AU6578286A - Hla-b 27, dna coding for it, and its use - Google Patents

Description

(CONVENTION. By one or mnore persons ar hC 1.1 ir 4 L

COMMON WE,

Paz en 11 ////llAIII IF'I

CONVENTION APPLICATION FOR A-PATENT

ltvert (in LtBEEURI-144WERKE AKTIENGES.ELLSClAFT, full Naflur cir Nametsol AppSlicant Or APIcants, of .D-355,0..arburg,,Federa.1 Republiz .of .Germany. followed by Address s).

2X Here hereby apply for the grant of a Patent for an invention entitled:" *insert Title s pf Invention. *HLA.B'' DNA QQJD.TNGF 17 AND. JTS.-USE S.0

00

*see

0 which is described in the accompanying complete specification. This application is a

*000(4) Here Isert Convention application and is based on the application numbered() oam of basi alContry or Conris and P13 5. 422 43 andP3

basic baseio for a patent or similar protection made in e .7 .e..aii dates on 28th, November 1985 and 21st December 1985

soso

ur address for service is Messrs. Edwd. Waters Sons, Patent Attorneys, Queen Street, Melbourne, Victoria, Australia.

DATED this- day of-

BEHRINGWERKE AKTI ENGE E.L$.QAE signa- ture W5 Of Alicant (s) or Seal of Cwriparsy and Stenoatures of Its Officers as) Prescribed )7y its Articese of Assoclalrn, James. Murray,,.,

Registered Patent Attorney

Form 7

COMMONWEALTH OF AUSTRALIA Patents Act 1952

DECLARATION IN SUPPORT OF A CONVENTION APPLICATION UNDER PART XVI. FOR A PATENT.

In support of the Convention application made under Part XVI. of the Patents Act 1952 by BEHRINGWERKE AKTIENGESELLSCHAFT of D-3550 Marburg, Federal Republic of Germany for a patent for an invention entitled: HLA-B 27, DNA CODING FOR IT, AND ITS USE

We, Dr. Heribert Bug, Amselweg 7, 3551 Niederweimar, Federal Republic of Germany Dr. Philipp Stein, Hbhenweg 28, 3550 Marburg, Federal Republic of Germany do solemnly and sincerely declare as follows: 1. We are authorized by BEHRINGWERKE AKTIENGESELLSCHAFT the applicant :for the patent to make this declaration on its behalf. 2. The basic application(s) as defined by Section 141 of the Act oWX made at Minchen in the Federal Republic of Germany under P 35 42 024.3 and S*..under P 35 45 576.4

Son November 28, 1985 and on December 21, 1985

by BEHRINGWERKE AKTIENGESELLSCHAFT 3 Hannelore Sz6ts, 18 Kiltrachinger Stra8e, D-8031 Gilching b) Elisabeth WeiB, 11 Dauthendey-Stra8e, D-8000 Minchen c) Christa Drner, 4 Massenhausener StraBe, D-8056 Neufahrn d) Margot Lang, 12 Geyerstra8e, D-8000 Miinchen e) Tomaso Meo, 18 Rue Quincampoix, F-75004 Paris Sf) Gert RiethmUller, 24 Hermann-Vogel-StraBe, D-8000 Miinchen a) f) Federal Republic of Germany

4X/are the actual inventor(s) of the invention and the facts upon which BEHRINGWERKE AKTIENGESELLSCHAFT is entitled to make the application are as follows: The said BEHRINGWERKE AKTIENGESELLSCHAFT is the assignee of the said Hannelore Sz6ts, Elisabeth WeiB, Christa Dbrner, Margot Lang, Tomaso eo., Gert Riethmuller 4. Te basic application referred to in paragraph 2 of this Declaration WaCX (were) the first application(s) made in a Convention country in respect of the invention the subject of the application. DECLARED at Marburg, Federal Republic of Germany this 13th day of November 1986

To the Commissioner of Patents BEHRINGWERKE AKTIENGESELLSCHAF

P okuris Prokurist ppa. Bug ppa. Stein

(12) AUSTRALIAN PATENT ABSTRACT (19) AU (11) AU-A-65782/86

(54) HLA-B 27, DNA CODING FOR IT, AND ITS USE (71) BEHRINGWERKE AKTIENGESELLSCHAFT (21) 65782/86 (22) 27.11.86 (24) 28.11.85 (31) 3542024 (32) 28.11.85 (33) DE 3545576 21.12.85 DE

