of
turbidity wa~ observed in th~ tub~ a~ter thi period,
the culture wa~examined for puri~y by microqcopic
: examination of a G~am stained slid~.
:: :
3 5
~: ,
wog2,,l9970 2 1 ~ ~ 0 8 8 PCI/US92/03284f~
The brot~ se~d culture was used as an inoculum
for one liter of sterile Di~co 13rucella broth containin~
596 horse s~ The inoculated culture was grown in a 3
liter flask by incubation at 35 d~g C in a
micro~erophilic atmosphere for 3 to 5 days. When a
moderate d~gre~3 of turbidi~y was ob~erved, the culture
was checked for purity as de!~;cribed above. One liter of
cultur~ genEarz~lly yielded an unwa hed c~ mount of
about 2 . O grams.
In order to isolat~ t~e protein allergens, the
living organisms from the liter c:ulture were pelleted by
centrifug~tion at 3, 000 RPM, 4 deg C for 15 minutes.
~hey were ;~ttenu~ ad and gros~ ly def atted by susp~ns ion
~nd vort~xing in ic~ c~ald ac~one f o~ lS ~inutes . The
att~nuated bac:teria WZIS then r~pelle~ed by si~nilar
: ~ c~n~i~ugation. T~ pal~Lat w~s r~g~usp~n~ed in 20 ml of
cold buffer co~taininy 50 ~ sodiu~ pho~phate, pH 7.3,
150 ~ N~Cl, 5 ~ EDl~A, 5 D~ E~;;T~, 100 Dlicrogra~s/ml PMSF
and 100 mi~:ro~a~/ml og b~næaDIidine. ~en ~a~ or 150-210
micron, ~cid~w~h~d glas~ b~ad~ (5ig~a, St. Lo~is, M0,
USA~: was ~dd-d ~n~ th~ ~usp-n~ion :~onic~t~d at se~ing ~-~
Noc, 7 u~ 400 lw~tt Bran~orl Sonif ier II ultran~onic
call di~rup~or with a r~gular tip. Th~a su~p~nsion was
:~ ~ 2~ u~ &oni~:a~d or :15 ~inut-s whl~ beinsl c:ooled in a
harlol ic~ ba~.~ T~e r~ulting ~$xtura wa$ ~hen
c~n~riu~d as abo~e: and l he~ ~upe~rnatan~ sav~d.
~: 30~: Th~ supern~tzln~ wa~; ce~trifuged for 1 hx at
~1~0,000 g. 4 d~g ~, in 21 B~c:~an ~ 40Ti rotor (~ckman,
Palo ~lto, CA, US~. To ~lne resulting superna~ant was
added û.456 ~/ml o~ R~Cl ~ (~ldrieh Ch~mical Co.,,
~: Milwauk~e l WiS ., usa) . Th~ ~olution W~5 lthen centrifuged
at 4 deg C for 48 ~rs. in a Beck~nan 70 Ti ro'co~ t~he
l~UC ~J r' i '''~ a o 8 JU~ ~99~
T ~,S 92/0328~
- 23 - .
first 24 hrs at 65,000 RPM and the second 24 hrs at
48,000 RPM). The supernatant contents of each gradient
tube were collect~d in ten equal fractions beginning at
the bottom of each tube. The pellet in each tube
representing most of the residual complex carbohydrates
and nucleic acids containing in the pregradient
supernatant was discarded.
~0 Ion Exchanqe Chromato~raphy
Each gradient fraction was dialyzed against 20
mM sodium phosphate buffer, pH 7.0, at 4 deg C using
dialysis tubing with a 1,000 MW cuto~f. An approximation
the protein content per fraction was made by
spectrophotometry at a wavelength of 280 nm. Ninety
percent of the detected protein was found in ~ractions 2
through 6, inclusive; these fractions were pooled. The
pooled fraotions were then loaded onto a Bio-Sil D~AE
: analytical anion exchange HPLC column (BioRad, Richmond,
CA, USA) and a 30 minute linear ~radient run achieving
100 percent Buffer B at the end of the gradientO The
equilibrating buffer (Buf~er A) was 20 mM Sodium
phosphate, pH 7Ø The salt containing buffer ~Buffer B)
was 20 mM sodium phosphate, pH 7.0, wi~h 1.0 M NaCl. The
~luted fractions wexe collected and the protei~ o~ each
quantified as before. The ~low-through ~void) fraction
containing macromol~cules and cationic molecules was
: loaded onto a Bio Sil SP cation exchange column (BioRad)
and run under the exact gr~dient conditions as for the
DEAE run. ~he resulting eluted fractions were also
quankified for protein.
