CA2109088A1 - Methods to detect and treat diseases caused by bacterial allergens - Google Patents
Methods to detect and treat diseases caused by bacterial allergensInfo
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- CA2109088A1 CA2109088A1 CA002109088A CA2109088A CA2109088A1 CA 2109088 A1 CA2109088 A1 CA 2109088A1 CA 002109088 A CA002109088 A CA 002109088A CA 2109088 A CA2109088 A CA 2109088A CA 2109088 A1 CA2109088 A1 CA 2109088A1
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
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Abstract
The invention provides materials and methods useful in the diagnosis and treatment of H.pylori induced gastric disease.
Included are polypeptides containing one ore more epitopes immunologically identifiable with epitopes of H.pylori proteins. These polypeptides are useful in assays which measure IgE in biological samples from putatively infected individuals. They are also useful for immunotherapy of infected individuals.
Included are polypeptides containing one ore more epitopes immunologically identifiable with epitopes of H.pylori proteins. These polypeptides are useful in assays which measure IgE in biological samples from putatively infected individuals. They are also useful for immunotherapy of infected individuals.
Description
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~'~
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, :
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~ `
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 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`
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 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~
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 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 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;
~~'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~'~
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, :
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~ `
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 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`
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 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~
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 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 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 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~.
:: :
~: , 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 -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 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 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 ~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.
, . .
.
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 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$.: APter 3 days a~ ~orQ luxuriant growth.~f cQloni~s:occurr~d. Thase colonie~ w~re used as :the inoc~lu~ for a~broth;~ed cultuse.
A;broth ~ed cult~ e w~ pr~pared by tran~ferring:~to several 10 ml scr~w-capped tubes 5 ml rile ~ruc~la broth with 5% horse ~ru~ (GI~C0 BRL), ,~
and colonies coll~`cted by swab ~ro~ th~ plate3~ All tu~e~ were in~ubated at 35 d~g C under ~ ~icroaerophilic : at~o~phere for ;3 ~to;5~day~ heavy degre~ of turbidity wa~ observed in th~ tub~ a~ter thi period, the culture wa~examined for puri~y by microqcopic : examination of a G~am stained slid~.
:: :
~: , wog2,,l9970 2 1 ~ ~ 0 8 8 PCI/US92/03284f~
The brot~ se~d culture was used as an inoculum for one liter of sterile Di~co 13rucella broth containin~
596 horse s~ The inoculated culture was grown in a 3 liter flask by incubation at 35 d~g C in a micro~erophilic atmosphere for 3 to 5 days. When a moderate d~gre~3 of turbidi~y was ob~erved, the culture was checked for purity as de!~;cribed above. One liter of cultur~ genEarz~lly yielded an unwa hed c~ mount of about 2 . O grams.
In order to isolat~ t~e protein allergens, the living organisms from the liter c:ulture were pelleted by centrifug~tion at 3, 000 RPM, 4 deg C for 15 minutes.
~hey were ;~ttenu~ ad and gros~ ly def atted by susp~ns ion ~nd vort~xing in ic~ c~ald ac~one f o~ lS ~inutes . The att~nuated bac:teria WZIS then r~pelle~ed by si~nilar : ~ c~n~i~ugation. T~ pal~Lat w~s r~g~usp~n~ed in 20 ml of cold buffer co~taininy 50 ~ sodiu~ pho~phate, pH 7.3, 150 ~ N~Cl, 5 ~ EDl~A, 5 D~ E~;;T~, 100 Dlicrogra~s/ml PMSF
and 100 mi~:ro~a~/ml og b~næaDIidine. ~en ~a~ or 150-210 micron, ~cid~w~h~d glas~ b~ad~ (5ig~a, St. Lo~is, M0, USA~: was ~dd-d ~n~ th~ ~usp-n~ion :~onic~t~d at se~ing ~-~
Noc, 7 u~ 400 lw~tt Bran~orl Sonif ier II ultran~onic call di~rup~or with a r~gular tip. Th~a su~p~nsion was :~ ~ 2~ u~ &oni~:a~d or :15 ~inut-s whl~ beinsl c:ooled in a harlol ic~ ba~.~ T~e r~ulting ~$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 -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 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 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 ~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.
, . .
.
Claims (27)
1. In a method of measuring IgE which binds immunologically to a bacterial allergen, wherein serum suspected of containing the IgE is reacted with an allergen extract of the bacteria coupled to a solid support, followed by washing and reacting with labelled anti-IgE, and detecting labeled anti-IgE bound to the solid support, the improvement comprising reacting the serum with a composition capable of removing IgA and IgG
from the serum prior to the reacting with the allergen extract of the bacteria coupled to the solid support, wherein the amount of composition used is sufficient to remove IgA and IgG which interferes with the IgE binding to the bacterial allergen.
from the serum prior to the reacting with the allergen extract of the bacteria coupled to the solid support, wherein the amount of composition used is sufficient to remove IgA and IgG which interferes with the IgE binding to the bacterial allergen.