(43) 4.6.87 (51)4 C07H 21/04 C12P 19/34 C12N 15/00 GO0N 33/545 GO1N 33/564 GO1N 33/577 CO7K 15/12

(72) HANNELORE SZOTS, ELISABETH WEISS, CHRISTA DORNER, MARGOT LANG, TOMASO MEO AND GERT RIETHMULLER

(74) WM (57) Claim

1. Genomic DNA and cDNA coding for HLA-B 27 and having nucleotide sequence I and II (Annex). 3. A diagnostic aid which contains DNA I or II or a characteristic part of these DNA sequences. 6. A process for the preparation of antibodies against HLA-B 27, which comprises immunization of experi- mental animals with HLA-B 27 and obtaining the serum containing the antibodies.

8. A diagnostic method which comprises bringing human genetic material into contact with a diagnostic aid as claimed in claim 3.

ND 5

105+65/31

Is@ tt iCCCTCcl&CiCciciccccilCiccACGcckccciccccccCc ic C ecciccccCCCCC ccCCTcTCc CTCTCTCCCTCCTTTCCICIACIGACCTCGCCCCCCA~~CACiTCC (y) not Are let Thr &is Pro Arm Tar Los Low Low Loa Lee TIp Cly Val Lee far Cie ,CCCCCCGCTCiCCTTcrlhiClcCCcAcCcACCciCCCcACTCiaSiTCTCCCcAGCiC~CCG ATO COG cIC &CC GCc CCC CC& &CC CIc Cic CrcOrCI T GOGOcOc CC&rC CCC CIC &CC GAG 72. IV $or Tar Irp &Is a rca act ACCTO OC CAI C CC TCCC G-ulc~cGICCGCGGQGAGAC~AACCCCCCA;-ACTGCCCTTCCCCCCCCCURC Mi two Sir not irm Tyr Th. Its Tor Sit Val Ser ir Pro Giv irt GIs Cie Pro rt the Ila iTrVal Cly Tyr Vol Asp Asp Tar Lee the Val Art Pie &is Sir ASP &is Cic TCC iTC AGOTAT TIC C&C &CC ICC arc rcc COG CCC ccc COC C44 GAG ccc COG TIC AIC ScC CIO CC TIC 01c GC C iCC cia TIC CIO &CC TrC CAL4CC CiCc CC

Ala Sir Pro art cie Ci. Pr. Art Ala Pro trp Ulu ci. Cie Civ Pro Ol. Tyr Trp Asp Art Gls Tir cis Ile Cys Lys &to Lys Ala Gas Tar Asp Are Cis Asp Leo ccc cc cc Ccc iCA Ia cAGAc4 ccccC AG TAT Icc GCi ccc CIO icA Cic SIC ic C CCC ccc ACiTGAG i Cia icr cia AcGcc c CCi ccc ACcc CA AGAG

Arg lTr Lou Lev art lvr Tyr t GIs Sar Cie Ala 0

Asp Tyr li. &Is Lev As@ cie IV Sor lie Tor Loa cAN Amm, Not lT Cir C~3 Asp Val Cly Pro Asp Cly Ari Lot Lot arm Cly Trr Its GIs Sip Ile Tyr Aip Gly Lys Cccrc iCOci &10 TT CC C rcr c&C iCC crc ca &ATr T Oc GAc GTc GOO ccc Ac COG ccc dc cic ccc CCU Tic ciC Cia Cic ccc Tic GAGC ccusCAT Tic iC Sap Los Sir $or irpTrAlaSp Tarliii i i 'is a Liv Arm &in Tyr Lee GIs Cly Cii Cy. Val Ca. GIs ila Tar GI Art Lys Tip Civ Ale Ala Arm e Is i Cie AC CIO ic icc icc iCC ccc cc s c cO C C cI ACCcc cia ccc cCC Ccc 1c GcAG icC rcccc cCIO CIa CI crc ic ccicT cr CG cu rCO Ai Gcc