CNBr a~ctivated paper discs were made
essentially by the method of Ceska. CesXa et al., J.
.
SUBâTIT~l~E SHEEr
IPEA/IJ~
WO 92~19970 ~ 2 1 0~`~ 8~8 PCr/US92/0328~ ~
--24 ~
Allergy and clin. ImmunO 49:1 (1912). More specifically,
paper discs (diameter 6 rnm) were cut with a punch from
5chleicher and Schuell 589 red ribbon filter paper. The
5 discs were allowed to swell for 30 minutes in wat~r. t~NBr
solution ( 5 per cent in wat~r~, was added and mixed with
a mechanical stirrer for 3 minutes in a water bath at 19
deg C. NaOH ( 1 M), wa~3 added dropwise to maintain the pH
in the range of 10. 0 to 10. 5. The suspension was
10 immediately poure~l into about a ten-fold excess of cold
NaHC03 solution (5 mN, 4 deg C). After thorough mixing,
the solution was decanted. The wash with NaHC03 solution
was repeated eleven times. The paper discs then were
washed twice each with 500 ml of 259~, 50%, and 75%
15 acetone in a srraded series, followed by washing four
times with 500 ml acetone (reagent grade, 4 deg C)O They
were then placed on a f ilter paper under hood ventilation
for 3 hours for dryingt packaged with dessicant pouches
in plastic bags, and stored at -20 deg C until use.
2 0 A su~icient volume was taken from each of the
:: elution sa~ples coll~cted duxing thl2 ion exchange runs
and diluted with 50 ml~ sodium c:arbonate buffer, pH 9. 6,
~: to yield a 3 ml solution co~3taining 300 micrograms o~ O
: pr:otein,. To ~ach was~add~d 30 CNBr activated paper discs
~nd the mixtur~ wa~ n ~laced under gentl¢ agita~ion
for 4~ at ~ deg :C in order to coval~ntly couple ~he
various proteins 1:o their respecti~e disc~;O The protein
as were washed and blocked with e~hanolamine as
described by C~ka, supra.
a~lP~e
.
~ 35
-
2109088
2/19970 PCT/~S~2~03
~25-
IgE specific for H. pvlori allergens was
assayed for using a modified RAST procedure. Part of the
proceduxe was essentially as described by Nalebuff et al.
(Nalebuff et al., Otolarygol Head Neck Surg 89:271
(1981).)~ More specifically, an aliquot o~ 100
microliters of serum wa~ incubated overnight with an
appropriate allergen disc and washed three times with 50
mM phosphate buffered saline (PBS), pH 7.3, containing
O.1% Tween 20. This was followed by a second o~ernight
in~ubation with I125-labelled anti-IgE specific _or the
D~-2 deter~inant. After being washed and prior to being
countsd/ the allergen discs were placed into fresh tubes
in a gamma countsr for the amount of time previously
selected by a time control. The time control consists o~
25 units of WHO standardizatio~ IgE that i run again~t a
PRIST anti-IgE di~c for the ti~e needed for th~ IgE to
:~ bind 25,003 counts. This time is used in the counting of
:~ ~ all subsequen~ tests.
Background level~ or individual patients were
de~ermined by running each protein A scrubbed serum (s~e
b~low) against 4 blank discs, and calculating a median
value r~presen~ing tha indiYidu~l's background. Values~
twic~:this b~ckgroun~ le~el or greater were de med
25~ positive. D-termining th~ indiv~dual backgrou~d level
~ for ~ach patient inGrea~e~ th~ preci~ion of the assay,
; ~ : i~ce i~ takes ;into~account t~e v~riability corresponding
dlr~ctly to tota~l serum IgE: ~not 3ust that specific for
the bacterial allergens).
~ 3 0 A~ shown in F1gure 1, in order to detect H.
:~ ~YlQ~i IgE,- it wa~es~ential ~o s~crub the serum samples
: to r~mov~ most IgG and IgA antibodie~ be~ore incubation
with discs containing ~ Py1o~i protein al}ergens.
~:~: Scrubbing wa3 by incubation with recombinank
~ 35 Proteih AlSepharose 5Zymed, S. San Francisco, CA, USA).
:
21090~8
WO 92/19970 PCl`/VS92/0328
--26--
More speci~ically, two ml of serum per one ml of Protein
A/Sepharose were incubated with agitation for 1 hr. The
suspension was then centrifug~d at 1500 RPM for 15 min.
and the serum supernatants collected.