2. The method of claim 1, wherein the allergen extract of the bacteria is a purified protein allergen.
3. The method of claim 1, wherein the composition is comprised of Protein A.
4. The method of claim 2, wherein the composition is comprised of Protein A.
5. A method of preparing purified protein allergen from bacteria comprising:
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing fraction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography.
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing fraction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography.
6. The method of claim 5, wherein the ion-exchange chromatography is on an anion exchange high pressure liquid chromatography (HPLC) column.
7. The method of claim 2, wherein the purified protein allergen is prepared by a method of preparing purified protein allergen from bacteria comprising:
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing fraction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography.
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing fraction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography.
8. The method of claim 2, wherein the purified protein allergen is prepared by a method of preparing purified protein allergen from bacteria comprising:
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing fraction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography, wherein the ion-exchange chromatography is on an anion exchange high pressure liquid chromatography (HPLC) column.
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing fraction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography, wherein the ion-exchange chromatography is on an anion exchange high pressure liquid chromatography (HPLC) column.
9. A composition comprised of a protein allergen prepared by the method of claim 5.
10. A composition comprised of a protein allergen prepared by the method of claim 6.
11. An immunotherapeutic method of treating an individual for a disease resulting from an allergic reaction to a bacterial infection comprising introducing into the individual a composition consisting essentially of protein allergens from the bacteria, wherein the conditions of the introduction are sufficient to alleviate the symptoms of the allergic reaction.
12. A method of determining whether an individual has an allergic response to Helicobacter pylori, the method comprising:
(a) providing serum from an individual suspected of containing IgE to H. pylori allergens;
(b) providing a composition consisting essentially of H. pylori protein allergens;
(c) reacting the serum of (a) with the composition of (b) under conditions which allow immunological binding between IgE and an allergen to which it is directed;
(d) detecting IgE-allergen complexes formed, if any, between IgE in the serum of (a) and a protein allergen in the composition of (b); and (e) thereby determining whether an individual has an allergic response to Helicobacter pylori.
(a) providing serum from an individual suspected of containing IgE to H. pylori allergens;
(b) providing a composition consisting essentially of H. pylori protein allergens;
(c) reacting the serum of (a) with the composition of (b) under conditions which allow immunological binding between IgE and an allergen to which it is directed;
(d) detecting IgE-allergen complexes formed, if any, between IgE in the serum of (a) and a protein allergen in the composition of (b); and (e) thereby determining whether an individual has an allergic response to Helicobacter pylori.
13. The method of claim 12, wherein the serum provided has been reacted with a composition capable of removing IgA and IgG from the serum in an amount sufficient to remove IgG and IgA which interfere with formation of the IgE-allergen complex.
14. The method of claim 13, wherein the composition is comprised of Protein A.
15. A composition consisting essentially of protein allergens of H. pylori.
16. A protein allergen of H. pylori coupled to a solid substrate.
17. The composition of claim 15 9 wherein the protein allergens of H. pylori are prepared according to a method of preparing purified protein allergen from bacteria comprising:
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing traction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography.
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing traction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography.
18. The composition of claim 15, wherein the purified protein allergens of H. pylori are prepared according to a method of preparing purified protein allergen from bacteria comprising:
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing fraction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography, wherein the ion-exchange chromatography is on an anion exchange high pressure liquid chromatography (HPLC) column.
(a) treating bacteria containing a protein allergen with acetone to remove lipid components;
(b) disrupting the acetone-treated bacteria in a solution comprised of buffer, salt, metal chelator, protease inhibitor, and benzamidine;
(c) separating a protein containing fraction from complex carbohydrates and nucleic acids;
(d) collecting a composition comprised of proteins which are of molecular weight at least about 1,000;
(e) separating the proteins of the composition of (d) by ion-exchange chromatography, wherein the ion-exchange chromatography is on an anion exchange high pressure liquid chromatography (HPLC) column.
19. A method of treating an individual for H.
pylori induced gastritis comprising introducing into the individual a composition comprised of a polypeptide which contains one or more epitopes that are immunologically identifiable with immunogenic epitopes of H. pylori, wherein the polypeptide is in an amount sufficient to relieve an allergic reaction to H. pylori in the individual, and wherein the composition is further comprised of a suitable excipient.
pylori induced gastritis comprising introducing into the individual a composition comprised of a polypeptide which contains one or more epitopes that are immunologically identifiable with immunogenic epitopes of H. pylori, wherein the polypeptide is in an amount sufficient to relieve an allergic reaction to H. pylori in the individual, and wherein the composition is further comprised of a suitable excipient.
20. The method of claim 19, wherein the composition is comprised of a purified protein allergen of H. pylori.
21. The method of claim 20, wherein the composition is comprised of an allergoid of an H. pylori protein allergen.
22. A diagnostic kit comprised of a polypeptide containing at least one epitope which is immunologically identifiable 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.