IrpLow irn art T Lot Ci. ila Civ Lyi Ole Tor Los Ole irg its 1 S4 Tad cIc CCC ici Tic CIO 0ia SAC ccc iAc c&a Acc cIO cC CCC cc a GYiCCiCCCCAIOOCCCCTTCccccccccAcriiidAicCCCCTCCCTcrC~CCACCicCACuieCC~AirACCcirc few.

i#is Pr. Ile $or Atp is Ci Ala Tkr Low Ais Cya Ip AI& LevGil Pue lTr Pre ip Pro Pro Lys Tor ma. Vol Tr Ale 216. ucicAICCiiiCCCCCtlaiiiilcrGCiCtlCCCircia AC ccc CC& Aid &CA cic cIa &CC cic cic CCCiiC ICI GiG CATcic ccc iCC CIA 0ac Oc Too cc CIa COX TIC Tic CcT AI*Cie Ile lTr Lsa Tar lip OleiAg Asp dly Cie Asp CIOio Ol Asp i Tor Ole Low lei Cie Tar irt Pro Ala Ct. *so Art Tor Pue 41% Lys lip Ala Ale Val lot 22TS acO cic ATCIC CIO cAC icc iC CO cccT ccc GAG ciC CAS ACT Cia GCc icr ci4 crr cTc cGAG iCC 4a CC& CC& CC& CAT ICA &CC TIC Ci. bi Icc CC& Oct ci0 cIG

lei Pra Sir Civ Cie Cie cii Armt T ar cyi Bi Val Cis Its Ole Ci, Li. Pro Lys Pro Lii Thr Lev irt irp A to".

11Val AaCiv Lou Ala Val Lee &Is Val Val Vi Is Gil Is Pre Sie Sat GIs Sir ltr Val Pre lie Val Civ iy &CCACacdlICCCclciOCCdcCCTCCCITdiccTiCCW SO ccc icr rcc CIa rcc icc crc CCC Arc crc cc &IT tT ;Ci cc ctc Oct Gc ci SCcCIS Gic arC SIC COS, Ala Val Val lie Val Not Ci If: art Lys .14e Set 4 ccT cIC crc CCT ccr CIC Ala ICI iCC Acc ASe iCC ici a cTiCCCilCCCaTcuCCCCTCCccTCTChcTTTiCTTCcciCTCCCcCTTcCcCCCCCTiACTTCTCCCTccSTictcciic:a

IV Giv Lys Civ cay $ir lT Sir as &IiC 74TCICCTcccmCCrCccicCicGOciitIICTTCCCiVIS OT CCMi44# 444 004 ic Tic ICY CIO aCT GCc I OTAriCTlcurccTCaCccitcaccail~cTciCCCiACCdCraTccCTcidrCCcAC ye Sir imp; Sir Ala cas civ St isp ati $or Lou Iii its STOP 7C7CCrCCCGCCTcTcACmdCGTClCTITTTTCTTCTCCIC CC &CC CAC ACT ccc cia ccc ICI cET CIO ICY c ciCcS iCi Sag iii crCAGiTCarCCCeTCrcrACicccCeCCCCcclcrT~

ACdTICldlclClCCClCclicdiccillil1icccClCCCCriFTITCSACIIOACCTCCCACCCTITITTCICIT~ci~ttaac~ccccCC~a

ACCCICICAYCldlc CciuCIclCmClC -CCCTTTTCCCAIACTCtTCASAOCCCGACCICYIITT&C~AGCSTACCACY~ATCCATSA

(11) AU-,A-65782/86 Annex II: cDNA sequ Ince (coding strand)

GGC TCC CAC TCC AIG ASS TAT TTLC CAC ACC TC GTG TCC CGC TTC ATC ACC GTG GGC TAC GTG GAC SAC ACG CTG TTC GCG AGT CCG AGA GAG GAG CCG CGG GCG CCG TGG ATA GAG GAC CGG GAG ACA CAG ATC ISO MAG GCC MAG GCA CAG ACT