Th~ results in Figure 1 were obtained by taking
two aliquots of the same s~rum from a patient with
document gastritis and H. ~vlori colonization, and
subjecting one of these aliquots to tha scrubbi~g
1~ procedure. The scrubb~d and unscrubbed samples.from
equivalent amounts of serum were then sub;ected to the
remainder of the ~AST prscedure using discs containing H.
pyl~i protein allergens, as desc~ib~d above. In the
figure, the serum IgE levels detected in the scrubbed
(open squares) and unscrubbed samples (closed circles)
are ~ompared. As s~en from the gr~ph, the ~crubbed
samples allowed the binding of IgE to the ~. pylori
p~otein all~rgens which had eluted fro~ the DE~E column
with a p~ak at fraction number 66. This binding was not
2 0 d2tected in the unscnlbbed sample . A repeated assay
yield~2d similar resulte.
Te~: ~on~ec:utive gastriti5/GI ulcer patients
that ~wQre disease po itive by endo~copy~ two p~tien~s
~uthout lesions by~ ~ endoscopy, and 12 apparently
a~y~p~omatic:control patients were tested using the
~odi~ied RAST proc~dure with scrubbing, a~ described in
. Example 2.
ten:di~ease positive patients had
mea~ura~le ~uant~ties of ~. ~xlQ~ specific Ig~ in their
s~r~. Tbe two nor~al endoqcopy p~tients were IgE
~ 35 negativ~, and SiY of twelve asymptomatic control su~jects
:
.
2~Q~088 16 Rec'd PCTIPT0 0 8 JUN ~99
PCT/U~ ~, 0328
- 27 -
wera also IgE positive to some of the HPLC eluted
proteins~ As shown in Figure 2, each IgE positive
patient appeared to react differently to the various
HPLC ~ractionated proteins.
The prevalence of IgE positive react~vity
toward the individual chromatographed ~ractions for each
positivQ patient in the "asymptomatic~' and "gastritis'~
patients was examined. There were several H. Pvlori
protein fractions to which the disease group patients
reacted with greater exclusivity than the "asympkomatic"
patients. This more exclusive reactivity was with D~:AE
fractions 59, 64, 66, 68, 72 and 74.
Figure 3 shows a plot of the net total IgE
im~nunological reactivity o~ serum from control and
gastritis patients using all available H. pylorl protein
fractions isolated from an HPLC DEAE column~ FigurP. 4 is
a plot of the net total IgE immunologlcal reacti~rity of
20 ~;2rum ~rom control and gastritis patients with the
proteins in fractions 59, 64, 66, 68, 72, and 74.
SUBSTITUTE SHE~T
iPEAJUS
21~9088
r- ~J ~ r y ~
W~ ~2/~g970 ~ P(~/VS92/032
--28 -
~ndustr1al Slqni~jLcan~ce
Polypeptides containing one or ~aore epitopes
5 immunologically identif iable with epitopes o~ H . Pylo~i
proteins ( including but not limited to puri~led
allergens, recombinantly or synthetically produced
polypeptides, and allergoids~ are useful in the di~gnosis
of H. }2~, induced gastric diseases, and may alss:~ be
lQ u~ful for tr~atment of these diseases. These
polypeptides are also useful for the produc:ltion o~
antibodie~;, both puri~Eied polyclorlal and monoc:lonal,
direc:t~dl towards epitopes of p~Lori. The anti~odies,
in their turn, are u~;eful in thQ puri~ic~tion of
15 polypeptides containir~g one or more epitop~s
immunologic~lly id~ntifiable with ~3pitopes of ~. ~Q~,
proteins. Monoclonal antibodi~s, in ~articular, are
useful in the productiorl of anti-idiotype ant~ bodies,
whie:h in turn" ar~ u~;eul for the delt~ction o~ antibodie~
20 containing specl~ic: epitopes of ~. ~;LQ~, and m~y al~;o
b~ useful in the production o~ vaccines ~or E~. E~
induced di~;eas~s.
The method~; dess::rib~d herein use one or more
polypeptides containing c~ne or ~nore epi~opes o~ ~.
25 ~LlSGL9 and d~ ct IgE :di 0eted to ~. ~C.~ zlllergens.
Th~ m~thod , particulz~rIy th~ modif ied RAST ~nethod, are
u~e~ul for th~ diagnosis Of ~ 2Y;LQ~ induced gastric
~8@~ 8.
, . .
.