23. A diagnostic kit according to claim 22, wherein the protein allergen is affixed to a solid substrate.
24. A composition comprised of a structural analog of an epitope of an H. pylori allergen, wherein the structural analog binds to an IgE paratope.
25. The composition of claim 24, wherein the structural analog is an anti-idiotype antibody.
26. A composition comprised of a purified polyclonal antibody directed to a polypeptide allergen of H. pylori.
27. A composition comprised of a monoclonal antibody directed to a polypeptide allergen of H. pylori.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US69323291A | 1991-04-26 | 1991-04-26 | |
US693,232 | 1991-04-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2109088A1 true CA2109088A1 (en) | 1992-10-27 |
Family
ID=24783856
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002109088A Abandoned CA2109088A1 (en) | 1991-04-26 | 1992-04-21 | Methods to detect and treat diseases caused by bacterial allergens |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0582672A4 (en) |
JP (1) | JPH06507494A (en) |
CA (1) | CA2109088A1 (en) |
WO (1) | WO1992019970A1 (en) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5567594A (en) * | 1991-04-26 | 1996-10-22 | Enteron, L.P. | Methods and compositions for the detection and treatment of diseases associated with antigens of microorganisms |
MX9301706A (en) * | 1992-04-13 | 1994-05-31 | Oravax Inc | VACCINE COMPOSITION FOR THE TREATMENT OF HELICOBACTER INFECTION. |
US5972336A (en) * | 1992-11-03 | 1999-10-26 | Oravax Merieux Co. | Urease-based vaccine against helicobacter infection |
EP0705426A4 (en) * | 1993-06-09 | 1998-07-08 | Quidel Corp | Antigen-specific one-step assays |
KR100333113B1 (en) * | 1993-07-27 | 2002-11-27 | 시에스엘 리미티드 | Treatment of h. pylori associated gastroduodenal disease |
US6406703B1 (en) | 1993-07-27 | 2002-06-18 | Csl Limited | Treatment of H. pylori associated gastroduodenal disease |
AU3814395A (en) * | 1994-10-20 | 1996-05-15 | Genelabs Diagnostics Pte Ltd | Helicobacter pylori diagnostic methods and kits |
AU5523196A (en) * | 1995-02-27 | 1996-09-18 | Enteron Limited Partnership | Methods and compositions for production of customized vaccines for diseases associated with antigens of microorganisms |
EP0794434B1 (en) * | 1995-09-22 | 2005-04-06 | Asahi Denka Kogyo Kabushiki Kaisha | Method for the analysis of allergen |
EP1872792A1 (en) * | 2006-06-29 | 2008-01-02 | Biotech Tools S.A. | A method for the production of hydrolyzed allergen |
RU2706685C2 (en) * | 2010-02-12 | 2019-11-20 | Лабораториос Лети, С.Л. | Method of producing allergen extract |
RU2568242C2 (en) * | 2012-04-24 | 2015-11-10 | Федеральное государственное бюджетное учреждение науки Институт молекулярной биологии им В.А. Энгельгарда Российской академии наук (ИМБ РАН) | Method of treatment of blood sample at immunological analysis of allergen-specific and total immunoglobulins e |
RU2587327C2 (en) * | 2014-11-19 | 2016-06-20 | Государственное бюджетное образовательное учреждение высшего профессионального образования "Российский национальный исследовательский медицинский университет им. Н.И. Пирогова" Министерства здравоохранения Российской Федерации (ГБОУ ВПО РНИМУ им. Н.И. Пирогова Минздрава России) | Method for diagnosis for in vitro of specific body sensitisation to bacterial allergens |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5328989B2 (en) * | 1974-05-27 | 1978-08-17 | ||
US4870053A (en) * | 1980-09-19 | 1989-09-26 | Roussel Uclaf | Novel compositions and processes |
US4849337A (en) * | 1983-01-31 | 1989-07-18 | Minnesota Mining And Manufacturing Company | Assaying allergen specific IgE levels with fluorogenic enzyme labeled antibody |
DE3708198A1 (en) * | 1987-03-13 | 1988-09-22 | Hoechst Ag | METHOD FOR ISOLATING BASAL MEMBRANE PROTEINS FROM HUMAN AND ANIMAL TISSUES |
US5091318A (en) * | 1990-04-13 | 1992-02-25 | Abbott Laboratories | Binding of allergens to a solid phase |
-
1992
- 1992-04-21 EP EP19920912647 patent/EP0582672A4/en not_active Withdrawn
- 1992-04-21 CA CA002109088A patent/CA2109088A1/en not_active Abandoned
- 1992-04-21 JP JP4511878A patent/JPH06507494A/en active Pending
- 1992-04-21 WO PCT/US1992/003284 patent/WO1992019970A1/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
EP0582672A1 (en) | 1994-02-16 |
EP0582672A4 (en) | 1994-06-29 |
AU667040B2 (en) | 1996-03-07 |
AU2003692A (en) | 1992-12-21 |
WO1992019970A1 (en) | 1992-11-12 |
JPH06507494A (en) | 1994-08-25 |
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