CTG CTC CGC TAC TAC AAC CAS AGC GAG GCC GG TCT CAC 110 TGC GAC GTG GGG CCG SAC 555 CGC CTC CTC CS C GGG TAC 130 MAG GAT TAC ATC GCC CTG MAC GAG SAC CTG AGC TCC TGS 150 CAG ATC ACC CAG CGC MAG TGG GAG GCG GC CGT GTG GCG 170 GAG GGC GAG TGC GTG GAG TGG CTC CGC AGA TAO CTG GAS 190 CGC GTG GAC ccc CCA AAG ACA CAC GTG ACC CAC CAC CCC 210 CTO AGG TGC TGG GCC CTG GGC TTC TAO COT GCG GAG ATC 230 GGC GAG SAC CAA ACT CAG GAC ACT GAG CTT GTG GAG ACC 250 TTC CAG AAG TGG GCA GCT GTG GTG GTG COT TOT SMAGAA 270 GTA CAG CAT GAG GGG CTG COG MAG CCC CTC ACC CTG AGA 290 ACC GTC CCC ATC GTG GGC ATT GTT GOT GGC CTG GCT GTC 310 GCT GTG GTC GCT GCT GTG ATG TGT AGG AGG MAG AGC TCA 330 ,340

TCT CAG GCT GCG TGC AGC GAC AGT GOC CAG GGC TCT GAT GTG TCT CTC ACA GCT TGA AMA GCC TGA

105+6 5S/

Form COMMONWEALTH OF AUSTRALIA

PATENTS ACT 1952-69

COMPLETE SPECIFICATION (ORIGINAL)

Class Int. Class Application Number: 9 579./

Complete Specification Lodged: Accepted: 0 Pe Published: ric:ity:

*.Rojated Ar;

aJme of Applicant: BEHRINGWERKE AKTIIENGESELLSCHAFT

dfress of Applicant: D-3550 Marburg, Federal Republic of Germany

oActual Inventor: HANNELORE SZOTS, ELISABETH WEISS, CHRISTA DORNER, MARGOT LANG, TOMASO MEO and GERT RIETHMULLER

EDWD. WATERS SONS, Address for Service 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled:

HLA-B 27, DNA CODING FOR IT, AND ITS USE

The following statement is a full descri.-tion Jf this invention, including the best method of performing it known to US

BEHRINGWERKE AKTIENGESELLSCHAFT HOE8/B023 K Dr.KL/mu

HLA-B 27, DNA coding for it, and its use

HLA-B 27 is a human antigen of class I among the human histocompatibility antigens. An increased occurrence of HLA-B 27 is regarded as a high risk factor for a group of rheumatic diseases, of which ankylosing spondylitis is the most important (Thomson, Theoret. Pop. Biol. (1981) 168-208).

It has been possible, by use of a suitable probe (from the 3'-untranslated region (Cosman, et al., Nature 10 295 (1982) 73-76) of an HLA-homozygous B cell line (R.A. Gatti et al., Tissue Antigens 13 (1979) 35-44)), to iso- late, identify and sequence the cDNA and the genomic S DNA for HLA-B 27. The nucleotide sequence is shown in the annex. Annex I shows the genomic DNA (coding strand) 15 and, over the exons, the amino acid sequence. Annex II shows the nucleotide sequence of the cDNA, which corres- ponds to exons 2 to 7 of the genomic DNA.

On the basis of these established DNA sequences, the ex- pert is able to use customary methods of chemical gene synthesis, for example by the phosphite method, to repro- duce the gene structure. It is also possible, knowing these nucleotide sequences, to synthesize probes and use them to obtain in a known manner, from a suitable cDNA "25 bank or genomic bank, the cDNA or the genomic sequence. In the case of chemical synthesis, it is possible to make use of natural cleavage sites (Annex III).

The DNA which has been obtained in this way can be in- corporated in a known manner into a vector, for example into one of the known and commercially available E. coli vectors such as pBR 322 or pBR 325, or the pUC vectors such as pUC 8, 9, 12, 13, 18 or 19, and be amplified in a suitable host, for example E. coli. The reisolated DNA can then in turn be used as a gene probe ("full length probe") for diagnostic purposes. The invention thus also relates to the use of the DNA as a diagnostic aid and for the production of a diagnostic aid and to a diagnostic method in which isolated human genetic material is exam- ined for the presence of a gene mutated where appro- priate coding for HLA-B 27, or the corresponding mRNA.

The invention furthermore relates to the use of the DNA for the preparation, by gene manipulation, of HLA-B 27, entailing, on the one hand, incorporation of the coding DNA in a manner known per se (European Patent A 0,090, 505) into an expression vector, and introduction of the latter into a suitable host cell or, on the other hand, direct introduction of genomic DNA into a eukaryotic 15 host cell, in each case expression there of the poly- peptide, and, likewise by methods known per se, isola- tion thereof. It is also possible in place of the iso- mo* lated genomic DNA to use an EcoRI fragment 6.5 kb in size or a corresponding clone from the gene bank used. S0.20 It is furthermore possible to employ the vector systems which are of proven use for eukaryotic host cells, in particular viral vectors or vectors specifically pro- duced for this use in yeast cells.

The HLA- 27 obtained according to the invention can be used as a diagnostic aid, for example to establish whether a human serum contains antibodies against HLA-B 27. Thus the invention also relates to a method of diagnosis for establishing the presence of antibodies against HLA-B 27, which comprises bringing a human serum which possibly con- tains these antibodies into contact with HLA-B 27.

The HLA-B 27 obtained according to the invention can also be used as antigen for raising HLA-B 27-specific antibodies. For this purpose, experimental animals such as mice, rabbits, sheep or goats are immunized in a customary manner, and the serum containing the antibodies which have been formed is obtained in a manner known per se.

The antigen is used in the following way to prepare mono- clonal antibodies: a cell producing antibodies against HLA-B 27, for example a mouse cell, is fused with a my- eloma cell, and the fused cell hybrids are cultivated in a known manner (for example by the methods described in U.S. Patents 4,196,265, 4,474,893 or 4,451,570).

The polyclonal and monoclonal antibodies thus obtained can be used in a manner known per se for diagnostic aids, the customary methods being used for this purpose. Thus, for example, it is possible to bring human serum into con- tact with these antibodies and thus establish the presence of increased amounts of HLA-B 27. Thus, it is possible to use human nucleated cells, preferably lymphocytes, in a 15 customary cytotoxicity test or a binding test which makes o* use of the antigen-antibody reaction, with the aid of a

marker, preferably a fluorescent dyestuff or an enzyme, to detect HLA-B 27.

Thus, a wide variety of diagnostic options is provided by the invention for establishing the presence of HLA-B 27 and the gene which codes for it. Hence the invention is an enrichment of the diagnostic options relating to the human antigens of class I.

Example 1

Using the vector pTCF (Grosveld et al., Nucl. Acids Res. (1982) 6715; Gene 13, (1981) 227), a genomic bank was set up from peripheral white blood cells from a healthy donor of HLA type HLA-A2/2, -85/27, -Cw2/3. This cosmid bank was probed with a cDNA probe specific for the HLA-B locus and derived from the 3'-untranslated region of the HLA-B 8 gene. DNA sequence analysis of 3'-Bgl II frag- ments which contain the entire HLA-B 27 gene and were subcloned in the vector pUC 13 was carried out essentially by the dideoxy method of Sanger, and only partly, in order to identify doubtful nucleotides, by the chemical sequencing method of Maxam Gilbert.

Mouse thymidine kinase-negative cells (tk cells), LD 1 (H-2k) were transfected with the cosmid DNA and the herpes tk gene contained in the vector pOPF (Grosveld et al., Nucl. Acids Res. 10, 6715) using the calcium phosphate transfer technique (Wigler et al., Proc. Natl. Acad. Sci. USA 76 (1979) 1373). Populations of transformed cells were se- lected by hypoxanthine/aminopterin/ thymidine selection (HAT), and stable Ltk+ transformed clones were identified. The human lymphoblastoid cell line LG-2 (HLA-A 2/2, B 27/27, Cwl/1) was selected as the positive control for HLA-B 27 expression. S **0

15 Example 2

9

To obtain polyclonal antibodies, experimental animals, for example rabbits, were immunized with HLA-B 27 prepared according to the invention. It is possible for this pur- 20 pose to couple the antigen by known methods to a carrier, Sfor example a protein, in order to intensify the immune response. The antigen, as such or bound to the carrier protein, can be injected into experimental animals, for example intravenously, intramuscularly or subcutaneously, together with an adjuvant, for example a silicate, an aluminate or an oil-containing adjuvant such as Freund's S. adjuvant, to obtain an improved immune response.

Example 3

a) To prepare diagnostic aids for the cytotoxicity test, the polyclonal or monoclonal antibodies which have been obtained according to the invention are diluted with a buffer solution, which contains a protective colloid, in such a way that their reactivity in the NIH cytotoxicity test is optimal.

b) To prepare reagents ready for use in the cytotoxicity test, the polyclonal or monoclonal antibodies obtained according to the invention are bound by adsorption onto plastic test plates, for example Terasaki microtest plates.

Example 4

To prepare diagnostic aids for the binding test, the poly- clonal or monoclonal antibodies obtained according to the invention are coupled to a fluorescent dyestuff, for example fluorescein isothiocyanate (FITC) or tetramethylrhodamine

isothiocyanate (TRITC), or to an enzyme, for example pero- Sxidase or alkaline phosphatase. I.

Example

To prepare diagnostic aids for detecting HLA-B 27 in human serum, the polyclonal or monoclonal antibodies obtained 20 according to the invention are bound by adsorption onto plastic test plates, for example microtiter plates. The enzyme-coupled polyclonal or monoclonal antibodies pre- pared as in Example 3 are uted to produce a test system, for example operating on the ELISA principle.

Example 6

To prepare diagnostic aids for detecting antibodies against HLA-B 27 in human or animal antisera, the HLA-B 27 prepared according to the invention is bound, for example by adsorp- tion, onto the surface of plastic test plates, and the anti- body against HLA-B 27 which is to be detected is determined by an antibody, which is enzyme-coupled for example, against human or animal immunoglobulin or IgG, for example by the ELISA principle.

noit Art Thr &Is Pro Ara Tar Lou Low Lou Lee Lev 7rp GAY &14 04L Ala Lou lairGls ICCCCCCTCCCACITCTALACICCCCACCCACCCICCCGG*CIC£CAATCTCCTCAGCCCCAC ATO CCG CIC ACC CO CCC COS &CC CdC IC C CTC CIC TOGCC GCGG CCCGC CIG &CC CAG Sao

OTQ6 ACLCC1T06OCT CCCTC 7CC 720AGGCAQGACAGCGGGCLCACG 2

It@ $mr Not LAr Tyr Pas @is Tar Set P.1 Sat Ara Pro GAY hra Cly Ci Pro Ara the Its Tar Val Cly Tyr Val &up Lsp Tar Low PA. l An.-Ph. &IS Ser &to Ala C ICC £76 AGOTAT TIC CAC &CC TCC 0T0 ICC CCO CCC: COG CCC CCO GAG CCC CGC TIC AMC &CC GIG CCC TACG CACC GC &CC CTG TIC GIG &GO ITC CAC ICC CAC CCC oil

&to See fro art Cl. Gls Pro Art &is fro trp Ile Clv Cl. Ci. ly Pro GIs Tyr Irp Lsp Ara GIs TierGIs Ile Cyi Ly's&Is Lys#to Cis Thr asp Art Cie asp Lou CCC SGT CCC &4A GAG GAG CCC CCU CCC CO ICC AT& GaO CL8 GAG OGG CCC GAG TAT CCGG CCCO GAG &CA Cat AIC TOO LAG CCC LACG C CAG ACT CatO CCLGA GAC C7C 415a

Lrt Tar Low Lev Avg Tyr Tyr Aso Cie Set Gas has 0 C~rCC GCOCCGCCCTAC C C~CIC AgCC GACCC a IMPCGTGGCSSTCCGGTGTCGGCGC

IV Sir Nis Tar Lev Cie Ana not Tyr GAY Cis ago Oal GAT fro asp Cly Art Lou Los Art Gil hT Sa Gs Sip Ala Tyr Asp Cli' Lys Asp lTr Ile Ala La hIsmClu CC ICT Cat &CC GIG CgO ALT £70 TAT 0CC l4G GAG O70 000 CCO GACGGCC CCC GIG CTC CCC CCU TAC Gat CAG Gat CCC TAG Gat G C LAG CAT TaG LTC CCC CICG AG a 1)

AsP Lev Sir Ser lrAi Tar Ala Asp Tar Ala Gle Ile Tar Cie Art Lyl Irp Clu Ala Ala Art Vli Ala Clu Gin Lou Art Ala Tyr Low Clie Civ Civ Gys Val Cis GAO 010 AGC ICC TOO &CC ctC CCC GAG &CC a" 001 GAO AIC &CC GAO CCC LAO TCC GAGLC CCC CG C GTO C GAG CAG GIG &A GCC TAC GIG GAG CCC GAG ICC 010 GAG AOss

a Irp Lee art ra Tyr Levis Aso Cl Lys Glv Tar Lou CIs Lrt a A ICC CTC CCC &CA TLC CTO GAO AAC 0CC LAO 040 AGO GIG CLO CCC 0CC C CTLCCACGGCCAGTGCGCCCTICCCC&TCTCCILIAGGCCCGGGATCCCCICCCCCACAGACGALAA&TCGAG IS416

Cr so Pro Pro Lys Thr Val Tar Big Nis Pro Ile Sir Lip Ike CII &II,Tar Lee AS Cis Irp At& LouClGA thetr Pro a) AGAGATGCSAAGGGCIGAIITCTGACICTTCCGATC~a hC CCC CC& &CA GAO 670 ACC CIC CAC CCC LbC TGI GAG CAT GAO CCC AG O I AG 0 CC TOG GCC CTGI CCC TIC TAO CCI 2164 Ul his Cay ile Tbr Low Tbr Irp Gls Art Asp Gly Cie Asp Cas Tar 01. Lsp Tar Cii Low Val Clu Tar Art Pro Ala Gly Asp ha Tar PbS GIs Lys Irp Ala &is Vli gas CU GAO 11C &CA GIG LOG TG4 GAG CC4 9AT GCC 0AO GAG GAL ACT OLO rat ACT GAO MI GIG GAG &CC MC CC& CCA, CC& GAT ACA ACC TIC CG hAG TCO CC& OCT GIG GIG 2270

Trp G 4 Vaa Pro Sir Gl Cie Cli Cl. Srt Tyr Iar Cy. Ile let Gie Nis Civ GAY Low Pro Lys Pro Lou Tar Lou Art Sole 0GIG TC CCTIC A Gag GIG GAt AGA TAC ACA TOC CAT 0TA CAG CAT GaO 000 CIG CCC AAC CCC GIG hCC CIC AGA ICC 6 G&AGCACCGGGAICLGGCCTCLTATCTOTGCCGGAAAGC

Is Pro Sir let Cis Sir Tar gla Pro IlieVli Cly Jim Val Gly Low Ala Val Low Ala lot Vl Val Ilv GIAl &G CGO ICY ICC CAO ICC LOG GTC COO LIC 010 CCC LII 611 CCI GGC 016 CCI GIG CIA @CA CII OC I 610£1 CC& 2526 AcCCGCTICGCLCcGTCLcGCCCCCTTCTTCCCTtCOITCGC,

z Ala fl Vli Ala Ile IA not Cyi Ara Art Lys Sat let 8 C CCIOTG1 TGT iC C ASAC SOC ICS C CTLCCCAACCCGIGLGCGTGGCCGTCCGITTICTCICCC&CTCCGCGTIICLAUCCCCACCTACAAGICTTGCCCCCATTACTCGLCSa 2663 CCI GIG GIG CCI

AL6 cCCTAGGccACcIICCAGGAOCCAOACITGI CCICGIGIIICCIGLICCCCIGCGTCIGAGCCIACTIGICGAAATICCTITTGGCICCACACCACGAGIICCTLCAGITCACCCC SP', C OC

Iv GAY Lys Gil GAY Sur Tyr Sur GCs&t Alt*i C Q; ICITCCTCCCACICCCCTCACAGG£AAITIGITTCCC£AG 61 CCL ASS GOA 000 AGC TAG IGI CAC CTCI60T GIA&GTATCGGCGICCOIICGaGCCACGGACCCCCIAITGGTCCTCTCCCGC 3103

is Sur Asp Sir III, Cln GAY Sor Lip Vl Sir Low Thr STOP 3235 TCICCCCGCCTCICACCACGICCIGTITITIICICIACICCAC CC AGC GatAOC, CCC Cat CCC 101 CAT GiG 101 dIC &CA CCI ICA ALL 0 CICACATTIICGCTCTAGCCICCCG000CCGGICI

CA TATCC CTCTGGITTCGCCGTCCCTASTT&IATACTAIACGSTCTAGCGTIIC~& 3635 CAACTCGLTAIAAAITOTIZC1C

0 CTCGGGTCICCA A ACCdICICCCCCGdCA8 Go ItGGCI$IGCCAI1CIG7GAICC ICA I GGCT IATICIGGI IAG ICC ItCACGCGCTCLCCTTCACICCICCITCTOICCT I GCCCIICACTCCLAAACGTICGCC .0 a a 0* 0* va 1S ACCC0ACCCICCAAGCCCCL a *0 iv. a.iv v .0 i iy'a

Annex II: cDNA sequence (coding strand)

GG C TCC AGG TAT TTC CAC TCC CCC GGC GG CCC CGC TTC GGC TAC GTG GAC AG AGG TTC GAG GGG GCG AGT GAG CCG CGG GCG TGG GAG GGG GCG TGG

GAC CGG ATC TGC AAG GCC GCA CGA GAG GAG ACG 100 CTG CTC AAC CAG AGC GAG GGG CTG CAG AAT GG C 120 GAG GAC GGG CGC CTC CGG GAG GAG GCC GG C 140 GAT CTG AAC GAG GAG AGG GGG GG GAG GCT 160 ATC AAG TGG GAG GCG CGT GAG CTG AGA CTG C :TG 180 GGC GAG TGG CTC CGC TAG GGG AAG GAG GAG

200 GTG AAG ACA GAG GTG CA C TCT GAG CAT ACG 220 AGG CTG GGC TTC TAG GG CTG ACG TGG GAT 240 CG GAG CAG GAG ACT GAG GTG CCA GCA GGA AGG 260 CAG GCT GTG GTG GTG TCT CAG AGA TAG CAT 280 CAG CTG CCG AAG CC ACC GAG GCG TCT TCG 300 GTC GGC ATT GTT GCT CTG GCA GTT GTG GGA 320 GTG GTG ATG TGT AGS AAG GGA AAA GGA TAG 330 340 TCT CAG GCT GCG TGC AGC GAG AGT GCC CAG GGC TCT GAT GTG TCT CTC ACA GGT TGA AAA

GCC TGA

Annex III: Cleavage sites in the cDNA

Endonuc Iease Cleavage site after nucLeotidle No./tripLet No. (coding strand)

Taq I 107 36 H in f I 123 41 SgL II 194 gL II 318 106 S. Kpn I 339/ 113 0* *5 Sac I 394/ 132 S. S S. Sau 3 A 422/ 141 f. **S 8 gL I 454/ 152 0* J 5 S Pvu II 465/ 155 0O *5 p P st I 539 180 0**e Sau 3 A 635 212 Pvu II 736 246 *S 5555 Sac I 937 313 Sd S S.

S S 555#SS S

S.

5* S. S~

THE CLAIMS DEFINING THE INVENT[ON ARE AS FOLLOWS: RXXXXXX'MXX

1. Genomic DNA and cDNA coding for HLA-8 27 and having nucleotide sequence I and II (Annex).

2. A process for the preparation of HLA-B 27, which comprises expression of the DNA of nucleotide sequence I or II (Annex) in a host cell.

3. A diagnostic aid which contains DNA I or II or a characteristic part of these DNA sequences. b0

4. A diagnostic aid which contains HLA-B 27.

5. A diagnostic aid which contains an antibody against HLA-B 27.

6. A process for the preparation of antibodies against HLA-B 27, which comprises immunization of experi- mental animals with HLA-B 27 and obtaining the serum containing the antibodies.

7. A process for the preparation of monoclonaL antibodies against HLA-B 27, which comprises fusion of a ,o cell which produces antibodies against HLA-B 27 with a Smyeloma cell.

8. A dia]nostic method which comprises bringing human genetic material into contact with a diagnostic aid as claimed in claim 3.

9. A diagnostic method which comprises bringing human serum into contact with a diagnostic aid as claimed in claim 4.

A diagnostic method which comprises bringing human serum into contact with a diagnostic aid as claimed in claim DATED this 26th day of November 1986. BEHRINGWERKE AKTIENGESELLSCHAFT EDWD. WATERS SONS PATENT ATTORNEYS MELBOURNE. VIC. 3000.